Fritchird, P.I. IMI. Iffectlreaess and Regulator? lama 1b Oil Spill
llore'-.dlitloo: Iiperi##ee« with the iiion faldei Oil Spill in Alaska, h:
liotreatient of Isdostrial aid Basardooi Waste. Morris I. Lerls and Michael I.
fiealt, Iditora, icGm-Illl, Km fork, I?. Pp. 26J-30J.
EPA/600/A-94/HO&
Chapter
12
Effectiveness and Regulatory
Issues in Oil Spill
Bioremediation: Experiences
with the Exxon Valdez
Oil Spill in Alaska
P. H. Prltchard
U.S. Environment*! Protection Agency
Environmental Research Laboratory
Sabine Island, Gulf Breeze, Florida
Abstract
The use of bioremediation as a supplemental cleanup technology in the
Exxon Valdez oil spill, in Prince William Sound, Alaska, has proven to
be a good example of the problems and successes associated with the
practical application of this technology. Field studies conducted by sci-
entists from the U.S. Environmental Protection Agency hav® demon-
strated that oil degradation by indigenous microflora on the beaches of
Prince William Sound could be significantly accelerated by adding fer-
tilizer directly to the surfaces of oil-contaminated beaches. Our results
from the application of an oleophilic fertilizer are presented as exem-
plary field and laboratory information. The fertilizer enhanced
biodegradation of the oil, as measured by changes in oil composition
and bulk oil weight per unit of beach material, by approximately
twofold relative to untreated controls.
These studies supported bioremediation as a useful cleanup alterna-
tive that was subsequently used by Exxon on a large scale, TTiey have
269

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270 Chapter Twelve
also generated a number of insightful lessons that have significant rel-
evance to future oil bioremediation efforts. This chapter discusses
these lessons and examines complications and difficulties in assessing
the effectiveness of bioremediation in the field.
Further field studies at a site involving an oil-contaminated beach
that was less energetic and higher in nonpetroleum organic matter and
using slow-release fertilizer granules applied at different concentra-
tions, contrastingly showed little effect of fertilizer application.
Precautions regarding extrapolation either from the laboratory to the
field or from field site to field site, are discussed.
As with many types of bioremediation, protocols are needed to gen-
erate consistent and relevant data sets for commercial processes that
will allow appropriate decisions to be made relative to the use of the
process or product in a field cleanup operation. The conceptual basis for
these protocols is a complicated matter and its development is signifi-
cantly influenced by field experiences such as the Exxon Valdez oil
spill. Discussion of these concepts provides an informative picture of
the problems and assumptions faced in making decisions about when
and how to apply a bioremediation technology.
The use of bioremediation for the cleanup of soils, sediments, and
aquifer materials contaminated with oil and petroleum hydrocarbons
has been extensively recognized (Lee and Levy, Bartha and Pramer).
Success has been possible because of the relative biodegradability of oil
and the knowledge that hydrocarbon degraders can be enriched in
many, if not most, types of environments (Levy, Atlas). In addition,
bioremediation is gaining acceptance as a viable technology; if used
prudently, it can provide efficient, inexpensive, and environmentally
safe cleanup of waste chemicals. Thus, the suggestion to use bioreme-
diation as a supplemental cleanup tool in the Exxon Valdez oil spill in
Prince William Sound, Alaska, was readily accepted as use of a new
technology ready for field demonstration (Pritchard and Costa). The
implementation of field studies to establish that oil degradation by in-
digenous microflora on the beaches of Prince William Sound could be
significantly accelerated by fertilizer application (Pritchard et al,,
1991), and the eventual large-scale application of fertilizer by Exxon as
part of their overall cleanup program, provided a number of useful
lessons and experiences that, if considered in the proper light, could
have considerable influence on future oil bioremediation efforts.
The emphasis of thi3 chapter will be on some of the difficulties and
problems associated with the fertilizer application and its effect on oil
degradation. I will concentrate primarily on the separate application of
an oleophilic fertilizer which occurred at a site called Snug Harbor on
Knight Island in Prince William Sound, and on the application of slow-
release fertilizer granules which occurred on Disk Island in Prince
William Sound. These applications provide contrasting results that are
Experiences with the Exxon Valdez OH Spill 271
instructive for both their success and failure. (Note that additional fer-
tilizer applications were conducted at these and other sites and sum-
maries of these results are available; Pritchard et al., 1991).
Closely linked to these field applications of fertilizers is the question
of which commercial products should be used and how the best ones
should be screened out. This includes not only fertilizers but also mi-
croorganisms and other oil-biodegradation-stimulating agents and con-
cepts. In Alaska, many of these commercial products could not be
considered because of the very short time available for field demon-
strations, but also because the data available for each product were so
variable and/or insufficient that reasonable selections could not be
made in a timely fashion. Subsequent to the Exxon Valdez oil spill,
however, the Office of Research and Development of the U.S.
Environmental Protection Agency (EPA) embarked on the develop-
ment of effectiveness and environmental safety testing protocols that
could be used to establish a consistent and relevant database upon
which decisions for the use of particular commercial products might be
based in the future. Some of these protocols have been developed and
are currently being validated with laboratory studies. The develop-
ment of a conceptual basis for these protocols has proven to be a useful
exercise that depends heavily on the lessons learned in Alaska and
other oil spills Describing, in part, that conceptual development here
provides an additional dimension to the regulatory complications that
come into play in the application of bioremediation to oil spill cleanup.
It is my hope that this information will stimulate others to carefully
consider the process by which bioremediation success is measured in
the field. It is this success issue, along with environmental safety as-
pects, that will be the key to good regulatory decision making.
Background
In any bioremediation effort, success will invariably involve a scientifi-
cally valid demonstration of process effectiveness and environmental
safety. Effectiveness, in the case of oil bioremediation, means establish-
ing that (1) removal or disappearance of the oil is attributable to bio-
degradation and not other nonbiological processes and (2) enhanced
biodegradation rates of oil are sufficiently better than natural rates to
justify expenditure of effort to implement the bioremediation process on
a large scale. Although environmental safety issues will not be ad-
dressed here, they too require considerable effort to verify the absence of
any adverse ecological effects associated with the fertilizer application.
In the Exxon Valdez oil spill, both aspects were crucial to the even-
tual acceptance of bioremediation by the public and state ai>d federal
regulatory agencies. Quelling skepticism, given the "subtleties* of a
biotechnological approach, indeed was and will continue to be a chal-

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272 Chapter Twelve
lenge in almost any bioremediation situation, whether it deals with oil
or other types of chemicals. Effectiveness and environmental safety is-
sues for bioremediation in Prince William Sound, where treatment was
centered on oil-contaminated gravel and cobblestone beaches, will, of
course, be considerably different than for oil-contaminated sandy
beaches, marshes, and wetlands. However, the lesson learned in terms
of demonstrating a viable cleanup technology will have no bounds.
Reflections on the initial assumptions by EPA scientists and their
colleagues as planning of the project commenced give rise to important
and useful insights. Several assumptions, discussed in the context of
what actually occurred in the field, provide instructive lessons that
could impact the responses to bioremediation at other spill sites. While
confidence provided by almost twenty years of accumulated research
data on oil biodegradation laid the groundwork for these assumptions,
we would have been naive not to expect some surprises!
Enrichments of oil-degrading microbial
communities
Clearly, it was reasonable to expect, even in the cold water tempera-
tures of Alaska, a significant enrichment of oil-degrading microorgan-
isms in the beach material following exposure to the oil. Research by
Atlas and his colleagues supported this idea (Atlas, 1981). As it turned
out, by early June 1989 (approximately 2 months after the spill), con-
centrations of oil degraders averaged around 10s per gram of oiled
beach material, which represented as much as a 10,000-fold increase in
the number of degraders relative to beaches that had not been con-
taminated with the oil. Studies by Lindstrom et al. (1991) have shown
similar trends, and an example of their results is shown in Table 12.1.
Enrichments of this magnitude suggested that oil was being degraded
(previous studies have demonstrated that Prudhoe Bay crude oil is
quite biodegradable), that some nitrogen and phosphorus were avail-
able to support growth of the hydrocarbon degraders, and that the cold
temperatures (10-16°C) were probably not overly restrictive to the in-
digenous microflora. The information not only implied the possibility of
nitrogen-limited biodegradation (i.e., a great excess of degrudablo or-
ganic carbon from the oil in the face of a finite supply of nitrogen in the
water), but also opened the possibility of accelerating oil biodegrada-
tion by overcoming this limitation alone.
In this case, we believed it was unnecessary to experimentally rever-
ify, through laboratory studies, the stimulatory effect of nitrogen on oil
biodegradation, even in oil-contaminated beach samples from Prince
William Sound, since previous experiences and numerous published re-
ports in the literature supported this as a sensible approach. Instead,
Experience* with the Exxon VaMaz Oil Spill 273
table 12.1 Median (n s 5 to 9 Samples) Hydroearbon-Oegrader MPN Microbial
Counts per Gram (Dry Weight) of Sediment for Treated (T) and Reference (R)
Plots'
Beach
site
Day
sampled
Median MPN
cells (1Q4) per
g of surface
sediment
Diff-
erent?t
Median MPN
cells (10*) per
g of subsurface
sediment
Diff-
erent?
T
R
T
E
KN-135B
0
262
4.24
No
1.66
1.63
No

2
4.79
1.58
No
1.02
0.47
No

4
15.50
4,20
No
10.30
1.00
Yes

8
1 56
15.60
No
10.10
2.27
No

15
15 60
9.75
No
16.20
2.34
No

52
13.70
23.40
No
75.40
36 00
No

56
139,00
17.90
Yes
582.00
9.78
Yea

70
14900
25.20
Yea
126.00
13,00
Yea

78
185.00
122.00
¦ No
170.00
117.00
No
KN-211B
0
0.96
4.63
No
4.60
1.63
No
2
77.00
127.00
No
193.00
2.23
Yes

4
9 55
48,00
No
81.93
80.35
No

16
45.40
44 44
No
46.22
97.49
No

31
23.94
98.78
No
99.80
24.95
No

45
30.83
53.23
No
33,18
25,32
No

102
18.10
8.51
No
3.72
0.96
No

112
3.19
11.70
No
8.51
1.28
Yes
KN-132B
0
24.90
23,00
No




2
155 00
21.70
No




4
77.70
16,00
No




8
160.00
37.10
No




16
97.30
15.70
Yes




29
28.00
16.00
No




43
135.00
0.59
Yes




60
84.10
1.78
Yea




95
117.00
53.20
Yes



•All values obtained for each day were subjected to a Mann-Whitney two-sample U teat to
determine whether the sampled populations were different at the 95% confidence level. KN-
135B was initially treated after day 0 and was refertiliied after day 52. KN211B was initially
treated after day 0 and refertiliied on day 42. KN132B was fertilized after day 0 and again on
day 40.
tStatiatieally significant differences are reported for surface and subsurface sediment*.
we reasoned that, in light of the magnitude of the problem at the time,
it was better to go directly to the field and conduct a practical demon-
stration of the same principle. Thus, in the very early stages of the oil
spill cleanup program in Alaska, the relatively simple measurement of
the number of oil degraders, along with the relevant literature infor-
mation, had provided a reasonable "first-line" indicator of oil bioreme-
diation feasibility.

