OCS Study 

BOEM 2015-051 

U.S. Department of the Interior 

Bureau of Ocean Energy Management 

Gulf of Mexico OCS Region

Cooperative Agreement 

Coastal Marine Institute 

Louisiana State University

Coastal Marine Institute

Biomass and Mass-balance Isotope 

Content of Seep Populations on the 

Upper Slope Gulf of Mexico, Determined

from Archived Samples
OCS Study

BOEM 2015-051

Published by

U.S. Department of the Interior

Bureau of Ocean Energy Management

Gulf of Mexico OCS Region

Cooperative Agreement

Coastal Marine Institute

Louisiana State University

Coastal Marine Institute

Biomass and Mass-balance Isotope

Content of Seep Populations on the

Upper Slope Gulf of Mexico, Determined

from Archived Samples

Authors

Robert S. Carney

Phillip Riekenberg

Brian Fry

December 2015

Prepared under BOEM Cooperative Agreement M10AC20008

by

Louisiana State University

Coastal Marine Institute

Baton Rouge, Louisiana 70803
iii

DISCLAIMER

This report was prepared under contract between the Bureau of Ocean Energy Management 

(BOEM) and Louisiana State University. This report has been technically reviewed by BOEM, 

and it has been approved for publication. Approval does not necessarily signify that the contents 

reflect the views and policies of BOEM, nor does mention of trade names or commercial 

products constitute endorsement or recommendation for use.

REPORT AVAILABILITY

To download a PDF file of this Gulf of Mexico OCS Region report, go to the U.S. Department of 

the Interior, Bureau of Ocean Energy Management, Environmental Studies Program Information 

System website and search on OCS Study BOEM 2015-051.

This report can be viewed at select Federal Depository Libraries. It can also be obtained from the 

National Technical Information Service; the contact information is below.

U.S. Department of Commerce

National Technical Information Service

5301 Shawnee Rd.

Springfield, Virginia 22312

Phone: (703) 605-6000, 1(800)553-6847

Fax: (703) 605-6900

Website: http://www.ntis.gov/

CITATION

Carney, R.S., P. Riekenberg and B. Fry. 2015. Biomass and mass-balance isotope content of seep 

populations on the upper slope Gulf of Mexico, determined from archived samples. U.S. 

Dept. of the Interior, Bureau of Ocean Energy Management, Gulf of Mexico OCS Region, 

New Orleans, LA. OCS Study BOEM 2015-051 63 p.

ABOUT THE COVER

The photograph taken from the submersible Johnson Sea Link shows a typical Bush Hill mussel 

bed. Bathymodiolus childressi cover a large portion of the exposed carbonate substrate and

Bathynerita naticoidea move about grazing on detritus and organic films.
v

ACKNOWLEDGEMENTS

This project would not have been possible without funding by the Bureau of Ocean Energy 

Management (BOEM) during both establishment of the specimen archive and the analyses 

presented herein. This report is derived in very large part from the 2012 Master’s thesis of Philip 

Riekenberg in the department of Oceanography and Coastal Sciences of Louisiana State 

University. Analyses were carried out in the laboratory of Dr. Brian Fry. The specimens in the 

archive were collected with the help of the Johnson SeaLink operating team and the captain and 

the crew of the Research Vessels Seward Johnson and Edwin Link. Conversations and 

collaboration with Chuck Fisher, Ian Macdonald, Craig Young, and Shawn Arellano increased our 

understanding of seep mussel-bed ecology. The study has benefitted from the project management 

provided by BOEM, especially Greg Boland, Dr. Maureen Mulino, and Dr. Agatha-Marie Kaller. 
vii

ABSTRACT

Methods are needed to assess the health of chemosynthetic communities so that anthropogenic 

impacts can be separated from natural changes associated with succession. The work reported 

successfully demonstrated the utility of analyses of stable isotopes δ13C, δ15N, and δ34S for the 

purpose of such an assessment. The specimens analyzed were contained in a LSU archive of 

material collected during BOEM-supported surveys in 1989–92 and NOAA-supported collecting 

in 2006. Two sites of know differences were examined. Bush Hill is at the junction of Green 

Canyon block 184/5 and Brine Pool NR-1 in Green Canyon 233. The former is a topographic high 

developed along a major fault. The latter is interpreted as a mud volcano located in a region of 

slope failure. The species analyzed were the foundation species of mussel Bathymodiolus 

childressi (tissue analyses = 253; shell analyses = 154) and an associated snail Bathynerita 

naticoidea (tissue analyses =247). The former depends on methanotrophic microbial symbionts in 

its gills for its primary nutrition although filter feeding is possible. The latter is fully heterotrophic 

and grazes on the surface of the mussel shell consuming any available detritus. 

Bush Hill mussels and snails differ greatly from the Brine Pool NR-1 in a manner consistent 

with thermogenic methane at Bush Hill and biogenic methane at the Brine Pool. As a carbon 

source, methane alone cannot explain an equally dramatic difference in δ15N and δ

34S values. The 

link among δ

15N, δ13C, and δ34S remains poorly understood but can be explained on the basis of 

four trophic resources being consumed. Bathymodiolus childressi is a mixotrophic rather than 

strictly chemoautotrophic foundation species. The four trophic resources modeled indicated that 

the mussel populations at both seeps consume methane through two symbiotic pathways and 

detritus through two additional pathways. The dominant detrital source appeared to be sulfide 

oxidizing microbes. The isotope compositions of all the sources except phytoplankton detritus are 

seep-specific. Sufficient methane-derived respiratory carbon was found in the shells for dead shell 

assemblages to be used for assessment of methane metabolism. Future research based on shells 

from dead seep communities may be able to trace the time course of the loss of the methane 

resource.

