BIOAUGMENTATION AND BIOSTIMULATION: A PARADOX BETWEEN
LABORATORY AND FIELD RESULTS
Kenneth Lee, Gilles H. Tremblay, and Johanne Gauthier
Marine Environmental Sciences Division, Fisheries and Oceans, Canada
Maurice Lamontagne Institute
P.O. Box 1000
Mont-Joli, Qebec, G5H 3Z4
Canada
Susan E. Cobanli
BDR Research Limited
P.O. Box 652, Station M
Halifax, Nova Scotia, B3J 2T3
Canada
Michael Griffin
National Environmental Technology Applications Center
University of Pittsburgh Applied Research Center
615 William Pitt Way
Pittsburgh, Pennsylvania 15238
ABSTRACT: In both shaker-flask and mesocosm-scale experiments,
a commercial oleophilic bioremediation agent containing biostimula-
tion (nutrients) and bioaugmentation (bacterial inocula) properties
was more effective in enhancing oil biodegradation rates than that of
no treatment and/or periodic inorganic nutrient addition. However,
similar results were not obtained from a subsequent 129-day field trial
conducted in a sand beach environment. In this case, periodic additions
of inorganic nutrients, with and without the commercial bioremedi-
ation agent, enhanced the number of heterotrophic bacteria and
microbial respiration rates within the oiled sediments. The commercial
product appeared to elevate the number of oil-degrading bacteria
within the oiled sediment between days 17 and 89. However, the addi-
tion of inorganic nutrients alone, on a periodic basis, was the most
effective means of enhancing the extent of oil biodegradation within the
residual oil and of reducing sediment toxicity. By retaining residual oil
and altering the physical and chemical characteristics of the treated
sediment, the oleophilic product suppressed both the rate and extent of
oil loss by tidal activity and biodegradation. This is not to say that the
use of the product was ineffective in protecting the environment or was
detrimental to it; the product does enhance natural biodegradation
rates, and it limits the transport of beached oil to more sensitive areas.
This study clearly illustrates the complexity associated with the selec-
tion of bioremediation agents, the need for improved experimental pro-
tocols for evaluating the performance and toxicity of bioremediation
agents, and the potential of nutrient enrichment as a bioremediation
strategy.
Bioremediation has been identified as the emerging oil spill
countermeasure of the 1990s, ever since its demonstration in the
1989 Exxon Valdez spill in Alaska (Prince, 1993; Bragg et al., 1994;
Swannell et al., 1996). Today, there are many proponents of the tech-
697
nique and vendors of products claiming to aid in the process. Funda-
mental research studies are under way to develop operational guide-
lines for various bioremediation strategies (Lee, 1995; Merlin, 1995;
Venosa et al., 1996). However, in the interim, the decision to bio-
remediate can be made on the basis of the available information on
the characteristics of the oil spilled and the site in question (Swannell
et al., 1996).
To aid spill responders in the selection and application of bioremedi-
ation agents, there is an immediate need for product efficacy tests, safety
assurance toxicity tests, information on application methodology and
operational limitations (what, how, when, and where), and cost-benefit
analysis. An international effort by regulatory agencies is under way
to establish cost-effective protocols to evaluate the efficacy and safety
of bioremediation products (Thomas et al., 1995; Blenkinsopp et al.,
1995). One of the most comprehensive examples of such a protocol is
the tier approach developed by the United States Environmental Protec-
tion Agency (U.S. EPA) in cooperation with the National Environ-
mental Technology Applications Center (NETAC) (National Environmen-
tal Technology Applications Center, 1993b). Each tier of the product
test protocol represents an increase in the amount, complexity, and cost
of the data generated (Table 1). This system provides users and regula-
tors with efficacy information and cost-effective vendor guidance and
feedback.
Bioremediation agents are based on bioaugmentation (addition of
specific oil-degrading biota) and/or biostimulation (e.g., addition of lim-
iting nutrients and/or oxygen). To meet the approval criteria for appli-
cation in the field, commercial bioremediation products in the United
States must pass both efficacy and toxicity tests (Environmental Pro-
tection Agency, 1994) and must be listed under the National Oil and
Hazardous Substances Contingency Plan (NCP) Product Schedule.
