Deep Water Horizon oil and methane carbon entered the food web in the Gulf of

Mexico
Author(s): A. Fernández-Carrera, K. L. Rogers, S. C. Weber, J. P. Chanton and J. P.
Montoya
Source: Limnology and Oceanography , Vol. 61, No. S1, Special Issue: Methane Emissions
from Oceans, Wetlands, and Freshwater Habitats: New Perspectives and Feedbacks on
Climate (2016), pp. S387-S400
Published by: Wiley

Stable URL: https://www.jstor.org/stable/10.2307/26628584

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LIMNOLOGY
and
OCEANOGRAPHY Limnol. Oceanogr. 61, 2016, S387–S400
C 2016 Association for the Sciences of Limnology and Oceanography
V
doi: 10.1002/lno.10440

Deep Water Horizon oil and methane carbon entered the food web in
the Gulf of Mexico
ndez-Carrera,1,2* K. L. Rogers,3 S. C. Weber,1,4 J. P. Chanton,3 J. P. Montoya1
A. Ferna
1
School of Biology, Georgia Institute of Technology, Atlanta, Georgia
2
Dpto Ecoloxıa e Bioloxıa Animal, Universidade de Vigo, Vigo, Spain
3
Department of Earth Ocean and Atmospheric Science, Florida State University, Tallahassee, Florida
4
Leibniz Institute for Baltic Sea Research Warnemu€ nde, Rostock, Germany

Abstract
The Deep Water Horizon (DWH) incident caused the release of an unprecedented amount of 13C- and
14
C-depleted oil and gas to the Gulf of Mexico (GoM), which formed surface slicks and deep oil/gas plumes
that extended laterally at 1000–1200 m. We conducted three research cruises between 2010 and 2012 to
study the potential assimilation of petrocarbon (C derived from oil and/or gas) into the GoM microbial food
web. In 2010, we found low d13C (230 to 225&) and D14C (2603 to 255&) values for suspended particles at
1000–1200 m depth as far as 289 km SW of the wellhead, providing direct evidence of the spatial extent of
the subsurface plumes. At those depths in 2010, methane and oil carbon accounted for up to 28% and 62%
of total particulate carbon (Csp), respectively. In the total area affected by the DWH, 80 6 56 to 104 6 91
tonnes (t) of methane-derived and 216 6 174 to 292 6 165 t of oil-derived carbon were incorporated into Csp.
In 2011 and 2012, the d13C values were distributed throughout the water column indicating that petrocarbon
was still present and recycling, especially in the section closest to the DWH, where oil supplied up to 53%
and 75% of Csp, respectively. Relatively low d15N (< 4&) values in suspended particles at 1000–1200 m in
2010 indicate stimulation of nitrogen fixation linked to methane oxidation in the months after the spill,
which accounted for up to 40% of the particulate nitrogen in the water column at those depths.

Between April and July 2010, the Deep Water Horizon
(DWH) blow-out and subsequent oil spill released up to 838
kilotonnes (kt) of crude oil and 363 kt of methane and gases
(Joye et al. 2011) to intermediate waters of the Gulf of Mexico
(GoM). The use of up to 7 3 106 L of dispersant, mainly Cor-
exit 9500A (Almeda et al. 2014), at the wellhead may have
ameliorated the development of surface oil slicks, but a
unique deep oil and gas plume formed at 1000–1200 m depth
(Camilli et al. 2010; Valentine et al. 2010; Spier et al. 2013)
that moved horizontally tens to hundreds of km, primarily to
the southwest of the DWH wellhead and with strong topo-
graphic steering (Camilli et al. 2010; Kessler et al. 2011).
Anthropogenic spills are not the only source of oil and gas
in offshore ecosystems of the Gulf. Thousands of natural gas
and oil seeps are distributed through the GoM (Garcia-Pineda
et al. 2010; Abbriano et al. 2011), creating an environment
*Correspondence: ana.fernandez@biology.gatech.edu and afcarrera@uvigo.es
Special Issue: Methane Emissions from Oceans, Wetlands, and Freshwater
Habitats: New Perspectives and Feedbacks on Climate
Edited by: Kimberly Wickland and Leila Hamdan
that selects for microbial communities able to tolerate and/
or degrade hydrocarbons. This preadaptation may have con-
tributed to the rapid and intense response of hydrocarbon-
degrading microbes to the sudden, extremely large input of
oil and gas to both surface and deep waters that occurred in
May and June 2010 (Hazen et al. 2010; Edwards et al. 2011;
Kessler et al. 2011; Mason et al. 2012; Valentine et al. 2012;
Gutierrez et al. 2013; Rivers et al. 2013). The community
response was sustained at lower rates, albeit above back-
ground levels, to at least the end of December 2010 (Crespo-
Medina et al. 2014). In general, the diversity of bacterial
groups within oil slicks decreased in the early stages of the
spill, with hydrocarbon degraders becoming more abundant
relative to other indigenous groups (Hazen et al. 2010; Mason
et al. 2012; Dubinsky et al. 2013; Rivers et al. 2013). In addi-
tion, laboratory bioassays have shown that the different
groups of microorganisms have contrasting sensitivities to
crude oil and/or dispersant. For instance, heterotrophic bacte-
ria are more resistant than nitrifiers to crude oil (Urakawa
et al. 2012), and the dispersant is toxic to some groups of
microzooplankton, with a synergistic negative effect when
combined with oil (Almeda et al. 2014). Together, these

