The biogeochemical cycling of methane in Ria de Vigo, NW Spain: Sediment processing and sea–air exchange

CH4 cycling in the Ría de Vigo

1

The biogeochemical Cycling of Methane in Ria de Vigo, NW Spain: Sediment

Processing and Sea-Air exchange.

Vassilis Kitidis1,*

, Louise Tizzard1, Günther Uher

1, Alan Judd

1, Rob C. Upstill-Goddard

1, Ian M.

Head2, Neil D. Gray

2, Gillian Taylor

2, †, Ruth Durán

3, Raquel Diez

3, Jorge Iglesias

3 and Soledad

García-Gil3

1 School of Marine Science & Technology, University of Newcastle, Newcastle upon

Tyne, United Kingdom. 2 School of Civil Engineering and Geosciences, University of Newcastle, Newcastle upon

Tyne, United Kingdom 3 Departamento de Xeociencias Mariñas e Ordenación do Territorio, Universidade de

Vigo, Spain † now at: Department of Human Evolution, Max Planck Institute of Evolutionary

Anthropology, Germany * Corresponding author ([email protected])

CH4 cycling in the Ría de Vigo

2

Abstract

Methane (CH4) concentrations were measured in the water column, in sediment porewaters,

and in atmospheric air, in the Ría de Vigo, NW Spain, during both the onset (April 2003) and at the

end of (September 2004) seasonal upwelling. In addition, CH4 concentration and stable isotopic

signatures (δ13

CH4) were measured in porewaters, and sediment methanogenesis and aerobic

oxidation of CH4 were determined in sediment incubations. Surface water column CH4 (2 m depth)

was in the range 3-180 nmol L-1

(110-8500 % saturation) and followed a generally landward

increase but with localised maxima in both the inner and middle Ría. These maxima were consistent

with CH4 inputs from underlying porewaters in which CH4 concentrations were up to 3 orders of

magnitude higher (maximum 350 μmol l-1

). Surface water CH4 concentrations were ~ three times

higher in September than in April, consistent with a significant benthic CH4 flux driven by

enhanced sediment methanogenesis following the summer productivity maximum. CH4 and δ13

CH4

in sediment porewaters and in incubated sediment slurries (20 oC) revealed significant sediment

CH4 oxidation, with an apparent isotopic fractionation factor (rc) ~ 1.004. Using turbulent diffusion

models of air-sea exchange we estimate an annual emission of atmospheric CH4 from the Ría de

Vigo of 18-44 × 106 g (1.1-2.7 × 10

6 mol). This estimate is approximately 1-2 orders of magnitude

lower than a previous estimate based on a bubble transport model.

CH4 cycling in the Ría de Vigo

3

Keywords

Methane;

Gas exchange

Sediment-water exchanges

Air-water exchanges

Gas fields

Gas seepages

Ría de Vigo

Spain

CH4 cycling in the Ría de Vigo

4

1. Introduction

Methane (CH4) is the next most important biogenic greenhouse gas after CO2; its current

atmospheric mixing ratio ~1.75 μatm (Dickinson and Cicerone, 1986; Wuebbles and Hayhoe, 2002)

is increasing at a variable rate that is not well understood but which reflects changing source and/or

sink strengths (Dlugokencky et al., 1994). The major sinks for CH4 are oxidation by hydroxyl

radical to CO and CO2 in the troposphere, and by O3 in the stratosphere. In contrast, CH4 sources

are more diverse and relatively poorly constrained. Wetlands are thought to be the dominant global

CH4 source, whereas the oceans are thought to be only a minor contributor (e.g. Bates et al., 1996).

However, recent work on coastal shelves and in estuaries indicates that these areas likely dominate

the marine CH4 cycle, prompting re-evaluations of the role of marine waters in the global budget

(e.g. Bange et al., 1994; Upstill-Goddard et al., 2000). CH4 in the marine environment is produced

via microbial methanogenesis in anaerobic environments, such as in sediments below the sulphate

reduction zone (Winfrey et al., 1981; Kelley, 2003; Middelburg et al., 2002), in the guts of

zooplankton and in faecal pellets (Marty, 1993; De Angelis and Lee, 1994), and in estuarine

turbidity maxima (Upstill-Goddard et al., 2000). Additionally, thermogenic sources may contribute

significantly to the marine CH4 budget, although they are poorly quantified (Judd et al., 1997;

Kvenvolden et al., 2001). CH4 in coastal marine systems is consumed by microbial oxidation in

sediments and in the water column (De Angelis and Scranton, 1993; Van Der Nat et al., 1997; Abril

and Iversen, 2002), and it is lost through sea-air exchange (Upstill-Goddard et al., 2000).

The stable isotopic signature of C in CH4 (δ13

CH4) can give insight into production and

consumption processes involved in CH4 biogeochemical cycling. Atmospheric CH4 has a δ13

CH4 of

-47 ‰ (Wuebbles and Hayhoe, 2002 and references therein) while both lighter (more negative

δ13

CH4) and heavier values have been reported from the marine environment reflecting

biogeochemical processing. Microbial methanogenesis can occur either via acetate fermentation or

CO2 reduction. The observed stable isotopic composition of biogenic CH4 reflects a balance

between the species composition of methanogens (Valentine et al., 2004), the supply of labile

CH4 cycling in the Ría de Vigo

5

organic carbon (Blair, 1998) and the isotopic signature of the substrate (Kotelnikova, 2002).

Methanogens preferentially utilise 12

C; hence the resulting CH4 is isotopically light, having a typical

δ13

CH4 of -60 ‰ to -97 ‰ (e.g. Popp et al., 1995; Boehme et al., 1996; Summons et al., 1998;

Tsunogai et al., 2002; Chan et al., 2005). By comparison δ13

CH4 from thermogenic sources is

typically -6 ‰ to -46 ‰ (e.g. Sassen and Macdonald, 1997; Sassen et al., 1999; Katz et al., 2002;

Tsunogai et al., 2005). Subsequent microbial oxidation of CH4 to CO2 gives rise to elevated δ13

C of

the residual CH4 in porewaters (Martens et al., 1999; Tsunogai et al., 2002) and in the water column

(Sansone et al., 1997; Cowen et al., 2002).