Recommendations for future research center around the management benefit of being able to 

establish the condition of the major foundation species. For mussel population, the existence of 

and the manner of feeding on the thiotrophic detritus can be undertaken in the field and in the 

laboratory. Application of stable isotope analyses to dead shell material should be undertaken to

gain an understanding of the causes of extinction for the many dead-shell assemblages encountered 

during the exploration for seep systems.
CONTENTS

LIST OF FIGURES ......................................................................................................................XI

LIST OF TABLES.......................................................................................................................XII

1. INTRODUCTION .................................................................................................................... 1

1.1. Statement of Purpose...................................................................................................... 1

2. BACKGROUND ...................................................................................................................... 2

2.1. Upper Louisiana Slope Seeps......................................................................................... 2

2.2. The Seep Mussel Bathymodiolus childressi................................................................... 4

2.3. The Seep Snail Bathynerita naticoidea.......................................................................... 7

2.4. Stable Isotopes of Biomass as an Analytical tool........................................................... 8

2.5. Condition Index as an Analytical Tool........................................................................... 9

3. DESIGN AND METHODS.................................................................................................... 11

3.1. Sampling....................................................................................................................... 11

3.2. Stable Isotope Analysis................................................................................................ 13

3.3. Estimation of Mussel Shell seep carbon incorporation ................................................ 14

3.4. General Data Analysis.................................................................................................. 14

4. RESULTS ............................................................................................................................... 16

4.1. Bathymodiolus childressi Tissue .................................................................................. 16

4.2. Mussel Shell Incorporation of Seep Respiratory Methane........................................... 38

4.3 Bathynerita naticoidea ................................................................................................. 40

5. DISCUSSION......................................................................................................................... 41

5.1 Answering the Initial three questions........................................................................... 41

6. CONCLUSIONS AND RECOMMENDATIONS ................................................................. 44

LITERATURE CITED ................................................................................................................. 46

APPENDIXES .............................................................................................................................. 51

APPENDIX A: MUSSEL AND SNAIL DATA TABLES .......................................................... 52

APPENDIX B: SUPPLEMENTAL DATA ANALYSIS TABLES............................................. 53
xi

LIST OF FIGURES

Figure 1. Position of Bush Hill and Brine Pool NR-1 relative to the complex slope 

geomorphology in the Gulf of Mexico. ..............................................................3

Figure 2. Bathymodiolus childressi is a dominant and foundation species at upper slope 

seep communities in the Gulf of Mexico............................................................4

Figure 3. Deposition of mussel shell.......................................................................................6

Figure 4. Bathynerita naticoidea is a small gastropod typically 1.2 cm in greatest 

dimension............................................................................................................7

Figure 5. Bathymodiolus childressi adductor mussel mass proportions and isotope value 

density plots for carbon (A & B), nitrogen (C & D), and sulfur (E & F), 

showing contrasts between Brine Pool and Bush Hill......................................18

Figure 6. Bush Hill Bed 1 compared to Bed 2 in 1989 and Bed 1A in 1991........................25

Figure 7. Brine Pool Positions for 1989 and 1991 boundaries added for clarity..................26

Figure 8. Brine Pool tissue values for1989, 1992, and 2006 with boundaries on 2006 for 

clarity. ...............................................................................................................27

Figure 9. Trophic mixing models as tetrahedrons surrounding Brine Pool (red) and Bush 

Hill (blue) data points in Cartesian space of the three stable isotopes data......29

Figure 10. Bush Hill between-bed and with-in bed proportional use of trophic resources 

presented as box and whisker plots with both sampling years pooled. ............32

Figure 11. Brine Pool trophic resource proportional use at pool ends and positions 

displayed as whisker and box plots by position 1991 and 1992 years pooled 

and 2006 omitted. .............................................................................................34

Figure 12. Bush Hill between-year and position within Bed 1 only in trophic resource use 

displayed as box and whisker plots. .................................................................36

Figure 13. Brine Pool position and year proportional use of trophic resources for all three 

years presented as box and whisker plots. ........................................................38

Figure 14. Percent of metabolic carbon in shell compared with size (LnHSV used)...........39

Figure 15. Plots of each pair of isotope values for all mussels (_M) and snails (_S) at Bush 

Hill (BH) and Brine Pool (BP). ........................................................................40

Figure 16. Trophic resources for Brine Pool and Bush Hill representing detrital (circles) and 

methane resources (hexagons) used by seep mussels.......................................43
xii

LIST OF TABLES

Table 1. Mean and standard deviation of condition index and glycogen content found for 

Bush Hill...........................................................................................................10

Table 2. Position and dates sampling design based on archived collection of mussels........12

Table 3. Brine Pool mussels separated into older and newer sampling................................17

Table 4. Bush Hill mussels parameters.................................................................................19

Table 5. Major Comparisons t-Tests between Brine Pool and Bush Hill.............................19

Table 6. Brine Pool mussel parameter correlations..............................................................20

Table 7. Bush Hill mussel parameter correlations................................................................20

Table 8. Within Brine Pool ANOVA for stable isotope spatial patterns..............................21

Table 9. Within Brine Pool ANOVA for stable isotope pool-end and year effects..............22

Table 10. Bush Hill mussel tissue stable isotope factorial ANOVA for spatial effects .......23

Table 11. Inter-Year and With-In Bed ANOVA at Bed 1 mussel tissue stable isotope 

values................................................................................................................24

Table 12. Estimated sources (vertices) of a four-source mixing tetrahedron .......................28

Table 13 Between-Seep t-Test comparisons with methane sources combined. ...................30

Table 14. Bush Hill ANOVA for each resource and position factors both years pooled .....30

Table 15. Brine Pool trophic resource ANOVA for position factors 2006 omitted .............33

Table 16. Bush Hill Bed 1 ANOVA of trophic use between years ......................................35

Table 17. Brine Pool among-year and pool-end trophic resource use ANOVA ..................37

Appendix A Table 1. Stable Isotope Values for Brine Pool Mussels...................................52

Appendix A Table 2. Proportions of four trophic resources for Brine Pool Mussels...........52

Appendix A Table 3. Brine Pool Elemental Mass of Nitrogen, Carbon and Sulfur as 

Milligrams and Mass Percent ...........................................................................52

Appendix A Table 4. Stable Isotope Values for Bush Hill Mussels.....................................52

Appendix A Table 5. Proportions of Four Trophic Resources for Bush Hill Mussels.........52

Appendix A Table 6. Bush Hill Nitrogen, Carbon and Sulfur Mass Percent .......................52

Appendix A Table 7. Mussel Shell Half Shell Volume, δ13C Shell, δ13C Tissue (‰) and 

Percent Metabolic Carbon for Bush Hill and Brine Pool .................................52

Appendix A Table 8. Brine Pool Bathynerites naticoidia Stable Isotope Data....................52

Appendix A Table 9. Bush Hill Bathynerites naticoidia Stable Isotope Data......................52

Appendix B Table 1. Brine Pool TukeyHSD post hoc Analyses of Position Effects on 

Stable Isotope Content of Mussel Tissue..........................................................53
xiii

Appendix B Table 2. Brine Pool TukeyHSD post hoc Analyses with Year, Position and 

Interaction Effects on Stable Isotope Content of Mussel Tissue......................53

Appendix B Table 3. Bush Hill TukeyHSD post hoc Analyses with Position Effects on 

Stable Isotope Content of Mussel Tissue..........................................................53