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 ndez-Carrera et al.
Ferna
findings suggest that microbial community functions changed
through time in areas affected by the oil spill.
The oil and gas released by the DWH oil spill represented
a substantial input of organic carbon to offshore waters of
the GoM. The carbon isotopic composition (d13C and D14C)
of particles and zooplankton collected near Mobile Bay
(> 100 km north of the DWH wellhead), and in Florida
waters during and after the spill suggests that oil and/or
methane carbon entered the microbial food web quickly
(Graham et al. 2010; Chanton et al. 2012; Cherrier et al.
2014). In addition to this direct impact on water column car-
bon budgets, the injection of oil and gas into the water col-
umn can affect other biogeochemical cycles. For example, in
sediments affected by natural methane seeps, aggregates of
anaerobic methanotrophic archaea (ANME-2) and sulfate-
reducing bacteria (SRB) are capable of using N2 as a source of
nitrogen, and this diazotrophy is linked to the oxidation of
methane (Miyazaki et al. 2009; Dekas et al. 2013). This cou-
pling between methane consumption and N2-fixation also
occurs at depth in the water column (Montoya et al.
unpubl.), linking the planktonic C and N cycles.
Here, we present measurements of the natural abundance of
carbon and nitrogen isotopes (d13C, D14C, and d15N) in sus-
pended particles collected from the water column during three
extensive surveys of the Northern Gulf of Mexico (2010–2012),
covering both the area directly affected by the DWH wellhead
leakage and the area potentially affected by the deep oil and
gas plume in 2010. We use these isotopes as a proxy for the
movement of oil and gas carbon into the microbial food web,
to assess the effect of the spill on deep-water diazotrophy, and
to document the westward extent of the subsurface plume. The
oil and gas released by the DWH spill were depleted in 13C rela-
tive to marine organic carbon (OC): d13Coil 5 227& (Graham
et al. 2010), d13Cmethane 5 257& and 261& (Cherrier et al.
2014; Crespo-Medina et al. 2014), and d13CmarineOC 5 221.0&
(Chanton and Lewis 2002; Chasar et al. 2005). Therefore, the
contribution of C derived from oil and gas (petrocarbon) to sus-
pended particles can be estimated using a three end-member
mixing model based on oil, methane and primary producers-
derived organic carbon. Our underlying hypothesis is that the
rapid response of microbial communities documented during
and after the DWH spill promoted the movement of petrocar-
bon into the microbial food web, thus lowering the d13C of sus-
pended particles with respect to marine organic carbon derived
from primary production.
Materials
We collected samples in the Northern Gulf of Mexico
(GoM) on cruises OC468 (21 August 2010–16 September
2010), EN496 (02 July 2011–27 July 2011), and EN515 (05
September 2012–15 September 2012) aboard the R/V Oceanus
(OC) and the R/V Endeavor (EN) (Fig. 1). Each cruise was
comprised of two legs, and sampled locations include the
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Deep Water Horizon oil and methane carbon
Deep Water Horizon (DWH) wellhead, areas affected by sur-
face oil slicks, the region of the main subsurface oil and gas
plume (1000–1200 m) detected in early June 2010 (Camilli
et al. 2010), and several natural cold seeps on the northern
continental slope. For this study, we delimited two main sec-
tions in our target area (Fig. 1): the DWH nearfield section,
which includes the stations that were likely influenced
directly by the DWH well itself (ca. 288–308N, 868–908W),
and the Southern section, which includes the stations in the
area potentially affected by the subsurface oil and gas plumes
(25.58–28.58N, 878–938W) that moved mainly to the south-
west at 1000–1200 m, but were also detected to the south-
east of the DWH wellhead at other depths (Spier et al. 2013).
Water samples were collected with 10 L Go-Flo (OC cruise)
or Niskin (EN cruises) bottles attached to a rosette equipped
with a Seabird CTD and a fluorometer, a transmissometer,
and oxygen sensors. Bottles were fired at 4–12 depths span-
ning the water column. Multiple casts were carried out at
each station to sample the entire water column, including
depths above, below and within the deep oil and gas plumes.
Particles were collected at each sampling depth by passing
1–20 L of water through pre-combusted (4508C for 2 h) 47
mm GF/F filters under gentle pressure. Filters were dried at
608C for at least 24 h, and stored over desiccant until analy-
sis of isotopic and elemental composition ashore. Once in
the lab, filters were trimmed to remove the particle-free
edge, and then cut into quadrants or halves, which were pel-
letized in tin capsules.
The natural abundance of carbon (C) and nitrogen (N)
stable isotopes was measured by continuous-flow isotope-
ratio mass spectrometry (CF-IRMS) using a Micromass Opti-
ma or an Isoprime 100 interfaced to a Carlo Erba elemental
analyzer (CE NC2500). All carbon and nitrogen abundances
are reported as d13C and d15N values (&) relative to Vienna
Pee Dee Belemnite (VPDB) and atmospheric N2, respectively.
The stability of the instrument and the contribution of any
blank to our measurements were checked using a size series
of elemental (methionine) and isotopic (peptone) standards
in each analytical run (Montoya et al. 2008).
The contributions of oil and methane to the d13C of sus-
pended particles (d13Csp) were estimated using a three mem-
ber mass-balance:
Csp 3d13 Csp 5Cbiomass 3d13 Cbiomass 1Coil 3d13 Coil
(1)
1Cmethane 3d13 Cmethane
where subscripts denote the source of C: sp for suspended par-
ticles, biomass for surface biomass, oil and methane for those
forms of petrocarbon. The contribution of organic matter
derived from surface production, Cbiomass, was estimated by mul-
tiplying the measured particulate organic nitrogen by the canon-
ical Redfield C : N ratio (6.625), and assuming that no nitrogen
was supplied by either oil or methane. Once the biomass contri-
bution was determined, the petrocarbon contributions, Coil and
 ndez-Carrera et al.
Ferna Deep Water Horizon oil and methane carbon

-93 -92 -91 -90 -89 -88 -87 -86 -85

OC468
31 EN496 31
EN515
DWH well

30 30
DWH
nearfield
29 29
Latitude

28 28

27 27

26 26
0 100 200 km Southern section

-93 -92 -91 -90 -89 -88 -87 -86 -85

Longitude

Fig. 1. Chart of stations sampled on cruises OC468 (21 August 2010–08 September 2010), EN496 (02 July 2011–27 July 2011), and EN515 (05 Sep-
tember 2012–15 September 2012). Black circles represent OC468, white inverted triangles represent EN496, and gray squares represent EN515. An
asterisk marks the position of the former Deep Water Horizon well.