The Ría de Vigo (Figure 1) is one of four large, submarine, incised valleys running east to west

along the north-west Iberian Peninsula, an area subject to strong seasonal upwelling from late

March to early October (Doval et al., 1997). Shallow seismic data reveal extensive acoustic

turbidity in the Holocene fill-sediments, indicating shallow gas accumulations; acoustic water

column targets and seafloor pockmarks indicative of sediment gas escape via ebullition are

characteristic (Garcia-Garcia et al., 1999; Garcia-Gil et al., 2002; Garcia-Garcia et al., 2003;

Garcia-Gil, 2003). A microbial origin for the shallow gas, consistent with the high sediment organic

carbon content (up to 12 % by weight), has been confirmed by the identification of CH4 (Garcia-Gil,

2003); an alternative thermogenic origin for this gas is inconsistent with the underlying geology

which is metamorphic and igneous basement (Garcia-Garcia et al., 1999; Garcia-Garcia et al.,

2003). The organic rich gassy sediments are mainly constrained within the main channel (Figure 1).

It has been speculated that these sediments might ultimately be a significant local source of

atmospheric CH4: an estimate of CH4 ebullition fluxes based on the density of acoustic plumes and

pockmarks concluded that ~0.1-4.1 x 109 g CH4 were emitted annually from the Ria de Vigo to the

atmosphere (Garcia-Gil et al., 2002).

Although both the underlying geology and the locations of major acoustic targets in Ría de

Vigo are well known from previous work, to date there has been no systematic study of its CH4

biogeochemistry and sea-to-air flux. We therefore made detailed biogeochemical measurements

CH4 cycling in the Ría de Vigo

6

during two seasons; at the onset (April) and at the end of (September), seasonal upwelling. Our

biogeochemical data include CH4 distributions in the water column, in sediment porewaters, and in

atmospheric air, stable isotopic signatures (δ13

CH4) in sediment porewaters, sediment

methanogenesis and potential aerobic oxidation of CH4 and sea-to-air CH4 fluxes determined using

a flux chamber and with gas exchange models.

CH4 cycling in the Ría de Vigo

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2. Methods

2.1 Study area

The Ría de Vigo (NW Spain) is a large coastal embayment approximately 30 km in length and

11 km wide at its mouth. It has a surface area of 178 km2, a maximum water depth of 50 m (Figure

1), and a spring tidal range ~2.3 m. The Islas Cíes lie on a north-south running fault at the mouth of

the Ría and mark the geographic boundary with the Atlantic Ocean. The Ría de Vigo including San

Simón Bay in the innermost part is tectonically controlled. Major freshwater inputs to San Simón

Bay are via the Verdugo-Oitabén and Alvedosa rivers. The Ría de Vigo can be divided into three

parts (Figure 1): a) the inner Ría (San Simón Bay to Rande Strait), b) middle Ría (Rande Strait to

Cabo de Mar) and c) outer Ría (Cabo de Mar to Islas Cíes). Seasonal upwelling, driven by northerly

winds plays a central role in the hydrography and biogeochemistry of the Ría de Vigo.

The general residual circulation is of the typical two-layer estuarine type in the inner Ría, with

low salinity water outflow at the surface and near bottom high salinity Atlantic inflow modified by

lateral circulation in the middle and outer Ría (Prego and Fraga, 1992; López et al., 2001; Souto et

al., 2003). River discharge is highest in winter and affects largely the inner part of the Ría which

behaves like a typical estuary (Prego and Fraga, 1992). The middle and outer parts of the Ría are

influenced by seasonal upwelling which generally lasts between March and October; during this

period intermittent upwelling events are punctuated by upwelling-relaxation events (Doval et al.,

1998). Nutrient rich and O2-depleted (>150 μmol L-1

) Atlantic Ocean bottom waters enter the Ría

during the upwelling events leading to primary production-remineralisation cycles in the water

column (Doval et al., 1998). Upwelled waters provide the dominant nutrient supply to

photosynthesis along a comparatively small terrestrial nutrient flux via river runoff (Prego,

1993;1994). Chlorophyll a reaches a maximum >8 μg L-1

during summer and declines to <1 μg L-1

during winter (Nogueira et al., 1997; Doval et al., 1998). Although the inner Ría is net

heterotrophic year-round (Prego, 1993b), overall the entire Ría de Vigo is net autotrophic, with a

mean rate photosynthesis ~350 mg C m-2

d-1

; this results in net atmospheric CO2 drawdown (Prego,

CH4 cycling in the Ría de Vigo

8

1993b; Borges and Frankignoulle, 2002). The resulting mean sedimentation flux of organic carbon

is estimated at 140 mg C m-2

d-1

(Prego, 1993). The sedimentation rate of organic carbon peaks

during the summer and can reach 540 mg C m-2

d-1

during an upwelling event (Gago et al., 2003).

Seasonal upwelling therefore plays an important role in regulating primary and bacterial

productivity in the Ría de Vigo water column and in regulating the supply of organic matter to

sediments.

2.2 Sample collection

Two field campaigns were carried out: i) between the 25th

and 27th

of April 2003 and ii)

between the 9th

and 15th

of September 2004, to coincide with the onset and the end of seasonal

upwelling respectively. Sampling of the middle and outer Ría was from B/O Mytilus while a small

boat was used in the inner Ría (San Simón Bay). In total, 84 water column samples and three

sediment cores (2 subtidal and 1 intertidal) were collected. Sampling locations, logged using GPS,

are shown in Figure 1. Discrete water samples were collected from the B/O Mytilus flow through

system, which had its inlet in the vessel’s bow at 2 m depth, and from just below the water surface

in the case of the small boat. Subsurface samples were collected over the full water depth using a 12

V DC submersible pump and flexible nylon tubing. Prior to sampling the tubing was flushed with at

least three times its internal volume in order to preclude sample cross contamination. B/O Mytilus

flow through samples and pumped samples from 2m depth showed agreement to better than ± 7 %

for dissolved CH4.

