&EPA
United States
Environmental Protection
Agency
National Health and Environmental
Effects Research Laboratory
Gulf Breeze, FL 32561-5299
Research and Development
EPA/600/S-97/007 April 1998
ENVIRONMENTAL
RESEARCH BRIEF
Development and Application of Protocols for Evaluation of
Oil Spill Bioremediation
J.E. Lepo1 and C.R. Gripe2
Abstract
Test systems that simulate oil slicks on open water or oiled
sandy beaches were developed to test the effectiveness of
commercial oil spill bioremediation agents (CBAs). Gravimetric
and gas chromatographic-mass spectrometric analytes (e.gv
selected n-alkanes, isoprenoids, and aromatic compounds) were
used to provide efficacy endpoints for comparing CBA-treated
test systems with untreated control systems. The resulting test
systems, and protocols for their use, .were evaluated using a
variety of CBAs. Aquatic chronic estimator toxicology tests
provided information on the environmental risks posed by the
bioremediation agent itself as well as by the effluent,from CBA-
treated test systems. Selected CBAs produced only minimal
losses of analytes in the open-water test system after 7 days
and somewhat greater, losses from the beach test system after
28 days. The use of a positive control consisting of selected oil
degrading bacteria and nutrients enhanced degradation of cer-
tain oil components. The environmental safety protocols were
also tested with a variety of CBAs; their intrinsic toxicity was
relatively low (>75 ppm), and effluent exiting open-water test
systems in which CBA and oil were allowed to interact was
toxic for only one out of six products. A variety of research
topics related to the development of CBA test system protocols
were also investigated.
'Center for Environmental Diagnostics and Bioremediation, University of West
Florida, Pensacola, FL 32514
HJ.S. Environmental Protection Agency, Gulf Ecology Division, Gulf Breeze, FL
32561 .
Introduction
Nutrient enrichment or seeding with microorganisms are ap-
proaches used to enhance the biodegradation rate of the oil
when bioremediation is considered as a treatment option for oil
spills. The U.S. Environmental Protection Agency (EPA) and
Exxon tested the potential for bioremediation by applying vari-
ous fertilizer formulations to oiled beaches that resulted from
the Exxon Valdez oil spill in Prince William Sound, AK, on
March 24, 1989. Analysis of the results suggested that this
approach enhanced biodegradation rates of the indigenous
microorganisms two- to three-fold. That study, as well as oth-
ers, stimulated interest in developing and testing a variety of oil
spill commercial bioremediation agents (CBAs) designed to
facilitate the biodegradation of spilled oil. Despite the prolifera-
tion of these CBAs, no standardized methods were available to
assess their efficacy and environmental safety. In this study we
report the development of test systems and protocols to mea-
sure the effectiveness of CBAs in various marine environments
as well as to evaluate the toxicity associated with their use.
The efficacy issue has many components^ The results from
consistent CBA efficacy tests for various environments (e.g.,
beach, marsh, open-water) can aid on-scene coordinators in
the selection of cost-effective technologies from the variety of
available commercial products used in the treatment of oil
spills. The efficacy tests should simulate, within reason, those
environmental variables that significantly affect performance of
a CBA. In addition, it is desirable to compare use of CBA-
enhanced degradation to that of intrinsic biodegradation (i.e.,
no intervention). Finally, efficacy assessments are dependent
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on the bioremediation endpoints selected which, in turn, may
be influenced by the toxicity of certain petroleum hydrocarbon
components as well as aesthetic factors.
There are a number of environmental concerns with the use of
biotechnology products for cleaning up oil spills in marine
environments. CBAs contain a variety of components, includ-
ing fertilizers, microorganisms, surfactants, enzymes or combi-
nations of these ingredients that may have direct, adverse
effects on resident organisms because of their toxicity. For
example, nitrogenous nutrients, frequently used to stimulate
biodegradation, are toxic to marine fauna, as are surfactants.
Various inert particulates (e.g., clay) used as carriers may also
be harmful to some organisms. Indirect effects of CBAs could
include (1) depletion of oxygen through eutrophication or in-
creased activity of oil-degrading microorganisms, (2) increased
bioavailability of and/or exposure to toxic oil components by
surfactants in the CBA or through microbiologically generated
surfactants, or (3) production of toxic metabolites from en-
hanced oil biodegradation.
