Chemosphere 90 (2013) 521526

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Chemosphere
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The primary biodegradation of dispersed crude oil in the sea

Roger C. Prince a,, Kelly M. McFarlin b, Josh D. Butler a, Eric J. Febbo a, Frank C.Y. Wang c, Tim J. Nedwed d
a
ExxonMobil Biomedical Sciences, Inc., Annandale, NJ 08801, United States
b
University of Alaska, Fairbanks, AK 99775, United States
c
ExxonMobil Research and Engineering Co., Annandale, NJ 08801, United States
d
ExxonMobil Upstream Research Co., Houston, TX 77252, United States

h i g h l i g h t s g r a p h i c a l a b s t r a c t

" Dispersed crude oil is rapidly and

extensively biodegraded in natural
seawater.
" This occurs without the need for
added nutrients or bacteria.
" This biodegradation extends to all
resolvable classes of hydrocarbons
except the hopanes.
" The approximate biodegradation
half-life is 1114 d.

5 46
minutes

a r t i c l e i n f o a b s t r a c t

Article history: Dispersants are important tools for stimulating the biodegradation of large oil spills. They are essentially
Received 30 April 2012 a bioremediation tool aiming to stimulate the natural process of aerobic oil biodegradation by dispers-
Received in revised form 3 July 2012 ing oil into micron-sized droplets that become so dilute in the water column that the natural levels of
Accepted 7 August 2012
biologically available nitrogen, phosphorus and oxygen are sufcient for microbial growth. Many studies
Available online 8 September 2012
demonstrate the efcacy of dispersants in getting oil off the water surface. Here we show that biodegra-
dation of dispersed oil is prompt and extensive when oil is present at the ppm levels expected from a suc-
Keywords:
cessful application of dispersants more than 80% of the hydrocarbons of lightly weathered Alaska North
Oil spill
Dispersants
Slope crude oil were degraded in 60 d at 8 C in unamended New Jersey (USA) seawater when the oil was
Biodegradation present at 2.5 ppm by volume. The apparent halftime of the biodegradation of the hydrocarbons was
Bioremediation 13.8 d in the absence of dispersant, and 11 d in the presence of Corexit 9500 similar to rates extrapo-
lated from the eld in the Deepwater Horizon response.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Large amounts of crude oil have been entering the worlds
oceans for millions of years, and a diverse group of microorganisms
has evolved to take advantage of this rich source of reduced carbon
(Prince, 2010; Prince et al., 2010). Much of the oil comes from nat-
ural seeps, and while some adheres to sediment particles and is de-
Corresponding author. Tel.: +1 908 730 2134; fax: +1 908 730 1199.
E-mail address: Roger.C.Prince@ExxonMobil.com (R.C. Prince).
graded on the sea bottom, some rises to the sea surface to form
slicks, and some disperses as small droplets in the water column
(Farwell et al., 2009). Other oil comes from anthropogenic sources,
both as runoff from land and from drilling and shipping accidents
(National Research Council, 2003). Oil is an unusual substrate in
two main regards. First, only a few molecules such as the smallest
saturates (e.g. methane, ethane and propane) and aromatics (e.g.
benzene, toluene, ethylbenzene and the xylenes) are signicantly
soluble so biodegradation of most oil components must take
place at the surface of the oil. Second, while oil provides a rich