On board B/O Mytilus, sea surface temperature (SST) and conductivity were measured with a

thermosalinograph (SBE21 SEACAT) inline with the flow through system, while hand held

instruments (Hanna Instruments, HI 147-00 and HI 8633) were used on the small boat.

Meteorological data (atmospheric pressure, wind speed and direction) were obtained from the B/O

Mytilus onboard meteorological package (Aanderra Instruments, sensors 3590, 2740, 2810 and

3455).

CH4 cycling in the Ría de Vigo

9

Headspace free water samples for CH4 analyses were collected in 100 mL gas tight glass

syringes fitted with gas tight, 3-way polycarbonate stopcocks. Samples were treated with 10 mmol

L-1

HgCl2 in order to arrest microbial activity (Upstill-Goddard et al., 1996; 2000) and stored in the

dark at 4 oC until analysis.

In April 2003 sediment cores were collected by gravity corer in the middle (core GC3; 42,262

ºN, 8,717 ºW) and outer (core GC1; 42,218 ºN 8,868 ºW) Ría (Figure 1). Water depth at both these

locations was ~40 m. Core GC3 was collected inside the gassy sediment fields and core GC1 was

collected from the edge of the outer gassy sediment fields. In September 2004 core SC1 (42,308 ºN,

8,621 ºW) (Figure 1) was collected from an intertidal area of San Simón Bay using a modified

suction corer (Méndez et al., 2003). In all cases core barrels were capped with rubber bungs and

transported to the University of Vigo for sectioning and analyses.

2.3 Upwelling Index

We obtained the monthly mean upwelling indices (UI) at 42.5 oN and 8.5

oW, available from

the US Pacific Fisheries Environmental Laboratory Live Access Server

(http://www.pfeg.noaa.gov/products/las.html). UI is computed from six-hourly pressure fields

prepared by the U.S. Navy Fleet Numerical Meteorological and Oceanographic Center on a global

spherical 1° grid. UI values for April/May 2003 and September 2004 were -35.5 and 8.8

respectively. Negative values indicate downwelling conditions, while positive values indicate

upwelling. UI values for April/May 2003 and September 2004 were -35.5 (downwelling) and 8.8

(mild upwelling) respectively (Figure 2). These are within the range of climatological monthly UI

means for the period 1967 – present; these indicate that April 2003 and September 2004 were

transitional months at the onset, and at the end of, upwelling respectively.

2.4 CH4 analysis

Water column samples were analysed for CH4 within 12 hours of collection by in-syringe

headspace equilibration (Upstill-Goddard et al., 1990) using CH4-free N2, followed by flame

ionisation detection gas chromatography (Shimadzu GC8A with a Shimadzu CR6A integrator).

CH4 cycling in the Ría de Vigo

10

Samples were injected onto a 2 m long, 3 mm internal diameter stainless steel column packed with

Porapak-Q (Jones Chromatography; 80-100 mesh) via a 6-port valve fitted with a 1 cm3

sample

loop. The loop was flushed with sample to at least 3 times its internal volume prior to sample

injection. Chromatographic separation was at 60o C with ultra high purity (UHP) N2 carrier. The

detector temperature was 120o C (Upstill-Goddard et al., 1996). Retention characteristics and

analytical precision (better than 2 % standard deviation from the mean for 5 injections of a 1.9 ppm

standard) were similar to those given in Upstill-Goddard et al. (1996). The gas chromatograph was

calibrated daily using two secondary CH4 standards prepared by pressure dilution (Upstill-Goddard

et al., 1990) and intercalibrated against gravimetric primary standards (BOC gases Ltd.). In situ

CH4 concentrations and percent saturations relative to air were calculated using data from

Wiesenburg and Guinasso, 1979).

Ambient air samples were collected by gas-tight syringe in the field and injected directly into

the gas chromatograph as detailed above.

2.5 Sediment profiles

Core barrels were sectioned in a N2-filled glove box within 24 hours of collection. A positive

internal pressure was maintained in the glove box throughout, in order to prevent ingress of O2

during core sectioning. Core GC1 was sub sampled at 3, 21, 35 and 66 cm depth, core GC3 was sub

sampled at 10, 13, 26, 33, 48 and 66 cm depth and core SC1 was sub sampled at 1, 6, 11, 21, 31, 41,

51 and 72 cm depth. Sediment was sub sampled. At each depth approximately 2 mL of sediment

were transferred into to a pre weighed 10 mL glass bottle (Sigma-Aldrich, Z113972) with a cut

syringe and used to determine porosity by weight loss on drying overnight at 60 oC. A 5 mL plastic

syringe was used in order to minimise gas loss and the tip was cut to allow sediment sampling.

Porewater CH4 analysis was by headspace-equilibration of sediment-slurries (e.g. Abril and Iversen,

2002) following the procedure for water column samples. Each sediment slurry was created in the

glovebox in order to maintain anoxic conditions; 3 mL of Milli-Q water (Millipore Corp., model

Q185), were bubbled for ~30 minutes with O2-free N2 in order to remove O2 and added to ~2 mL

CH4 cycling in the Ría de Vigo

11

wet sediment (transferred with a cut syringe) in a pre weighed 10 mL glass bottle, which was then

sealed with a butyl rubber septum (Sigma-Aldrich, Z166065). Following headspace equilibration 1

mL of headspace was displaced with a gas tight syringe fitted with a luer needle pushed through the

rubber septum, by injecting Milli-Q water through a second needle. The headspace sample was then

diluted further with CH4-free N2 and injected onto the chromatographic column. Following analysis,

the residual sediment samples were dried and reweighed, and the porewater volume calculated by

adjusting for sample porosity. To determine the procedural blank 3 mL of Milli-Q were added to

replicate glass bottles without sediment and treated as above. A small, but detectable blank was

presumably due to residual CH4 dissolved in the Milli-Q water; the blank was <1 % of the lowest

CH4 concentration measured in sediment porewaters.