To address these needs, the U.S. EPA, through a cooperative
agreement with the National Environmental Technology Appli-
cations Center (NETAC) at the University of Pittsburgh, estab-
lished a panel of scientists from government, academia, and
industry to oversee the development of a tiered system of
protocols that provide increasingly more complex and environ-
mentally realistic data. These protocols are intended to serve
as guidelines for CBA suppliers to enable them to provide
technical information on their products to potential buyers and,
in the process, to aid in the development and commercializa-
tion of oil spill bioremediation technology [1]. In the Base Tier,
the vendor provides information on product safety including
formulation and unacceptable chemical or biological compo-
nents. Tier I is a feasibility assessment concerning the
manufacturer's production capabilities, a description of how the
product will be used, and information on previous usage. Tier II
efficacy (developed at the U.S. EPA Risk Reduction Engineer-
ing Laboratory, Cincinnati, OH) monitors oil biodegradation in a
closed, shake-flask test system in which the oil is physically
agitated; environmental safety is evaluated by determining the
toxicity of the CBA itself as well as the CBA combined with the
water-soluble fraction of oil. Tier III flow-through test systems
are designed to simulate oil spills in open-water, sandy beach,
or marsh environments (the latter developed at the U.S. EPA
Environmental Research Laboratory, Athens, GA); effluents
can be monitored for petroleum hydrocarbons or toxicity. Tier
IV testing is an actual field evaluation of the protocol test
systems conducted on a controlled release of oil or a "spill of
opportunity."
This project represented a cooperative research effort between
the University of West Florida Center for Environmental Diag-
nostics and Bioremediation, Pensacola, FL, and the U.S. Envi-
ronmental Protection Agency, Gulf Ecology Division, Gulf
Breeze, FL. The major focus was the development of efficacy
protocols for Tier III open-water and beach scenarios, as well
as environmental safety protocols for these, plus the marsh
environmental and Tier II. These protocols were published in
draft form in a manual by NETAC [1, 2]. Ancillary research
conducted as a part of this project in areas such as analytical
chemistry, microbiology, and toxicology provided the technical
support for these protocols; most of the research documenting
protocol considerations and development is detailed in the
Research Products Cited section of this summary.
Protocol Development
Tier Test Systems
Generic values were selected in the developmental phases of
the test systems for parameters such as temperature, turbu-
lence, salinity, and source of seawater. An artificially weath-
ered Alaskan North slope crude oil (ANS5213) was chosen
because it seemed likely that a number of days would pass
before a CBA would be applied to an oil spill, and the prepara-
tion of this oil removed many of the low boiling point compo-
nents that would likely volatilize over such a period. Both the
control containing oil and a treatment with oil plus a CBA were
conducted in triplicate. At the end of the test period, oil resi-
dues in the test systems (open water and beach sand) and
effluents were extracted with methylene chloride, weighed, and
analyzed gas chromatographic-mass spectrometry (GC/MS).
Efficacy of oil bioegradation in Tier III was measured at test
termination by statistical comparison (P<0.05) of the reductions
in chemical endpoints (e.g., oil residue weight and selected n-
alkanes, isoprenoids and aromatic compounds) from oil-con-
taining test systems with and without a CBA.
Open-Water Test System
The Tier III open-water test system, illustrated in Figure 1,
provides an intact, undisturbed oil-on-water slick in a flow-
through design [3]. The test container is a 500 ml glass jar
sealed with a septum and lid. A magnetic stirrer spins a small
stir bar in the bottom of the jar to mix the water column with
minimal disturbance to the slick. A constant flow of seawater is
delivered under an ANS521 oil slick by a peristaltic pump.
Water for toxicity testing is withdrawn by a second pump
through Teflon®4 tubing to a reservoir, while a third pump
forces air under the water surface for aeration and displaces
seawater through a tube positioned at the air/water interface.
This water sample is acidified to stop further biodegradation
and later extracted and analyzed for oil residues. The system
is operated continuously for 7 days after CBA addition.
Beach Test System
The Tier III oiled beach test system (Figure 2) provides a
sandy beach substratum colonized by microflora indigenous to
seawater and simulated tidal fluxes [4]. The system consists of
an exterior 600 ml glass beaker containing a 250 ml fluorocar-
bon beaker filled with sand to which oil is added (Figure 2).
Holes drilled through the bottom of the inner beaker allow
water to flow between the beakers during tidal exchanges; a
fluorocarbon screen in the bottom prevents loss of sand. The
test systems are clamped on an orbital shaker and rotated at
70 rpm to simulate a gentle wave action, -tf £«,
The oil was treated by the "521" process according to the Draft International
Standard ISO/DIS (1989) method: the crude is heated to 374°F under atmo-
spheric pressure; the system is then cooled and placed under partial vacuum
(20 mm Hg) for the final distillation to an atmospheric equivalent of 521 "F; the
distillate is discarded and the residue, designated ANS521, was supplied by
NETAC to laboratories involved with protocol development research.