However, vendor claims and impressive product demonstrations in the
laboratory do not guarantee effectiveness in field applications (Lee and
Levy, 1987; Venosa et al., 1991). To address this discrepancy, we field-
698 1997 INTERNATIONAL OIL SPILL CONFERENCE
Table 1. The U.S. Environmental Protection Agency/National
Environmental Technology Applications Center tier approach
for bioremediation product testing
Base tier Basic information: Product safety, regulatory status,
acceptability of its chemical and biological components
Tier I Focus on feasibility: Contains information on proposed
use, potential effectiveness, safety certification
Tier II Laboratory scale data: Product efficacy and safety data
obtained with a standard shake flask testing protocol
Tier III Microcosm-simulated field test demonstration: Simula-
tion of environmental scenarios (e.g., open water,
marshes, beaches, etc.)
Tier IV Field demonstration: Product efficacy and safety in a
field application setting
tested the efficacy of a commercial bioremediation agent containing
both bioaugmentation and biostimulation properties. This agent is listed
in the NCP Product Schedule (tier II). Comparisons of this product to
that of no treatment and nutrient enrichment with low-cost inorganic
agricultural fertilizers alone were also made.
Experimental materials and methods
Venture Condensate, produced from an offshore exploratory well off
the coast of Nova Scotia, Canada, was weathered in the laboratory by
aeration to a specific gravity of 0.81 for use in the experiment.
PRP (Petrol Rem, Incorporated, Pittsburgh, Pennsylvania) was cho-
sen to represent commercial bioremediation products in this study. It is
an oleophilic, microencapsulation, bioremediation delivery system for-
mulated for the treatment and removal of petroleum spills on water,
sand, and pavement. This product is amongst the 15 bioremediation
products listed in the current NCP Product Schedule (tier II). PRP con-
tains mineral nutrients and nonpathogenic bacteria within spherical par-
ticles made from plant-derived natural products.
The agricultural fertilizer mix used in this study was formulated from
granular forms of ammonium nitrate (N:P:K) 33-0-0 (C-I-L Canada)
and triple superphosphate 0-46-0 (International Mineral and Chemical
Corporation).
Bacterial numbers were determined with a modification of the minia-
turized most probable number (MPN) method (Brown and Braddock,
1990). Serial ten-fold dilutions of a homogeneously mixed 1:10 sub-
sample were inoculated into sterile 24-well tissue culture plates con-
taining Bushnell Haas broth and a sheen of Venture Condensate for the
enumeration of oleoclastic (oil-degrading) bacteria and Marine Broth
for the enumeration of total heterotrophs. After 3 to 4 weeks of incuba-
tion at the interstitial water temperatures recorded at the time of sample
collection, the plates were scored for the presence of microbial growth.
A computer program was used to determine the most probable number
of bacteria per gram of sediment and its 95% confidence limits (Klee,
1993).
Bacterial respiration was monitored with a closed circuit respirome-
ter (Micro-Oxymax respirometer; Columbus Instruments International
Corporation, Columbus, Ohio) containing a single-beam nondispersive
infrared detector to quantify CO2 concentrations (Czekajewski et al.,
1994). In this automated system, air between a reference chamber and
the head space of the sample chambers is sequentially passed through
the detector (with air return to the same chambers). To evaluate the effi-
cacy of the bioremediation agents in the laboratory, Venture Conden-
sate, PRP, and/or the agricultural fertilizer mix was added to sediment
samples at predetermined concentrations (Table 2) prior to making
experimental test slurries by mixing 50 g beach sand (from the experi-
mental field site) with 15 mL seawater in 250-mL chambers. The cham-
bers were incubated at 12C in the dark with gentle shaking to simulate
natural tidal activity and to maintain an equilibrium between the head
space CO2 and the sediment slurry. Changes in head space CO2 concen-
tration were measured in replicate samples for each experimental treat-
ment to calculate the mean CO2 production rate over specific time inter-
vals. During the field trial, CO2 production was monitored in slurries
Table 2. Experimental treatments used in the field study.
Treatments were replicated four times in a complete block design
(a sample of each experimental treatment is contained within a
complete experimental block), as described by Venosa et al. (1996).
Treatment (g/kg wet sediment)
Additives UC V V 1 NP V 1 PRP V 1 NP 1 PRP
V 12.85 12.85 12.85 12.85
N 0.23 0.23
P 0.02 0.02
PRP 1.30 1.30
N 5 nitrogen (ammonium nitrate 33-0-0, N-P-K); P 5 anhydride of
P2O5 (triple superphosphate 0-46-0, N-P-K); PRP 5 Petroleum Reme-
diation Product; UC 5 unoiled control; V 5 Venture Condensate
prepared with sediment recovered from each experimental enclosure (50
g of sediment with 15 mL of seawater in 250-mL sample chambers). The
sample chambers were incubated in the dark under ambient seawater
temperatures with gentle shaking to simulate natural tidal activity. To
compute CO2 production rates (L/h), changes in the head space gas of
each chamber were measured over a period of approximately 15 hours,
at sampling intervals ranging from 55 to 65 minutes.