0045-6535/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2012.08.020
522 R.C. Prince et al. / Chemosphere 90 (2013) 521526
source of carbon and energy, it contains no signicant amounts of
biologically available nitrogen or phosphorus essential for micro-
bial growth. Together these mean that the biodegradation of signif-
icant amounts of oil in aerobic environments is likely to be limited
by the available surface area of the oil, and the presence of nutri-
ents. The biodegradation of oil on shorelines has been enhanced
by the judicious application of fertilizers to the oil (Bragg et al.,
1994; Prince and Bragg, 1997), but adding nutrients to slicks on
the open ocean is not usually appropriate in view of the likely com-
petition for uptake by planktonic photoautotrophs. Instead the
preferred approach for stimulating the biodegradation of oil slicks
and subsea releases is to add chemical dispersants to substantially
increase the surface to volume ratio of the oil, and allow the oil to
disperse so that the natural background levels of biologically avail-
able nitrogen or phosphorus are adequate for microbial growth
(National Research Council, 2005). For example, dispersants were
used on a large scale following the spill from the Sea Empress in
South Wales (Lunel et al., 1997), and more recently at the leaking
wellhead of the Deepwater Horizon blowout in the Gulf of Mexico
(National Commission on the BP Deepwater Horizon Oil Spill and
Offshore Drilling, 2011).
Despite these large-scale uses, and many tests of the effective-
ness of dispersants in dispersing crude oil, from laboratory through
large tanks, eld trials and actual use (e.g. Mukherjee and Wrenn,
2009; Li et al., 2009a,b; Belore et al., 2009; Lewis et al., 2010), few
papers have addressed the question of the biodegradation of dis-
persed oil under environmentally relevant conditions. Oil slicks of
light to medium oils spread rapidly until they are only approxi-
mately 100 lm or less in thickness (Lehr et al., 2002). Applying dis-
persant to such a slick in 1-m waves causes nearly immediate
mixing of dispersed oil into the top 11.5 m of the water column
(Delvigne and Sweeney, 1988), resulting in immediate dilution by
a factor of about 10 000 to give an average oil concentration of
100 ppm (Belore et al., 2009). The dispersed droplets are so small,
typically 10100 lm in diameter (Li et al., 2011), that they are en-
trained in the water column as discrete droplets that do not coalesce
but diffuse to ever more dilute concentrations. It is technically chal-
lenging to do biodegradation experiments at such dilutions, and no
published work that we have found (Bruheim et al., 1999; Lind-
strom and Braddock, 2002; MacNaughton et al., 2003; Venosa and
Holder, 2007; Zahed et al., 2010) comes very close.
We report here the biodegradation of fresh and lightly weath-
ered crude oil (the latter mimicking exposure at sea for 24 h) with
and without the dispersant Corexit 9500 (Nalco, 2011), at concen-
trations approaching those seen following a real spill in natural
seawater with no added nutrients or bacteria. This mimics the
conditions for natural biodegradation, but at the expense of
demonstrating the physical effectiveness of the dispersant at
such concentrations, and in stirred laboratory experiments, Alaska
North Slope crude oil disperses quite well with no additions. We
show that oil is extensively degraded under such conditions, and
that Corexit 9500 exerts no inhibitory effects.
2. Materials and methods
Seawater was collected from the New Jersey shore in April 2010
and January and April 2011 (winter conditions, salinity = 28 ppt,
temperature = 8 C). Nitrate and phosphate levels were below
detection limits with simple laboratory colorimetric tests, but are
likely to have been near 7 and 0.5 lM respectively (Louanchi and
Najjar, 2001). The seawater was transferred to a cold room at
8 C and aerated for 24 h prior to assembly of the experiments in
5 L carboys. Four liters of seawater were placed in each carboy,
and were stirred with a large magnetic stirrer to generate a 2 cm
vortex. We tried two methods of adding oil to these vessels, with
indistinguishable biodegradation results. In the rst, Alaska North
Slope crude oil was weathered by evaporation at laboratory room
temperature in a hood until it had lost 20% of its weight. Corexit
9500 was added at 5% to an aliquot, and 10 lL of the weathered
oil or weathered oil plus dispersant was added to each carboy
(both in triplicate) with a positive displacement pipette to yield
2.5 ppm oil by volume. As shown below, these experiments re-
vealed no substantial difference in the extensive biodegradation
seen after 60 d. In the second pair of experiments, 10 lL of fresh
Alaska North Slope crude oil was added to the 4 L batches of sea-
water without dispersant: the dispersed oil was generated by oat-
ing 1 mL of oil on 1 L of seawater in an aspirator vessel. The water
was stirred vigorously with a magnetic stir bar to generate a 2 cm
vortex, and 67 lL of Corexit 9500 was dropped on the slick with a
positive displacement pipette. The oil instantly dispersed through-
out the water column, and after 2 min of stirring the stirrer was
turned off and the contents allowed to settle for a few minutes.
Dispersed oil was drawn off from the bottom of the aspirator bot-
tle, and 10 mL of the dispersed oil/water mixture was added to 4 L
of seawater. In this case enough replicate vessels were assembled,
in two separate campaigns, that duplicate carboys of oil with and
without Corexit could be sacriced at 0, 4, 6, 11, 24 and 41 d.
At the designated time, oil was extracted from each carboy,
three times, with methylene chloride, dried with sodium sulfate,
and analyzed by gas chromatography coupled with mass spec-
trometry (Douglas et al., 1992). Care was taken to prevent com-
plete evaporation of the methylene chloride so as not to lose
volatile oil components during extraction. Oil biodegradation was
followed with respect to 17a(H),21b(H)-hopane as a conserved
internal marker within the oil (Prince et al., 1994). Apparent
half-times of the loss of analytes were calculated as before (Prince
et al., 2007). Two-dimensional GC followed earlier methods (Wang
et al., 2003), but used a ame ionization detector; the rst column
separated the oil components by boiling point, the second by
polarity. This approach separates the oil into eight major classes:
3-ring aromatics, 2-ring aromatic + 1-saturated ring or 5-saturated
rings, 2-ring aromatics or 4-saturated rings, 1-ring aromatic + 1-
saturated ring or 3-saturated rings, 1-ring aromatic or 2-saturated
rings, 1-saturated ring, alkanes, and hopanes.
3. Results
Fig. 1 shows total ion chromatograms of the initial oils and oils
extracted after biodegradation with the natural indigenous micro-
biota and nutrients at 8 C. The top trace is the original oil before
addition to the seawater; the sample with Corexit had this added
to the neat oil, although the experiment used oil dispersed by addi-
tion of the dispersant to the oil on the water surface, as described
above. It is noteworthy that the initial sample with dispersant
shows prominent peaks near 28 min that are not seen in the sam-
ples extracted from the water phase these are some of the surfac-
tant molecules (Nalco, 2011) that are not extractable from water
with methylene chloride. On the other hand, the features near
14 min can be attributed to the hydrocarbon solvent of the disper-
sant (Nalco, 2011) these are extracted from water with our pro-
tocol, as shown in the samples labeled Day 0. These samples were
extracted within 10 min of the initial assembly of the experimental
systems, and it is noteworthy that in this time the samples without
dispersant had lost most hydrocarbons smaller than naphthalene.
The evaporative loss was much reduced in the dispersed sample,
presumably because the hydrocarbons were in the bulk water
phase.
The oils were extensively and rapidly degraded, with both the
alkanes responsible for the sharp peaks and the unresolved mate-
rial showing substantial losses. Very similar extensive biodegrada-
 R.C. Prince et al. / Chemosphere 90 (2013) 521526 523