Sediment sub samples for microbial incubations (13 cm and 66 cm for GC3, and 10 cm and 66

cm for GC1) were stored in the dark at 4o C prior to the incubation experiments.

2.6 Stable isotopic analysis

During the headspace equilibration procedure additional subsamples were collected in He-

flushed, pre-evacuated 10 mL vacutainers (IsoChem, 116117EVAC) and stored for subsequent

stable isotopic analysis of δ13

CH4 at the University of Newcastle using a PDZ Europa Ltd, Trace

Gas II (TGII) preparation unit attached to a PDZ Europa Ltd 20/20 isotope ratio mass spectrometer.

Each gas sample was flushed from the 10 mL vacutainer with high purity helium (99.999% purity)

at 21 mL min-1

for 500 seconds via a Gilson 220 autosampler. The gas stream was passed through a

liquid N2 cryotrap in order to retain non-methane hydrocarbons. CO in the gas stream was then

oxidized to CO2 by passing it through ShutzeTM

reagent and subsequently removed as CO2 in a

CarbosorbTM

scrubber tube. Remaining CH4 in the gas stream was oxidised to CO2 in a combustion

furnace at 1000ºC over a Ni-Pt catalyst. The catalyst was regenerated with short O2 pulses (> 15

seconds) after each sample. Following combustion, water vapor was removed through a NafionTM

membrane and the CH4 derived CO2 was cryofocused by passing the sample stream through a series

of cryotraps before injection into the mass spectrometer. Cryofocusing was achieved by lowering a

CH4 cycling in the Ría de Vigo

12

1 m long coil of steel tubing (1/16 ″ o.d.) into liquid N2 for 500 seconds followed by immersing a

second coil into liquid N2 for a further 110 seconds. The isotopic signature of CH4 derived CO2 was

monitored at m/z = 44, 45 and 46 and its δ13

C value was compared with a laboratory CO2 standard

pre-calibarated against international standard IAEA-NBS22. The total run time was 20 minutes. All

isotope ratios expressed in standard δ notation are relative to V-PDB.

2.7 Sediment incubations

Sediment incubations designed to provide information on microbial methanogenesis and

potential aerobic CH4 oxidation (MO) commenced within 10 days of sample collection.

Approximately 2 mL of wet sediment from each depth were transferred into replicate pre weighed

10 mL glass bottles and capped with rubber septa in the glove box. For MO incubations 2 mL of

low nutrient seawater (0.2 μm filtered surface water from the South Atlantic Gyre, collected in

2000) were added to create a slurry. The slurries were briefly exposed to air (to allow O2 into the

headspace) and resealed before 100 L of 99 % CH4 (Sigma-Aldrich, 29,547-7) were injected

through the septa to give approximately 1 % CH4 (v./v.) in the headspace. Empty bottles (no

sediment) were treated as above and used to assess leakage during the experiments. For

methanogenesis determinations, ~2 mL of wet sediment were transferred to the bottles as above.

Controls were treated similarly except that 0.5 mL of a 1 mmol L-1

bromoethane sulphonic acid

(BES) solution were added in order to inhibit methanogenesis. CH4 concentration was monitored

during 10 day incubations at room temperature (~20 oC). Incubations of this nature are susceptible

to artefacts relating to prior sample storage, differences between ambient and incubation

temperatures, and substrate depletion. As such any quantitative derivations drawn from them, such

as rates of bacterial processes, should be viewed cautiously. We therefore choose to subsequently

discuss these data only qualitatively.

2.8 Sea-Air flux estimates

A 54 L floating chamber with a surface area 0.3 m2 was deployed by diver in the inner Ría

during September 2004 (Figure 1). The chamber was deployed at 10 a.m. (local time) at low tide

CH4 cycling in the Ría de Vigo

13

and the concentration of CH4 in the headspace was monitored hourly over a 9 hour period in order

to provide direct estimates of sea-to-air CH4 fluxes. The chamber was anchored to the sea floor with

enough slack to allow for tidal differences in water depth over the deployment period. The

headspace volume change over the course of the experiment was <2 % of the initial headspace

volume.

We additionally estimated sea-to-air CH4 fluxes (FSA) based on surface water concentrations

according to:

4CHkF wSA (1)

where kw is the gas transfer velocity and ΔCH4 is the sea-air concentration difference. Various

relationships relate kw to wind speed. The most commonly used such parameterisations are those of

Liss and Merlivat (1986) (equations 2 and 3) and Wanninkhof (1992) (equation 4):

66.0

60017.0

c

w

Suk , u < 3.6 m s

-1 (2)

5.0

60065.985.2

c

w

Suk , 3.6 m s

-1 < u ≤ 13 m s

-1 (3)

5.0

2

66031.0

c

w

Suk (4)

where u is the wind speed at 10 m (8.2 m s-1

and 3.3 m s-1

for April 2003 and September 2004

respectively) and Sc is the Schmidt number of CH4 (868 at 15 oC and 746 at 18

oC).

CH4 cycling in the Ría de Vigo

14

3. Results

3.1 Hydrography

During both April 2003 and September 2004 salinity ranged from <25 in the inner Ría to ~34-

35 in the outer Ría. During April 2003 sea surface temperature ranged from 15 oC in the outer Ría

to 16 oC in the inner Ría. Corresponding values during September 2004 were 18

oC and 21

oC.

Partial thermohaline stratification was more pronounced in the Ría de Vigo during April 2003

compared to September 2004 indicating contrasting hydrographic regimes (Figure 3). Low salinity

surface water was observed in all depth profiles from the outer and inner Ría during April 2003

(Figures 3b, 3h and 3e respectively). In contrast salinity profiles collected in September 2004

showed that the water column was well mixed with respect to salinity (Figures 3k, 3q and 3n).

Partial stratification in September 2004 was evidenced by warmer surface water in the outer and

middle Ría (Figures 3l and 3r respectively). The observed temperature and salinity distributions

were therefore consistent with upwelling during April 2003 and downwelling during September

2004. An alternative explanation may be increased freshwater runoff entering the inner Ría during

April 2003. Wind driven upwelling during this period may have enhanced outwelling of freshwater

from the inner Ría.