Cmethane, were estimated by mass balance (Eq. 1). As isotopic end
members, we used d13Cbiomass 5 221.0& (Chanton and Lewis
2002; Chasar et al. 2005); d13Coil 5 227.0& (Graham et al.
2010); and d13Cmethane 5 257 and 261& (Cherrier et al. 2014;
Crespo-Medina et al. 2014). We applied this mass-balance
approach only to the samples with d13Csp < 221& (i.e., those in
which a significant contribution of oil or gas was most likely).
In addition, filter sections from 18 samples from the 2010
cruise (OC468) collected in the area affected by the subsurface
oil and gas plumes were analyzed for the natural abundance of
radiocarbon (14C). Individual filters were combusted and puri-
fied to CO2 following the methods of Peterson et al. (1994).
The resultant CO2 was sealed in a 6 mm ampule and sent to
either Woods Hole National Ocean Sciences Accelerator Mass
Spectrometry (NOSAMS) Center or Lawrence Livermore Nation-
al Laboratory Center for Accelerator Mass Spectrometry (CAMS).
The D14C data were corrected for the blank of the filter
and the reagents using the following equation:

D14 Cmeasured 3 Cmeasured 2 D14 Cfilter 3 Cfilter 2 D14 Creagent 3 Creagent

D14 Csp 5 (2)
Cmeasured 2 Cfilter 2Creagent

The blank filter had a D14Cfilter 5 2550 &, with a size (Cfil-
ter) of 0.830 lmol CO2 and the reagent blank was
D14Creagent 5 2255 & with a size (Creagent) of 0.167 lmol
CO2. The size of the samples (Cmeasured) ranged from 5 to 37
lmol CO2. We also note that dissolved organic carbon
(DOC) can be adsorbed to the glass fiber filters during the fil-
tration process (Gardner et al. 2003; Griffith et al. 2012), so
our measurements could reflect some contamination by
DOC. However, given that open ocean DOC is generally less
14
C-depleted than petrocarbon (Benner et al. 1997; Griffith
et al. 2012), the net effect would be to dilute the petrocar-
bon signature in our samples, so our estimates of the contri-
bution of oil/gas to particles are effectively conservative
lower limits.

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 ndez-Carrera et al.
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Fig 2.

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 ndez-Carrera et al.
Ferna Deep Water Horizon oil and methane carbon

Fig. 2. (see prior page). Vertical distribution of d13C (A, C, D, E) and D14C (B) of suspended particles for all cruises, and relation of d13C vs. D14C (F)
for samples from the Southern section of our 2010 cruise (OC468). Circles in A and crosses in B, F represent cruise OC468 (21 August 2010–08 Septem-
ber 2010), inverted triangles in C represent cruise EN496 (02 July 2011–27 July 2011), and squares in E represent EN515 (05 September 2012–15
September 2012). The symbols in panel D represent the d13C at the stations located further to the southwest of the DWH well in the Southern section
(south of 278N and East of 888W) of the 2010 cruise. In A, C, and E, gray symbols are the stations in the Deep Water Horizon section, white symbols are
stations in the Southern section. The gray box in panels A–E defines the depth of the oil and gas plume developed in 2010. The line in panel F shows
the best fit linear regression used to estimate the composition of petrocarbon (Eq. 6). Note that a D14C of 21000& corresponds to d13C 5 235.9&.

To estimate the contributions of recently fixed carbon, endmember. In brief, we solved the regression line for d13C
methane, and oil sources to suspended particles using d13C when D14C 5 21000& and then used the mass balance
and D14C values, we created a matrix using the following equation:
three-equations:
d13 Cregression 2d13 Coil
15fbiomass 1foil 1fmethane (3) fmethane 5 (7)
d13 Cmethane 2d13 Coil