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274 Chapter Twalva
Mineralization test as an indicator of oil
degradation activity
Depending on the environmental situation involved in the oil spill,
other first-line indicators of bioremediation potential might be desir-
able. There is always a tendency, where the local public is involved, to
generate "site-specific" information, at the very least to generate a bet-
ter comfort factor. A discussion of these indicators here, therefore, is
worthwhile, even though they were not used initially in Alaska.
Circumstances are likely to occur in other spills in which information
on the enrichment of oil degraders in the contaminated areas alone is
not sufficient for initial decision-making purposes.
Mineralization studies involving measurements of total C02 produc-
tion can provide excellent first-line information. The approach pro-
vides, quick, relatively unequivocal time course data suitable for
testing different treatment options (e.g., effects of adding nitrogen). If
natural oil degradation is occurring in contaminated beach material,
then considerable amounts of C02 should be produced from oil miner-
alization relative to a control containing uncontaminated beach mate-
rial. Several laboratory systems can be used for this type of
measurement. Biometer flask systems (Bartha and Pramer, 1965),
which are designed to trap C02 in side-arms containing an alkaline
solution, can be adapted to measure these mineralization rates of oil
on contaminated beach material (Mueller et al., 1992). Commercially
available respirometric systems, such as the Micro-Oxymax™
(Columbus Instruments, Columbus, Ohio) can also be used. The Micro-
Oxymax™ system, which can be adapted to standard laboratory shake
flasks, also measures oxygen consumption. The procedure entails plac-
ing oil-contaminated beach material and its associated microbial com-
munity (mixed sand and gravel in the case of Prince William Sound)
directly in the flasks and flushing fresh seawater in and out as a simu-
lation of the tidal exchange (Mueller et al., 1992). An example of the
data generated from such a mineralization test is shown in Fig. 12.1.
This experiment was performed with oil-contaminated Prince William
Sound beach material, taken considerably after our bioremediation
field demonstration had begun. The respirometric flask method ia in-
dicative of the short-term tests that can be initially conducted as a
data-gathering exercise for a particular oil spill. Note the enhancing ef-
fect of adding nitrogen fertilizer (Fig. 12.1). By comparing rates of CO.,
production, an estimate of the extent of enhancement of oil biodegra-
dation can be obtained. The system can be easily adapted to test beach
material from the other oil spill sites.
Since any other type of organic matter in the beach material can also
produce C02, care must be taken to assure that one is measuring oil
Expsrlencaa with tha Exxon Valdex Oil Spill 275
500
400
300
200
100
-100
— o
CL.
-400
-500
Incubation time (hr)
—O— Laval 1	—o—uvata
Laval«	—*— PWS mmi —*— Siama
Legend 	
Lawdt * 350 pfxn W7Q ppm P
i«»«2.35ppmNff ppmP
Laval 3 ¦ 3 S ppm WO 7 ppm P
Laval 4 » o 35 ppm N/0 0/ ppm P
pws * Pnrx* Wtfluum Sound
Figure 12.1 Fertilizer specific activity S02 consumption, CO, production) for six
treatments over time.
mineralization. In addition to running control flasks with uncontami-
nated beach material as indicated above (with and without added ni-
trogenous nutrients) and ensuring that mineralization in the presence
of the oil is considerably above background, one can also add a radiola-
beled hydrocarbon. If oil degradation is active, the production of radio-
labeled CO., should be extensive and immediate. We have found that
phenanthrene works well because it was rapidly and completely ab-
sorbed to the oil (Mueller et al., 1992; Pritchard et al., 1991). The tidal
cycle washed out any phenanthrene remaining in the aqueous phase,
and subsequent CO., production was then mainly from the bacterial
communities associated with oil surfaces.

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276 Chapter Twelve
Oil ipill bioremediation as a finishing step
We also made the very important initial assumption that bioremedia-
tion would be most effective as a finishing step in the cleanup program.
As it turned out, the physical washing procedure employed by Exxon
removed the bulk of the oil, but it left the beach material still quite con-
taminated and aesthetically unpleasing, and the oil ecologically avail-
able. Aa there were very few follow-up alternatives, bioremediation
then became intriguing as a finishing step. Without the initial removal
of this bulk oil, bioremediation may not have been tenable in the con-
text of Alaska. That is, even if oil biodegradation was quite active, it is
largely a surface-oriented process and it would take extended time
(there was a relatively small window in the summer months in Alaska
when water temperatures are amenable to oil biodegradation). For
quicker results, the oil must first be distributed throughout the beach
material to increase the surface-to-volume ratio. And since tilling was
unreasonable on most of the cobblestone beaches in Alaska, the more
bulk oil removed by a physical cleanup process and the more the resid-
ual oil dispersed into the beach material, as was accomplished by the
physical washing procedure, the more effective bioremediation would
likely be. Similar considerations would be required for other types of
beach material.
In highly porous beaches, as found in Prince William Sound, oil dif-
fuses to some extent into the gravel, thus increasing the contaminated
surface area. The physical washing process may enhance this aspect.
On a sand or mud shoreline, however, where porosity is much lower,
most of the oil will predictably be concentrated at the surface. Initially,
the contaminated beach material may very well be physically removed.
But the remaining contaminated beach material can potentially be
treated effectively by bioremediation, again as a finishing step. In this
case, tilling may be used as a mechanism to further disperse the re-
maining oil into the beach material, increasing the surface-to-volume
ratio, and improving the success of bioremediation. Tilling also aerates
beach material and helps disseminate added fertilizers, thus prevent-
ing the availability of oxygen or inorganic nutrients from becoming a
major limiting factor to bioremediation. In Alaska, because of the
highly porous nature of the beaches, the high oxygen concentrations in
the cold seawater, and the flushing effect of 5-m tides, oxygen limita-
tion was never a consideration.
Choice of Fertilizer Formulations
We assumed for Alaskan beaches that nitrogen (and phosphorus) had
to be applied in a manner which would passively expose the oil-de-
grading microbial communities to the elevated nutrient concentrations
Experiences with the Exxon Valdaz Oil Spill 277
TABLE 12.2 Description of Fertilizers Tested


Commercial Name


Wood ace
Customblen
Inipol EAP 22
Manufacturer
Vigoro Industries
Sierra
Chemical Co.
Elf Aquataine
Form
Briquette
Granule
Liquid
Size
5 x 5 x 5 cm
2- to 3-mm diameter
	
N source
Isobutyraldehyde-
diurea
Ammonium
nitrate
Urea
N:P:K
14:3:3
28:8:0
7.3:2.8:0
Specific gravity
1.8
1.8
0.996 g/mL
Viscosity
	
	
250 cSt
Application rate on 986 gf'm2
12-m x 35-m plots
100 g/m*
284 g/mJ
Method of application Net bags
(11.8 kg. ea.)
Fertilizer
spreader
Backpack
sprayer
Test areas
Snug Harbor
Snug Harbor
Passage Cove
Snug Harbor
Passage Cove
over an extended period. Given the large tidal cycle and significant
wave action, fertilizer materials placed on the beach surface would
likely wash away in a few days. To overcome this problem, two types of
slow-release fertilizers were initially considered; solid pelletized for-
mulations and liquid oleophilic formulations. Characteristics of each
fertilizer considered are summarized in Table 12.2.
Summarizing some of the criteria used to make the selections is in-
structive, as this could help in making fertilizer selections in the fu-
ture. The three main criteria were (1) ease of application and potential
to retain position on the beaches, (2) nutrient release characteristics,
and (3) physical durability over time. As it turned out, fertilizer gran-
ules seemed to best fit the criteria (Pritchard et al., 1991), They were
easy to apply over a large surface area and were found to stick tightly
to the oiled beach material and worked their way down under cobble
where they were difficult to dislodge. Nutrient release characteristics,
which were determined in simple laboratory test systems (Venosa et
al., 1990; Glaser et al., 1991), showed that much of the nitrogen (am-
monia and nitrate) and phosphorus (phosphate) release from the gran-
ules occurred in the first 24 to 72 h (Fig. 12.2). However, sufficient
quantities continued to be released steadily for considerable periods
thereafter. Thus, as the tides wash in and out of the beach, nutrients
should be distributed to the microbial communities associated with the
oil for a period of 2 to 3 weeks or longer. Although the physical condi-