Oil analysis was carried out by gas chromatography/mass spec-
troscopy (GC/MS). Archived sediment samples (frozen immediately
after collection at 215C in hexane-rinsed glass jars) were dried,
extracted with dichloromethane, concentrated, and solvent-exchanged
into hexane prior to injection into a Hewlett-Packard 5890 Series II gas
chromatograph equipped with an HP 5972A mass selective detector
(MSD). The instrument was operated in the selected ion-monitoring
(SIM) mode to quantify specific saturated hydrocarbons, polynuclear
aromatic hydrocarbons (PAHs), and sulphur heterocyclic constituents.
All analyte data were normalized to a conservative compound C2-chrys-
ene. Nutrient concentrations of the interstitial water within experimen-
tal enclosures were determined with a Technicon Autoanalyzer (Tech-
nicon Industrial Systems, 1972, 1973).
Acute toxicity of sediments was determined by measuring the fluo-
rescence of the bacteria Vibrio fischeri following exposure to serial dilu-
tions of suspended sediment recovered from each experimental enclo-
sure, as described in the Microtox Solid-Phase Test (SPT) protocol
(Microbics Corporation, 1994). The inhibitory concentration (as ppm of
wet sediment) that induces 50% reduction (IC50) of bioluminescence rel-
ative to a control population was determined from the dose response
curve.
Experimental design
Laboratory experiment. An experiment with sediment slurries in
seawater was conducted in the laboratory using the respirometry system
to determine the effectiveness of PRP and/or inorganic nutrient addi-
tions to enhance the mineralization of weathered Venture Condensate
stranded within sand beach sediments collected from the intertidal zone
of a sheltered cove (Long Cove) located on the eastern shore of Nova
Scotia, Canada (Lee and Levy, 1987).
Field experiments. The field site was established at Long Cove on
July 7, 1994. In-situ enclosures were constructed of 243-m mesh Nitex
fabric and filled with 8 L of coarsely sieved, wet sand (to remove sea-
weed and shell debris) from the immediate area. Weathered condensate
was added to selected enclosures at 3% (v/v) concentration. Subsamples
were collected on day 0, before the enclosures (20 in total) were buried
5 cm below the beach surface. The enclosures were attached to wooden
stakes 1.7 m apart in a row parallel to the water line, midway between
the high- and low-water mark. On day 7, the 20 experimental enclosures
were recovered and subsampled for chemical and microbiological
analysis with stainless steel spatulas, after homogeneous mixing of the
sediments from each enclosure in individual stainless steel trays. Biore-
mediation agents (see Table 2) were homogeneously mixed into the sed-
iments with stainless steel garden trowels prior to redeployment of the
 1997 INTERNATIONAL OIL SPILL CONFERENCE 699

in-situ enclosures. The four experimental treatments and the unoiled
control were replicated (4 times) in a complete block design (Venosa
et al., 1996). In an attempt to ensure optimal nutrient concentrations for
bioremediation, inorganic nutrients (NP) were reapplied after each sam-
ple collection event between July 15 and November 14, 1994 (days 7,
17, 33, 46, 67, 89, 101, and 129).
Results and discussion
PRP was chosen as a representative example of the commercial biore-
mediation agents for the following reasons: (1) it is listed under the cur-
rent U.S. Environmental Protection Agency National Contingency Plan
Product Schedule, January 1996; and (2) an independent nonprofit third
laboratory has demonstrated its effectiveness in enhancing the biodegra-
dation rate of a diesel oil slick in a mesocosm test system that simulated
a stream environment (Figure 1) (National Environmental Technology
Applications Center, 1993a).
Laboratory experiment. Respirometry has been demonstrated to be
an effective tool for demonstrating the performance of bioremediation
agents in enhancing mineralization of crude oil (Venosa et al., 1993;
Swannell et al., 1994). In the laboratory experiment conducted with
slurries of marine sediment, PRP with the addition of inorganic nutri-
ents (V 1 NP 1 PRP) was found to be more effective in the short term
(,200 hours) than inorganic nutrient addition alone at the same N and
P concentrations (V 1 NP) to enhance CO2 production rates within sed-
iment slurries containing weathered Venture Condensate (Figure 2).