+ Corexit 9500 1:15 tion was seen when the experiment used 20% weathered crude oil
as shown in Fig. 2A. These losses can be quantied using
17a(H),21b(H)-hopane as a conserved internal marker within the
Initial oil (Prince et al., 1994). Although hopanes are eventually biode-
graded (Prince and Walters, 2007), this process does not appear
to have happened in our experiments (Fig. 2B), although there
% % was biodegradation of the C28 and C29 tricyclic terpanes after
Days 60 d. If 17a(H),21b(H)-hopane had been degraded in our experi-
loss loss
ments, our estimates of the extent of biodegradation would be
0 18 12 underestimates. The biodegradation of representative alkanes
and aromatics in the oil are shown in Fig. 3. The essentially com-
plete loss of all the alkylnaphthalenes likely includes some evapo-
rative losses, but the losses of the other aromatics can be
4 24 24
principally attributed to biodegradation (Prince and Walters,
2007). Unfortunately GC/MS cannot readily quantify all the indi-
vidual hydrocarbons of crude oil. The major groups of compounds
6 25 30
can, however, be separated using two-dimensional gas chromatog-
11 51 64 raphy with ame ionization detection, as shown in Fig. 4. Fig. 5
compares the initial oil with samples of weathered oil after 60 d
24 69 of biodegradation. It is clear that all the resolved components have
77
undergone substantial loss, with the exception of the hopanes. This
41 81 includes the aromatic compounds with up to at least three rings,
82
the saturated ring compounds with up to at least ve rings, mul-
ti-ring compounds with both aromatic and saturated rings, and
5 46 5 46
minutes minutes the linear and branched alkanes.
We have enough replicates (n = 10) that we can calculate the
Fig. 1. Total ion chromatograms of fresh crude oil without and with 6.7% Corexit median apparent half-lives of primary biodegradation of some
9500 at the beginning of the incubation, and after the indicated days of stirring in individual analytes, and these are listed in Table 1. The apparent
unamended seawater at 8 C. The initial sample with Corexit is actually a sample of
fresh oil plus Corexit, while the experimental samples had the dispersant added to
oil oating on water as described in Section 2. A dispersant