"Mention of trade names does not constitute endorsement by the U.S.
Environmental Protection Agency.
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Pump
(800 ml/day)
Microcosm & stir plate
synchronous motor
Figure 1. Tier III simulated open-water oil spill test system.
Two tidal cycles per day are simulated by adding and removing
seawater from the test systems via timer-controlled peristaltic
pumps. ANS521 oil is added to the beaker containing the sand
at high tide after cycling for one week to allow microbial
colonization. A glass cylinder is inserted into the sand in the
center of the fluorocarbon beaker to prevent oil from seeping
down the Inside of the fluorocarbon beaker. Water is drained
from the space between the glass and fluorocarbon beakers to
ensure that it passes through the oiled sand as it exits the test
system. Effluent water is collected in a bottle to be used for
toxicity tests, or is acidified to stop microbial activity and stored
for extraction later. The test is conducted for 28 days after
addition of the CBA.
Validation of Tier III Test System Efficacies
Using CBAs and Positive Controls
The ability of the open-water system to measure the effective-
ness of bioremediation agents was evaluated using eight CBAs
that represented a variety of bioremediation technologies (e.g.,
nutrient, dispersant, microbial) [3, 5]. Occasionally some end-
points such as total weight of oil were significantly reduced in
the treated systems compared with the controls. However,
such differences were very small, sometimes amounting to
only 1 %, and of low environmental significance. Positive con-
trols (see next section) were developed, partially in an effort to
evaluate the test systems under more ideal conditions; daily
addition of oil-degrading microorganisms and nutrients to the
open-water system resulted in the greatest biodegradation of
oil components, including a statistically significant weight loss
and decreases in 30 of the 66 QC/MS analytes. Thus, we
conclude that the test system itself was capable of giving a
measurable response, although its accuracy in modeling actual
field conditions remains to be evaluated. These results may
indicate that the recommended application rates of CBAs are
not sufficient to produce substantial changes in oil bio-
degradation. Daily or more frequent additions may be unten-
able in some open-water field situations (e.g., large area spills);
however, spills of a more confined nature may be reasonably
treated with higher or more frequent applications. The beach
test system was evaluated with 3 CBAs and a positive control
[4]. Substantially greater losses of n-alkanes, some aromatics,
and recoverable oil (gravimetric) were noted in CBA- and
positive control treated test systems when compared with open-
water systems; controls generally lost about 6% of the oil by
weight, while systems treated with a CBA or a positive control
that utilized either nutrients or oil-degrading microorganisms
and nutrients lost approximately twice as much oil. Two prod-
ucts were tested twice, allowing us to examine replication of
the protocol.
Tier III Efficacy Related Research
One of the project goals was to examine how various factors
influenced the ability of the protocols to measure CBA effec-
tiveness. The studies focused on microbiological issues related
to assemblages of oil-degrading bacteria, as well as the effect
of oil quantity on biodegradation.
Research that characterized artificial microbial assemblages
and natural oil-degrading consortia [5, 6] was based on two
efficacy protocol issues: optimization of test system response
through positive controls and development of an artificial sea-
water. Positive control treatments consisting of nutrients or
nutrients plus oil-degrading microorganisms were developed
as CBA surrogates to establish baseline performance for the
Tier III test systems. This was particularly important for test
system validation after tests with a number of CBAs failed to
elicit substantial changes in the selected efficacy endpoints;
positive control treatments were found to increase biodegrada-
tion, but not substantially. The concept of an artificial seawater
containing microorganisms capable of degrading both alkane
and aromatic oil components was an important protocol con-
sideration in order to avoid the necessity of shipping quantities
of natural seawater to laboratories where CBAs were tested,
as well as to increase the consistency of the test media for
conducting the efficacy protocols. Although the evaluation of
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microbial CBA efficacy would not necessarily require that an
artificial seawater contain microorganisms, nutrient CBAs oper-
ate on the premise of stimulating indigenous oil-degrading
microorganisms and thus would require competent populations
of such organisms to perform effectively.