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m Chapter Twelve
too
Each point represents one water exchange
Total phosphorus
Nitrate
o-
Ammonia
100
Time (d)
Rflum 12.2 Cumulative release of ammonia and nitrate from SIERRA CHEMICAL gran-
ules in static flask equipments.
tion of the granules slowly deteriorated on contact with seawater, we
observed granules on the beaches with fertilizer inside 2 to 3 weeks
after application. The fertilizer granules were ultimately used by
Exxon in combination with the liquid oleophilic fertilizer for all of the
large-scale applications in Prince William Sound.
Fertilizer briquettes were also found to be satisfactory based on the
above criteria (Venosa et al., 1990). These are approximately the size of
charcoal briquettes and contain organic sources of nitrogen and phos-
phorus that slowly hydrolyze to release urea and phosphate over time.
Nutrient release characteristics of the briquettes were very similar to
those of the granules. Although the briquettes maintained their physi-
cal integrity much better, if broadcast over the beach surface, they were
easily moved around by the tides and waves, resulting in very hetero-
geneous distributions. Consequently they had to be packaged in seine
net bags and secured to the beach with metal stakes. Although this was
effective, it presented significant logistical problems at the time of their
consideration for use on a large scale. The briquettes were, however,
used as part of our field demonstration in bioremediation, and appeared
effective in enhancing oil biodegradation (Pritchard et al., 1991).
Rather than broadcast fertilizer granules and briquettes onto the
beach surface, they could also be buried in the beach material, say in
Experiences with the Exxon Vakfez OU Spill
279
trenches running parallel to the water line in the contaminated inter-
tidal zone. Depending on the porosity of the beaches, tides and inter-
stitial water movement could effectively distribute the released
nutrients to the bacteria. Because of the physical integrity of the bri-
quettes, they would be most suitable for this type of application. A
burial approach was initially considered in Alaska but never really
tested, again because of perceived logistical restrictions (later to be-
come unfounded). However, fertilizer granules were applied by Exxon
and the State of New York in this manner to a sandy beach on PraJl's
Island (located in an estuary southwest of New York City) that was
contaminated with diesel fuel. Initial reports (Madden, 1991) sug-
gested that the application was successful in enhancing diesel oil
biodegradation. Distribution of nutrients will, of course, depend on the
hydrodynamics of interstitial water in the beach, and, in many cases,
specific information will be lacking. Rather than proceeding based on
these limited successes, one could quite easily perform a pilot study, of
several days' duration, to actually measure movements of nutrients in
interstitial water (Madden, 1991).
Pragmatically, the best criteria for determining how much slow-re-
lease fertilizer to place on a given beach were to apply as much as pos-
sible without exceeding toxic concentrations of ammonia and/or
nitrate. These nutrients are toxic (96-h LCM) to marine invertebrates
(good sentinel bioassay for most sensitive species) at concentrations in
the water of around 10-15 ppm (Pritchard et al., 1991). Our experience
has been that one will face adverse environmental effects thresholds
long before the demand for nitrogen by the oil-degrading microbial
communities is saturated. Keep in mind that the toxic effect threshold
should take into account the initial burst release of nutrients associ-
ated with these types of fertilizer formulations. Alternatively, deter-
mining fertilizer application rates baaed on the quantity of oil present
in the environment is made difficult because of the tremendous het-
erogeneity in oil distribution and concentration frequently encountered
in the field.
Oleophilic fertilizer
The concept of oleophilic fertilizers is based on the use of organic
sources of nitrogen and phosphorus in a liquid carrier that is miscible
with oil. In theory, when the liquid carrier is applied, the nutrients es-
sentially dissolve into the oil and thereby keep them in contact, for sus-
tained periods, with the bacteria growing on the oil's surface. Several
types of oleophilic fertilizers have been successively tested in both lab-
oratory and small-scale field experiments. Most of these were designed
and tested with the idea of treating oil on the surface of water rather
than oil on beach material. Pioneering studies by Atlas and Bartha