d13 Csp 5fbiomass 3d13 Cbiomass 1foil 3d13 Coil 1fmethane 3d13 Cmethane
D14 Csp 5fbiomass 3D14 Cbiomass 1foil 3D14 Coil 1fmethane 3D14 Cmethane
where f is the fraction of each carbon source in the sample
(Bauer et al. 2002; Cherrier et al. 2014). The isotopic end-
members for D14C were 141& for recently fixed marine car-
bon (D14Cbiomass) in the GoM (Chanton et al. 2012), and
fossil 21000& for methane and oil. The stable isotope end-
members for d13C were as noted above.
We did not include the carbon delivered by the Mississip-
pi river in either of the models (Eqs. 1, 3-5) for the following
reasons. The d13C of the particulate organic carbon associat-
ed with the outflow has been reported to range from
223.3& to 226&, while the D14C ranges from 286& to
2223&, (Cherrier et al. 2014). As we will show, these D14C
values fall relatively near the modern carbon production val-
ues. In our three-way model (Eqs. 3-5), using the terrestrial
signal instead of the modern signal resulted in negative con-
tributions to particulate organic carbon in most of our shal-
low samples, because the samples are more similar to the
modern signal than to the terrestrial signal. Thus, to the
extent that Mississippi river inputs affect our surface sam-
ples, we assumed that our marine production values repre-
sent an upper limit of this influence.
A second approach to estimate the percent of recently
fixed (frf) carbon and fossil carbon in the Csp used a radiocar-
bon mixing model:
D14 Csp 2D14 Cpetrocarbon
frf 5
D14 Crf 2D14 Cpetrocarbon
with endmembers of D14Crf 5 141& and D14Cpetrocarbon
21000&. The petrocarbon content was then simply (1 – frf).
To compare our estimate of petrocarbon content to the three
equation matrix, the individual methane and oil estimations
were added together.
We then used a linear regression of d13C vs. D14C (Fig. 2F)
to determine the composition of the petrocarbon
where d13Cregression 5 235.9&, d13Coil 5 227& (Graham et al.
(4) 2010) and d13Cmethane 5 257 or 261& (Cherrier et al. 2014;
Crespo-Medina et al. 2014). This allowed us to determine
the relative fraction of the fossil particulate organic carbon
(5) from methane (fmethane) and oil sources (1 – fmethane).
All statistical analyses were performed using R 3.1.1
(R Core Team 2014). Non-parametric tests were used because
the raw variables did not follow a Gaussian distribution. The
correlation between variables was explored using the Kendall
tau coefficient, the differences between sections were tested
using the Kruskal–Wallis rank sum test, and for the statistical
significance of means we used a Wilcoxon rank sum test.
Results
Suspended particle d13C
In 2010 (cruise OC468), the vertical distribution of the
natural abundance of 13C in suspended particles (d13Csp)
showed a wide range of variation through the water column
with values between 230.1& and 212.8& (Fig. 2A). In the
upper 400 m, d13Csp spanned between 226.0& and 217&,
with a few higher values ranging up to 212.8& and an aver-
age of 221.1 6 1.9& (mean 6 SD, n 5 157) for the whole
cruise. The lowest values were found at the depth of the sub-
surface oil and gas plumes (1000–1200 m), where d13Csp
ranged between 230.1& and 219.6&, with an overall aver-
age of 224.9 6 3.0& (mean 6SD, n 5 31), which is statistical-
ly different from the d13C of particles in the upper 400 m of
the water column (Wilcoxon rank sum test, p < 0.01). In gen-
eral, d13Csp values at depth were slightly lower in the South-
ern section relative to the DWH nearfield section, as
illustrated by the stations farthest to the southwest of the
(6)
DWH wellhead in the Southern section, where d13Csp consis-
tently tended to be more negative with depth (Fig. 2D). We
applied our three-member mass-balance approach (Eq. 1) to
depths between 1000 m and 1500 m to account for both Csp
produced within the oil and gas plume, and for Csp poten-
tially exported from the plume itself, a pathway consistent
with the low d13Csp values we found immediately below the
plume in 2010 (Fig. 2A). Our mass balance suggests that up
to 28% of suspended particle carbon (Csp) was supplied by

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 ndez-Carrera et al.
Ferna Deep Water Horizon oil and methane carbon

Fig 3.

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 ndez-Carrera et al.
Ferna Deep Water Horizon oil and methane carbon

Fig. 3. (see prior page). Vertical distribution of d15N (A, C, E) and C : N ratio (B, D, F) of suspended particle. Circles represent cruise OC468 (21
August 2010–08 September 2010), inverted triangles represent cruise EN496 (02 July 2011–27 July 2011), squares represent cruise EN515 (05 Septem-
ber 2012–15 September 2012). Gray symbols are the stations in the Deep Water Horizon section, white symbols are stations in the Southern section.
Dashed lines in the C : N panels represent the Redfield C : N ratio (6.625). The gray box defines the depth of the oil and gas plume developed in 2010.

