&EPA
United States
Environmental Protection
Agency
National Health and Environmental
Effects Research Laboratory
Gulf Breeze, FL 32561
Research and Development
EPA/600/S-97/008 March 1998
ENVIRONMENTAL
RESEARCH BRIEF
Effectiveness and Safety of Strategies for Oil Spill Bioremediation: Potential
and Limitation, Laboratory to Field
J. E. Lepo1 and C. R. Gripe2
Abstract
Several additional research efforts were identified during the
development of test systems and .protocols for assessing the
effectiveness and environmental safety of oil spill commercial
bioremediation agents (CBAs). Research that examined CBA
efficacy issues included: (1) development of oil-degrading micro-
bial assemblages for use as positive controls or indigenous
microbial flora, (2) assessment of the effect of oil quantity on
extent of oil biodegradation, (3) investigation of an apparent
anomaly in relative susceptibility of classes of hydrocarbons to
biodegradation, and (4) evaluation of the effect of emulsifi-
cation on oil biodegradationi Environmental safety research
explored the use of toxicological endpoints as an alternative to
analytical chemical endpoints in addition to techniques for
investigating the toxicity of water-soluble fractions of oil. Mo-
lecular microbiological tools were developed to study the mi-
crobial ecology of oil spill habitats, to detect potential indicators
of oil/CBA effects on key ecological processes, such as nitro-
gen fixation in the rhizosphere, as well as to enumerate indig-
enous microorganisms important for bioremediation efficacy
(i.e., hydrocarbon-degrading bacteria). Finally, field studies al-
lowed assessment of oil biodegradation efficacy in a more
realistic context without the constraints of laboratory test sys-
tems.
Introduction
Over the last 10 years, an increase in the development of
commercial bioremediation agents (CBAs) designed for clean-
ing up oil spills has provided a variety of choices to on-scene
1 Center for Environmental Diagnostics and Bioremediation, University of West
Florida, Pensacola, FL 32514. ,
2 U.S. Environmental Protection Agency, Gulf Ecology Division, Gulf Breeze, FL
32561.
oil spill coordinators, but no standardized procedures existed
for selection of appropriate technologies. Two of the more
important issues in the selection process are the effectiveness
and environmental safety of the CBA. In an earlier project, we
developed flow-through test systems that modeled oil spills on
open-water and sandy beaches in order to evaluate CBAs. The
open-water test system consisted of a 500-ml sealed glass jar
with a constant flow of seawater under a slick of weathered oil.
The beach test system provided a sandy beach substratum,
colonized by seawater microorganisms, inside a 250-ml fluoro-
carbon beaker receiving two tidal cycles per day. Effluents
from both systems were collected for oil residue analysis and
toxicity determinations. Gravimetric and gas chromatographic-
mass spectrometric analyses (GC/MS) of residues extracted
from the test systems provided endpoints for comparing the
effectiveness of biodegradation of oil by various CBAs with
untreated controls. Coupled with the development of efficacy
protocols that used these test systems were environmental
safety protocols, designed to evaluate the risk of CBA use to
marine and estuarine fauna. Survival and growth of a crusta-
cean (Mysidopsis bahia, mysid) and a fish (Menidia beryllina,
inland silverside) were measured in a 7-day exposure to a CBA
by itself, as well as to the CBA in the presence of a sublethal
water-soluble oil fraction. The mysid test included a measure of
fecundity. To evaluate the possibility of increased toxicity as a
result of CBA and oil interaction (e.g., increased oil availability
or toxic oil metabolites), mysids were exposed for 7 days to
effluent from the efficacy test systems.
During the development of CBA protocols, factors were identi-
fied that limit oil degradation effectiveness, and approaches
were developed to better assess CBA efficacy and safety. This
project summarizes the results of research to address these
questions. The studies have been grouped into four broad
categories: factors that affect or limit bioremediation efficacy,
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environmental safety endpoints, microbial ecology of oil spill
habitats, and field assessments of bioremediation.
This project was a cooperative research effort between the
University of West Florida Center for Environmental Diagnos-
tics and Bioremediation and the U.S. Environmental Protection
Agency, Gulf Ecology Division. Collaboration with a subcon-
tractor, AEA Technology in the United Kingdom, provided bio-
remediation research in a field setting, as well as related
research with microcosms, characterization of oil-degrading
microbial communities, and effects of emulsification.