3.2 CH4 in the water column

Table 1 summarises mean CH4 concentrations and salinities in surface waters of the outer,

middle and inner Ría and near the mouth of the Verdugo-Oitabén River. Ría de Vigo surface water

was everywhere supersaturated in CH4 relative to atmospheric air; concentrations ranged from 3

nmol l-1

(101 % saturation) to 180 nmol L1.(8500 % saturation) (Figure 4). CH4 concentrations from

same surface water locations were on average 3–fold higher during September 2004 (490-8500 %

saturation) than in April 2003 (101-1850 %).

Surface concentrations generally increased landwards during both seasons (Figure 4) and

decreased with salinity (Figure 5). However dissolved CH4 was non-conservative over the salinity

ranges sampled in both field campaigns, indicating significant CH4 sources to the water column in

CH4 cycling in the Ría de Vigo

15

both the inner and middle Ría (Figure 5). During April 2003 dissolved CH4 was highest in the

Rande Strait (47 nmol L-1

) connecting the inner and middle Ría (Table 1 and Figure 4). In

September 2004 dissolved CH4 was highest near the mouth of the Verdugo-Oitabén River (180

nmol L-1

), which was not sampled during April 2003 (Figure 4).

Vertical profiles from April 2003 showed a marked increase in dissolved CH4 concentration

with depth in the inner Ría (Figure 3d: 37 nmol L-1

at the surface; 56 nmol L-1

at the bottom and

Figure 3g: 40 nmol L-1

at the surface; 65 nmol L-1

at the bottom), whereas in the outer Ría the

change in CH4 with depth was relatively slight (Figure 3a: 4 nmol L-1

at the surface; 5 nmol L-1

at

the bottom). Similar results were obtained in the outer Ría in September 2004 (Figure 3j: 5 nmol L-1

at the surface; 4 nmol L-1

at the bottom), while the middle Ría de Vigo depth profile showed a

decrease in CH4 concentration with depth (Figure 3p: 67 nmol L-1

at the surface; 43 nmol L-1

at the

bottom). Although the September 2004 depth profile of CH4 from the inner Ría showed the same

trend of increasing CH4 with depth as in April 2003, the September 2004 concentrations were

higher (Figure 3m: 95 nmol L-1

at the surface; 115 nmol L-1

at the bottom). Water column maxima

were observed in the vicinity of a) the halocline in the inner and outer Ría in April 2003 (Figures 3g

and 3a: 73 nmol L-1

and 6 nmol L-1

respectively) and b) the thermocline in the middle and outer Ría

in September 2004 (Figures 3p and 3j: 71 nmol L-1

and 10 nmol L1 respectively).

3.3 CH4 in sediments

In April 2003 porewater dissolved CH4 was ~ 7-37 μmol L-1

in the outer Ría (core GC1)

(Figure 6a) and 3-350 μmol L-1

in the middle Ría (Core GC3) (Figure 6b). There was an overall

increase in CH4 with depth in both cores. Most notably the CH4 concentration at 66 cm in ‘in gas

field’ core GC3 was an order of magnitude higher than in core GC1. In the intertidal core from the

inner Ría (Core SC1, September 2004) dissolved CH4 was in the range of 1-235 μmol L-1

and in

contrast to both the central and outer Ría during April 2003 distinct concentration maxima were

evident just below the surface and at 31 cm (Figure 6c)

CH4 cycling in the Ría de Vigo

16

Porewater CH4 was progressively 13

C depleted (isotopically lighter) with depth; δ13

CH4 was in

the range -44.7 ‰ V-PDB to -51.5 ‰ V-PDB for GC1 (Figure 6a) and -42.5 V-PDB to -55.0 ‰ V-

PDB for GC3 (Figure 6b). Methane oxidation (MO) was only observed in sediments from GC3,

while methanogenic activity was only observed in the uppermost cm of SC1. Figure 7 shows the

depletion of CH4 (99 % loss) in the headspace of vials containing sediment slurry from GC3

collected at 13 cm sediment depth within the 10 day incubation period. A decrease in CH4 was also

observed in the control and subsample collected at 66 cm sediment depth, but this was small by

comparison (~20 %) and was probably due to leakage of CH4 from the vials.

3.4 Sea-Air flux

The observed ubiquitous surface CH4 supersaturations imply that the Ría de Vigo is a strong

local source of atmospheric CH4. In the floating chamber (Figure 7) the CH4 flux estimated from the

change in headspace CH4 with time was 63 μmol CH4 m-2

d-1

. Sea-to-air CH4 fluxes determined

using the relationships of Liss and Merlivat (1986) and Wanninkhof (1992) are summarised in

Table 2 based on mean CH4 concentrations for the inner, middle and outer Ría listed in Table 1. The

flux of CH4 to the atmosphere increased landwards by 2 orders of magnitude and was generally of

the same order of magnitude during both field campaigns owing to the lower wind speed in

September 2004 despite the higher CH4 concentration (Table 2). The estimated sea-air flux for the

inner Ría for September 2004 was 47-84 μmol m-2

d-1

. This was in good agreement with the

measured sea-air flux (63 μmol m-2

d-1

) derived from the floating chamber data.

CH4 cycling in the Ría de Vigo

17

4. Discussion

4.1 Upwelling conditions

Stratification in the Ría de Vigo is mainly caused by salinity rather than temperature gradients

during the upwelling season (Alvarez-Salgado et al., 2000); saline Atlantic Ocean water

predominates in deeper waters, with a less saline layer influenced by terrestrial runoff near the

surface (López et al., 2001; Souto et al., 2003). Our observed temperature and salinity distributions

(Figure 3) were therefore consistent with upwelling during April 2003 and non-upwelling

conditions during September 2004, in contrast to the upwelling index (UI) values (Figure 2). The

disparity between these indications of upwelling (temperature-salinity distribution in the water

column versus UI) may be due to differences in the timescale over which they were examined. As

the UI represented monthly mean values it did not reflect acting over the shorter time-scales relating

to water column measurements of temperature and salinity. Given that the flushing time of the Ría

de Vigo is in the order of one week, it seems reasonable to assume a faster response time to

changing wind conditions for temperature and salinity than for UI. We therefore concluded that the

April 2003 and September 2004 field campaigns were carried out under upwelling and non-

upwelling conditions respectively.