Appendix B Table 4. Bush Hill TukeyHSD post hoc Analyses with Position, year and 

interaction Effects on Stable Isotope Content of Mussel Tissue ......................53

Appendix B Table 5. Bush Hill TukeyHSD post hoc Analyses of Four Mussel Tissue 

Trophic Source Positions with Position Effects ...............................................53

Appendix B Table 6 Brine Pool ANOVA with Position Effects in Stable Isotopes of Mussel 

Tissue with Five Outliers Excluded..................................................................53

Appendix B Table 7. Brine Pool TukeyHSD post hoc Analyses of four Mussel Tissue 

Trophic Source Positions with Position Effects ...............................................53

Appendix B Table 8. Brine Pool TukeyHSD post hoc Analyses of four Mussel Tissue

Trophic Sources with Year, Position and Interaction Effects...........................53

Appendix B Table 9. Bush Hill TukeyHSD post hoc Analyses of four Mussel Tissue 

Trophic Sources for Bed 1 with Year, Position and Interactions Effects.........53
1

1. INTRODUCTION

1.1. STATEMENT OF PURPOSE

The upper slope off Louisiana exhibits a very complex topography caused by tectonic 

movement of salt layers on the long term and slope failure events on the shorter term (Figure 1). 

This combination of factors produces a region with vast areas of typical benthic soft bottom and

embedded chemically-extreme communities shaped by various combinations of hydrocarbon and 

brine seepage (Roberts 2011). The purpose of this study is to better understand the natural variation 

of the trophic state within seep mussel populations, based on the stable isotope analysis of a 

relatively large number of specimens from two geochemically contrasting sites, Bush Hill and the 

Brine Pool NR1. 

The work reported in this study is a modest effort with no at-sea component to meet the 

information need of how the ecological status of chemosynthetic seep communities might be 

assessed. Rather than undertake new expensive sampling, we have used archival material at 

Louisiana State University’s Department of Oceanography and Coastal Sciences (DOCS). The 

tool of choice is stable isotope analysis looking at the important isotopes 13C, 15N and 34S. These 

isotopes and the total mass of the elements C, N, and S may provide a measure of condition. The 

species examined are the foundation mussel species and an abundant gastropod. Selected mussel 

shell and soft tissues are also examined to see if information can be gained from the shell remains 

at inactive seeps. Samples dating to 1989, 1991, and 1992 have been augmented with material 

from 2006. Because all analyzed material was collected before the Deepwater Horizon accident,

the data reported here is a baseline for future assessment and monitoring.

The questions directly asked within this study are:

 Does the mass percent of carbon, nitrogen, and sulfur in tissue of the seep mussel

Bathymodiolus childressi provide a useful tool for assessing the overall condition of

populations?

 Does the stable isotope composition of carbon, nitrogen and sulfur in mussel tissue

provide a useful indication of trophic condition of a population capable of both

chemosynthesis and heterotrophic feeding?

 Is the determined trophic condition of a foundation species also reflected in a consumer

organism, such as the snail Bathynerita naticoidea, in the same system?

 Does analysis of mussel shells have the potential of providing information on the

trophic condition that might be used to assess past seepage condition?
2

2. BACKGROUND

2.1. UPPER LOUISIANA SLOPE SEEPS

2.1.1 Chemosynthetic Systems and the Atypical Deep Sea

Between 1984 and 1985, species related to hydrothermal-vent fauna were reported from the 

eastern Pacific and the Gulf of Mexico cold continental slopes and recognized as being supported 

by the seepage of methane and sulfides (Paul et al. 1984, Kennicutt et al.1985, Kulm et al. 1986). 

Increased and more effective exploration of slopes has verified that similar seep systems are a 

relatively common habitat that exists as a geochemical continuum (Levin 2012). Chemosynthetic 

seep communities were first reported in the northern Gulf of Mexico in 1985 (Kennicutt et 

al.1985). Boland (2010) provides a good historical review of participants and publications. 

Following subsequent BOEM studies (MacDonald et al. 1995), these systems were afforded 

protection as a potentially sensitive habitat. Notice to Lessees (NTL) 2009-G40 and previous 

NTL’s recognize that chemosynthetic communities are widespread and vary in condition and 

ecological importance. Criteria for recognizing the level of importance of an individual seep 

location, however, remain incompletely determined but somehow related to density and 

biodiversity of the systems.

The seeps in the Gulf are the most studied in the world, due to the extensive oil and gas 

development in the region. The initial discoveries off Louisiana occurred during industry and 

Bureau of Ocean Management (BOEM)-supported investigations. A full history has been given 

by Boland (2010). Since the discoveries, various aspects of the biology, chemistry, and geology 

of Gulf seeps have been investigated, supported primarily by BOEM but also the National Oceanic

and Atmospheric Agency (NOAA) and the National Science Foundation (NSF). Cordes et al. 

(2009) provide an informative review on the ongoing research.

Because of the depth limitation of the Johnson Sea Link (JSL) submersible, which was operated 

at that time by the Harbor Branch Oceanographic Institute (HBOI), the most intense investigation 

of the northern Gulf Upper Continental Slope was above 1000 m. JSL was a cost effective and 

highly reliable vehicle with a depth limit of 1000m. JSL provided an unequaled panorama of the 

bottom and a very large capacity for samples. Unfortunately, HBOI terminated the submersible 

program, and JSL is no longer available.

The specimens analyzed in this study were collected at either Bush Hill (Green Canyon 185) 

or Brine Pool NR-1 (Green Canyon 233) (see Figure 1). The sites lie between longitudes 91o 50.0 

W and 91o 10.0W and latitudes 28o 0.0N and 27o 30.0N. A seismic survey of this area was carried 

out with a 20x20m per pixel resolution 6–8 May 2012 by the R/V Okeanos Explorer. Bathymetry, 

bottom backscatter, and water column backscatter indicative of methane bubbles were recorded. 

The region has been sculpted by salt diapers, withdrawal basins, and slope failure scars (Figure 1). 

All of these features are typical of the northern Gulf slope. The region contains numerous bubble 

plumes and high amplitude seafloor (Shedd et al. 2011).
3

Figure 1. Position of Bush Hill and Brine Pool NR-1 relative to the complex slope geomorphology in the Gulf 

of Mexico. 

2.1.2. Bush Hill Green Canyon 184/85

Bush Hill (Long -91.5082o Lat 27.7811o) is a topographic high rising to a depth of ~445 m,

located on the western flank of a shelf canyon that appears to be a large slope failure feature (Figure 

1). Figure 1 is a 20m resolution digital terrain model courtesy of the Coastal and Ocean Mapping 

Center at the University of New Hampshire and the NOAA Ocean Exploration Program. It lies 

over a secondary fracture to a large growth fault associated with salt tectonics (MacDonald et al. 