Factors Affecting/Limiting Efficacy
As evaluation of the test systems progressed, it became appar-
ent that the CBAs selected to test the efficacy protocols did not
seem to cause substantial losses of petroleum hydrocarbons.
This raised a number of issues regarding factors that may
affect or limit bioremediation or our ability to measure
bioremediation effectiveness. A significant fraction of the re-
search studied some of these ancillary issues.
Through the use of simple shake-flask systems, more complex
open-water and sandy beach "microcosms," and actual field
trials, it was possible to address various environmental factors
that influence biodegradation of oil in environmental spills. This
section focuses on research of those factors: artificial assem-
blages of microorganisms for development of positive controls
and artificial seawater; the order of degradation of oil compo-
nents; and the extent of biodegradation as influenced by the
amount of oil, or emulsification.
Artificial Microbial Assemblages for Positive
Controls or as Components of Artificial
Seawater [1,2]
We developed a set of standard bioremediation treatments to
be used as "positive controls" in order to investigate the effects
of environmental parameters on biodegradation of oil in the
elected standard environments. The ability to promote consis-
tent biodegradation of target analytes of crude oil, including
polycyclic aromatic hydrocarbon (PAH) constituents, was a
major requirement of positive control regimes. Selected micro-
organisms that were applied along with inorganic nutrient supple-
ments (nitrogen and phosphorus) was one type of positive
control. The microorganism fraction included strains that de-
grade PAHs combined with strains selected for degradation of
n-alkanes and production of biosurfactants. The selected strains
were tested for interstrain compatibility. A non-microbial posi-
tive control consisted of only inorganic nutrients. These con-
trols were used to refine test systems and examine the effects
of environmental parameters. These same microbial strains
may also appropriately serve as surrogate indigenous back-
ground flora to be included in an artificial seawater to reduce
the dependence on collection and shipment of natural seawa-
ter for CBA testing. A collaboration with Environment Canada
was established to allow exchange of oil-degrading microbial
strains for evaluation at each laboratory.
Relative Susceptibility to Biodegradation of
Hydrocarbon Compound Classes [3,4]
Much of the oil biodegradation literature supports the concept
that PAHs are generally more recalcitrant than the more easily
degraded n-alkanes. However, we observed substantial deple-
tion of fluorene, phenanthrene, dibenzothiophene, and,other
PAHs in the active control treatments of test systems that
simulated oiled beaches. These active controls consisted of
.Gulf of Mexico seawater (with no added microorganisms or
nutrients) pumped through the test systems in simulated tidal
cycles over a 28-day period. One possibility was that these
PAHs, which are orders of magnitude more soluble than the n-
alkanes, dissolved in the seawater and were washed out of the
test systems with the tides, perhaps aided by biosurfactants
produced by oil-degrading microorganisms. However, PAHs
were not detected in the pooled test system effluents. The
greater disappearance of PAHs relative to n-alkanes was en-
hanced by the addition of nutrients (inorganic N and P). Micro-
cosm sediment core experiments performed by collaborators in
the United Kingdom produced a similar pattern of degradation,
only to an exaggerated degree: nutrient-treated cores showed
moderate n-alkane depletion, but levels of PAHs were below
the detection limit of GC/MS.
In a further attempt to resolve the issue of PAH loss via
degradation versus wash-out (facilitated by biosurfactants), we
examined oiled beach microcosms with sterile synthetic sea-
water. Triplicate treatments included sterile control, 10 ppm of
a bacterially produced rhamnolipid biosurfactant added to the
seawater, or biweekly inoculation of the microcosms with two
marine bacteria that produce biosurfactants but degrade only
n-alkanes. Test systems inoculated with the alkane-degrading
microorganisms exhibited depletion of the n-alkanes, but es-
sentially all of the aromatic analytes were still recoverable from
the oiled sand; we were able to recover both alkane and PAH
analytes from the other two treatments. This suggests that the
compound class of lower PAHs is preferentially degraded by
microorganisms indigenous to natural seawater under aerobic
conditions.