100
- +

A + Corexit 9500 1:20

loss
%

Days

% %
0 loss loss
0
TOTAL n C17 n C18 n C28
60 82 88 total saturates pristane phytane

5 46 5 46
minutes minutes B
100
B C23-tricyclic terpane
C29-hopane
C30-hopane
C28-tricyclic terpanes
C29-tricyclic
terpanes
loss
%

Day 0

C24-tricyclic terpane
C31-hopanes
C25-tricyclic terpane
C26-tricyclic terpanes C32-hopanes
C33-hopanes
Day 60 0
C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2

28 43 Naphthalenes Dibenzothiophenes
minutes Phenanthrenes Chrysenes

Fig. 2. (A) Total ion chromatograms of articially weathered crude oil without and
with 5% Corexit 9500 at the beginning of the incubation, and after 60 d of stirring in
unamended seawater at 8 C. The Corexit was added to the oil prior to addition to
the ask. (B) m/z = 191 chromatograms of the initial oil and biodegraded samples
with Corexit.
Fig. 3. Loss of (A) total resolved hydrocarbons, total saturates (measured as m/
z = 57) and representative alkanes, and (B) representative polycyclic aromatic
hydrocarbons in the samples of Fig. 2. The Cn nomenclature indicates the number of
alkyl carbons on the aromatic nucleus; C3 for example, includes trimethyl, methyl
ethyl, propyl and isopropyl forms. The error bars are standard errors.
524 R.C. Prince et al. / Chemosphere 90 (2013) 521526

been reports that dispersants inhibit the biodegradation of polycy-

2nd Diimension Retention Time (Seconds)