During the development of test system protocols, quantities of
oil capable of generating a 0.5 mm thick slick, if evenly spread
over the surface, were typically added to both open-water and
beach test systems. Additional research was conducted com-
paring the results of oil bioremediation with a positive control of
selected oil-degrading bacteria and nutrients, using one-tenth
the typical quantity of oil for open-water and one-fifth the oil for
beach test systems. Gravimetric results indicated this reduction
of oil resulted in an approximately 3-fold increase in oil loss
from the low-dosed open-water system and a 2-fold increase in
loss from the beach system [7, 8].
Environmental Safety of CBAs
Tier II
Concern for intrinsic product toxicity is addressed at the Tier II
level [9] with two 7-day chronic estimator exposures of a
crustacean (Mysidopsis bahia, mysid) and a fish (Menidia
beryllina, inland silverside). The mysid test has three end-
points— survival, growth, and fecundity — while the silverside
test focuses on survival and growth. In this tier, CBA toxicity is
also assessed in the presence of a sublethal water-soluble
fraction (WSF) of oil to examine the potential of synergistic
interactions. During protocol evaluation, test cost concerns
became a major consideration; this issue was addressed by
comparisons of endpoint sensitivities associated with various
acute and short-term chronic toxicity tests [9].
Evaluation of the Tier II protocol with five CBAs [9] suggested
that these products exhibited relatively low toxicity, because
none were toxic below 75 mg/L. Mysids were more sensitive
than silversides for the two products that were tested with both
organisms.
Tier III
Important ecotoxicological considerations for CBA use in ma-
rine environments include the possibility of toxic metabolite
production or enhanced toxicity of oil through increased oil
bioavailability. These are addressed at the Tier III level, which
allows CBA and oil to interact in flow-through test systems;
toxicity is monitored [10] with a mysid 7-day chronic estimator
test on the effluent from the open-water, beach, and marsh test
systems. The relatively conservative dilution modeled by the
test systems increases the sensitivity of the protocol for pre-
dicting toxicity. To assess the nature of toxicity from CBA/oil
interactions, one would first ascertain that the toxicity of efflu-
ent from test systems containing oil and a CBA exceeded that
of the effluent from control systems containing only oil. If it
does, and this toxicity is more than would be expected from
similar exposures to CBA alone (from Tier II tests), generation
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of toxic metabolites or oil/CBA synergistic effects would be
suggested.
The Tier III environmental safety protocol was evaluated by
testing the toxicity of effluents exiting from the open-water test
system treated with six different CBAs. For five of the six
CBAs, the effluent as prepared for toxicity testing was not
toxic; the sixth CBA open-water effluent was toxic, but the
effects were less than would have been expected from Tier II
intrinsic toxicity tests [10, 11]. Effluent exiting the marsh test
system exceeded the toxicity expected from the CBA alone
and was reproducible.
Environmental Safety Related Research
Several peripheral but fruitful lines of investigation came from
our pursuit of acceptable criteria for adequate effectiveness or
safety of CBAs. The concepts addressed by this research are
discussed below with the appropriate citations.
The selection of efficacy endpoints based on analytical chemis-
try (gravimetric and selected GC/MS analytes) emphasizes
aesthetic as well as toxicological considerations of oil spills. An
ecological-based evaluation dependent on the ability of benthic
organisms to recolonize oiled beaches after bioremediation
represents another measurement of CBA success. An applica-
tion of such an endpoint was attempted by adapting a 10-day
amphipod (Leptocheirus plumulosus) sediment toxicity test.
This test evaluated toxicity associated with the use of CBAs on
oiled beaches, including potentially toxic metabolites, after the
28-day CBA efficacy test [11, 12]. This test organism actually
proved to be too sensitive, as exposure to oiled sediment, with
or without bioremediation agents, inhibited burrowing at the
end of the test.
Salt marsh areas represent productive, sensitive ecosystems
that are particularly vulnerable to the effects of oil spills due to
their coastal location. These complex, stratified communities
are probably not amenable to most physical approaches for
removal of spilled oil, such as scraping of the intertidal sedi-
ment, and thus could be viewed as potential candidates for
bioremediation approaches. The role of microbial communities
in the maintenance of salt marshes exceeds that of most other
marine environments where spills could occur, such as sandy
beaches, indicating a need for characterization of microbial
communities. To investigate the effects of hydrocarbon pollu-
tion on bacterial diversity in the rhizosphere and rhizoplane of
selected wetland ecosystem plants, fatty acid profiles and
molecular probes for selected conserved genes were utilized
[13, 14].