Effect of Amount of Oil on Biodegradation [2,5]
The degree of oil biodegradation in the open-water and sandy-
beach systems was evaluated with a range of oil doses. We
used periodic applications of a positive control that supplied
inorganic nitrogen and phosphorus and two marine bacteria
capable of degrading n-alkanes and a range of aromatic com-
pounds. The amount of oil typically used (referred to here as
"high-oil") modeled a slick of 0.5-mm nominal thickness: 1.9 ml
for the beach and 2.5 ml for the open-water systems; the
respective "low-oil" doses were 0.38 ml and 0.25 ml. Gravimet-
ric results indicated that after 28 days, the beach low-oil inocu-
lated treatment lost an average of 22.5% weight, while the
high-oil, inoculated treatment lost only 11.3%. The open-water,
low-oil inoculated treatment lost 19.1%; the high-oil, inoculated
lost 2.9%. Thus, the lower doses of oil were more highly
degraded in terms of total oil weight lost. In addition, more of
the recalcitrant GC/MS analytes were affected, and to a greater
degree, by this positive control treatments than in high-oil dose
treatments.
Effects of Emulsification on In Situ Oil
Biodegradation [6]
Depending to a large extent on weather conditions, spilled oil
may undergo emulsification to varying degrees as wind and
wave action mix seawater into the oil slick. The effect of
emulsification on the biodegradation rate of Arabian Light crude
oil was studied by dosing microcosms designed to mimic a fine
sediment beach with two oii-in-water emulsions: 25% and 50%
artificial seawatenoil (v:v). The bioremediation strategy incor-
porated the weekly additions of inorganic sources of nitrogen
and phosphorous. The results showed that emulsions with a
higher concentration of water were more resistant to biodegra-
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dation and that addition of external sources of nitrate and
phosphate was not effective in enhancing rates of biodegrada-
tion over background rates. Conversely, emulsions with a lower
water content were more amenable to biodegradation and the
rate of breakdown could be significantly enhanced by the use
of inorganic fertilizers. This suggested that emulsification may
be a key factor influencing the rate at which oil spilled; at sea is
biodegraded, when it is subsequently washed ashore. The
ability of responders to enhance this degradation by using
bioremediation will depend on the level of emulsification.
Environmental Safety Research
It is important to assess the environmental impact of the
application of biotechnology products to oil spills in marine
environments. CBAs contain a variety of components, includ-
ing fertilizers, microorganisms, surfactants, enzymes or combi-
nations of these ingredients, and may themselves be toxic to
resident organisms.
Various inert particulates (e.g., clay) used as carriers may also
be harmful. Indirect effects of CBAs could include oxygen
depletion through eutrophication or increased activity of oil-
degrading microorganisms, increased bioavailability of toxic oil
components from CBA-associated microbiologically generated
surfactants, or enhanced production of toxic oil degradation
metabolites. Research described here examines the use of
toxicology as an alternate endpoint to analytical chemistry for
evaluating efficacy, as well as selection of oil:water ratios for
preparing a water soluble fraction (WSF) of oil for toxicity
testing.
Use of Toxicological Endpoints as Alternatives
to Bioremediation Efficacy Endpoints [1,7]
A 10-day amphipod (Leptocheirus plumulosus) sediment toxic-
ity test (American Society for Testing and Materials, E1367-92)
was adapted to evaluate increased toxicity that might be asso-
ciated with the formation of toxic metabolites in the beach test
system following the 28-day CBA efficacy test. The test has
two endpoints: survival and the amphipod's ability to rebury
itself at the end of the 10-day exposure period. However, we
observed that oiled sediment, whether subjected to
bioremediation or not, was toxic to this test organism, thus
preventing accurate assessment of any added toxicity due to
metabolites from bioremediation. .
Further research examined whether reduction in the toxicity of
oiled sediments through bioremediation could be used to re-
duce effective mortality (reburial) as an alternative to chemical
analysis of oil residues. The addition of as little as 40 or 100
mg of oil to beach test systems increased the effective mortal-
ity of L plumulosus to 69% and 79%, respectively. Oiled beach
test systems that were treated with oil-degrading microorgan-
isms and nutrients showed significant losses of oil residue
weights relative to the untreated control; moreover, such treated
microcosms showed substantial and significant reductions in
the target analytes as determined by conventional GC/MS
analyses, indicating that the remediation was a "success."
However, we could find no differences in the effective mortality
of the test organisms between the bioremediated systems and
the untreated, oiled controls. It could be that oil components
had been metabolized to equally toxic compounds, or that
reburial of the amphipods was influenced by characteristics of
the oil that may be unaltered by the bioremediation treatment
(e.g., the ability of resins and asphaltenic compounds to stick
to the amphipods). Thus, although the results indicate that
significant reductions in analytical chemical endpoints do not
necessarily correlate with decreased toxicity and perhaps should
be reexamined, additional research will be required to develop
the very sensitive amphipod test into a useful indicator of
efficacy.