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280 Chapter Twelve
(Atlas and Bartha, 1973), demonstrated that the addition of paraf-
finized urea and octylphosphate to Prudhoe Bay crude oil on the water
surface significantly enhanced the biodegradation of the oil. Similar
success has been reported for a commercial product, Victawet 12, or 2-
ethylhexyl-dipolyethylene oxide phosphate (Bergstein and Vestal,
1978), for several natural sources of lipophilic nitrogen and phospho-
rus, such as soybean lecithin and ethyl allophanate (Olivieri et al.,
1978), and for MgNH
-------
202 Chapter Twelve
Visual changes. Test beaches at Snug Harbor, where Inipol was ap-
plied as part of a field demonstration of its effectiveness, produced
some surprising visual results (Pritchard and Costa, 1991). These
beaches were moderately contaminated with oil and had not been sub-
jected to the physical washing process at the time of our test. They were
selected as representative of those that received the physical washing.
Visually, the cobble areas had a thin coating of sticky oil covering the
rock surfaces and mixed sand and gravel under the cobble. Oil did not
penetrate more than a few centimeters below the gravel surface. In
some areas, small patches of thick oil and "mousse" (oil/water/air mix-
ture in colloid form) could be found.
Approximately 10 to 14 days following oleophilic fertilizer applica-
tion, reductions in the amount of oil on rock surfaces were visually ap-
parent. The change was particularly evident from observations in
aircraft where the contrast with oiled areas surrounding the plot was
dramatic, etching a "clean" rectangle (12 x 28 m) on the beach surface.
The contrast was also impressive at ground level; there was a precise
demarcation between fertilizer-treated and untreated areas. At this
time, the untreated control plots appeared unaltered visually.
Close examination showed that much of the oil on the surface of the
cobble was gone, yet considerable amounts of the oil remained under
the cobble and in the mixed sand and gravel below. Remaining oil was
not dry and dull as was the oil on the untreated control beach, but ap-
peared softened and wetter. It was also very sticky to the touch, with
no tendency to come off the rocks. At the time of these observations, no
oil slicks or oily materials were observed leaving the beach during tidal
flushing.
We believe that visual disappearance of oil on the cobble surface 2 to
3 weeks following Inipol application was largely due to biodegradation
and not a chemical washing phenomena. Chemical data to support this
belief are presented below. In addition, we tried to force the chemical
washing effects by adding large concentrations of Inipol repeatedly on
several miniplots in Snug Harbor, and it did not affect oil removal; a
period of at least 2 to 3 weeks was required to see any "cleaning" effect
regardless of the amount of Inipol applied. The application of aqueous
fertilizer solutions (tested at a different beach), which contained only
inorganic chemicals and no organic-surfactant-like materials, also pro-
duced the "cleaning" effect in about the same time period, further sup-
porting the role of biodegradation (Pritchard et al., 1991). Finally,
experiments performed by Exxon researchers (R. Prince, S. Hinton,
and J. Bragg, personal communications) have shown that, in specially
designed tests to measure the effectiveness of a variety of commercially
available chemical rock washers, Inipol was ineffective. Also, they have
observed in microcosm studies that Inipol seemed to become more
tightly associated with the beach material; that is, the oil had much
Experiences with the Exxon Valdaz OH Spill 283
more of a tendency to move to the glass walls of the microcosms in the
absence of Inipol.
Six to eight weeks after fertilizer application, the contrast between
the treated and untreated areas on the cobble beach had lessened.
Reoiling of the Inipol-treated beach from oiled subsurface material
and/or the concurrent slow removal of oil on the surface of the beach
material surrounding the treated areas was probably responsible for
this decrease in contrast. Toward the end of the summer season, the
area used for the bioremediation studies became steadily cleaner, in-
cluding the control plots. Several storms and more frequent rainfall, as
well as natural biodegradation, undoubtedly contributed to these
changes.
Overall, rapid oil disappearance brought on by the application of the
oleophilic fertilizer made these beaches more compatible with local
wildlife (less tendency for fur and feathers to become oiled). These dra-
matic changes occurred in a shorter period of time than the limited
changes noticed in untreated plots, and possibly helped accelerate bio-
logical recovery of the intertidal area.
Measures of Effectiveness
Obtaining definitive information on the role of biodegradation in the
removal of oil residues from beach material, or from any complex envi-
ronmental matrix for that matter, is a difficult task. In general, for oil
spill bioremediation, one has to produce both qualitative information
on changes in oil composition that are indicative of biological processes
and quantitative information on decay rates of oil, or some of its hy-
drocarbons, that are also indicative of biological processes. Qualitative
information establishes the extent to which biodegradation has oc-
curred; however, with a complex chemical mixture like Prudhoe Bay
crude oil, the removal of more than just a few short-chain hydrocarbons
(representing only a small percentage of the oil), and removal of more
than just the aliphatic fraction of the oil (i.e., leaving behind aromatic,
heterocyclic, and branched hydrocarbons, polar chemicals, etc.), is de-
sirable.
Simultaneously the quantitative information establishes that the
enhancement of oil biodegradation by the fertilizer treatments was suf-
ficient to merit full-scale operation; generally a two- to threefold en-
hancement over the untreated controls will probably be acceptable to
many decision makers and regulatory groups, but this is not based on
a comprehensive database. Both types of information, however, were
difficult to obtain in the field because of many different problems en-
countered.
Any bioremediation testing program that is based on analytical tech-
niques involving ¦ U .uo.ippearance of oil residues or the disappearance
-------
284 Chapter Twelve
of hydrocarbons resolvable by gas chromatograph is open to scientific
criticism because several environmental fate processes (including pho-
tosynthesis, physical dissolution, chemical washing, volatility, etc.) can
affect or contribute to this disappearance phenomenon. To confront
these potential criticisms, we chose an approach that integrated sev-
eral analytical procedures with several key assumptions. First, we as-
sumed that the disappearance of several target hydrocarbon groups
could be used as definitive indicators of biodegradation. We assumed
further that strong indicators of biodegradation would be associated
with substantial changes in the composition of several fractions in the
oil, particularly selected aromatic hydrocarbons. We would thus at-
tribute these compositional changes to biodegradation.
Second, we assumed that if a correlation between changes in hydro-
carbon composition and changes in residue weight of the oil could be es-
tablished, disappearance rates of the residue weights could be used as
the primary quantitative measure of fertilizer effect (i.e., significant
differences between treated and control plots). The rate of information
could then be used to estimate cleanup effectiveness over extended
time periods.
However, some discussion as to why these parameters were selected
is in order because the criteria for what constitutes biodegradation of
oil are complicated and controversial. Many studies have considered
measurements of reductions in aliphatic hydrocarbon concentrations
as generally indicative of biodegradation, but their value is often ques-
tioned because these hydrocarbons are (1) frequently the most readily
degradable fraction, (2) the least toxic, and (3) often only a small per-
centage (by weight) of the oil. Measuring compositional changes in the
aromatic fraction adds a further dimension, as these hydrocarbons are
less readily degradable and potentially more chronically toxic.
However, most of the common procedures for measuring the aromatic
hydrocarbons are based on mass spectral analysis which concentrates
on only 10 to 20 selected compounds, representing only a very small
fraction of the total aromatics. Whether these selected aromatic hydro-
carbons are good surrogates for the degradation of the rest of the aro-
matic compounds has not been established. However, if one
concentrates only on the aromatic hydrocarbons and shows that they
degrade, one has the advantage of being able to assume that aliphatic
hydrocarbons will almost certainly be extensively degraded as well.
Regardless of compositional changes, it seems reasonable to require
that bioremediation, as a worthy cleanup tool, should effect the re-
moval of bulk material; changes in composition without much change
in oil residue removal seem to present only half the picture. Moreover,
oil biodegradation under optimized conditions in the laboratory will re-
sult in as much as a 40 to 60 percent reduction in the total weight of oil
Experiences with the Exxon Valdei Oil Spill 285
(Atlas and Bartha, 1973) and therefore it does not seem unreasonable
that some reduction in oil residue can be expected, even under field
conditions. The ultimate measure of biodegradation would be to frac-
tionate the oil into aliphatic, aromatic, heterocyclic, polar, and asphal-
tene fractions and determine weight loss of each of these fractions.
Most of these fractions can be analyzed by gas chromatography to de-
termine qualitative changes in composition. This analytical procedure
has been used by Westlake and hia colleagues in several studies
(Jobson et al., 1972).
Changes in the normal-alkane-to-branched-
alkane ratios
To provide perspective on measuring effectiveness in bioremediation at
the level of a field demonstration, the results' frM Alaska with the
oleophilic fertilizer are instructive. Chemical analysis of the oiled
beach material, exposed and unexposed to the oleophilic fertilizer, was
accomplished by collecting beach material according to a block design
(21 samples taken at each sampling time, 7 each in contiguous blocks
along a line in the high-, mid-, and low-tide zones of the beach) and
then extracting samples (with methylene chloride) from the cobble sur-
face and from the mixed sand and gravel under the cobble. The weight
of oil recovered (measured in milligrams per gram of beach material)
was determined gravimetrically and oil composition was determined
by injecting extracts into a gas chromatograph following standard an-
alytical procedures (Pritchard et al., 1991).
Changes in composition were determined first by examining the re-
solvable alkanes. Historically, this has been done by calculating the
weight ratio of a hydrocarbon that is known to readily biodegrade (gen-
erally the CI 7 and CI 8 normal alkanes) to one that is slower to biode-
grade (generally the branched alkanes pristane and phytane), which
chromatograph very close to the n-C17 and n-C18 alkanes) (Atlas). We
generally focused on the n-C18/phytane ratio because pristane is some-
times found naturally in seawater. The ratio concept is based on the
idea that most nonbiological fate processes (physical weathering,
volatilization, leaching, etc.) will not produce differential losses of
aliphatic and branched hydrocarbons that have similar gas chromato-
graphic, and correspondingly, chemical, behavior. Support for this con-
cept can be found in the biogeochemical studies on oil (Kennicutt,
1988). However, since the branched alkanes do in fact biodegrade
(Prinik et al., 1977; Mueller et al., 1992), they need only to degrade
more slowly than the straight-chain alkane to take advantage of the
ratio method. Clearly, in this case, the measure of biodegradation will
be conservative.
-------
286 Chapter Twelve
Focusing on the effects of the oleophilic fertilizer Inipol EAP™ 22, the
results in Fig. 12.3a show that, following an initial lag, extensive decay