The Tier II environmental safety protocol includes an estima-
tion of CBA toxicity in the presence of an oil WSF. Such
studies traditionally require mixing oil to water in a 1:9 ratio for
a period of time, discarding the oil and using the WSF. Our
investigation of the toxicity of crude oil WSFs with different oil-
to-water ratios suggests that traditional methods may be un-
necessarily wasteful of oil and that similar toxic effects could
be observed with ratios as high as 1:499 [15].
Most toxicity test organisms are selected for their ease in
culturing or collecting, sensitivity, and suitability for testing
protocols (e.g., test system size, tolerance to salinity or par-
ticles, etc.). They generally have some ecological importance
for their role in food webs, but seldom have commercial signifi-
cance. One task focused on the development and test of
sensitivity of blue crab larvae (Callinectes sapidus) for toxicity
testing, providing an economically relevant toxicity test species
that could be used to evaluate oil or CBA toxicity. The blue
crab larvae were more sensitive than two other test organisms,
grass shrimp (Palaemonetes pugio) larvae and mysids
(Mysidopsis bahia), when tested with an anionic surfactant,
sodium dodecyl sulfate [16].
Conclusions
The open-water and beach test systems were evaluated for
their ability to assess CBA efficacy using both commercial
products and positive controls. There are substantial barriers to
effective performance of oil-spill CBAs, among them dilution
rates, nutrient and biomass limitations, and a limited time in
which a CBA can remain in contact with the oil spill. Some
CBAs effected significant changes in one or more targeted
hydrocarbons relative to the control. Oil losses from treated
beach test systems were greater than from open-water sys-
tems, but no substantial decreases in oil residue weights were
associated with CBA treatments.
The CBAs used to evaluate the environmental safety protocols
generally exhibited low intrinsic toxicity (Tier II) and low or no
toxicity in effluent from the efficacy open-water test system.
One notable exception was an increase in effluent toxicity
associated with the application of a CBA to oil in the marsh test
system. Although use of these toxicity testing procedures does
not ensure that identical results would occur in the field, or that
all possible environmental effects would be predicted (e.g.,
mutagenicity, teratogenicity, or community/ecosystem level ef-
fects), this protocol provides a consistent means for evaluating
safety of CBAs under standard conditions that model some of
the important parameters of an open-water spill scenario.
Efficacy information generated from these protocols coupled
with assessments of toxicity for CBAs should provide useful
information to an on-scene coordinator considering the selec-
tion of various CBAs.
Acknowledgments
Validation of the effectiveness protocol for Tier III open-water
and beach test systems, as well as the ecotoxicology for Tier II
and Tier III, was performed through cooperative agreement
CR-818991 between the University of West Florida Center for
Environmental Diagnostics and the U.S. Environmental Protec-
tion Agency (EPA) Laboratory at Gulf Breeze. The EPA Project
Officer was P. H. Pritchard. K. Ranga Rao was Principal
Investigator. Some aspects of this research were also sup-
ported through cooperative agreement CR-822236 with the
EPA. Anthony Mellone coordinated the analytical chemistry
component of the project for UWF. The following people con-
tributed ideas and technical assistance during the development
of this project: Ahmet Bulbulkaya, Wanda Boyd, Mike Bundrick,
Peter Chapman, Jim Clark, Carol Daniels, Barbara Frederick,
Tim Gibson, Wallace Gilliam, Jeff Kavanaugh, Joanne
Konstantopolis, Tony Mellone, Len Mueller, Neve Norton, Jim
Patrick, Parmely Pritchard, Bob Quarles, George Ryan, Mike
Shelton, Scott Spear, Phil Turner, Ling Wan, Vicki Whiting,
Diane Yates, and Shiying Zhang.
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Research Products Cited
1. National Environmental Technology Application Corporation
(NETAC). 1993. Evaluation Methods Manual for Oil Spill
Response Bioremediation Agents. (August, 1993) University
of Pittsburgh Applied Research Center, Pittsburgh, PA
15238.
2. National Environmental Technology Application Corporation
(NETAC). 1991. Oil Spill Bioremediation Products Testing
Protocol Methods P Manual. (October, 1991) University
of Pittsburgh Applied Research Center, Pittsburgh, PA
15238.
3. Lepo, J. E., B. Frederick, G. P. Norton, T. Mellone, M.
Shelton, S. Spear, P. H. Pritchard, and C. R. Gripe.
Evaluation of Protocols to Assess Efficacy and
Environmental Safety of Commercial Oil-Spill Bio-
remediation Agents: Open-Water Test System—Efficacy
Test. Manuscript in preparation.