4.2 Water column CH4

The observed concentrations (3-180 nmol L-1

) and non-conservative behaviour of CH4 in the

Ría de Vigo are consistent with conditions in other temperate estuarine systems that reflect both low

salinity CH4 input and its in situ production (Bange et al., 1998; Bussmann and Suess, 1998;

Sansone et al., 1999; Upstill-Goddard et al., 2000; Abril and Iversen, 2002; Middelburg et al.,

2002). The more saline surface and near bottom waters of the outer Ría in April 2003 were

relatively weakly supersaturated in CH4, consistent with vertical mixing of deeper water, low CH4

Atlantic inflow. By the time this input had reached the inner Ría however, it must have been

involved in significant mixing with additional CH4 sources, as evidenced by the strong vertical CH4

gradients observed. Such progressive landward enrichment of bottom water CH4 is consistent with a

CH4 cycling in the Ría de Vigo

18

sediment CH4 input. In contrast, during September 2004 deep water CH4 enrichment was only

observed in the inner Ría; there was generally little change with depth in water column CH4

concentration in the outer Ría, consistent with a well mixed water column evidenced by the salinity

profiles.

Unlike the deep water CH4 distributions, the water column maxima observed during both field

campaigns may be attributed to lateral advection or additional water column sources, such as

microbial methanogenesis in the anaerobic cores of particles in the halocline/pycnocline, as

observed in other nearshore basins (Sieburth and Donaghay, 1993) and in the open ocean (Scranton

and Brewer, 1977; Karl and Tilbrook, 1994). The spatio-temporal coincidence of the CH4 maxima

with the location of the halocline/thermocline (Figure 3) where biological activity and or

accumulation of particles would be expected to be high lend support to this hypothesis.

Unfortunately, there were no ancillary data to support either high particle abundance (e.g.

particulate organic carbon, transmissometer data) or biological activity (e.g. Chlorophyll a

concentration, microbial counts or incubations) and therefore the hypothesis of pelagic

methanogenesis must for now remain speculative.

4.3 Porewater CH4

Porewater CH4 concentrations (<0.35 mmol L-1

) in the Ria de Vigo are in general agreement

with those found previously in most other freshwater, estuarine and coastal locations (Adams et al.,

1982; Martens et al., 1998;1999; Abril and Iversen, 2002; Whiticar, 2002; Carini et al., 2003), but

are higher than those in some others of lower organic content (e.g. Abril and Iversen, 2002). The

observed CH4 concentration gradients reflect production at depth, consistent with the geophysical

evidence (Garcia-Garcia et al., 1999), and imply significant sediment-water exchange. The highest

porewater CH4 concentrations observed were insufficient for the formation of free gas (>5.1 mmol

L-1

), consistent with geophysical evidence that suggests free gas accumulation in the Ría de Vigo at

sediment depths ranging between 1.5-5 m (Garcia-Gil et al., 2002).

CH4 cycling in the Ría de Vigo

19

The highest CH4 concentration found at 66 cm sediment depth for core GC3 had a δ13

CH4

value of -55 ‰ consistent with a microbial origin (Cicerone and Oremland, 1988). The lack of any

observed methanogenic activity in the sediment slurry incubations from GC1 and GC3 during April

2003 (<66 cm depth) leads us to believe that these samples originated above the zone of active

methanogenesis, hence this zone must have been deeper than 66 cm below the sediment surface.

Although methanogenesis has often been observed in the uppermost 5-10 cm of freshwater and

estuarine sediments (Kerner, 1993; De Graaf et al., 1996; Bange et al., 1998; Avery and Martens,

1999; Hellings et al., 2000; Avery et al., 2003), in such situations low ambient salinity and hence

low sulphate may facilitate the establishment of methanogenic bacterial activity at such shallow

depths.

Although the porewater-water column CH4 concentration gradient implied significant fluxes

from the sediment to the overlying water, the CH4 profiles obtained in April 2003 showed apparent

removal of CH4 from the porewaters between 20 and 66 cm (Figures 6a and 6b). Hence the benthic

flux of CH4 is mediated by consumption within the sediment, most likely by aerobic and anaerobic

microbial oxidation. Aerobic oxidation of CH4 was observed during incubation of GC3 surface

sediment (13 cm), confirming the presence of a microbial sink for CH4 evidenced by the isotopic

data (Figure 6). Based on the relationship of Cowen et al. (2002), we calculated the kinetic

fractionation constant of CH4 oxidation rc:

1000

1000

Src (5)

where S is the slope of the regression of δ13

C-CH4 against log10 [CH4]. The fractionation

constant for both cores was 1.004, in agreement with previously reported fractionation constants

(1.004-1.012) from the Columbia River estuary and hydrothermal plumes (Sansone et al., 1999;

Cowen et al., 2002). Microbial oxidation of CH4 is therefore an effective barrier to the efflux of

CH4 from sediments to the overlying water of the Ría de Vigo. Similar removal of CH4 by

microbial oxidation has been found to eliminate the sediment – water efflux of CH4 at Eckernförde

CH4 cycling in the Ría de Vigo

20

Bay in the Baltic Sea and the Tommeliten cold seep in the North Sea (Martens et al., 1999;

Niemann et al., 2005). It is likely that aerobic oxidation at the sediment-water column boundary

further moderated the benthic flux of CH4.