2003). Laser line scanning of ~28,000 m2of the top of the feature found Bathymodiolus childressi

mussel clumps to cover 72 m2, exposed carbonates 610 m2and tubeworm bushes 332m2

(MacDonald et al. 2003). As is typical of many seep features, the living chemosynthetic fauna

covers a relatively small area. In this case ~2% of the mound. Bush Hill was the first seep site 

directly observed by submersible on the Louisiana upper slope in 1986. It lies at the juncture of

Green Canyon Blocks 184 and 185 and is in the Jolliet development field.
4

2.1.3. Brine Pool NR-1 Green Canyon 233

Brine Pool NR-1 (Long -91.2792o Lat.27.7238o) was found during seafloor surveying with the 

nuclear research submarine NR-1 of the U.S. Navy (MacDonald et al. 1990). It has been interpreted 

as a brine-filled low relief expulsion feature in a larger mud volcano (MacDonald and Peccini 

2009). Newer bathymetry shows the pool is on the eastern flank of a 9 m high hill in a region 

marked by many slope-failure scars (Figure 1). The pool is ringed by a mat of the mussel

Bathymodiolus childressi. Neither tubeworms nor exposed carbonates occur in the immediate area 

of the pool but they have been encountered in the surrounding terrain.

2.2. THE SEEP MUSSEL BATHYMODIOLUS CHILDRESSI

Figure 2. Bathymodiolus childressi is a dominant and foundation species at upper slope seep communities 

in the Gulf of Mexico. 

Bathymodiolus childressi Gustafson et al. (1998) (Figure 2; Image courtesy of the Chemosynthetic 

Ecosystems Project, Natural History Museum of Rotterdam. J. Trausel and F. Slieker 

photographers) is the dominant mussel at upper-slope seep sites in the Gulf where it is sufficiently 

abundant to be a foundation species providing physical substrate as well as a food source for other 

biota. Based on molecular evidence there is a single interbreeding population in the Gulf (Carney 

et al. 2006). As of 2010, 51 species in the genus Bathymodiolus have been described strictly from 

hydrothermal and cold seep environments (Saeter et al. 2010). Molecular phylogenetics suggests 

that the genus is probably polyphyletic and is in a genus/species cluster with Adipicola (nine

species) and Idas (30 species) which are associated with organic falls such as wood and whale 

carcasses (Duperron 2010; Lorion 2010). B. childressi is a distinct species in this larger complex 
5

which includes Atlantic and Pacific members. The distributions of Atlantic members delineate the 

Atlantic Equatorial Belt (AEB) Cold Seep Faunas (Olu et al. 2009). B. maritanius is a closely 

related species found at West African seeps on the Nigerian and Moroccan continental slopes 

(Genio et al. 2008). Initially considered a Gulf endemic distributed between 450 and 2200m depth, 

the species has been reported from the submarine canyons of the U.S. northeast slope (Demopoulos 

et al., 2014; Wagner et al., 2014) and additional range extensions are likely.

Though most species of Bathymodiolus harbor thiotrophic microbes in enlarged gill tissue, B. 

childressi is nearly unique in harboring only methanotrophic symbionts as far as the current level 

of sampling has demonstrated (Duperron et al. 2007; Duperron 2010, Kellermann et al. 2012). 

The metabolism of methane-oxidizing symbiotic microbes places a high oxygen demand on the 

host mussel (Childress and Girguis 2011), which is met by maintaining a flow of oxygen-rich 

ambient seawater across the gills. In effect, all species of Bathymodiolus are capable of filter 

feeding even though the in situ ecology of the genus appears to be primarily chemoautotrophic 

(Page et al. 1990, Young and Pile 1999).

2.2.1 Mussel Shell Deposition

An answer to the question of whether mussel shells record the history of methane seepage

experienced by Bathymodiolus childressi depends upon the details of shell deposition. When 

CaCO3 shells are deposited by aquatic invertebrates, the main source of CO2 in carbonate is 

dissolved inorganic carbon (DIC) in the water. This is in stark contrast to air breathing animals in 

which the main source of CO2 for deposited hard parts (bone, shell) is respiratory carbon, which 

causes these hard parts to be isotopically representative of the δ13C of their diet. This difference 

in respiratory carbon input between air breathing and aquatic animals is a result of atmospheric 

CO2/O2, ratios being about 30 times lower than those in aquatic settings, resulting in air 

breathing animals having significantly less availability of CO2 from the air as they take up O2

(McConnaughey et al. 1997) .

Aquatic animals do, however, incorporate some respiratory carbon into shell material; hard parts 

range from 7–15% in mollusks and as much as ~30% in fish otoliths. Although the molluscan 

amount is small, detection of that small amount in chemosynthetic mussels is facilitated by the fact 

that the methane-derived respiratory carbon is so very distinctive (-40–80‰ δ13C). Successful 

detection, however, requires adjustment for kinetic isotope fractionations that exist as the different 

crystalline forms of CaCO3 (calcite or aragonite) develop in the shell. Romanek and colleagues 

(1992) used two different fractionations for aragonite and calcite of 2.7‰ and 1‰, respectively,

for the enrichment that occurs as CO3

=

becomes incorporated into the shell from the extra pallial 

fluid (EPF) (Figure 3; from McConnaughey and Gillikin 2008).

In this study, we have used an estimate of 90% calcite and 10% aragonite composition of the shells 

that agrees with the findings of Demina and colleagues (2012) about the range of calcite compared 

with aragonite composition of shells found in vent related Bathymodiolus species and results in a 

fractionation factor of 1.17‰. The EPF undergoes an alkalization through the use of an ion 

transporter called Ca2+ Atlases, which pumps protons away from the site of calcification. This 

process creates an increase in pH of 0.5 units, resulting in a shift in the predominant species of 

CO2 from HCO3

-

to CO3

=

as a result of deprotonation. This process results in a 4–6 fold increase 

in the concentration of CO3

=

in the extra pallial fluid (EPF) compared with the surrounding tissues 

(McConnaughey and Gillikin 2008). This results in a depletion of HCO3 in the surrounding tissues 
6

and sets up a diffusion gradient by which CO2 is constantly entering the tissues and the EPF to 

maintain the super saturation of CO3

=

set up by the slight alkalizination of the EPF.