4. Lepo, J. E., B. Frederick, G. P. Norton, T. Mellone, M.
Shelton, S. Spear, P. H. Pritchard, and C. R. Gripe.
Evaluation of Protocols to Assess Efficacy and
Environmental Safety of Commercial Oil-Spill
Bioremediation Agents: Beach Test System — Efficacy
Test. Manuscript in preparation.
5. Lepo, J. E. 1993. Evaluation of Tier III Bioremediation
Agent Screening Protocol for Open Water Using
Commercial Agents. University of West Florida/U.S.
Environmental Protection Agency, Gulf Breeze
Environmental Research Laboratory. EPA/600 1-93/001.
6. Norton, G. P., D. F. Yates, M. P. Hancock, and J. E. Lepo.
1994. Isolation and Characterization of Marine Bacteria for
Use as Surrogate Indigenous Petroleum Hydrocarbon
Degrading Microflora. Annual Meeting, American Society
for Microbiology, Las Vegas, NV. 23 - 27 May 1994.
Abstract no. Q-45.
7. Lepo, J. E., S. Zhang, N. Norton, J. Kavanaugh, C. R.
Gripe, P. H. Pritchard. Effect of Oil Dosing in Beach and
Open-Water Microcosms on the Degree of Biodegradation.
Manuscript in preparation.
8. Zhang, S. G. P. Norton, and J. E. Lepo. 1995. The
Biodegradation of Differing Amounts of Crude Oil in Open
Water and Beach Laboratory Simulations. Annual Meeting,
American Society for Microbiology, Washington, DC. 21 -
26 May 1995. Abstract no. Q-61
9. Kavanaugh, J .L., C. R. Gripe, C. B. Daniels, A. Bulbulkaya,
P. K. Turner, and J. E. Lepo. Evaluation of Protocols to
Assess Efficacy and Environmental Safety of Commercial
Oil Spill Bioremediation Agents: Agent Toxicity. Manuscript
in preparation.
10. Kavanaugh, J. L., C. R. Gripe, C. B. Daniels, W. Boyd, V.
K. Whiting, and J. E. Lepo. Evaluation of Protocols to
Assess Efficacy and Environmental Safety of Commercial
Oil-Spill Bioremediation Agents: Open-Water Test System
—Toxicity Test. Manuscript in preparation.
11. Kavanaugh, J. L., C. R. Gripe, C. B. Daniels, W. T. Gilliam,
R. Araujo and J. E. Lepo. 1996. Some Aspects of the
Environmental Safety of Commercial Oil Spill
Bioremediation Agents. Biotechnology Risk Assessment:
Proceedings of the Biotechnology Risk Assessment
Symposium, June 6-8, 1995, Pensacola, FL. Morris Levin,
Chris Grim, and J. Scott Angle, Editors. University of
Maryland Biotechnology Institute, College Park, MD. Pp.
391-407.
12. Lepo, J. E., C. R. Gripe, and P. H. Pritchard. 1994.
Effectiveness and Safety of Strategies for Oil Spill
Bioremediation: Potential and Limitations. Symposium on
Bioremediation of Hazardous Wastes: Research,
Development and Field Evaluations. San Francisco, CA,
June 28 - 30, 1994. EPA/600/R94/075, pp. 80-86.
13. Frederick, B. A., D. F. Folse, and J. E. Lepo. 1994.
Physiological and Genetic Adaptation of Salt-Marsh
Rhizosphere Communities To Oil Stress. Annual Meeting,
American Society for Microbiology, Las Vegas, NV. 23 -
27 May 1994. Abstract no. N-182.
14. Frederick, B. A., D. E. Weber, and J. E. Lepo. 1993.
Ecological Impact of Oil Spills on Spartina alterniflora and
on the Diversity of Their Microflora in Artificial Sediments.
Annual Meeting, American Society for Microbiology, Atlanta,
GA. 16-20 May 1993. Abstract no. Q-278.
15. Kavanaugh, J. L., J. E. Lepo, C. R. Gripe, A. Bulbulkaya,
C. B. Daniels and T. Mellone. The Toxicity of Crude Oil
Water Soluble Fractions Prepared with Different Oil-to-
Water Ratios. Manuscript in preparation.
16. Whiting, V.K., G.M. Gripe, and J. E. Lepo. 1996. Effect of
Sodium Dodecyl Sulfate on Newly Hatched Blue Crab,
Callinectes sapidus, and Other Routinely Tested Estuarine
Crustaceans. Arch. Contam. Toxicol. 31:293-295.
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