4.4 Seasonal changes in water column CH4: a seasonal sediment source?

CH4 concentration in surface waters in September 2004 was on average 3-fold higher than in

April 2003 (Table 1). The April-September contrast in surface water CH4 is unlikely to be due to

increased water column methanogenesis in September 2004 as this would have resulted in more

pronounced subsurface maxima in depth profiles. Increased freshwater inputs are also unlikely to

have contributed to the observed differences in the central and outer parts of the Ria as evident from

the non-conservative, convex mixing behaviour with salinity which suggests significant inputs of

CH4 at these locations (Figure 4). It is conceivable that an increase in surface water CH4

concentration was attributable to increased residence time in the Ría de Vigo. Wind driven

upwelling reduces residence time by enforcing the residual, 2-layer circulation and thereby

enhancing outwelling from the Ría to the adjacent shelf. Thereby, we would expect higher CH4 in

the water column to coincide with non-upwelling conditions as was the case in September 2004. A

previous modelling study of residual flows in the Ría de Vigo found that the typical residence time

varied between 2.5-4.1 days for the middle and 2.8-5.5 days for the outer Ría respectively (Alvarez-

Salgado et al., 2000). Extremes of 1.4 and 11 days for the middle Ría and 1.7 and 130 days for the

outer Ría have been reported during high river discharge in winter and in the absence of wind in

autumn respectively (Alvarez-Salgado et al., 2000). However, such conditions were not

encountered during our study and therefore we considered only typical variability in residence time.

Under such conditions, increased residence time would therefore typically account for 40-50 % of

the observed increase in CH4. Finally, the observed differences may be due to enhanced

methanogenesis in sediments prior to sampling in September 2004.

Seasonal variation in surface water CH4, similar to that found in the present study, with higher

concentrations in the summer has been reported for the Baltic and Southern North Seas (Bange et

CH4 cycling in the Ría de Vigo

21

al., 1994; Bange et al., 1998). Bange et al. (1998) found that CH4 concentrations in surface waters

of the southern Baltic Sea in September were up to one order of magnitude higher than in April and

they interpreted this to reflect increased methanogenic activity in the underlying sediments during

the summer in response to elevated sediment temperatures and a seasonal input of relatively

undegraded particulate organic matter. The temperature dependence of methanogenesis in the Ria

de Vigo is unknown, however available data from freshwater and estuarine sediments (Bange et al.,

1998; Bange et al., 1998; Avery et al., 2003) indicate an increase in temperature from 15 oC to 18

oC between April and September to be consistent with a 20-40 % increase in methanogenesis.

Assuming that increased residence time and temperature enhanced methanogenesis may account for

an average of 45 % and 30 % of the observed increase in CH4 respectivelly, the remaining 25 %

must be due to enhanced methanogenesis from the summer input of labile organic carbon to

sediments. In the Ria de Vigo the majority of autochthonous carbon production during upwelling

(~68 % or ~0.5 g C m-2

d-1

) sinks as particulate organic carbon (Doval et al., 1997; Gago et al.,

2003), and presumably stimulates microbial activity at the sediment surface. Consistent with this,

methanogenesis and high porewater CH4 were observed in surface sediments (uppermost 1 cm)

from core SC1 in September 2004 but not in cores GC1 and GC3 collected during April 2003.

Hence we attributed the majority (>50 %) of the observed differences in CH4 to enhanced

methanogenesis in sediments of the Ría de Vigo during September 2004.

4.5 Sea-Air flux

The measured sea-to-air flux of CH4 from the floating chamber data agrees rather closely with

the calculated fluxes from the turbulent diffusion models for the inner Ría during September 2004.

This is an interesting result given that the use of floating chambers to calculate sea-to-air fluxes has

been criticised on the grounds that they modify wind and water induced turbulence. However for

the conditions we observed, i.e. the absence of breaking waves and low to moderate wind speeds

(<10 m s-1

), such errors are minimised (Kerner et al., 2003), although the chamber may have

interfered with the tidal current and induced additional sea surface turbulence since it was tethered

CH4 cycling in the Ría de Vigo

22

to the sea floor. Additional concerns relate to the accumulation of gas in the headspace; this can

reduce the water-air concentration gradient (ΔC) and thereby suppress the sea-to-air flux. To

minimise this problem floating chambers are usually deployed for much shorter periods than the

several hours used in our work (e.g. Borges et al., 2004). During our deployments ΔC, varied by

<10 %; hence we do not anticipate significant errors to have arisen in this way. Indeed, our rationale

for using long deployment times was that the resulting data would be more suited for comparison

with the results from the turbulent diffusion models, for which the appropriate timescales are hours

to days.

Our flux estimates provide a basis for estimating the annual emission of atmospheric CH4 from

the Ría de Vigo. Although our data do not provide full seasonal coverage they do suggest that wind-

driven upwelling largely controls the annual variability in water column CH4 concentrations

through enhancing sedimentary methanogenesis (see section 4.4). Given that our data span the onset

and the end of the upwelling period, they cover the period during which the highest fluxes are

expected; hence they allow a preliminary upper estimate of the annual CH4 flux from the Ría de

Vigo.

Assuming a mean area weighted flux for each of the inner, middle and outer Ría, the annual

efflux of atmospheric CH4 from the Ría de Vigo is 18-44 × 106 g (1.1-2.7 × 10

6 mol). This is ~2

orders of magnitude lower than a previous estimate of 0.1-4.1 x 109 g a

-1 for CH4 ebullition fluxes

based on the density of acoustic plumes and pockmarks (Garcia-Gil et al., 2002), and is comparable

to the annual sea-air flux of CH4 from the Humber estuary (36 × 106 g a

-1) estimated by Upstill-

Goddard et al. (2000). The discrepancy between our estimate and that of Garcia-Gil et al., 2002) is

not surprising given that the turbulent diffusion models exclude bubble mediated sea-to-air CH4

fluxes. In shallow nearshore regions such as the Ria de Vigo bubble residence times in the water

column may be very short. Consequently, significant CH4 and CO2 may vent to the atmosphere in

rising bubble plumes but remain unaccounted for by conventional flux estimates that are based on

dissolved gas distributions alone. Interestingly however, the agreement between our flux chamber

CH4 cycling in the Ría de Vigo

23

and turbulent-diffusion estimates of CH4 exchange suggest that at least during the measurement

period, CH4 ebullition rates were relatively low at the chamber deployment site. The estimate of

Garcia-Gil et al. (2002), likely represents an upper limit for CH4 emission as it takes no account of

CH4 converted back into CO2 via aerobic and anaerobic microbial oxidation in sediments and the

water column. Further efforts to constrain the emission of CH4 from the Ría de Vigo to the

atmosphere should focus on seasonal and inter annual variability of both the dissolved and bubble

fluxes.