There was no difference in mineralization rates following any of the
treatments for approximately 80 hours because of a lag phase that may
have been caused by toxic components in the condensate eventually lost
by evaporation or by an adaptation period required by the microbiota
found in both the natural environment and in the PRP product formula-
tion. The results from this experiment clearly demonstrated that the
commercial product with inorganic nutrient amendments (V 1 NP 1
PRP) stimulated microbial activity, as shown by the significant increase
in mineralization rates between 80 and 150 hours in the three replicate
chambers (see Figure 2). These CO2 mineralization rates were sustained
at this elevated rate for the duration of the study (320 hours). For the
remaining treatments with the inorganic fertilizer alone (V 1 NP), the
PRP alone (V 1 PRP), and the oiled control (V) mineralization rates
increased at a slower rate after a lag period of approximately 120 hours.
Nutrients are clearly a limiting factor controlling the attainable CO2 pro-
duction rates, because the rates in the chambers that did not receive addi-
tional inorganic nutrient supplements declined after 180 hours. Further-
more, the oiled sediments treated with inorganic nutrient (V 1 NP)
supplements alone achieved the same mineralization rates as those in
combination with PRP (V 1 NP 1 PRP) by approximately 210 hours.
Field experiment. The field experiment was carried out for 129 days
(July 7 to November 14, 1994). Analytical results obtained within the
first few weeks of the experiment showed a high level of variability in
the interstitial nutrient concentrations of one of the experimental blocks.
Detailed salinity measurements correlated these results with extensive
groundwater runoff over a small section of the beach. All experimental

Figure 1. PRP efficacy test results for the biodegradation of a surface diesel oil spill in a
freshwater mesocosm. Results are expressed as the amount of individual compounds
remaining relative to the conservative marker C3-phenanthrene. Experimental results are
obtained with the untreated control slick on day 0 (A) and day 14 (B ); the PRP-treated slick,
on day 0 (C ) and day 14 (D).
700 1997 INTERNATIONAL OIL SPILL CONFERENCE
Figure 2. Result of laboratory PRP efficacy test on intertidal
marine sand beach sediments containing indigenous bacteria.
Microbial activity (respiration) is inferred from CO2 production
rates.
data from this block were rejected, and final data analysis was based on
three replicate samples for each treatment, each collected from a sepa-
rate enclosure.
The number of heterotrophic bacteria within the enclosures (Figure
3A) declined immediately following oil addition (day 0). However, full
recovery was achieved by day 7, since bacterial numbers in the oiled
Figure 3. Time-series changes in the total number of hetero-
trophic bacteria (A); oil-degrading bacteria (B ); and interstitial
water temperature (C )
sediments were higher than those found in the unoiled control sediment.
Semicontinuous additions of inorganic nutrients stimulated bacterial
numbers to a higher level within the oiled enclosures with or without the
addition of PRP. Total numbers of heterotrophic bacteria observed in
the oiled enclosures treated with inorganic nutrients (V 1 NP; V 1 NP
1 PRP) were higher than in the oiled enclosures treated with (V 1 PRP)
or without (V) the commercial product. Data plotted with 95% confi-
dence limits (see Figure 3A) showed no significant increase in het-
erotrophic bacterial numbers following the addition of PRP to the oiled
sediments.
The numbers of oil-degrading bacteria increased rapidly in response
to the addition of the weathered condensate (see Figure 3B). There
was a trend in the data to suggest that PRP elevated the number of
oil-degrading bacteria within the oiled sediment enclosures between
day 17 and day 89 (see Figure 3B). The stimulatory effect of PRP
to sustain higher numbers of potential oil-degrading bacteria is
probably due to the nutritional value of the inorganic and organic
constituents within the product and not the addition of microbial
inocula, since differences arising from the addition of PRP to the oiled
sediments were not observed until day 17. This hypothesis is also in
line with previous mesocosm test results, which indicated that the
most prevalent organisms in the PRP formulation were not hydrocar-
bon degraders (National Environmental Technology Applications
Center, 1993a). It is important to note that, although numerous
companies offer bioremediation products based on the addition of
microbial inocula, there has been no conclusive evidence that these
products offer any advantage over indigenous organisms present in the
open environment (Lee and Levy, 1987; Swannell et al., 1996). The
seasonal decline in the MPN of oil-degrading bacteria (see Figure 3B)
may be correlated with temperature (see Figure 3C) and/or the loss of
bulk oil.