Figure 3. Deposition of mussel shell.
7

2.3. THE SEEP SNAIL BATHYNERITA NATICOIDEA

Bathynerita naticoidea is an abundant heterotrophic snail at upper slope seeps that has a tight but 

poorly understood connection with the mussels (Figure 4; image courtesy of Life Desk E. Thomas 

photographer). It is closely associated with Bathymodiolus childressi and feeds with its scraping 

radula on the shell of the mussel consuming any mucus films, microbes and detrital material. It is 

a heterotroph but not a mussel predator. Rather, it can be seen as an integrator of all particulate 

food sources present within the mussels beds. Thus its isotopic values may reflect mussel 

production through a mucus food source, free-living microbes and photosynthetically-derived 

detritus.

Figure 4. Bathynerita naticoidea is a small gastropod typically 1.2 cm in greatest dimension. 

Gastropods are common members of hydrothermal vent and seep communities ; 218 species 

are recognized in the most recent compilation (Sasaki et al. 2010). Upper-slope seeps in the Gulf 

are occupied by Bathynerita naticoidea. Initially described from Bush Hill specimens by AH. 

Clarke (1989). The snail appears to be restricted to the upper-slope seeps and is always in 

association with Bathymodiolus childressi. Naticoidea is the only species in the genus. The animal 

is abundant, survives collection, and stays alive in cold aquaria at ambient pressure. As a result, it 

has received modest research attention and has been the subject of two theses (Zande 1994; Van 

Gaest 2006).
8

Nutrition is strictly heterotrophic and no symbiotic microbes have been found. The snail 

browses on the surface of B. childressi with its scarping radual. It may consume exuvia, byssal 

fibers and periostracum from the mussel and any detrital material on the shell. That detritus can be 

both bacterial and algal (Zande and Carney 2001). Specimens from Bush Hill had a significantly 

lower average size that those at the Brine Pool. Most animals are ~1.2 cm in largest dimension. In 

trials carried out by Zande (1994), eggs contained within cases underwent full development to the 

swimming veliger stage without the case opening. Van Gaest (2006) repeated the experiment but 

found that the case did open and development took place in the plankton; this gives the snail the 

potential for long-distance dispersion (Young et al. 2012). The relationship between B. childressi

and B. naticoidea remains poorly understood. In simple behavioral trials, the snails showed 

significant preference for experimental habitats containing water from B. childressi aquaria 

(Dattagupta et al. 2007). This was not a response to a simple stimulant, such as methane. 

2.4. STABLE ISOTOPES OF BIOMASS AS AN ANALYTICAL TOOL 

2.4.1. Basic Carbon, Nitrogen and Sulfur Isotope Analysis

Stable isotopes are those element forms that exist in the natural environment and are not 

radioactive in nature. They are commonly referred to as HCNOS isotopes in biological studies 

indicating they are the isotopic forms of hydrogen, carbon, nitrogen, oxygen, and sulfur and are 

commonly used to identify biological processes in natural systems (Fry 2006). Stable isotopes of 

carbon and nitrogen have been used in many ways: to delineate food webs and decipher who is 

eating whom (Peterson and Fry 1987) and when coupled with sulfur the stable isotopes of carbon 

and nitrogen have identified the relative transience of fish in estuaries (Fry and Chumchal 2011). 

There are many current applications for these tools, because they allow for the relatively cheap 

tracing of elements through both biological and physical processes without the need for expensive 

or radioactive tracers. However, this study addresses only the use of naturally occurring carbon, 

nitrogen, and sulfur.

Stable isotope measurements are reported in δ (del) notation. That is, they are reported in 

standard notation as: δ = ((HF/LF)sample) / ((HF/LF)standard)-1)) * 1000 the fractional abundance 

(

HF/LF) of heavy and light isotope of both the sample and the standard. The del notation is then in 

the permil (‰) basis, that is, parts per thousand, which allows for the amplification of very small 

differences into a more familiar and manageable scale. The standards that samples are measured 

against are: for carbon, PeeDee Belemnite (PDB) and for nitrogen, air, and sulfur, Canyon Diablo 

Troilite (CDT)(Fry 2006). These standards have been meticulously established to provide lab to 

lab continuity and quality assurance for stable isotope analysis and are currently maintained 

through the International Atomic Energy Agency.

2.4.2. Trophic Mixing Estimation from Three Isotopes

The cause of scatter in stable isotope data is most often considered to be the mixing of multiple 

food sources and metabolic isotope fractionations. For this reason, mixing models are often 

developed that allow estimation of the trophic resources being used. When the isotopic content of 

a specimen is considered to be the sum of fractional contributions from multiple sources, 

calculation of those contributions provides an ecologically useful means of data analysis and 

interpretation (Phillips 2012). The computational requirements are that the isotopic values are 

modeled as a series of linear equation, as below.
9

A x δ13Csource1 + B x δ13Csource2 + C x δ13Csource3 + D x δ13Csource4 = δ13Cmeasured

A x δ15Nsource1 + B x δ15Nsource2 + C x δ15Nsource3 + D x δ15Nsource4 = δ15Nmeasured

A x δ34Ssource1 + B x δ34Ssource2 + C x δ34Ssource3 + D x δ34Ssource4 = δ34Smeasured

And, the proportional contribution of resources sum to 1.0.

A + B + C + D = 1.0

Unique solutions can be determined for the contributions, so long as the number of sources is 

not greater than one more than the number of variables (isotopes). Obtaining those solutions using 

linear algebra requires first that a square matrix of isotope (CNS) composition of the sources be 

established, inverted, and multiplied by a rectangular matrix of sample isotope values. The 

calculations were carried out in Microsoft© Excel© , using the built in array functions 

MINVERSE and MMULT.

There are various approaches for establishment of the basal sources. In this study, the approach 

of Wissel and Fry (2005) was used; it considered prior knowledge of highly probable sources in 

the two seep systems and sources consistent with the pattern of isotope values and the ecology of 

B. childressi. Spatially, the four basal sources define the vertices of a tetrahedron in a Cartesian 

space with the axes δ13C, δ15N and δ34S. All samples that consist of a mixture of these four sources 

must lie within that tetrahedron. This improves on the estimation of sources and requires adjusting 

initial vertices until all sample points are corralled. This trial and error process of source adjustment 

began with 2-dimension examination of each isotope pair and then refining the tetrahedron in a 3-

dimension view. The steps were carried out using OriginPro® 8.6 using X, Y, Z scatter plots and 

trajectory graphing functions. 