methane at depths between 1000 m and 1500 m in the South-
ern section. The contribution of methane to Csp averaged 7% 6
5% (mean 6 SD, n 5 8, d13Cmethane 5 261&) to 18% 6 7%
(n 5 12, d13Cmethane 5 257&) at those depths. On the other
hand, oil contributed as much as 24% of Csp at depths between
1000 m and 1500 m in the Southern section, but up to 62% in
the DWH nearfield section at those depths. The average range
for oil contribution to Csp in the DWH nearfield section at
1000–1500 m was 39% 6 16% (n 5 8, d13Cmethane 5 257&) to
47% 6 14% (n 5 8, d13Cmethane 5 261&).
In 2011 (cruise EN496) and 2012 (cruise EN515), the
range of variation of d13Csp in the water column was similar
to that of the 2010 cruise (229.7& to 216.4&), but the ver-
tical distribution was markedly different (Fig. 2C,E). In both
years, we measured low d13Csp values (< 225&) throughout
the water column of the DWH nearfield section, with a clear
shift to lower d13C values in suspended particles (< 223&)
below 300 m in 2012 (Fig. 2E). A wider range of variation
also appeared in deep waters below 1500 m depth in 2011
and 2012 (Fig. 2C,E), although this may reflect the greater
number of samples taken at those depths during this cruise
in comparison to the 2010 cruise. In the Southern section in
2012, we found consistently low values around 225& below
200 m. These samples were collected near the GC600 site
(27.378 N, 90.568 W), a very active seep field in the GoM,
which provides a measure of the potential contribution of
petrocarbon from natural inputs of oil and gas to suspended
particles. The whole cruise averages for d13Csp in the upper
400 m were similar to that from 2010 (221.6 6 1.4& in
2011, and 223.0 6 2.1& in 2012). The averages at the depths
of the former oil and gas plume (1000–1200 m) were
222.1 6 2.4& in 2011, and 225.6 6 3.6& in 2012.
In the 2011 and 2012 cruises, the estimated contribution
of methane to Csp (Eq. 1) through the water column
decreased in both sections relative to that of 2010. In 2011,
methane contributed between 0% and 16% to suspended
particles, with a whole cruise average of 4% 6 3% (n 5 89,
d13Cmethane 5 257&), while in 2012 we found that methane
carbon contributed 0–28% of Csp, with a whole cruise aver-
age ranging between 4% 6 4% (n 5 17), and 7% 6 5%
(n 5 75), depending on choice of end members for methane.
On the other hand, the contribution of oil carbon to sus-
pended particles was maintained over time. In 2011 (cruise
EN496) the oil contribution ranged between 0% and 53%
(n 5 89), with a cruise average from 13% 6 9% (n 5 89) to
20% 6 9% (n 5 89). In 2012 (cruise EN515) the contribution
of oil to suspended particles was between 0% and 75%
(n 5 199), averaging from 25% 6 15% (n 5 86) to 33% 6 14%
(n 5 90).
Suspended particle D14C
The radiocarbon content of samples from the 2010 cruise
(OC468) varied with depth, with the most depleted samples
found within the depth range of the subsurface plume
(Camilli et al. 2010; Valentine et al. 2010) (Fig. 2B). The
D14C signatures ranged from 2603& to 7.7& (n 5 18). Sam-
ples from the upper 200 m of the water column were less
14
C depleted, and ranged from 264& to 7.7& (n 5 5). Sam-
ples taken from 840 m to 1200 m ranged from 2603& to
248& (n 5 13), with an average D14C of 2283 6 186&
(mean 6 SD, n 5 13). Within the plume at depths between
1000 m and 1200 m, the D14C values ranged from 2603& to
255&, with an average of 2357 6 195& (mean 6 SD, n 5 8).
The sample with the most depleted signatures of D14C and
d13C (2603& and 230.1&, respectively) was from a depth of
1050 m.
We estimated the contribution of different carbon sources
(recently fixed, methane, and oil) for each sample using two
methods where we incorporated the radiocarbon data either
through the three equation matrix (Eqs. 3-5) or through the
radiocarbon mixing line (Eqs. 6 and 7). Using the three
equation matrix, we estimated that between 0% and 16% of
suspended particle carbon was derived from fossil methane,
averaging 3% 6 3% (n 5 5) for shallow samples (< 200 m)
and 7% 6 6% (n 5 13) for deep samples (840–1200 m), while
0–46% was derived from oil, averaging 5% 6 3% (n 5 5) for
shallow samples and 23% 6 14% (n 5 13) for deep samples.
Modern carbon made up 38–95% of the Csp, averaging
92% 6 2% (n 5 5) for shallow samples and 70% 6 18%
(n 5 13) for deep samples. The total fossil carbon in each
sample ranged from 0% to 62%, with shallow depths averag-
ing 8% 6 2% and deep samples averaging 30% 6 18%.
We found a significant linear correlation between d13C
and D14C for samples from cruise OC468 in 2010
(d13C 5 220.968 1 0.015 3 D14C, R2 5 0.89, p < 0.01, Fig. 2F).
The petrocarbon endmember, calculated by extending the
linear regression in Fig. 2F to D14C 5 21000&, had a d13C of
235.9&. Using the d13C of the two petrocarbon endmem-
bers, oil 227& (Graham et al. 2010) and methane 257 or
261& (Cherrier et al. 2014; Crespo-Medina et al. 2014) we
estimate by mass balance that 27% of the fossil carbon end-
member was derived from methane, while 73% was derived
from oil.
Suspended particle d15N
The vertical distribution of the d15N of suspended par-
ticles (d15Nsp) in the water column was similar for all of the
cruises, with low values and greater variability in surface
waters and a shift to more positive and somewhat less

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 ndez-Carrera et al.
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Fig 4.