The addition of weathered condensate to the sediment sustained a sig-
nificant increase in sediment respiration rates for approximately 50 days
(Figure 4). During this period, a small but significant increase in micro-
bial activity was observed with the addition of the PRP. A sustained
level of high microbial activity was realized by the addition of inorganic
nutrients, and the optimum result was obtained with a combination of
the PRP and the inorganic nutrient.
Detailed GC/MS analysis was conducted to identify the efficacy of
the various treatments to enhance oil biodegradation rates. Changes in
oil composition can be used to monitor biodegradation if a hydrocarbon
component can be identified that will not be biodegraded to a significant
degree over the time scale of monitoring. Decreases in the ratio of
biodegradable hydrocarbons to the stable component can then be used
to monitor loss over time by material balance.
Hopanes have been used in previous marine oil spill bioremediation
field trials as a stable marker (Bragg et al., 1994; Venosa et al., 1996).
Although hopanes can be biodegraded, the rate is negligible compared
to that of most other hydrocarbon components. Unfortunately, we were
unable to use this compound as a conserved internal biomarker in this
study because its concentration in the condensate was at or near the
detection limits of our instrumentation. We thus used C2-chrysenes
Figure 4. Time-series changes in microbial activity within oiled
sediments following experimental treatments as determined by
CO2 respiration rates

which are also considered nonbiodegradable markers (Environmental
Protection Agency, 1994) or highly degradation resistant (Wang et al.,
1995). The chrysene series exhibited a pronounced increase relative to
other components within the oil because of their very low solubilities in
water and high resistance to degradation, as indicated by the decrease in
the relative ratios of the sum of napthalenes, phenanthrenes, dibenzo-
thiophenes, and fluorenes to the sum of chrysenes (for samples V 1 NP
and V, day 129, the relative ratios decreased to the range of 0.3 to 30.0
from 79.0 for the source oil).
In this study the structural identification of C2-chrysenes was based
on mass data, GC retention data with reference standards, and literature
data. We monitored the primary ion for C2-chrysenes, ion 256 (Envi-
ronmental Protection Agency, 1994) during GC/MS SIM analysis. By
looking at the distribution profiles (patterns) for ion 256, we observed
numerous peaks corresponding to various C2-chrysene isomers and/or
homologs. Four peaks (Figure 5), identified by their Kovats retention
index (KI), were found in all samples: C2(1), KI 2683; C2(2), KI 2707;
C2(3), KI 2719; and C2(4), KI 2730. Among them, C2(1) was the most
resistant to degradation. To account for bulk oil loss, all the GC/MS
data, including those for total C2 chrysenes, are presented as the percent
analyte remaining relative to the selected C2(1) internal marker. As evi-
dent from the results obtained on day 67 and day 129, there was a sig-
nificant decrease in both the aliphatic and aromatic compounds in all
cases with the addition of the bioremediation agents under study (Fig-
ures 6 and 7).
The most effective treatment was periodic inorganic nutrient addi-
tions alone (V 1 NP) followed by periodic nutrient additions with PRP
(V 1 NP 1 PRP) and PRP alone (V 1 PRP). The data suggested that
the full potential of PRP was limited by nutrient availability. Based on
visual observations of the normalized data to the conserved biomarker,
the extent of biodegradation for the residual oil appears to be less with
the use of PRP supplemented with inorganic nutrient amendments
(V 1 NP 1 PRP) than with treatment with the same concentrations of
inorganic nutrient alone (V 1 NP). This is due to the oleophilic nature
of the commercial product, which prevented the physical loss of oil
from the sediments, and to the high proportion of wax esters of biogenic
origin within the PRP formulation, which coelute with n-alkanes from
n-C27 to C33.
To demonstrate the effectiveness of the various treatments to enhance
bioremediation and/or to prevent the loss of oil from the sediments, for
day 129, on a percent basis relative to that of the oiled control plots (V)
Figure 5. Distribution of C2-chrysenes (m/z 256 mass fragmen-
togram). Numbers in parenthesis correspond to each of the four
C2-chrysene isomers and/or homologs, identified by the Kovats
retention index (KI), that were found in all samples: C2(1),
KI 2683; C2(2), KI 2707; C2(3), KI 2719; and C2(4), KI 2730.