2.5. CONDITION INDEX AS AN ANALYTICAL TOOL

A condition index (CI) is intended to be a measure of the health of organisms. In an 

environmental management context, it can be used as a tool in the assessment of the sensitivity of 

a population to impact and as an indication of an impact. Numerous CI have been developed and 

can be generally categorized as either morphological or physiological (Hayes and Shonkwiler 

2001). The former have the potential of being non-invasive and non-lethal. The latter require tissue 

sampling but may be better linked to the processes actually determining health. The CIs are often 

a simple ratio or a more complex statistical value. The use of shell-to-soft tissue comparisons is a 

common CI for bivalves that is suggested by the variable water content of the flesh of bivalves 

such as oysters. A flesh:shell mass ratio attributed to Freeman (1974) is a common CI in bivalve 

aquaculture (Alunno-Bruscia et al. 2001). 

The assessment of the individual condition of chemosynthetic mussels and determination of 

the spatial variability of that condition were pioneered by Smith (1985) at the initial hydrothermal 

discovery site in the Galapagos Rift. It was established that the ratio of soft tissue dry mass to 

internal shell volume was greater within dense central patches than small peripheral patches of the 

vent mussel Bathymodiolus thermophilus. Smith's method of measuring condition and spatial
10

contrasts has been widely applied with some modification to other chemosynthetic systems 

including those in the Gulf . 

Condition of the seep mussel Bathymodiolus childressi has been assessed at Bush Hill (Nix et 

al. 1995) and the Brine Pool (Smith et al. 2000) following the overall approach of Smith (1985) 

but using ash-free dry weight rather than simple dry weight as the tissue measure. Those results 

are presented in Table 1. The condition was maximum at the inner edge of the Brine Pool mussels 

(Smith et al. 2000) and decreased slightly at the outer edge. The lower condition at the outer edge 

was still markedly higher than found at Bush Hill (Nix et al. 1995). Two additional sites had 

conditions between those of Bush Hill and the Brine Pool and were attributed to site-specific 

differences in the supply of methane. These determinations provide a basis of comparison with the 

isotope and total carbon, nitrogen and sulfur measurements obtained in the current study. 

Table 1. Mean and standard deviation of condition index and glycogen content found for Bush Hill 

Site Date of

sample

Condition Index

Ash-free dry mass/shell volume

Glycogen %

Of Wet Mass

Bush Hill 1991 0.04 ± 0.01 0.88 ± 0.63

1992 0.05 ± 0.02 1.13 ± 0.88

1993 0.05± 0.01 0.73 ± 0.44

Brine Pool Inner 1993 0.12 ± 0.02 3.1 ± 0.9

1994 0.13 ± 0.02 2.8 ± 1.0

1995 0.10 ± 0.3 1.7 ± 0.6

Brine Pool Middle 1 1993 0.10 ± 0.01 3.2 ± 0.4

1994 0.10 ± 0.03 1.9 ± 0.04

1995 0.09 ± 0.02 1.6 ± 0/8

Brine Pool Middle 2 1994 0.12 ± 0.02 2.8 ± 0/9

Brine Pool Outer 1993 0.11 ± 0.02 3.0 ± 0.7

1994 0.10 ± 0.02 2.2 ± 0.8

1995 0.06 ± 0.02 0.9 ± 1.9
11

3. DESIGN AND METHODS

3.1. SAMPLING

The specimens analyzed in this study were collected and archived primarily between 1989 and 

1992 as parts of “Chemosynthetic Ecosystems Study” or Chemo-I study (MacDonald et al. 1995) 

and “Stability and Change in Gulf of Mexico Chemosynthetic Communities” (MacDonald 2002). 

Faunal composition was being examined, and the proposed sampling design for those studies was 

to have had replicated collections partitioned by substrate (mud or rock) and position (edge or inter 

portion of a mussel bed) and nested within four seeps. In the field, the mussels did not cooperate. 

Rock predominated as a substrate and some sites had too few mussel beds to allow for the desired 

replication. A simpler comparison among sites and among locations, however, proved very 

informative. Compositional monotony was encountered at the four sites (Carney 1994). The spatial 

and temporal design that could be examined from the archived samples is presented in Table 2.
12

Table 2. Position and dates sampling design based on archived collection of mussels

SEEP Feature Position Year Specimens JSL Dive

Bush Hill Bed 1 Edge 1991 17 3139

1992 14 3269

10 3269

Interior 1991 21 3139

1992 8 3269

3 3269

Bed 2 Edge 1992 6 3274

Interior 1992 6 3274

6 3274

3 3274

Bed 1A Edge 1991 15 3139

Interior 1991 13 3139

3 3139

Bush Hill Subtotal = 125

Brine Pool North Interior 1989 20 2598

1991 14 3145

Edge 1989 13 2598

1991 5 3145

Scattered 2006 11 3521

South Interior 1989 13 2598

1991 8 3145

Edge 1989 9 2598

Scattered 2006 35 3522

Brine Pool Subtotal = 128

Total Specimens = 253

At Bush Hill, three mussel beds were sampled and designated Bed 1, 2, and 1A (close 

proximity to 1 and possibly an extension of it). At each bed, fauna was collected at an outer edge 

and an inner portion. Only Bed 1 was sampled over two successive years. The Brine Pool has

essentially a single large mussel bed or ring. It was sampled at its northern and southern ends. At 

each end, collections were made at an Inner area extending over the brine and an Outer edge over 

mud. The Brine Pool sampling was repeated over a two-year interval. The Brine Pool was 

resampled during a NOAA-supported program in 2006 and provided a single longer-term set of 

samples. That 2006 sampling did take place at the northern and southern ends of the Brine Pool 

but was scattered rather than targeted the outer edge and inner portion. Snails were analyzed from 

Bush Hill and Brine Pool along with two sites, GC-272 and GC-234, which had also been studied 

in earlier investigations.
13

In an ideal sampling design seeking spatial and temporal effects, there should be a large number 

of replicates for each factor. Separate seeps, mussel beds within seeps, position within those beds,

and mussel specimens should be heavily replicated. Time series sampling should sample the same 

number of replicates over several years. Unfortunately, deep seeps do not lend themselves to ideal 

sampling. The composition, size, number, and persistence of beds are highly different at each site. 

Much opportunistic sampling must take place. Replication must be low, and it may not be practical 

to resample the same bed over time. Indeed, because sampling removes animals, the systems are 

modified through time. Spatial and temporal trends from such sampling should be interpreted with 

caution.

All megafauna sampling used the grab of the Johnson Sea Link (JSL) submersible. The JSL

grab is an integral part of the hydraulic manipulator on JSL. It consisted of two semi-cylindrical 

jaws 19.2 cm long and 17.8 cm wide when open, which rotate closed about a common axis. The 

bite of the JSL grab samples 341 cm2square meters of bottom. Each sample consisted of two 

scoops of the grab. Samples were sorted at sea and stored in 5% formaldehyde. Upon completion 

of the Chemo-I and Chemo-II studies, specimens in 5% formaldehyde were archived at Louisiana 

State University.