12 clic aromatic hydrocarbons (Lindstrom and Braddock, 2002) and

sulfur containing species such as the dibenzothiophenes (Foght
A
et al., 1983). Neither effect is seen in our experiments.
B
8 C 4. Discussion
D
H
Many earlier experiments (e.g. Bruheim et al., 1999; Lindstrom
E and Braddock, 2002; MacNaughton et al., 2003; Venosa and Holder,
4 F 2007; Zahed et al., 2010) used rather higher concentrations of oil
and additional nutrients to allow its degradation. A classical med-
G ium, used by many, is that of Bushnell and Haas (1941), which pro-
n C15 n C20 n C25 n C30
n C10 vides 25 mM bioavailable nitrogen as a combination of nitrate and
ammonium, and 13 mM phosphate. The medium was developed
0 20 40 60 80
during World War II for studying biodegradation in fuel storage
1st Dimension Retention Time (minutes) facilities, and it is really quite inappropriate for mimicking marine
systems where the levels of nutrients are many orders of magni-
Fig. 4. Two-dimensional GC chromatogram of the initial, partially weathered oil.
The rst dimension separates by boiling point, the second by polarity. A: 3-ring tude lower. Such high levels might well alter the relative compet-
aromatics series. B: 2- ring aromatic + 1-saturated ring or 5-saturated ring series. C: itive advantage of the microbial populations. Even nearshore
2-ring aromatics or 4-saturated ring series. D: 1-ring aromatic + 1-saturated ring or Atlantic waters contain only a few micromolar available nitrogen
3-saturated ring series. E: 1-ring aromatic or 2-saturated ring series. F: 1-saturated (Levitus et al., 1993; Louanchi and Najjar, 2001; Pelegri et al.,
ring series. G. alkane series representative n-alkanes are identied. H: hopane
series.
2006; While and Haines, 2010), and less than micromolar phos-
phate, and most of the worlds oceans have only nanomolar con-
centrations of nutrients (Patey et al., 2010). Clearly these natural
levels of nutrients, and the native biota of fresh seawater are ade-
12
quate for substantial biodegradation of dilute hydrocarbons.
A We note that our measurements detect only the hydrocarbons
in crude oil, and do not address the potential biodegradability of
the asphaltenes and resins (Tissot and Welte, 1984). Furthermore,
2nd Dimension Retention Time (Seconds)

4 the disappearance of analytes minimally indicates only the initia-

12
B biomass in our experiments Yerushalmi and Guiot (1998) con-
4
12
C the most readily biodegraded would likely already have been con-
4
0 20
1st Dimension Retention Time (minutes)
Fig. 5. Two-dimensional GC chromatogram of the initial, partially weathered oil
with dispersant (A), and the biodegraded oils with (B) and without (C) dispersant.
Components attributable to the dispersant (Nalco, 2011), not present in Fig. 5, are
enclosed in ellipses.
half-lives are calculated from the amounts remaining at various
times, and they do not include the clear lag-times apparent in
Fig. 1. Nevertheless they provide an indication of how rapidly oil
can disappear from the environment when it is dispersed at low
concentrations. As expected (Douglas et al., 1996), phenanthrenes
and dibenzothiophenes were degraded at essentially similar rates,
while chrysenes were more resistant to biodegradation, with or
without dispersant. It is noteworthy that all the isomers of the al-
kyl aromatics of Fig. 3 are degraded 3-methylchrysene is appar-
ently the least degradable methylchrysene, but its degradation was
clearly underway at the end of the experiment (Fig. 6). There have
tion of the biodegradation process commonly known as primary
biodegradation not their ultimate biological oxidation to water
and CO2. Quite likely much of the carbon is still in the microbial
cluded that the biomass yield of microbes growing on gasoline var-
ied from 0.43 mg/mg to 0.69 mg/mg, and it seems reasonable that
these values might be representative of many degradable hydro-
carbons. Beolchini et al. (2010) estimated a value of 0.39 mg bio-
mass/mg of hydrocarbons in harbor sediments, which likely
contain signicant amounts of recalcitrant hydrocarbons since
sumed. It has long been recognized that hydrocarbon-degrading
organisms incorporate hydrocarbons into cellular lipids (Davis,
1964; Mazzella et al., 2005) and storage products (Klein et al.,
2008; Manilla-Prez et al., 2010; Rontani, 2010). These enter the
ocean foodweb, and may eventually give rise to enhanced sheries
(Levy and Lee, 1988).
40 60 80
5. Conclusions
Our experiments demonstrate that even under late winter con-
ditions, in our case a water temperature of 8 C, the primary bio-
degradation of oil in seawater can be reasonably rapid, with no
additional nutrients, when the concentration of oil is in the few
ppm. These are the concentrations likely to occur after a successful
application of dispersant (National Research Council, 2005). Recent
data from the Gulf of Mexico suggesting the similarly rapid biodeg-
radation of oil dispersed at the sea bottom from the Deepwater
Horizon blowout (Hazen et al., 2010), with a lag of several days be-
fore biodegradation got underway (Reddy et al., in press), are fully
concordant with our ndings.
It is important to recognize that the simple comparison of the
rate of biodegradation in the presence and absence of dispersant
in Table 1 is somewhat misleading in judging the effectiveness of
dispersants. As we have shown, the biodegradation of dispersed
 R.C. Prince et al. / Chemosphere 90 (2013) 521526 525