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 ndez-Carrera et al.
Ferna
Fig. 4. (see prior page). d15N vs. d13C (A, C, E) and C : N ratio vs. d13C (B, D, F) of suspended particle. Circles represent the OC468 cruise (21
August 2010–08 September 2010), inverted triangles represent the EN496 cruise (02 July 2011–27 July 2011), squares represent EN515 (05 Septem-
ber 2012–15 September 2012). Gray symbols are the stations in the Deep Water Horizon section, white symbols are stations in the Southern section.
Dashed lines in the C : N panels represent the Redfield C : N ratio (6.625).
Fig. 5. d15N vs. d13C of suspended particles at 1000–1200 m depth in
the Southern section during OC468 cruise (21 August 2010–08 Septem-
ber 2010). The solid line shows the best fit linear regression (model II)
estimated with R (d15N 512.66 1 0.29 3 d13C, R2 5 0.20, p 5 0.014,
n 5 24).
variable values below 300–400 m depth (Fig. 3A,C,E).
Although the range of d15Nsp was large on all cruises (23.9&
to 10.5& in 2010, 25.6& to 11.7& in 2011, and 21.3& to
10.6& in 2012), most of the values fell between 5& and
10&. In the upper 400 m of the water column, d15Nsp varied
between 0& and 10& in 2010, 25& and 10& in 2011, and
between 21& and 11& in 2012. This range of variation nar-
rowed to values between 5& and 10& in deeper waters, and
down to 0& and 10& below the depth of the 2010 subsur-
face oil/gas plume. At most of the stations sampled in 2010,
d15Nsp in deep waters below 500 m was lower in the DWH
nearfield section than in the Southern section but at the
depth of the oil/gas plume (Fig. 3A).
Suspended particle C : N ratio
The vertical distribution of carbon to nitrogen (C : N)
ratios in suspended particles differed between cruises (Fig.
3B,D,F). In 2010 (cruise OC468), the DWH nearfield section
and the Southern section were distinctly different. While
C : N ratios tended to increase with depth in the upper
1500 m of the DWH nearfield section, it varied roughly sym-
metrically around the Redfield C : N (6.625) ratio in the
Southern section with values between 3.8 and 7.6 (Fig. 3B).
In contrast, no obvious trend was found with depth in 2011
(cruise EN496), although values in the DWH nearfield sec-
tion were generally higher than those in the Southern
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Deep Water Horizon oil and methane carbon
section, especially in deep waters between 500 m and
1500 m (Fig. 3D). C : N ratios above Redfield (> 10) were
measured throughout the water column in 2012 (cruise
EN515), especially in the DWH nearfield section, where
C : N increased with depth (Fig. 3F).
Statistics
For the 2010 cruise (OC468), d15Nsp showed a weak but
significant correlation with d13Csp (s 5 0.151, p < 0.01,
n 5 256, Fig. 4A) and C : N ratio was weakly correlated with
d13Csp (s 5 0.199, p < 0.01, n 5 256, Fig. 4B). The correlation
between d15N and d13Csp is higher for the samples between
1000 m and 1200 m depth in the Southern section
(s 5 0.362, p < 0.05, n 5 24, Fig. 5). In contrast, our 2011 data
set (cruise EN496) showed no significant correlations
between d15N and d13Csp (p > 0.05, n 5 244, Fig. 4C) or C : N
ratio and d13Csp (p > 0.05, n 5 244, Fig. 4D). Since the correla-
tions for the 2010 cruise as a whole were weak, we treated
our suspended particles as a mixture of phytoplankton, bac-
teria and detritus and did not apply a lipid correction (Gra-
ham et al. 2010). In 2012 (cruise EN515), d15N and C : N
were significantly correlated with d13Csp (s 5 0.215,
s 5 20.418, respectively, p < 0.01, n 5 109, Fig. 4E,F).
When considering the sections within each cruise, the
Kruskal–Wallis tests revealed that d13C differed significantly
between the DWH and the Southern sections both in 2010
(p < 0.01, n 5 256), and 2011 (p < 0.01, n 5 247), but not in
2012 (p > 0.05, n 5 109). On the other hand, d15Nsp and
C : N values differed significantly between sections for the
2010 cruise (p < 0.01, n 5 256).
Discussion
The Deep Water Horizon (DWH) blow-out was the largest
oil spill in U.S. history (Abbriano et al. 2011). The unprece-
dented amount of oil and gas released over 86 d following
the explosion produced an immediate response by the indig-
enous microbial communities (Hazen et al. 2010; Valentine
et al. 2010; Edwards et al. 2011; Kessler et al. 2011; Mason
et al. 2012; Gutierrez et al. 2013; Rivers et al. 2013), which
may have transferred substantial amounts of oil and gas-
derived carbon (petrocarbon) into the microbial food web.
Our results are consistent with other studies focused on shal-
lower waters off the coast of Alabama and Florida (Graham
et al. 2010; Chanton et al. 2012; Cherrier et al. 2014) in sug-
gesting that this carbon was rapidly incorporated into small
particles by June 2010, and that it was still being recycled
through the microbial food web 2 yr after the well-head was
capped (Fig. 2A,C,E).
 ndez-Carrera et al.
Ferna
The oil and methane released by the DWH had character-
istically low d13C values of about 227& for oil (Graham
et al. 2010) and 257& to 261& for methane (Cherrier et al.
2014; Crespo-Medina et al. 2014). These values are different
from that of typical surface primary production, which usu-
ally ranges between 222& and 220& (Chanton and Lewis
2002; Chasar et al. 2005). Therefore, we expect that biologi-
cal processes using these hydrocarbons as a source of carbon
will lower the d13C of organic matter below that of surface
primary production. The quick response of methanotrophic
and oil-degrading bacteria and archaea to the sudden supply
of both oil and gases between 1000 m and 1200 m depth in
May–June 2010 (Camilli et al. 2010; Valentine et al. 2010)
appears to have driven the movement of the hydrocarbons
into the microbial food web, generating the consistently low
d13C (< 225&) of suspended particles (d13Csp) at those
depths in the Southern section in August 2010 (Fig. 2A),
which were detected as far as 289 km southwest from the
DWH well (Fig. 2D). Using a three-member (surface biomass,
oil, and methane) isotope mass-balance approach, we esti-
mate that as much as 28% of particulate carbon was derived
from methane in that section at depths between 1000 m and
1500 m. In addition, the DWH methane and oil was radio-
carbon dead, with highly depleted radiocarbon values
(D14C 5 21000&) relative to that of Gulf dissolved inorganic
carbon (DIC), which had a D14C of 141& in 2010 (Chanton
et al. 2012). Radiocarbon analysis provides further evidence
that methane and oil have been incorporated into the
microbial food web via suspended particulate organic carbon
and zooplankton (Chanton et al. 2012; Cherrier et al. 2014),