Preparation of Water-Soluble Fractions (WSFs)
of Crude Oil for Toxicity [8]
The toxicity of crude oil components occurring in the aqueous
environment is of special importance in the consideration of the
environmental impact of crude oil spills on water. Although
considerable research has been conducted to determine the
toxicity of aqueous solutions containing dissolved and/or par-
ticulate oil to aquatic organisms, methods for preparing aque-
ous media in these studies vary substantially. Mixing time,
mixing energy, oil properties, oil-to-water ratio, temperature,
light conditions, and properties of the water used may affect
the composition of oil components in the water phase. Most
studies prepare a WSF by layering oil on water and mixing the
two phases together for a designated period; after separation,
the water phase is removed as the test solution. A clear
relationship between the oil-to-water ratios used for WSF and
the chemical and toxicological effects is not apparent in the
current literature. We prepared WSFs of both weathered and
fresh Alaskan North Slope crude oil with a range of oil-to-water
ratios. Toxicities of WSFs in a series of acute and short-term
chronic toxicity exposures of the mysid, Mysidopsis bahia,
were tested, resulting in no apparent differences among oil-to-
water ratios ranging from 1:9 to 1:499. This study suggests that
petroleum hydrocarbon components distributed into the water
column reach saturation, and their effects on submerged aquatic
biota would not be expected to change substantially over a
wide range of oil pollution levels, and that high oil:water ratios
may be unnecessary.
Microbial Ecology of Oil Spill Habitats
The microbial ecology of areas that may be impacted by oil
spills and, potentially, CBAs relates to both an environmental
safety issue (environmentally significant communities that could
be adversely affected) as well as an efficacy concern (pres-
ence of oil-degrading microorganisms). This section describes
the development of tools to assess diversity of microbial com-
munities with respect to important ecological functions, such as
nitrogen-fixation, and to enumerate hydrocarbon-degrading
microorganisms whose activity could be stimulated with appro-
priate amendments.
Effects of Oil Pollution/Bioremediation on
Bacterial Diversity [9,10,11]
Salt-marsh and wetlands ecosystems comprise some of the
more sensitive and biologically active ecosystems on earth.
The proximity of salt marsh ecosystems to oil-related activities
increases the potential for contamination in these ecologically
sensitive habitats. We studied the effects of sediment oiling on
marsh plants and their rhizosphere microflora by growing
Spartina atterniflora seedlings for 4 weeks in autoclaved or
unautoclaved artificial sediments mixed with oil and inoculated
with a characterized microbial rhizoflora culture from a natu-
rally occurring S. alterniflora colony. At harvest, we examined
the short-term physiological adaptation of the rhizosphere by
microbial fatty acid profiles and the genetic diversity and activ-
ity of ecologically significant enzymes (glutamine synthetase,
glutamate synthase, and glutamate dehydrogenase). Strongly
conserved genetic regions for glutamine synthetase and
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glutamate dehydrogenase have been identified for probing
restriction-digested DNA. Effects on the plants were assessed
by measuring chlorophyll quantity and quality, root and shoot
dry weights, and mortality. Branched-chain fatty acid methyl
esters representative of inoculated rhizosphere communities
decreased in oiled sediments. Plants grown in oiled sediments
had significantly reduced biomass and chlorotic leaves, and
autoclaved sediments supported more vigorous plant growth.
Gene probe analyses and other methods were developed in
order to detect changes in diversity of subsets of microbial
communities associated with the rhizosphere of wetlands in
response to stress of oil contamination or bioremediation ef-
forts. Microorganisms that catalyze nitrogen fixation were stud-
led, since these populations were among those likely to be
affected by remediation strategies involving application of
bioavailable nitrogen. Although we used these technologies on
model wetlands systems, they would be applicable to other
matrices. We attempted to correlate measurements of the
appropriate microbial activities (e.g., acetylene reduction, am-
monium assimilation, environmental nitrogen fluxes) with oil
dose.