in the n-C18/phytane ratio occurred through time for cobble surface
samples. A decay also occurred on the untreated control beach but at
about half the rate on the fertilizer-treated beach (Fig. 12.36). Despite
the large variability around the median values, slopes of the decay
curves (following the June 17 sampling and not including the
September 9 sampling) were statistically different from zero and from
each other at the 95% confidence interval. Based on the assumptions
described above about the meaning of the ratio changes, biodegrada-
tion was occurring on both beaches and was enhanced by the applica-
tion of the fertilizer. Note that oil had already undergone
biodegradation prior to fertilizer application as the ratio for unde-
graded weathered Prudhoe Bay crude was around 2.0. Also, large de-
creases in the ratios were invariably linked to considerable reduction,
if not complete removal, in the concentrations of the resolvable (by gas
chromatography) alkanes, n-C17 to n-C30.
The large variability in the ratios shown in Fig. 12.3 (that is, many
samples showed evidence of biodegradation while others showed very
little) was a function of the highly heterogeneous distribution of oil on
the beach. Possibly the same amount of biodegradation was occurring
in each sample, but since biodegradation takes place on the oil's sur-
face, a grab-sampling procedure (which was almost unavoidable in this
case) necessarily encompasses sufficient quantities of undegraded oil
from below that surface to dilute the measure of biodegradation.
Much less change in the n-Cl8/phytane ratio, if any, occurred in the
mixed sand and gravel under the cobble for the oleophilic-fertilizer-
treated beach and the untreated control (Fig. 12.4a and 12.46). As
striking, however, was the unexpected difference in the initial ratio be-
tween the cobble surface samples and the mixed sand and gravel sam-
ples (t = 0 sampling, June 8, 1989). In both cases, substantial
biodegradation of the oil had occurred prior to fertilizer application but
the degradation was much more pronounced in the mixed sand and
gravel samples. Why the ratio was so much lower in the mixed sand
and gravel was not clear. With less total oil concentration overall in
these samples initially, biodegradation was possibly more apparent be-
cause of less dilution from undegraded oil during sampling.
Biodegradation of phytane
Following the logic set out above, the absence of a change in the n-
C18/phytane ratios through time for the mixed sand and gravel sam-
ples suggested that oil biodegradation was not occurring despite its
degraded state prior to fertilizer application. The initial low ratio may
Experience* with the Exxon Valdez Oil Spill 287
i
CO
o
c
15
11
08 Jun 89 24 Jun 89 10 Jul 89 26 Jul 89 11 Aug 89 27 Aug 89 12 Sep 89
(a)
3r
is
S.
.e
a
ao
5
1
1
18
18
08 Jun 89 24 Jun 89 10 Jul 89 26 Jul 89 11 Aug 89 2? Aug 89 12 Sop 89
Sampling data
| o Analysis value
Median
(f)
figure 12.3 Changes in n-ClS/phytane relationships over time at treated and control
cobble beaches.
-------
289 Chapter Twtivt
o
!'
9
c
08 Jun 89 24 Jun 89 10 Jul 89 26 Jul 89 11 Aug 89 27 Aug 89 12 Sep 89
(a)
CO
O
c
13
10
08 Jun 89 24 Jun 89 10 Jul 89 26 Jul 89 11 Aug 89 2? Aug 89 12 Sep 89
Sampling data
o Analysis value
Median
ib)
Figure 12.4 Changes in n-C18/phytane relationship over time at treated and control
sand and gravel beaches.
Experiences with the Exxon Valdai: Oil Spill 289
100
c
e
o
£
o
n-G18
c
40
IS
CL
20
phytana
100
0
20
40
60
80
Days
Rgure 12.5 Changes in the concentration of phytane and n-Cl 8 (expressed as percent
change relative to the t = 0 median concentration) in oil samples from mixed sand and
gravel (under the cobble) from Inipol-treated beach plots in Snug Harbor.
have limited subsequent degrees of observable change. However, part
of this effect can be explained by another unexpected complication.
Examination of phytane itself in the mixed sand and gravel under the
cobble on the Inipol-treated beach showed that its decay was as fast as
that for n-C18 alkane (Fig. 12.5). Consequently, either biodegradation
was not occurring (i.e., some nonbiological process was removing both
hydrocarbons simultaneously) or phytane was actually being degraded
as fast as the n-018, Phytane degradation is not common, but it does
occur (Pirnik et al., 1977; Mueller et a)., 1992), and we have easily iso-
lated phytane-degrading microorganisms from the beach material in
Prince William Sound. Thus, microbial communities on Alaskan
beaches may have a very pronounced ability to degrade branched alka-
nes, and the concept of using phytane as an internal biological marker
in that case becomes compromised. The cobble surface samples also
showed significant decreases in phytane through time, but at a slower
rate than the n-C18, thus giving the observed decay in the ratio.
Alternatively, results from the mixed sand and gravel samples under
the cobble suggested that possibly Inipol was acting in a chemical man-
-------
290 Chapter Twelve
ner (surfactant effect) to remove aliphatic and branched hydrocarbons.
However, n-Cl8 and phytane disappeared at essentially the same rate
in mixed sand and gravel samples from the untreated control beach
{data not shown). Since there was no possibility of a chemical effect on
the control plot, one would have to conclude that phytane removal was
primarily due to biodegradation.
Compositional changes In aromatic
hydrocarbons
At this point we can conclude that biodegradation of the aliphatic frac-
tions of oil was occurring on the samples taken from the cobble surface,
and, quite possibly, in the mixed sand and gravel samples as well. As
stated above, we believe that biodegradation of the aliphatic fraction
was not sufficient in itself to establish that bioremediation was effec-
tive. Thus, another approach for examining the overall biodegradation
of the oil was to perform selective mass ion spectrometry following gas
chromatographic analysis. A variety of aromatic hydrocarbons, which
are perhaps more difficult to degrade than the aliphatic hydrocarbons,
can be examined with this method (Kennicutt, 1988; Rowland et al.,
1986). This is important not only because it tracks another degradabie
fraction of the oil, but because certain polycyclic aromatic hydrocar-
bons (PAHs) are known to be procarcinogens under specific conditions.
Observing their removal from the oil would therefore imply a reduction
in potential adverse ecological effects. Whether this toxicity issue is
really relevant (due to improbable exposure scenarios), it was nonethe-
less a factor to be considered in effectiveness assessments. Further-
more, as the low solubility of the PAHs makes them difficult to
degrade, they can be used as the measure of extensiveness of oil degra-
dation. Mass spectral analysis of a variety of aromatic and heterocyclic
hydrocarbons in several samples of oil with greatly reduced n-Cl 8/phy-
tane ratios and aliphatic hydrocarbon concentrations is shown in Table
12.4. The selected aromatic hydrocarbons represent a group of methyl-
substituted homologs that are found close to the mass number of each
parent chemical structure (based on known standards). The values in
the table are normalized to hopane (17 alpha, 21 beta), a multiring
cyclic alkane (C30). Hopane and its homologs, which are quite resistant
to biological attack, have been used for some time as conserved inter-
nal biomarkers in oil by the geochemists (Kennicutt, 1988). However,
unlike the n-Cl8/phytane ratio, the relative changes cannot be at-
tributed to biodegradation with as much confidence; differential decay
between a hydrocarbon and hopane could be due to nonbiological pro-
cesses since there may be considerably less chemical similarity be-
tween the target hydrocarbon and hopane. Nonetheless, hopane
Experiences with the Exxon Valdez Oil Spill 291
TABLE 12.4 Relative Concentrations (Mean* and Standard Deviation) of Aromatic,
Heterocyclic, and Cyclic Hydrocarbons Normalized to Hopane
Prudhoe Bay crude Unfertilized beach Fertilized beach
n-C18
52.9
1.14(1.43)
0.96 (0.78)
Phyturiu
28.3
13.80(2.30)
0.63 (3.63)
C3/Naphthalenes
31.9
0.16(0.13)
0.08 (0.04)
G3/Kluorenes
5.30
1.74 (0.38!
1.01 (0.89)
03/Phenanthrenes
10.0
5.40(0.25)
3.36(1.35)
Dibenzothiophene
5.33
0.07 (0.03)
0.04 (0.03)
Ca/Dibenzuthiopherie
9.49
5.34 (0.64)
3.42(1.43)
Chrysenes
1.22
0.89(0.04)
0,71 (017)
C3/Chryaeuea
2.49
1.13(0.22)
1.03(0.44)
Nurliupane
0.56
0.62 (0.93)
0.59 (0.06)
Stearanes
5.5
5.91 (0.50)
5.15 (0.42)
*N = 8; randomly selected from the 7/29/89 sampling; all samples 50 milligrams of extracted
oil residue per milliliter.
provides a consistent standard to normalize the concentrations of aro-
matic hydrocarbons.
From Table 12.4, the relative difference in the amount of each group
of homologs between the beach samples and Prudhoe Bay crude oil in-
dicates the degree of compositional change that occurred in the sam-
ples on the beach. Samples from the Inipol-treated beach with very low
n-C18/phytane ratios also showed large changes in many of the aro-
matic hydrocarbons, Norhopane and stearanes are equally as resistant
to biodegradation as hopane, and their consistency throughout
strongly supports the concept of hopane as a conserved internal biolog-
ical marker. Again, there was no way to explicitly state that the
changes in the aromatic and heterocyclic hydrocarbons were due to
biodegradation, but the suggestion is strong. In fact, many of the
higher-molecular-weight PAHs are unlikely to be affected by chemical
or physical processes, and therefore biodegradation may be the only
mechanism that might explain their disappearance. Also, these sam-
ples were taken from the beach approximately 78 days after the appli-
cation of the Inipol fertilizer; any residual chemical effect from the
fertilizer that could cause these changes in composition was equally
unlikely.
If we assume that nonbiological fate processes fsuch as leaching or
dissolution) remove groups of aromatic hydrocarbons in the order of
their solubility, the most soluble being removed first, then any excep-
tion to that trend could be attributed to biodegradation since solubility
is not the sole characteristic determining susceptibility to microbial at-
tack. Examination of Table 12.4 shows that there are several cases
-------
292 Chapter Twelve
where decreases in aromatic hydrocarbon concentrations in the field
samples (relative to Prudhoe Bay crude oil) are greater for hydrocar-
bons that are more insoluble. For example, there was a greater de-
crease in C3-chrysenes than there was in chrysene itself, and there was
Akh!, rr8"^'116 C3-fluorenes than the C3-phenanthrenes.
Although these differences could be an artifact of the sampling and/or
analysis, it can be argued that biodegradation was the only process
where this differential effect is a possibility. Thus, in most samples
where substantial degradation of the oil has occurred (as measured by
H-^H/Phytane ratios), there was also a concomitant decrease in
many high-molecular-weight aromatic hydrocarbons.
Relationship of compositional change to
residue weight change
The next step was to establish a relationship between compositional
changes and the loss of oil residues. A positive correlation supports the
idea that loss of oil residues was due to biodegradation processes.
igure 12.6 shows a plot of the changes in residue weight against
changes in the n-C18/phytane ratio. A good positive correlation was ap-
120
2 80 -
i = -4 0964 + 1.1507x R"2 = 0.950
y = 9.2128 + 0.56433* R*2 = 0.944
O Control
• Inipol
20 40 60 80 100 120
n-C18/ptiytane ratio
ngur» 12.6 Relationship of changes in oil residue weight to changes in the n-ClS/rhvt»n»
bie surfa^a for	f thd'a" va,u« t = 0) from oil samples taken firo^cob-
pointaat the lowwleft comirand.lultreated ^«ch plots in Snug Harbor. Data
pom« at tne lower left corner of the graph represent samples taken late in the sampling
Experience* with tha Exxon Valdez OH Spill 293
TABLE 12.5 Rat* Analysis of Natural Log-Trantformed OH Residue Weight*
(mg/g) In Cobble Surface Sample* (July 8,1989 to July 29,1989 only) for Test
Beaches at Snug Harbor
Beach
Slope
(std, dev.)
Significance of slope,
greater than zero
N T-value P*
Half-life,
days
Time to remove
90%, days
Inipol
-0 016
80
-2.4 0.02
44
146
treated
(0.007)