1997 INTERNATIONAL OIL SPILL CONFERENCE 701
on day 0, for the n-alkanes between n-C12 and C26 (to avoid the contri-
bution of the wax esters in PRP), only 11.0% of the oil remained in the
oiled control enclosure; 0.1% of the oil remained in the enclosure treated
with inorganic nutrients alone (V 1 NP); 5.4% of the oil remained in
the plots treated with the inorganic nutrients with the PRP addition
(V 1 NP 1 PRP); and 25.3% was found in the plots treated with PRP
alone (V 1 PRP).
Interstitial nutrient concentrations within sediments (determined prior
to nutrient readditions) suggest that higher rates of oil biodegradation
could have been achieved in this study, since the demand for nitrogen
and phosphorus exceeded supply (concentrations reduced to that of or
below the unoiled control) up to day 90 (Figure 8).
Semicontinuous additions of the agricultural fertilizer mix did not
increase sediment toxicity, since nutrient overloading did not occur
(Figure 9). Consistent with the GC/MS results, acute toxicity studies
also demonstrated the effectiveness of inorganic nutrient additions in
accelerating the recovery from risk of oil exposure. There was no evi-
dence of acute toxicity following the addition of PRP to sediments
alone at the concentrations used in the field (IC50 value . 80,000 ppm).
However, because of its intrinsic oil retention properties, the natural
rates of toxicity reduction in oiled sediments were suppressed by treat-
ment with PRP.
Conclusion
Until recently, relevant performance data were lacking to support the
use of bioremediation as a general oil spill countermeasure technique.
Application information in remedial engineering was limited for biore-
mediation technologies, and developers could not demonstrate that their
products were as effective or as economical as existing methods. How-
ever, controlled experiments are expanding our knowledge base, and
operational guidelines are now under development.
In this study, scientific evidence collected in laboratory and meso-
cosm-scale tests showed some advantage in using a commercial biore-
mediation agent with biostimulation (nutrients) and bioaugmentation
(bacterial inocula) properties over no treatment and/or fertilization
with inorganic nutrients. However, results from a 129-day field trial
conclusively demonstrated that inorganic nutrient addition in a semi-
continuous manner was the most effective method of enhancing the
extent of oil biodegradation for the residual oil and of reducing sedi-
ment toxicity. This is not to say that the commercial product is inef-
fective or detrimental to the environment, since it does enhance nat-
ural oil biodegradation rates and limits the transport of beached oil to
more sensitive areas. This study illustrates the complexity involved in
the selection of bioremediation agents (because they may have more
than one mode of action), the need for improved experimental proto-
cols for the evaluation of bioremediation agents (including field tri-
als), and the potential of using nutrient enrichment as a bioremedia-
tion strategy.
Biography
Dr. Kenneth Lee is a research scientist with the Department of Fish-
eries and Oceans, Canada. His research focuses on the relationship
between biogeochemical processes and the microbial transformation of
contaminants in estuarine and coastal environments. He has conducted
field studies on marine oil spill bioremediation for over 12 years.
Acknowledgments
This research was funded by the Panel of Energy Research and Devel-
opment (PERD), Canada, and the Toxic Chemicals Program under Fish-
eries and Oceans, Canada. The authors gratefully acknowledge the tech-
nical assistance of D. Guay, S. Kirby, and K. Saunders.
702 1997 INTERNATIONAL OIL SPILL CONFERENCE

Figure 6. Changes in the composition of n-alkanes pristane and phytane in response to exper-
imental treatments at day 67 and day 129. To account for bulk physical losses of oil attributed
to current and/or tide activity, the results are expressed as the percent analyte remaining rela-
tive to an internal conserved marker highly resistant to biodegradation.
 1997 INTERNATIONAL OIL SPILL CONFERENCE 703

Figure 7. Changes in the composition of aromatic compounds in response to experimental

treatments at day 67 and day 129. As for the n-alkanes, results are expressed as the percent
analyte remaining relative to an internal conserved marker highly resistant to biodegradation.
704 1997 INTERNATIONAL OIL SPILL CONFERENCE
Figure 8. Time-series changes in the concentration of interstitial
water nutrients NH4 (A), PO4 (B ), and NO2 1 NO3 (C ) within the
experimental enclosures
Figure 9. Time-series changes in the acute toxicity of sediments
(IC50) within the experimental enclosure replicates
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