A method to assess the community structure of nitrogen-fixing
bacteria in the rhizosphere was developed. Total DNA was
extracted from the macrophytic plants' root zones (Spartina
altemiffora and Sesbanla macrocarpa) by bead beating and
was purified by CsCI-EtBr gradient centrifugation. The average
DNA yield was 5.5 ng g-1 of soil and was of sufficient purity for
PCR amplification of n//H. [a-32?] dCTP was incorporated into
the PCR reaction and n/7H PCR products were restriction
digested. Restriction Fragment Length Polymorphism (RFLP)
analysis of the amplified sequences revealed differences in the
community structure of nitrogen-fixing rhizobacteria of the field-
collected salt marsh plant, Spartina alterniflora, and of a labo-
ratory cultured Sesbanla macrocarpa. Soil inoculation experi-
ments were used to determine the efficiency of the methods,
and amplified n/VH DNA could be detected when 104 cells each
of Vibrio natriegens and Azotobacter vinelandii were added per
gram of soil. Restriction patterns produced by each species
were detected at 10* cells g-1 soil. These results indicate that
RFLP analysis of amplified n//H sequences from rhizosphere
communities may provide information on species composition
and reveal shifts in diversity.
Development of Molecular Methods to Monitor
Hydrocarbon-Degrading Bacteria [12]
Methods were developed for the molecular biological analysis
of hydrocarbon-degradation genes during an oil spill
bioremediation field trial at Stert Flats, Somerset, UK (see next
section, Field Research). PCR primers were developed that
would specifically amplify a diverse range of mefa-cleavage
dioxygenase genes (xy/E, nahC, bphC, mpd, mpd\ and a gene
encoding a component of the alkane monoxygenase gene
(a//cB) from cultivated microorganisms and from nucleic acids
extracted from environmental samples. (These primers can
also be used to generate polynucleotide gene probes useful for
the analysis of cultured bacteria and environmental nucleic
acids.) We were unable to enumerate toluene- or naphthalene-
degrading bacteria from an oil spill site by dilution plate meth-
ods, suggesting that the populations of these organisms at the
Stert site were low. Our initial plan to concentrate on the
diversity of mefa-cleavage genes in these bacteria was there-
fore modified to encompass the analysis of total hydrocarbon-
degrading bacteria obtained either by dilution plate methods or
by most probable number (MPN) methods. We could not suc-
cessfully use the gene probe methods on samples from the
MPN plates and thus used dilution plating on oil agar, followed
by colony blot procedures to examine the hydrocarbon degra-
dation genes in the cultivated fraction of hydrocarbon-degrad-
ing bacteria. This revealed a predominance of bacteria contain-
ing only a//cB-like genes; no aromatic ring-cleavage dioxygenase
genes were ever detected in colony blots.
A method to extract DNA suitable for enzymatic amplification
was developed and used on samples of beach sediment from
the Stert site. When this had been achieved successfully, DNA
isolated from selected plots at the Stert site was challenged
with our complete suite of primers and probes specific for
catabolic genes involved in aromatic and aliphatic hydrocarbon
degradation. The results obtained were strikingly different from
the colony hybridization procedures. It proved very difficult
indeed to detect a//cB-like genes using a combined PCR-gene
probe assay, while xy/E-like genes were readily detectable in
the beach sediments. Other mefa-cleavage dioxygenases were
less widespread and nahC-, mpd- and mpdl-like genes were
not detected. Some plots were shown, however, to contain
genes similar to the bphC gene.
As part of the method development for the project, a large
number of naphthalene- and toluene-degrading bacteria were
isolated from river water and sediments and subjected to PCR
and gene probe analysis with primers and probes specific for
xyE.-, nahC- and bp/rC-like genes. In addition, the bacterial
strains were characterized using random amplification of poly-
morphic DNA-PCR (RAPD-PCR). This showed that consider-
able diversity existed in the naphthalene and toluene-degrad-
ing bacteria from the river water and sediments. However,
almost all the strains characterized harbored genes homolo-
gous with the well-characterized xy/E and nahC genes.
With the development of nucleic acid-based methods to study
microbial ecology here and in other laboratories, there is po-
tential to expand our knowledge of both the microbial popula-
tions involved in bioremediation and their catabolic genes.
While detection of specific genes associated with particular
catabolic activities is useful, determining the expression and
activity of these genes would be of far greater value. Methods
to do this have been developed recently by others. Application
of this to petroleum hydrocarbon bioremediation offers exciting
possibilities for the elucidation of changes not only in microbial
populations but also their activities and how these relate to
observed changes in hydrocarbon degradation.