Untreated
-0.006
65
-0.56 0,58
124
411
control
(0.010)




'Only the Inipol rata is significantly different from isra at the 95% confidence level.
parent. The results are interesting because a rapid reduction in the
ratio could occur without a significant change in oil residue weight.
However, this was not the case, and we can conclude that once degra-
dation of the aliphatic fraction commences, so does biodegradation of
many of the other fractions of the oil, at different rates.
Good quantitative information on biodegradation can now be ob-
tained. Since decay rates for the oil residues appeared to be first order,
half-lives of the oil can be calculated (Table 12.5). Application of
oleophilic fertilizer caused a greater than twofold increase in the dis-
appearance of oil residues on the cobble surfaces as compared to the un-
treated control. The difference was statistically significant despite the
variability in the data. No difference in the oil residue decay rates was
detected in the mixed sand and gravel.
Based on the discussion above we would attribute the greater rate of
decay on the fertilizer-treated beach to an enhancement of biodegrada-
tion from the provision of nitrogen and phosphorus nutrients.
Interestingly, the enhancement effect of the fertilizer appeared to be
sustained for as long as 90 days. This time period was well beyond that
in which nutrients would be released or in which the fertilizer might
have a chemical washing effect. Thus, "priming* the biodegradation
process with a little bit of nutrient seemed to go a long way. One can
generalize and say that over a 120-day period (i.e., the maximum win-
dow for Alaska in which water temperatures are >10°C and thereby ad-
equate for oil biodegradation), bioremediation would remove
(assuming linearity) approximately 4 times more oil from the cobble
surface than would disappear on untreated control beach. Thus with an
initial concentration of 1.0 milligram of oil per gram of beach material
(cobble surface), biodegradation can potentially remove most of the oil
in a single summer season. This was consistent with our visual obser-
vations. The absence of any effect on oil residues in the mixed sand and
gravel under the cobble suggested that oil may not have been Bpread in
-------
294 Chapter Twelve
a thin enough layer over the beach material to allow bioremediation to
have an effect during this testing period. Or possibly the Inipol was un-
able to provide nutrients to this area of the beach—i.e., it was primar-
ily acting at the beach surface.
Nutrients and microbial blomass
Following the application of the oleophilic fertilizer, interstitial water
samples were taken during several tidal cycles to determine if in-
creased concentrations of nitrogen and phosphorus could be observed.
Water samples were taken using a modified root feeder apparatus
which sampled water 10-15 cm below the surface of the mixed sand
and gravel. Sampling was conducted 2,10, and 30 days after fertilizer
application. Elevated nitrogen concentrations were seen only in the
day 2 sampling (Table 12.6), but in areas of the test beach, very high
concentrations were observed. However, the variability was quite large
with somewhat of a bias toward one side of the treated area. If all of the
nitrogen in the fertilizer was released at once into a hypothetical body
of water overlying the beach test plot at high tide, one would expect
concentrations of approximately 200-300 |im N. Obviously, these con-
centrations were reached in some areas of the beach. Given that three
tidal cycles had occurred prior to this sampling, much of the nitrogen
in the Inipol fertilizer was probably released in the first few days. This
corresponds with the nutrient release data generated from laboratory
studies described above. Thus, the enhancing effect of the oleophilic
fertilizer on oil biodegradation may have been the result of an initial
pulse of nutrients rather than a sustained concentration of nutrients
over extended periods. Other laboratory and field data support this
possibility (Pritchard et al., 1991).
Increases in oil biodegradation rates as a result of fertilizer applica-
tion should also result in increases in the number of oil-degrading bac-
teria. To determine if this was the case, beach samples (mixed sand and
TABLE 12.6 Ammonia Nitrogen (|iM) In Interstitial Water Samples Taken
on an Incoming Tide, 2 Days Following Application of Oleophilic Fertilizer
on a Cobble Beach in Snug Harbor