and possibly into higher organisms along the Gulf coast
(Wilson et al. 2016). The three equation matrix (Eqs. 3-5)
used to analyze the radiocarbon and stable isotope carbon
values in the Southern section in 2010 produced estimates of
methane contributions to particulate carbon ranging from
0% to 16%, which is generally consistent with the 13C iso-
tope mass-balance approach (0 2 28%). From this three equa-
tion approach, we also estimated that the contribution of
recently fixed carbon ranged from 38% to 95%, averaging
70% 6 18% for deep samples, while oil contributions were
estimated to range from 0% to 46%, averaging 23% 6 14%
for deep samples.
The results of our second approach, using a radiocarbon
mixing line (Eqs. 6 and 7), agreed with the three equation
matrix for estimating the contribution of total fossil carbon
in each sample. We used the linear regression shown in Fig.
2F, to estimate the d13C of petrocarbon when
D14C 5 21000&, producing a value of d13C 5 235.9&. Using
the isotopic composition of methane and oil, we estimated
that methane comprised about 27% of the petrocarbon end-
member while oil comprised the remaining 73%.
Samples collected from within the deep hydrocarbon
plume in 2010 showed depletion in both d13C and D14C sig-
natures (Fig. 2A,B). As stated above, these results are
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Deep Water Horizon oil and methane carbon
consistent with previous studies of d13C and D14C in Mobile
Bay (> 100 km north of the well-head) and the DeSoto Can-
yon, which showed a rapid transfer of hydrocarbon to small
particles and zooplankton (Graham et al. 2010; Chanton
et al. 2012; Cherrier et al. 2014). In the months following
the end of the spill, methane dispersed through the water
column in the Northern Gulf, and methane oxidation rates
decreased substantially, although the rates remained above
background levels for an extended period (Crespo-Medina
et al. 2014). This suggests that a continuous input of carbon
derived from DWH methane into the microbial food web
was still possible in a broad region of the Northern Gulf,
again contributing to low values of d13Csp. Indeed, as a con-
servative approach, we used the range of average contribu-
tions of methane for the entire 2010 cruise (7% 6 5% to
9 6 8, Eq. 1) scaled to the total area exposed to oil
(11,200 km2) for more than 20% of the duration of the spill
(Hu et al. 2011; Macdonald et al. 2015) to estimate that
between 80 6 56 and 104 6 91 t of methane-derived carbon
was incorporated into suspended particles roughly 1 month
after the well was capped.
Our observation of 13C-depleted suspended particles at a
station located 289 km to the southwest of the DWH well
(27.168N, 90.718W, Fig. 2D), provides direct and unambigu-
ous evidence of the spread of the subsurface oil and gas
plumes for hundreds of kilometers in the summer of 2010.
Previous studies have used indirect evidence to estimate the
spatial extent of the deep oil and gas plumes. For instance,
Kessler et al. (2011) report oxygen depletions as far west as
928W, and state that they tracked submerged hydrocarbon
intrusions for 500 km southwestward from the well using
fluorescence and oxygen anomalies. Valentine et al. (2014)
state that the plume extended for 300 km to the southwest
based on distributions of hopane in sediments. Other
authors report that the plume spread in the neighborhood of
50 km southwest of the wellhead (Camilli et al. 2010; Reddy
et al. 2012; Spier et al. 2013). To our knowledge, our study
provides the only direct evidence for the presence of petro-
carbon hundreds of kilometers to the southwest of the DWH
wellhead.
In addition to the deep plume, oil-derived carbon from
surface oil slicks could produce an imprint in suspended par-
ticles in the euphotic zone that might persist well after the
slicks themselves. This memory effect can explain the broad
distribution of low d13Csp values (230& to 225&) we
observed, especially in the DWH region, during the 2011
and 2012 cruises (Fig. 2C,E), when oil accounted for up to
53% and 75% of Csp (Eq. 1), respectively. Using the cruise
average contribution of oil to suspended particles (18% 6
15% to 25% 6 14%, Eq. 1), we estimate that between
216 6 174 and 292 6 165 t of DWH oil-derived carbon was
present in suspended particles in 2010, and that some of this
DWH petrocarbon was still present in particles in 2011 and
2012.
 ndez-Carrera et al.
Ferna
Other factors might also contribute to low d13C values in
2011 and 2012. High river discharge during spring could
result in very negative d13C (< 225&) in organic matter dur-
ing summer or autumn (Chanton and Lewis 1999). These
inputs could partly contribute to the low d13Csp values mea-
sured in surface waters (< 15 m depth) in the DWH region,
where lower surface salinities (25–34 p.s.u., data not shown)
were found on all of our cruises (Fig. 2A,C,E). In addition,
two of the Southern section stations sampled during 2010
(cruise OC468) and all stations sampled in 2012 (cruise
EN515) were in/near the GC600 lease block (27.378 N, 90.568
W), one of the most active seep fields in the GoM. In 2010,
only samples taken at the depth of the plume
(1000 2 1200 m) were 13C depleted (d13Csp < 225&), sugges-
ting that naturally released oil and gas made only a minor
contribution to the low d13Csp observed that year at depth.
In 2012, the deep profiles near site GC600 (Fig. 2E) showed a
consistent 13C depletion below 500 m and no vertical struc-
ture resembling the 2010 deep plume. This overall subsurface
depletion in 13C, around GC600 in 2012 appears to reflect
episodic natural releases of oil and gas that produce d13C
profiles quite different from those we found in 2010 in the
immediate aftermath of the DWH spill. This mechanism is
unlikely to be an important factor in the DWH section,
mainly in the Mississippi Canyon lease block region, which
does not contain any seep fields as persistent as those at
Green Canyon (Garcia-Pineda et al. 2016).
The variation of d15Nsp in surface water (0 2 200 m) clear-
ly reflects the influence of the major sources of nitrogen for
primary producers in the Northern Gulf. On the one hand,
nitrogen fixation (diazotrophy) is an important process in
the GoM, as evidenced by the large and frequent blooms of
the diazotrophic cyanobacterium Trichodesmium (Capone
et al. 1997; Holl et al. 2007). Diazotrophy appeared to supply
appreciable amounts of isotopically depleted N (Montoya
et al. 2002) to the euphotic layer, lowering d15Nsp to values
close to or below 0& (Fig. 3A,C,E), especially in the Southern