Field Research [6,13,14,15,16]
Laboratory research on bioremediation efficacy strategies may
suffer from the limitations of laboratory constraints. With this in
mind, field studies guided by results from sediment column
microcosm experiments were conducted. These studies incor-
porated an additional endpoint, respirometry, to assess the
effectiveness of stimulating indigenous oil-degraders with nutri-
ents.
A field evaluation of the use of bioremediation to treat oiled fine
sand in the intertidal zone of Stert Flats (Somerset, UK) was
conducted, and the use of in situ respirometry and analytical
chemistry to monitor bioremediation success was evaluated.
Early experimental studies had shown that superficial oil is
rapidly removed from Stert Flats, with tidal action removing or
depositing 0.05 - 0.10 m of fine sand in a single tidal cycle.
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Thus, only oil found at depth as a result of penetration or burial
by sediment deposition is persistent. To evaluate the feasibility
of bioremediation to treat this stranded subsurface.oil, a subse-
quent field trial was conducted using inorganic sources of
nitrogen and phosphate. Arabian light crude oil (weathered and
emulsified with 25% seawater) was added to selected plots at
a coverage of 4 l-trr2. Regular addition of nutrients (sodium
nitrate and potassium dihydrogen orthophosphate) was made
throughout the 3-month experiment, beginning 1 week after oil
application. The application rate was determined by separate
laboratory studies using columns of sediment from the field
site. The success of the bioremediation strategy was determi-
ned by chemical analysis of the residual hydrocarbons and
monitoring of carbon dioxide evolution in situ. The! results
suggest that inorganic fertilizer did stimulate the biodegrada-
tion and mineralization of oil buried in the aerobic zone of fine
sediments.
Conclusions
Earlier studies indicated that application of bioremediation tech-
nologies to simulated oil spills in open-water and beach model
systems did not result in high oil biodegradation rates.: Further
advancement of this technology requires a full understanding
of its limitations as well as sound approaches to overcome
them. We hope these studies will advance more valid criteria
for assessing the effectiveness and safety of bioremediation
approaches to environmental oil spills, and that our conclu-
sions can be extrapolated from these laboratory model sys-
tems and field trials to actual environmental spills. We believe
that the results of the development of a consensus for efficacy
and safety criteria endpoints will provide better guidelines for
developers of CBAs to improve their products.
Acknowledgments
Research conducted on the effectiveness and safety of strate-
gies for oil spill bioremediation was performed through coop-
erative agreement CR-822236 between the University of West
Florida Center for Environmental Diagnostics and the U.S.
Environmental Protection Agency (EPA) Laboratory at Gulf
Breeze. The EPA Project Officer was C. Richard Gripe. The
Principal Investigator was Dr. Joe Eugene Lepo; Dr. K. Ranga
Rao was Co-Principal Investigator. Some aspects of this re-
search were also supported through cooperative agreement
CR-818991 with the EPA. Anthony Mellone coordinated the
analytical chemistry component of the project for the University
of West Florida. The following people contributed ideas and
technical assistance during the development of this project:
Ahmet Bulbulkaya, Mike Bundrick, Peter Chapman, Carol
Daniels, Tim Gibson, Wallace Gilliam, Barbara A. Miller, Parmely
H. Pritchard, Jeff Kavanaugh, Len Mueller, Neve Norton,
Katharine Ruopp-Edwards, Mike Shelton, Phil Turner, Diane
Yates, and Shiying Zhang. :
Key Personnel in the United Kingdom included Richard P. J.
Swannell of the United Kingdom's Atomic Energy Authority
(AEA) Technology, who, as the lead overseas collaborator,
coordinated the preliminary microcosm work and the field trials.
He was assisted by David Mitchell, also of AEA Technology.
Dr. Ian Head of the University of Newcastle Upon Tyne di-
rected an investigation of the diversity of genes encoding
mete-cleavage of aromatic hydrocarbons; the work served as a
master's thesis project for Mr. Kristian Daly. Analytical chemis-
try analyses for the overseas component of the project were
conducted by Dr. David Martin Jones, also at the University of
Newcastle Upon Tyne. Dr. Kenneth Lee of Canada's, Depart-
ment of Oceans and Fisheries also collaborated on the field
experiments.
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