Block*

1
3
5
7
High-tide zone
57
300
10
4
Mid-tide zone
410
61
3
6
Low-tide zone
190
3
2
3
*Blociu were 5 m long and 4 m wide running end-to-end parallel to the water line
and covering three parallel zones, each 4 m wide running 9ide-to-side up the beach.
Bloclu 2, 4, and 6 in each zone were not sampled.
Experiences with the Exxon Valdez Oil Spill 295
gravel) were analyzed using an MPN (most probable number) proce-
dure (Pritchard et al., 1991) in which changes in the physical consis-
tency of the oil were monitored as an indication of oil biodegradation.
There was no significant difference between the control and treated
beaches over the 3-month sampling period (data not shown). However,
as indicated above, the concentrations of oil degraders were very high
to start with, and, with the large variability observed in the data, in-
creases of approximately two orders of magnitude were needed to be
significant. In addition, increases in biomass could be obscured by
sloughing of the cells or predation by protozoa. Field studies the fol-
lowing summer (1990) were finally able to demonstrate significant in-
creases in hydrocarbon degraders but only in the beach subsurface
(Lindstrom et al., 1991).
Disk Island Field Study
A portion of the northwestern shore of Disk Island (located between
Ingot and Knight Islands) was chosen as a study site in the summer of
1990, one year after the oil spill. The study was designed to obtain dose-
response information for fertilizer application. Fertilizer granules were
selected because it was relatively easy to apply different concentrations
of the granules in a controlled manner.
The study site was chosen because it was one of the few remaining
large areas with moderately to heavily contaminated beach material
that could be reasonably used for experimental purposes. The beach
area chosen for study, which had not been through the physical wash-
ing process used by Exxon, had a shallow slope with little wave activ-
ity. Oil contamination was surface and subsurface and was packed into
the mixed sand and gravel beach material more densely than observed
on other types of beaches in Prince William Sound.
Different amounts of fertilizer granules were applied to plots as
shown in Fig. 12.7. The 100 g/m2 application rate was the concentration
of granules applied on a large scale by Exxon. Prior to the fertilizer ap-
plication, samples of beach material were homogenized and placed in
sampling baskets located in each plot. These sampling baskets were
then harvested periodically to determine the effect of the fertilizer on
oil biodegradation. The homogenization reduced variability in oil con-
centrations and therefore greatly simplified sampling efforts.
Changes in the concentration of ammonia following application of
the fertilizer granules are shown in Fig. 12.8. These data were obtained
from sampling wells that were driven into the beach material to allow
sampling of the interstitial water with incoming and outgoing tides.
The highest concentrations of ammonia were seen with the highest
-------
296 Chapter Twelve
*! 251b
•«W
»» j	, *2'5B -
•toOOO"" ,60°"OOpm-
Flgur* 12.7 Disk Island fertilizer specific activity plot, map and rate of CUSTOMBLEN
granule application.
concentrations of granules applied (rate 4; 1000 g/m2), but this concen-
tration was not sustained For more than I to 2 days. In fact, ammonia
concentrations approached background levels 5 to 10 days after appli-
cation, regardless of the fertilizer granule concentration applied.
Clearly, if a dose response is to be observed, it will result from an ini-
tial pulse of nutrients rather than sustained concentrations through
time. Results for the release of phosphate and nitrate were similar.
An examination of the decrease in the n-C18/phytane ratio for sam-
ples taken from the beaches at different times following initial appli-
cation of the fertilizer showed that there was essentially no
enhancement of biodegradation, as the extent of decrease was not
greater than that seen on the control, untreated plots. A comparison of
the ratios for a control plot and the plot receiving the highest concen-
tration of fertilizer granules is shown in Pig. 12.9. Biodegradation was
obviously occurring (i.e., a steady decrease in the ratios), but it did not
seem to be stimulated by the fertilizer. This was a startling result be-
cause it reveals that not all beach conditions may be equally amenable
to bioremediation. Because of the low-energy features of the beach and
the more compact nature of the beach material, mass transport limita-
tions (availability of nutrients and/or oxygen) may have become a sig-
nificant problem, and this is a key factor to be considered in using
bioremediation on other types of oil-contaminated beaches. We are also
aware that the beach material contained quantities of humic material;
this may have interfered with the oil-degrading microbial communities
either as a competing sink for available oxygen or as a degradable car-
bon source that was preferable to petroleum hydrocarbons.
This experience provides a lesson regarding the use of laboratory
tests as an indicator of the potential for bioremediation. If it was a
Experiences with the Exxon Valdai OH Spill 287
3r
1 *\
f\\
i \
I \
\ \A .^.
§4 ¦V
r-»~y-*"	t _ —	mmm*=Q
22 Jun 90	29 Jun 90	06 Jul 90	13 Jul 90
Sample collection date
Legend x Control 1
« Control 2
a Rate 1
o Rate 2
A Rale 3
o Rate 4
(a)
3r
e
a
A.
—a.
22 Jun 90	29 Jul 90	06 Jul 90	13 Jul 90
Sample collection date
legend x Control 1
« Control 2
~ Rate 1
o Rate 2
A Rale 3
o Rate 4
(b)
Figure 12 J (a) Changes in ammonia concentration over time for the incoming tide for all
plots for the Disk Island Fertilizer Application Rate Study. (6) Changes in ammonia con-
centration over time for the outgoing tide for all plots for the Di»k Island Fertilizer
Application Rate Study.
-------
298 Chapter Twelve
0.9
0.8
1	07
2
§ 0.6
I M
5 0.4
f 0.3
0.2
0.1
O.Ohj	I
20 Jun 90 28 Jun 90
06 Jul 90
14 Jul 90
22 Jul 90
30 Jul 90
Sample collection date
Legend oo o Oiled homogenale &&& Basket sample •-++ Median value
(a)
1.0
0.9
o.a
I °-7
8
i 0.6
| 0.5
S 0.4
f 0.3
0.2
0.1
00b	i__
20 Jun 90 28 Jun 90
14 Jul 90
06 Jul 90
22 Jul 90
30 Jul 90
Sample collection dale
Legend coo Oiled homogenale a a a Basket sample «-»-• Median value
Flgura 12.9 (a) Changes in the n-Cl S.'phyt&ne ratio over time for the 500 g/m4 fertilizer
application for the Disk Island Fertilizer Application Rate Study. (6) Changes in the n-
ClS/phytane ratio over time for untreated central plot nuinliur I for the Dink Inland
study.
Experiences with the Exxon Vaidaz OH Spill 299
mass transport phenomenon that effected successful bioremediation at
Disk Island, removing beach material to the laboratory and conducting
tests similar to those described above will likely not reveal the limita-
tions inherent in the field. Most of these laboratory tests involve shake
flasks, which by design optimize mass transport, and one would there-
fore expect that samples may show unrealistically high activities rela-
tive to the field. This is illustrated in mineralization studies performed
in conjunction with the Disk Island study. Beach material from the
sampling baskets, when placed in biometer flasks (see above), showed
mineralization activities that reflected an enhancement effect due to
the presence of the fertilizer (Fig. 12.10). Some of the total CO, pro-
duction may have been from "nonpetroleum" organic material present
in the beach material; however, similar studies using radiolabeled hy-
drocarbons revealed that stimulation of oil degradation by the fertilizer
was probably occurring in these flasks. There also appears to be a dose-
response relationship in these results, suggesting that doubling the
fertilizer concentrations did not double the oil biodegradation rate as
measured by mineralization. Clearly this relationship was not realized
in the field.
Thus the stimulatory effect observed in the laboratory was not re-
flected in the field. The flask studies did, however, indicate that some
mass transport limitation was affecting the bioremediation in the field.
E
SL
C
0
1
I
03
50g/m:
O
u
3
Untreated
control
Q.
m
CC
0.005 0.01 0.015 0.02 0.025 0.03
Fertilizer application rate
0
100 200 300 400 500 600 700 800 900 1000
Rate of fertilizer application (g/m2)
Figure 12.10 Plot of rate of OCX, production veraua rate of fertilizer concentration.
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300 Chapter Twelve
The only way to protect from this extrapolation problem is to perform
microcosm studies in which intact samples of beach material can be
studied in the laboratory under conditions similar to those in the field.
Such systems were in fact developed and tested during the Alaskan oil
spill project (Pritchard et al., 1991), but they involve considerably more
complexity, time, and expense.
EPA Program in Oil Spill Bioremediation
Protocol Development
Closely tied to the field studies just described is the question of which
commercial products, whether they are microorganisms, nutrients,
surfactants, or others, could or should be used in a bioremediation con-
text, As we mentioned above, in Alaska, many of these commercial
products could not be considered because of the very short time frame
for field demonstrations, but also because the data available for each
product were so variable and/or insufficient that reasonable selections
could not be made in a timely fashion. The development of effectiveness
and environmental safety testing protocols to be used in establishing a
consistent and relevant database, upon which decisions for the use of
particular commercial products might be based, is now under way
within the Office of Research and Development of the EPA. The con-
ceptual basis for these protocols has proven to be complicated to for-
mulate because of the need to keep the scope of the testing within
reason. However, the problems encountered in environmental variabil-
ity, as illustrated above, make it very difficult to devise protocols that
will ultimately provide the "right" kind of information to allow appro-
priate regulatory decisions to be made. A review of the initial concep-
tualizing we have carried out to date is in fact quite informative.
Obviously, as these protocols are tested and validated, these concepts
will have to be modified, so information presented here is not to be con-
sidered as final guidelines.
The protocols for determining the effectiveness of oil bioremediation
products must ultimately contain the following components:
1.	Simplified "expert system* (basically decision trees) for use by regu-
lators that will encourage rational consideration of peripheral fac-
tors that are keys to the success of bioremediation on open waters
(Tier I—decision trees)
2.	A screening test that allows the relative effectiveness of different
bioremediation products to be assessed in terms of their ability to
promote significant biodegradation of oil under a standard set of lab-
oratory test conditions (Tier II—screening information)
Experience* with the Exxon Valttez CHI Spill 301
3.	A procedure for extrapolating laboratory information to the field on
a site-specific basis using definitive kinetic and dose-response infor-
mation that is integrated with simplified and quantitative predic-
tive frameworks (Tier III—field extrapolation information)
4.	A procedure for the use of flow-through microcosm systems to de-
termine the relative effect of different commercial bioremediation
products on oil slicks under the environmental conditions that are
likely to be experienced during oil spills (Tier III—field extrapola-
tion information)
5.	Guidelines for the performance of appropriate controlled field stud-
ies in artificial enclosures to clearly establish the fate of the oil dur-
ing bioremediation (Tier IV—direct field demonstration)
It is prudent to focus on only one portion of the protocol development
because of space limitations. I will provide the concept for the testing
that would be implemented under the site-specific extrapolation (item
3) and the microcosm testing (item 4) components of the protocol.
However, a brief description of the concepts behind a Tier II testing
protocol will be given first as a means to develop comparisons.
The purpose of Tier II testing is to determine the ability of a partic-
ular bioremediation product to promote significant biodegradation of
oil under a standard set of laboratory testing conditions. It is not de-
signed to address effectiveness of a bioremediation product under site-
specific conditions.
It is further assumed that the Tier II tests are to be divided into
parts: that which measures the activity of biological products and that
which examines the effectiveness of nonbiological products. Tests for
biological products involve adding the product directly to an oil slick in
a proportion recommended by the manufacturer. Other supplements
recommended by the manufacturer are also added. It is assumed that
the microbial flora of natural water is not required since most of the bi-
ological activity is provided by the product. Tests are conducted with
an<3 without added nutrients in the water and with sterile (autoclaved)
and nonsterile products. Flasks are incubated by shaking at 20°C. If
the product is found to be effective, results are compared with a stan-
dard data set that is developed from research information specifying
the minimum amount of product activity (oxygen uptake profile and
changes in oil chemistry) required to make the product effective as a
bioremediation agent.
For the testing of nonbiological products, it is assumed that they gen-
erally involve some mechanism or procedure for stimulating the oil
biodegradation capabilities of natural microbial flora. In most cases
this involves the rapid enrichment of the oil-degrading microorganisms
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302 Chapter Twelve
within the total bacterial population. Since at the Tier II level of test-
ing it is not appropriate to consider site-specific factors that affect the
activity or enrichment of oil-degrading microorganisms, a standard
mixture of bacterial pure cultures that degrade all of the major frac-
tions of the oil tested needs to be used. Alternatively, a specific sam-
pling site can be selected as a consistent source of natural populations
of bacteria. However, there is no guarantee that the responsiveness of
a water sample from this site will remain constant, and thus the use of
a mixture of pure cultures has several advantages: (1) It allows one to
store the bacteria over long periods and preserve their activity so that
it is the same each time it is used; (2) it eliminates the variances that
are likely to occur if natural samples are used as inocula, thereby elim-
inating the requirement to run reference products each time a test is
conducted; (3) optimal conditions of enrichment are employed, thus giv-
ing an evaluation of the bioremediation product under ideal conditions.
If it does not work under these conditions, then it is very unlikely to
work in the field, where the conditions will be a lot less ideal.
Effectiveness of the product will be measured against a standard
time, as established by practical conditions at a spill site and by the
time it will take to enrich the mixed culture to a point where it will af-
fect the oil. The oil may break up on the surface of the water relatively
quickly as a result of the product addition. The test system is designed
not only to monitor this event but also, as a closed system, to allow the
fate of the oil to be followed for several weeks thereafter. If significant
degradation is seen during this incubation period, then it will be as-
sumed that oil leaving the water surface as a result of adding the prod-
uct will in fact be degraded. Research now carried out under the Oil
Spill Research Program will provide a verification for this assumption.
Concept and development of Tier III testing
protocols for open water
The appropriateness of oil bioremediation products that are proposed
for use in open sea bioremediation must be tested in the laboratory
under conditions that are reasonably representative of field conditions.
Although testa of this nature can become quite complex, microcosm sys-
tems that model special features that might be key to the success of oil
bioremediation on open waters have been designed. By far the key fea-
ture of the microcosms is the incorporation of a dilution capacity in the
microcosms that would simulate the field. Flow-through microcosms
that contain an oil slick on the water surface are used in this part of the
protocol to assess
1. The tendency of a bioremediation product to remain with the oil long