section offshore. On the other hand, nitrate from deep
water, which has a global average d15N of 4.8& (Liu and
Kaplan 1989; Sigman et al. 1997), likely supported a substan-
tial fraction of primary production at most of our stations,
particularly in the DWH nearfield section of the 2010 cruise
(Fig. 3A). In addition, nitrogen carried by the Mississippi riv-
er (Turner and Rabalais 2013) may also have contributed to
plankton nutrition in this area, which is relatively near the
Mississippi estuary, and whose nitrate d15N could range
between 21.4& and 12.3& as a result of the different land
uses across the basin (Chang et al. 2002).
Below the surface layer, we found surprisingly low d15Nsp
values (< 5&) in most of our samples at 1000–1200 m depth
in 2010, suggesting a significant input of isotopically deplet-
ed N to the planktonic community in these deep waters.
Low d15N values at depth were less common in 2011
and 2012, when very few samples showed values < 5&
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Deep Water Horizon oil and methane carbon
(Fig. 3C,E). A variety of processes may generate low d15N val-
ues in particles, but previous studies have reported potential
and actual heterotrophic diazotrophy by methanotrophic
archaea in natural methane seep sediments (Dang et al. 2009;
Miyazaki et al. 2009; Dekas et al. 2013), which seems to be
controlled by substrate (methane) availability. It is likely that
diazotrophy similar to that in seep sediments would have
been promoted by the high methane concentrations preva-
lent in the water column during the DWH incident (Hazen
et al. 2010; Valentine et al. 2010; Joye et al. 2011; Crespo-
Medina et al. 2014), leading to the observed low d15N of par-
ticulate organic nitrogen in 2010. Pure culture toxicity bioas-
says also provide evidence that heterotrophic bacteria, which
are less sensitive to oil than methane-oxidizing bacteria, may
have increased in abundance as result of the DWH oil spill
(Urakawa et al. 2012). Given the dramatic increase (> 7 orders
of magnitude) in methane flux into the wellhead region (Joye
et al. 2011), the significant correlation between d15Nsp and
d13Csp at 1000 2 1200 m in the Southern section (s 5 0.362,
p < 0.05, n 5 24, Fig. 5) suggests an increase in heterotrophic
nitrogen fixation by methanotrophs in intermediate waters
during the DWH oil spill. This coupled methanodiazotrophy
would lower d15Nsp at depth by contributing 15N-depleted
(d15N 5 22&) nitrogen to organic matter. In the Southern
section, we used a two-end-member isotope mixing model
(Montoya et al. 2002) with nitrate (d15N 5 4.8&) and diazo-
trophic nitrogen (d15N 5 22&) as nitrogen sources and esti-
mated that diazotroph N could account for up to 40% of the
particulate nitrogen at 1000–1200 m depth in 2010.
The vertical distribution of the C : N ratio of suspended
particles was clearly different between our cruises (Fig.
3B,D,F), showing greater increases with depth in 2010 (cruise
OC468) and 2012 (cruise EN515) than in 2011 (cruise
EN496). This increase in C : N ratio with depth in 2010 is
especially apparent in the DWH nearfield section, with val-
ues as high as double the Redfield ratio at depth. In June
2010, bacterial communities within the surface oil slicks
responded to the oil input by increasing their respiration
rates but not their biomass (Edwards et al. 2011). The meta-
bolic shift reflected an increased stress due to enhanced
phosphorous demand in a largely nutrient depleted environ-
ment. Under these conditions of dramatically increased car-
bon availability, the incorporation of carbon would have
increased substantially relative to other nutrients, leading to
the elevated C : N ratios that we measured during the 2010
cruise. Assimilation or adsorption of oil by particles will also
result in lower d13Csp values and should lead to negative cor-
relation between C : N ratio and d13C (i.e., the more depleted
the d13C, the higher the C : N ratio). In fact we find a weak
but positive correlation (s 5 0.199, p < 0.01, n 5 256, Fig. 4B),
suggesting that other processes, including high respiration
rates and high C assimilation, had a significant impact on the
d13C of particles. In contrast, we found C : N ratios in the
Southern section of the 2010 cruise that were lower than the
 ndez-Carrera et al.
Ferna
Redfield ratio, especially at the depths of the subsurface
plume (Fig. 3B). Interestingly, many of the samples with a
low C : N ratio also had low d13C and d15N values, again sug-
gesting that methanotroph-associated diazotrophy supported
balanced growth of deepwater microbial populations, thereby
linking the C and N cycles at depth.
Conclusions
The quick response of bacterial communities to the
release of oil and gas by the Deep Water Horizon blowout
led to the movement of 13C and14C-depleted oil/methane-
derived carbon into particles during summer 2010, when at
least 216 6 174 to 292 6 165 t of oil carbon and 80 6 56 to
104 6 91 t of methane carbon were present in suspended par-
ticles. We further document the impact of the plume on sus-
pended particle at depths between 1000 m and 1200 m as
much as 289 km southwest of the DWH wellhead in 2010.
That C input persisted in the system in subsequent years
(2011 and 2012) in the whole region, presumably via recy-
cling through the planktonic microbial food web. The
extremely large amounts of methane released to the water
column also appeared to somehow stimulate methanotroph-
associated diazotrophy at depths between 1000 m and
1200 m in the region of the Southwest Plume, which in turn
contributed to the relatively low d15Nsp values we found at
those depths in 2010.
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R/V Endeavor for their help and support during operations at sea. AFC was sup-
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This research was made possible by a grant from The Gulf of Mexico Research
Initiative. Data are publicly available through the Gulf of Mexico Research Ini-
tiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearch-
initiative.org (R1.x132.134:0073, R1.x132.134:0074, R1.x132.139:0016
and R1.x132.139:0017). This is ECOGIG contribution #363.
Conflict of Interest
None declared.

Mickle, and J. P. Chanton. 2016. Tracing the intrusion of

Submitted 01 September 2015
fossil carbon into coastal Louisiana macrofauna using nat-
Revised 14 March 2016; 05 July 2016
ural 14C and 13C abundances. Deep-Sea Res. II Top. Stud.
Accepted 17 August 2016
Oceanogr. 129: 89–95. doi:10.1016/j.dsr2.2015.05.014
Associate editor: Leila Hamdan

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