CH4 cycling in the Ría de Vigo

24

Acknowledgements

We would like to thank the captain and crew of the B/O Mytilus for their assistance with fieldwork

and Charles Belanger (Iso-Analytical Ltd.) for assistance with the stable isotope analysis. This work

was supported by the UK Natural Environment Research Council (grant NER/A/S/2001/01017) and

Ministerio de Ciencia y Tecnología of Spain (grant REN2003-03233/MAR).

CH4 cycling in the Ría de Vigo

25

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CH4 cycling in the Ría de Vigo

33

Table 1: Average CH4 concentration (nmol L-1

) ± standard deviation (number of samples in

parenthesis) and average salinity (in brackets) in in each of the outer, middle and inner parts of the

Ría de Vigo during April 2003 and September 2004 field campaigns.

Field Campaign Dissolved CH4 (nmol L-1

), respective salinity in brackets

outer middle inner Verdugo-

Oitabén

April 2003 11±10 (11), [33] 26±12 (15), [32] 39±5 (8), [28] no data

September 2004 34±28 (5), [35] 70±16 (5), [34] 112±39 (13), [31] 180 (1), [19]

CH4 cycling in the Ría de Vigo

34

Table 2: Estimated sea-air CH4 fluxes for the inner, middle and outer part of the Ría de Vigo based

on gas exchange models parameterised according to: a) Liss and Merlivat (1986) and b)

Wanninghoff (1992).

inner middle outer

Area (× 106 m

2) 24 46 108

April 2003

Dissolved CH4 (nmol L-1

) 39 26 11

Sea-Air flux (μmol m-2

d-1

) 84a – 167

b 53

a – 107

b 1

a - 3

b

September 2004

Dissolved CH4 (nmol L-1

) 112 70 34

Sea-Air flux (μmol m-2

d-1

) 47a – 89

b 8

a – 60

b 1

a – 10

b

CH4 cycling in the Ría de Vigo

35

Figure captions

Figure 1: Sample locations and areas of known gassy sediment (shaded area, from Garcia-Garcia et

al., 1999) during a) April 2003 and b) September 2004. The boundaries of the inner (San Simón

Bay to Rande Strait), middle (Rande Strait to Cabo de Mar) and outer (Cabo de Mar to Cíes Islands)

Ría de Vigo are marked in a. (top). Circles denote discrete underway surface sampling points,

triangles denote positions of water column depth profiles and stars denote positions of sediment

cores.

Figure 2: The monthly mean (1967-present) upwelling index (UI) and UI values during our field

campaigns. Data obtained from http://www.pfeg.noaa.gov/products/las.html

Figure 3: Water column depth profiles of dissolved CH4 (squares) salinity (circles) and temperature

(triangles) during April 2003 for water column profiles P1 (a-c), P2 (d-f) and P3 (g-i) and

September 2004 for water column profiles P1 (j-l), P2 (m-o) and P3 (p-r).

Figure 4: Distribution of dissolved CH4 concentrations in surface waters of the Ría de Vigo during

a) April 2003 and b) September 2004.

Figure 5: Distribution of CH4 concentration with salinity in surface samples of the Ría de Vigo.

Figure 6: Distribution of porewater CH4 (circles) and δ13

CH4 values (triangles) in sediments for

GC1 (a.), GC3 (b.) and SC1 (c.). δ13

CH4 data were not collected for SC1.

Figure 7: Incubation experiment following aerobic CH4 oxidation in subsamples of sediment from

core GC3. Loss of CH4 with incubation time was only observed in the headspace of vials containing

subsample from 13 cm sediment depth (grey diamonds, while the control and subsample from 66

cm depth remained relatively unchanged. Error bars represent the standard deviation of CH4

concentration in the headspace of replicate vials (n=3).

Figure 8: CH4 mixing ratio in the floating chamber headspace (solid dots) and water depth at the

deployment site (solid line).

CH4 cycling in the Ría de Vigo

36

GC1

GC3

P1

P2

SC1

P1

P3

P2

P3

San Simón B.

C. de Mar

Cíes Is.

Rande St.

Figure 1: Kitidis et al., Methane cycling in the Ría de Vigo

CH4 cycling in the Ría de Vigo

37

-200

-150

-100

-50

0

50

100

Jan

FebM

arA

prM

ay Jun

Jul

Aug Sep O

ctN

ov Dec

Month

Up

wel

lin

g I

nd

ex

Average 1967-2004

This study

Figure 2: Kitidis et al., Methane cycling in the Ría de Vigo

CH4 cycling in the Ría de Vigo

38

Figure 3: Kitidis et al., Methane cycling in the Ría de Vigo

CH4 cycling in the Ría de Vigo

39

Figure 4: Kitidis et al., Methane cycling in the Ría de Vigo

CH4 cycling in the Ría de Vigo

40

0

50

100

150

200

250

18 22 26 30 34Salinity

CH

4 (

nm

ol

L-1

)

Sep-04

Apr-03

Figure 5: Kitidis et al., Methane cycling in the Ría de Vigo

CH4 cycling in the Ría de Vigo

41

Figure 6: Kitidis et al., Methane cycling in the Ría de Vigo

CH4 cycling in the Ría de Vigo

42

0

3

6

9

0 50 100 150 200 250

Incubation time (hours)

CH

4 i

n h

ead

spac

e (p

ptv

)

GC3-13 cm

control

GC3-66 cm

Figure 7: Kitidis et al., Methane cycling in the Ría de Vigo

CH4 cycling in the Ría de Vigo

43

0

2

4

6

8 12 16 20Time

CH

4 (

pp

m),

dep

th (

m)

Figure 8: Kitidis et al., Methane cycling in the Ría de Vigo