3.2. STABLE ISOTOPE ANALYSIS

In 2010, the archived samples from Bush Hill and Brine Pool NR-1 were opened, washed in a 

deionized water (DI) bath for a period that did not exceed 24 hours, dissected and dried at 60°C 

for a minimum of 12 hours until completely dry. Mussels were separated into three parts: mantle, 

abductor muscle, and an outer portion of shell. The adductor muscle portions were processed using 

a WIG-L-BUG (Dentsply International) into a fine powder and then loaded with vanadium 

pentoxide catalyst into the sampler for CNS analysis. The shell portion of the sample was dried, 

periostracum removed, then ground into a fine powder, treated with bleach for 12 hours to remove 

organic material, rinsed copiously with DI, then dried again and loaded with vanadium pentoxide 

catalyst in the sampler for analysis.

Mussel and snail size was quantified by caliper measurements of maximum shell length and 

shell height to the nearest millimeter. Mussel half shell volume was measured using small glass 

beads to completely fill half of the shell and then weighed. Glass bead weight was then graphed 

versus shell height and the resulting function y = 0.7953e^(0.1835 * shell height (mm)) with an R2

value of 0.9842 was used to calculate volume for the half shell of each individual. A subsample of 

the specimens was used to establish this relationship.

Mussel and snail tissue samples were processed for CNS isotope analysis using the methods 

described by Fry (2007), reporting δ13C ‰ compared with PeeDee Belemnite (PDB), δ15N ‰ 

compared with atmospheric nitrogen (AIR), and δ34S ‰ compared with Canyon Diablo Triolite 

(CDT). The analytical confidence levels are 0.1‰ for both δ13C and δ15N, and 0.4‰ for δ34S. 

Laboratory check standards were interspersed within samples in a range of weights within each 

run in order to ensure consistency within and between the sample runs. Shell samples were run in 

the same manner as tissue samples, but only carbon isotope values were kept, because the sulfur 

and nitrogen isotope values were highly variable. These fluctuations in data were an effect of the 

extremely small sample size that remained after the bleach treatment. A check for the effects of 

formaldehyde on CNS isotopes were performed using fresh commercial Mytilus sp. obtained 

locally by performing analysis on preserved and unpreserved portions of each individual (n=6). 

Due to the extremely depleted nature of δ13C in cold seep animals that are dependent on methane 
14

as a carbon source, this effect is likely an enrichment and likely to have a much smaller effect on 

the overall (‰) values due to the large range in observed δ13C values.

3.3. ESTIMATION OF MUSSEL SHELL SEEP CARBON INCORPORATION

The formula used to assess methane-derived respiratory carbon shells in this study is:

F(δ13CTissue) + (1-F)( δ13CDIC) = δ13CShell – Δ.

 Where F is equal to the rate of incorporation of respiratory carbon, 

 δ

13CTissue is the value of the tissue of each mussel.

 (1-F) is the remainder of the carbon not provided through respiration, 

 δ

13CDIC is the value of seawater DIC (0.69‰; Fu 1998).

 δ

13CShell is the value of the shell of each mussel.

 Δ is a fractionation factor associated with the kinetic isotope effects between the 

crystalline structures of calcite and aragonite. 

3.4. GENERAL DATA ANALYSIS 

The eight variables for mussel and snail tissue consisted of shell length, dry tissue sample mass, 

δ

13C, δ15N, δ34S, total C mass, total N mass, and total S mass. For mussel shells, there were two 

parameters: shell and δ13C. Exploratory data analysis consisted of plots of values and examination 

of correlation matrices. Data tables are provided in Appendix I and statistical analyses contained 

in Appendix II. Simple univariate parametric tests were carried out, when appropriate, to test the 

significance of an apparent relationship. Due to the large sample size some relationships are 

statistically significant but explain only a minor amount of data variance.

The most algebraically complex task was assessing the percentage of respiratory carbon 

incorporated into the shell (F). It is calculated using the formula from McConnaughey and 

colleagues (1997), which calculates F as a function of the respective contributions from respiratory 

sources (obtained using the δ

13C mussel abductor tissue values) and the dissolved inorganic carbon 

p

o

o

l

.

T

h

e

f

o

r

m

u

l

a

u

δ

13C shell - Δ = (δ13Ctissue * F ) + (δ13CDIC * (1 – F))

 Where δ

13Ctissue is the measured abductor tissue value for the individual mussel and 

serves as the end member value of the respiratory carbon incorporated into the shell. 

 δ

13Cshell is the measured value of the shell for the individual mussel. 

 δ

13CDIC is 0.69 ‰ of the surrounding dissolved inorganic carbon pool and is the 

predominant end member for carbon incorporated into the shell. 

 Δ is 1.17‰ as a fractionation factor correction for the difference in carbon fractionation 

between the formation of the two predominate crystalline forms of CaCO3, calcite and 

aragonite. This correction assumes a 90% composition of calcite in the shell with a 

10% composition of aragonite and is in line with previous measurements in mytilid 
15

mussels (Demina et al. 2012) and utilizes fractionation factors associated with the 

formation of aragonite and calcite (Romanek et al. 1992). 
16

4. RESULTS

The data used in the analyses presented below are contained in nine linked Microsoft© Excel©

files in Appendix I. Three tables for the Brine Pool present stable isotope content, trophic source 

proportional mixing, and biomass elemental mass proportion for C, N, and S (Appendix I; Tables 

1, 2, and 3). Similar values for Bush Hill mussel tissue are also provided (Appendix I; Tables 4, 5,

and 6). Shell respiratory carbon incorporation is provided in Appendix I Table 7. Stable isotope 

values for the snail Bathynerites naticoidea Brine Pool and Bush Hill are in Appendix I Table 8 

and Appendix I Table 9, respectively. All files are in the general form of a data frame for analysis 

in the R statistical package. In addition to the measured variables factors there are also two position

factors. Position factor 1 is beds at Bush Hill and North or South end at the Brine Pool. Position 

factor 2 is Inner and Edge. At the Brine Pool, Inner is adjacent to the brine.

Each specimen analyzed has an Archive ID based on the dive number of either JSL I or II. The 

following number from 1 to 12 is the sample storage chamber on the submersible. Any other 

following number refers to an actual storage container in the Louisiana State University specimen 

archive. At the time of collection the chamber number for each sampling location was recorded in 

a cruise log.