Table 1
Initial concentrations and median apparent half-lives of primary biodegradation of some individual analytes. The concentrations assume full solubility in the total volume.

Initial concentration (ppb) Median apparent half-life (d) Median apparent half-life with dispersant (d)
Total detectable hydrocarbons 13.8 11.0
Total saturates (m/z = 57) 15.6 11.6
17a(H),21b(H)-hopane 0.48
Heptadecane 9.24 1.9 1.8
Pristane 4.87 4.7 3.4
Octadecane 5.48 2.5 2.2
Phytane 3.36 5.3 3.8
Triacontane 1.85 13.1 7.8
Naphthalene 1.35 2.3 1.9
C1-naphthalenes 2.96 2.0 1.7
C2-naphthalenes 3.73 1.9 1.7
C3-naphthalenes 2.88 1.9 1.8
C4-naphthalenes 1.46 2.1 1.9
Fluorene 0.20 3.1 2.6
Phenanthrene 0.56 2.6 2.2
1-Methylphenanthrene 0.36 2.8 2.4
2-Methylphenanthrene 0.27 2.9 2.5
3-Methylphenanthrene 0.22 3.0 2.6
9-Methylphenanthrene 0.41 3.5 2.5
C2-phenanthrenes 1.48 7.9 9.7
C3-phenanthrenes 1.05 18.9 22.4
C4-phenanthrenes 0.63 28.8 35.7
Dibenzothiophene 0.49 2.7 2.4
1-Methyldibenzothiophene 0.17 3.7 4.5
2/3-Methyldibenzothiophenes 0.27 3.0 3.5
4-Methyldibenzothiophene 0.51 2.8 3.5
C2-dibenzothiophenes 1.43 7.8 7.6
C3-dibenzothiophenes 1.26 19.5 21.2
C4-dibenzothiophenes 0.69 33.3 35.9
Benz[a]anthracene 0.03 26.6 17.8
Chrysene 0.10 30.8 36.2
1-Methylchrysene 0.02 14.3 14.3
2-Methylchrysene 0.02 6.3 11.2
3-Methylchrysene 0.11 27.1 36.5
4/6-Methylchrysenes 0.02 11.3 12.4
C2-chrysenes 0.25 28.1 28.5
Sum 16 EPA priority pollutants 2.24 4.5 4.0

The Cn nomenclature indicates the number of alkyl carbons on the aromatic nucleus; C3 for example, includes trimethyl, methyl-ethyl, propyl and isopropyl forms. 2- and
3-methyldibenzothiphenes co-elute, as do 4- and 6-methylchrysenes. 5-methylchrysene is below detection. Most of the EPA priority pollutants (Keith and Telliard, 1979) are
below detection limits in our experiments; the predominant components are naphthalene and phenanthrene.

by oxygen access. The role of dispersants is not to stimulate the

dispersant
100 - + biodegradation of dispersed oil, but to allow the dispersion. We
show here that the biodegradation of dispersed oil is essentially
3-methyl
unaffected and certainly not inhibited by the presence of disper-
sants, and is rapid and extensive.
2-methyl
4/6-methyl
loss

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