enough to be effective
Experiences with the Exxon Vatdei Oil Spill 303
2. The potential of a product to emulsify the oil and cause it to be dis-
persed
Microcosm systems that allow the application of a bioremediation
product and the necessary supplements to a contained oil surface and
then allow seawater to flow under the slick at different velocities and
turbulences to create the necessary dilution capacity estimated from a
particular field situation will be used. This testing will determine the
extent to which the product and the supplements will stay associated
with the oil. It will not of course indicate that similar dilution will occur
in the field, but it will provide a consistent method to screen, on a rel-
ative basis, products and the efficacy of their application strategy. If,
for example, a product (or the required supplement) is rapidly removed
from the oil slick in the microcosm, it will almost certainly be removed
much faster in the field. A product that appears to be effective (i.e., ap-
pears to stay with the slick and affect the fate of the oil) would be one
considered for further testing. In addition, flow and turbulence condi-
tions in the microcosms can be varied to provide a range of conditions
under which the effectiveness of the product could be evaluated.
The Tier III testing is based on biodegradation kinetics and involves
the use of microcosm studies to determine the effect of certain key en-
vironmental parameters on the ability of the bioremediation product to
enhance the biodegradation rates of oil. Kinetic information is very im-
portant for protocols dealing with the treatment of oil on the open
water because it will always be a question of how fast the product will
work under a specific set of environmental conditions relative to the
rate at which the oil slick is being dispersed naturally.
The use of flow-through microcosms is necessitated by a requirement
to determine the effectiveness of a product under conditions that can-
not be modeled in a flask study or Tier II level testing. These conditions
are defined specifically as the following:
1.	Presence of an intact oil layer floating on the surface of a water col-
umn
2.	Ability to impart and control water column turbulence during test-
ing with the intact oil layer
3.	Continuous input of water containing significant concentrations of
particulates in the water column
4.	Flow-through conditions to allow water exchange and dilution
under the oil layer
5.	Temperature, particularly as it affects the physical nature of the oil
6.	Concentrations of inorganic nutrients in the flow-through water
7.	Microbialalow water
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304 Chapter Twelve
In the consideration of microcosm testing at the Tier III level, several
points must be considered carefully.
1.	Microcosm testing is expensive for the vendor because it requires
rather elaborate testing facilities, extensive analytical chemical
analysis, and sophisticated means of interpreting the resulting
data. Thus, the testing should be kept to a minimum.
2.	Because of this expense and elaborateness, microcosm testing can-
not be used to examine the influence of a large number of ecological
or oil spill conditions on the effectiveness of the bioremediation
product.
3.	Microcosms by definition are designed to simulate certain environ-
mental conditions more realistically than simpler tests, such as
shake flasks. Therefore, the specific conditions to be modeled in the
microcosm must be critically evaluated such that the microcosm
testing does not produce data that is more effectively and efficiently
obtained in simpler systems.
4.	When considering open-water application of a bioremediation prod-
uct, it must be realized that modeling conditions typical of a spill
site will be very complex, to the point that it is questionable how ef-
fectively certain conditions can be appropriately modeled in micro-
cosms. For example, the effectiveness of a bioremediation product
will likely be a function, in part, of wave and turbulence conditions.
Developing these conditions in a microcosm is difficult. In addition,
extrapolation of microcosm information to the field is complicated
because these conditions will vary on a day-by-day or even hour-by-
hour basis. A range of turbulence/wave conditions could be tested in
the microcosm, but this must be limited to testing extremes because
of the constraints of cost and time when using microcosms.
5.	Finally, it must be kept in mind that Tier III testing involves putting
information on the "shelf* to be used at the time of the spill (time is
too short following the spill to conduct microcosm-type tests). Thus
the shelf data must be of such a nature that it can be used effective-
ly in making decisions at the time of the oil spill.
As mentioned above, one of the most critical factors when performing
microcosm studies is the ability to interpret the resulting data. The
larger the database available on the product performance, particularly
in terms of kinetics, the better one will be able to use the microcosm re-
sults and extrapolate the information to a site-specific situation.
Because each site is different, it is impossible to have information "on
the shelf* for a particular product that will deal with every site.
Therefore, one has to make a decision on how to generalize the testing
Experience# with the Exxon Valdei Oil Spill 305
approach. This can be accomplished either by testing waters from sev-
eral designated areas (Atlantic Coast versus Pacific Coast or Gulf
Coast, northern waters versus southern water, protected bays versus
open bays, wetlands versus marshes, etc.) or by examining the effect of
selected environmental parameters that encompass, in a general way,
all of the conditions in these different areas and then extrapolating the
general results to site-specific conditions that can be determined at the
time of the spill. The latter approach seems to be the most reasonable.
Consequently, a protocol must be developed based on an initial deci-
sion as to the major factors that will most likely affect the performance
of the product in treating oil on open waters wherever the spill takes
place. In general, the most important factors will be
1.	Temperature
2.	Turbulence
3.	Salinity
4.	Background concentration of inorganic nutrients
5.	Suspended particulates
6.	Background microbial activities
7.	Type and concentration of oil
Then it must be determined if the factors can be measured in the field
at the time of the spill (within 1 to 2 days). For those that likely can be
measured, quantitative relationships can be established between the
factor and the product performance. A good example is turbulence. The
effectiveness of the product can be established under three different
turbulent conditions: high, medium, and low. The data is graphed (ef-
fectiveness versus turbulence expressed in Reynolds number or the
equivalent), and a relationship established (linear, exponential, etc.)
using statistical techniques. Once the relationship is known, then per-
formance of the product for a particular site condition can be predicted.
That is, a general indication of turbulent conditions at the spill site will
be obtained and the graph will be used to determine the effectiveness
of the product.
Since ultimately one will likely be dealing with several environmen-
tal factors at once and several specialized environmental conditions, a
simple calculation framework can be used. A protocol should stipulate
that, for any product, the key environmental factors that will affect
performance for any site will be turbulence, temperature, microbial ac-
tivity, type of oil, nutrient concentrations in the water column, and par-
ticulates in the water column. No other factors need be considered as it
is assumed that they will be insignificant in the overall decision to use
-------
306 Chapter Twelve
or not to use the product. Thus every product will have "data on the
shelf that relates these key environmental factors to performance of
the product.
Summary and Conclusions
The results from our field demonstration in oil spill biorernediation in
Prince William Sound indicate that the oleophilic fertilizer Inipol
EAP™ 22 served as an effective nutrient source for oil-degrading mi-
crobial communities. It enhanced oil biodegradation, as measured both
by changes in oil composition and oil residue weights, by as much as
twofold relative to the untreated controls. This was enough of a re-
sponse to merit incorporation of biorernediation, on a large scale, into
the remedial action plan for oil-contaminated beaches in Prince
William Sound. Despite this enhancement effect,, the importance of its
oleophilic nature is still unclear, at least for oil-contaminated beach
material from Prince William Sound. However, our studies report the
belief that the visual observation of oil removal from the beaches S to
10 days following application of the Inipol was due largely to biorerne-
diation and not to a chemical washing effect.
Overall, rapid oil disappearance brought on by the application of the
oleophilic fertilizer made these beaches more compatible with local
wildlife (less tendency for fur and feathers to become oiled). These
changes occurred in a shorter period of time than those limited changes
observed in untreated control plots, and possibly helped accelerate bi-
ological recovery of the intertidal area.
We have also summarized some of the lessons associated with mea-
suring biorernediation success in the field. Effective measures of
biodegradation and interpretation of resulting data, given the highly
variable nature of field studies, have been emphasized as a key element
in this success. In addition, the complications and difficulties that
arose during our use of biorernediation efforts in Alaska were also dis-
cussed. Hopefully this will help guide similar applications of biorerne-
diation at future spills.
References
Atlas, R. and R. Bartha. 1973. Stimulated biodegradation of oil siicka using oleophilic fer-
tilizers. Environ. Set, Technol. 7:538-541.
Atlas, R. M. 1981. Microbial degradation of petroleum hydrocarbons: Ad environmental
perspective. Microbiol. Reu. 45:180-209.
Bartha, R. and D. Pramer. 1965. Features of a flask and method for measuring the per-
sistence and biological effects of pesticides in soil. Soil ScL 100:68-70,
Chianelli, R. R., T. Aczel, R. E. Bare, G. N. George. M, W. Genowiu, M. J. Grossman, C.
E. Haith, F. J. Kaiser, R. R. Lessard, R. Liotta, R. L. Mastracchio, V. Minal-Bemero,
R. C. Prince, W. K. Bobbins, E. I. Stiefel, J. B. Wilkinson, S. M. Hinton. J. R. Bragg, 3.
Experiences with the Exxon Valdez OH Spill 307
J. McMillem, and R. M. Atlas. 1991. Biorernediation technology development and ap-
plication to the Alaskan spill. In Proceedings 1991 Oil Spill Conf, Am. Petroleum
Inst., Washington, D C., pp. 545-555.
Bergatein, P. E. and J, R. Vestal. 1978. Crude oil biodegradation in arctic tundra ponds.
Arctic 31:159-169.
Glaser, J. A., A. D. Venosa, and E. J. Opatken 1991. Development and evaluation of ap-
plication techniques for the delivery of nutrients tu contaminated shoreline in Prince
Willium Sound. In Proceedings 1991 Oil Spill Conf., Am. Petroleum Inst.,
Washington, D C., pp. 556-562.
Halmo.G. 1985. Enhanced biodegradation of oil. In 1983 Proceedings Oil Spill Conf., Am.
Petroleum Inst., Washington. D C., pp. 531-537.
Jobson, A. M., F. D. Cook, and D. W. S. Westluke. 1972. Microbial utilization of crude oil.
Appl. Microbiol. 23:1082-1089.
Kennicutt, M. C. 1988. The effect of biodegradation on crude oil bulk and molecular com-
position. Oil Chem. Pollut. 4:89-112.
Lee, K. and E, M. Levy. 1987. Enhanced biodegradation of light crude oil in sandy
beaches. In 1987 Proceedings, Oil Spill Conf., Am. Petroleum Inst., Washington, D C.,
pp. 411^179.
Lindstrom, J, E., R. C. Prince, J. C. Clark, M. J. Grossman, T. R. Yeager, J. P. and E. J.
Brown. 1991. Microbial populations and hydrocarbons biodegradation potential in fer-
tilized shoreline sediments affected by the T/V Exxon Valdez oil spill. Appl. Environ.
Microbiol. 57:2514-2522.
Madden, P. C. 1991. Final Report Frail's Island Biorernediation Project. Exxon Res. and
Engineering, Florham Park, N.J. 82 pp.
Mueller, J. G , S. M. Resnick, M. E. Shelton. and P. H. Pritchard. 1992. Effect of inocu-
lation on the biodegradation of weathered Prudhoe Bay crude oil. J. Ind. Microbiol. In
press.
Olivien, R., P Bacchin, A. Robertiello, N. Oddo, L. Degen, and A. Tonolo. 1976. Microbial
degradation of oil spills enhanced by a slow-release fertilizer. Appl. Environ.
Microbiol. 31:629-634. (57)
Olivieri, R , A. Robertiello, and L. Degen. 1978. Enhancement of microbial degradation
of oil pollutants using lipophilic fertilizers. Marine Pollut. Bull. 9:217-220. (59)
Pirnik, M. P., R. M. Atlas, and R. Bartha. 1977. Hydrocarbon metabolism by
Brevibacterium erythragenes Normal and branched alkanes. J. Bacterial.
119:868-878.
Pritchard, P. H„ C. F. Costa, and L. Suit. 1991. Alaska Oil Spill Biorernediation Project.
U.S. EPA, Office of Res. and Dev. Report, EPA/600/9-91/046a, 522 pp. Washington,
DC.
Pritchard, P. H. and C. F, Costa. 1991. EPA's Alaskan oil spill bioremediatioD project.
Environ Sci, Technol. 25:372-379.
Rowland, S. J., R. Alexander, R. i. Kazi, D. M. Jones, and A. G. Douglas. 1986. Microbial
degradation of aromatic components of crude oils: A comparison of laboratory and field
observations. Org. Gtochem. 9:153-161.
Sveum, P. and A. Ladousse. 1989. Biodegradation of oil in the Arctic: Enhancement by
oii-soluble fertilizer application. In 1989 Proceedings Oil Spill Conf, Am. Petroleum
Inst., Washington, D C., pp. 439-446.
Tramier, B. and A Sirvimt. 1983. Enhanced oil biodegradation: A new operational too) to
control oil spills in Proceedings 1983 Oil Spill Conf, Am. Petroleum Inst.,
Washington, D C., pp. 155-219.
Venosa, A. D , J. R. Haines, J. A. Glaser, E. J. Opatken, P. H. Pritchard, and C. F. Costa.
1990. Biorernediation treatability trials using nutrient application to enhance cleanup
of oil contaminated shoreline. In Proceedings 83rd Air and Waste Management
Association Annual Meeting, Air and Waste Management Assoc., Pittsburgh, Pa., pp.
90-22.3.
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(I'l.EAS!: READ INSTRUCTIONS ON THE REVERSE BEE-ORE COMPLE'
WSfgW/A-94/205
2
3. RECIPIENTS
. TITLE AND SUBTITLE
FFECTIVENESS AND REGULATORY ISSUES IN OIL SPILL BIOREMEDIATION:
XPERIENCES WITH THE EXXON VALDEZ OIL SPILL IN ALASKA
5 REPORT DATE
G. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
H. Pritchard
0. PERFORMING ORGANIZATION REPORT NO.
i. PERFORMING ORGANIZATION NAME AND ADDRESS
J.S. Environmental Protection Agency, Environmental Research Laboratory,
Sabine Island, Gulf Breeze, Florida
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
i2, SPONSORING AGENCY NAME AND ADDRESS
U.S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
GULF BREEZE, FLORIDA 32561
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
'a, Biotreatment of Industrial and Hazardous Waste. Morris A. Levin and Michael A. Gealt (ed.), McGraw-Hill Book Co., New York, p. 269-307
16. ABSTRACT
The use of bioremediation as a supplemental cleanup technology in the Exxon Valdoz oil spill, in Prince William Sound, Alaska, has proven to be a
good example of the problems and successes associated with the prac tical application of this technology. Field studies conducted by scientists
from the U.S. Environmental Protection Agency have demonstrated that oil degradation by indigenous microflora on the beaches of Prince William
Sound could be significnatly accelerated by adding fertilizer directly to the surfaces of oil-contaminated beaches. Our results from the application
of an oleophilic fertilizer are presented as exemplary field and laboratory information. The fertilizer enhanced biodegradation of the oil, as measured
by changes in oil composition and bulk oil weight per unit of beach material, by approximately twofold relative to untreated controls. These studies
supported bioremediation as a useful cleanup alternative that was subsequently used by Exxon on a large scale. They have also generated a
number of insightful lessons that have significant relevance to future oil bioremediation efforts. This chapter discusses these lessons and examines
complications and difficulties in assessing the effectiveness of bioremediation in the field.
17. KEY WORDS AND DOCUMENT ANALYSIS
A. DESCRIPTORS
B. IDENTIFIERS/OPEN ENDED TERMS
C. COSATI FIELD/GROUP



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RELEASE TO PUBLIC
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21. NO. OF PAGES
38
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