4.1. BATHYMODIOLUS CHILDRESSI TISSUE 

Results for shell length, δ13C‰, δ15N‰, δ34S‰, C%, N%, and S% (element mass percent)

are presented as density plots (Bowman and Azzalini 2003) in Figure 5 and in Tables 3 and 4 with 

descriptive statistics. The percent values are based on molar percent of the dried tissue samples.

4.1.1. C, N, and S % Mass Differences

At Bush Hill C, N, and S comprised 47.34± 2.91, 12.88± 0.94, and 1.28±0.14 molar percent 

of the dried tissue leaving an ash component of 38.5%. During the first two samplings at the Brine 

Pool, the C, N, and S values were 48.70± 2.48, 12.46± 0.92, and 1.33± 0.10. Samples collected in 

2006 had respective values of 47.75± 3.79, 12.94± 1.06, and 1.53± 0.18. The ash component at the 

Brine Pool in 1989–1991 was 37.5% in 2006. Inspection of density plots (Figure 5) confirm that 

there are overlapping ranges but differences in the modal value being most obvious in the case of 

sulfur.

In spite of the relatively similar values, the molar percent of the tissue sample contributed 

by total nitrogen, carbon, and sulfur (Tables 3 and 4) were significantly different (α = 0.05) 

between the two seeps during the earlier 1989–1992 samplings. When the older and 2006 

samplings at the Brine Pool were compared, only the sulfur percentage had changed significantly; 

it was higher at the more recent time.

4.1.2. Stable Isotope Differences

With respect to between-seep differences, the stable isotope results confirm the previous 

studies that were based on smaller sample sizes (MacAvoy et al. 2003). Bush Hill and Brine Pool 

NR-1 represent widely separate situations primarily reflective of the underlying methane source 

(Figure 5B). Isotopic carbon results were consistent with a biogenic methane source at the Brine 

Pool and a thermogenic methane source at Bush Hill. The δ13C values at Bush Hill occur over a 

range of -36.2‰ to -46.2‰ with a mean of -38.65‰ . δ

15N has a range of 6.4‰ to -0.42‰ with a 
17

mean of 4.34‰ and δ34S a range of 9.0‰ to -12.28‰, mean 1.28‰ (Figure 5D and 5F). At Brine 

Pool NR-1 the ranges are δ13C -49.9‰ to -75.0‰ mean of -60.1, δ15N 4.4‰ to -18.0‰ Mean of 

and δ34S 13.9‰ to -1.1‰ mean of. There is no overlap in δ13C values at each seep although the 

two ranges approach each other at their extremes. The δ15N values have minimal overlap and are 

largely distinct at each seep. Five specimens collected at the northern outer edge of the Brine pool 

were outliers with respect to δ15N; they had values similar to Brine Pool mussels.

Table 3. Brine Pool mussels separated into older and newer sampling 

Statistic Length cm δ

15N δ13C δ34S %N %C %S

Brine Pool 1991-1992 n=82

Mean 8.86 -14.06 -60.10 10.01 12.46 48.70 1.33

StDev 2.49 4.82 4.92 3.47 0.92 2.48 0.10

Skewness -0.45 3.12 -0.62 -1.48 -0.55 0.34 -0.45

Kurtosis -0.93 8.99 -0.45 1.49 -0.32 6.71 0.85

Minimum 3.0 -17.82 -75.01 -1.12 10.2 38.3 1.0

Maximum 12.9 4.42 -49.88 13.92 14.1 59.9 1.6

Range 9.9 22.24 25.12 15.04 3.9 21.6 0.6

Brine Pool 2006 n=46

Mean 8.65 -16.08 -62.88 10.82 12.94 47.75 1.53

StDev 2.82 1.02 4.54 1.19 1.06 3.79 0.18

Skewness -0.26 0.78 0.10 -1.22 -0.52 -0.91 -1.50

Kurtosis -0.98 0.62 -1.13 1.30 0.03 1.26 2.95

Minimum 2.9 -17.99 -70.73 7.24 10.0 36.2 0.9

Maximum 13.1 -12.94 -54.30 12.61 14.8 54.6 1.9

Range 10.2 5.05 16.43 5.37 4.8 18.4 1.0
18

Figure 5. Bathymodiolus childressi adductor mussel mass proportions and isotope value density plots for 

carbon (A & B), nitrogen (C & D), and sulfur (E & F), showing contrasts between Brine Pool and 

Bush Hill. 

Descriptive statistics for the seven parameters measured are presented in Table 3 for the Brine 

Pool and Table 4 for Bush Hill. The isotope values echo the distinctiveness found between the 

density plots of the two seeps (Figure 5). 
19

Table 4. Bush Hill mussels parameters

Statistic Length cm δ

15N δ13C δ34S %N %C %S

Bush Hill 1989-1992

N=125

Mean 6.81 4.34 -38.65 0.69 12.88 47.34 1.28

StDev 1.65 1.17 1.91 3.94 0.94 2.91 0.14

Skewness -0.33 -0.66 -1.16 0.05 -0.31 2.41 0.27

Kurtosis -0.56 0.10 1.29 -0.21 3.00 22.33 2.07

Minimum 2.7 0.42 -46.24 -12.38 9.9 36.2 0.8

Maximum 9.8 6.42 -36.22 9.00 16.3 68.1 1.8

Range 7.1 6.0 10.02 21.38 6.40 31.9 1.0

Table 5. Major Comparisons t-Tests between Brine Pool and Bush Hill

Length cm δ

15N δ13C δ34S %N %C %S

Test of Equal Means between Brine Pool versus Bush Hill

t 6.5938 33.9449 37.6748 17.89 3.2344 3.5751 2.7226

P value <0.00001 <0.00001 <0.00001 <0.00000 0.00146 0.000441 0.007043

Test of Equal Means Older Brine Pool versus 2006 Brine Pool

t 0.4368 3.651 3.2226 1.9379 2.5809 1.5266 7.0011

P value 0.6633 .0004304 .001717 0.0552 0.01161 0.1315 <0.00001

Significant differences α=.05 highlighted in yellow

Correlations among length, the three isotopes and the three elemental percentages are presented 

in Tables 5, 6, and 7. These show a combination of similarities and differences. Half-shell volume 

(HSV) has a relatively large positive correlation, δ

13C only at the Brine Pool where much larger 

animals were included in the archive. At Bush Hill, HSV showed no significant correlations. The 

correlations among the three isotopes were all significant and of the same sign at both seeps, 

although the magnitude varied. Correlations among elemental percentages were always positive; 

this indicates that these three elements increase and decrease together.
20

Table 6. Brine Pool mussel parameter correlations

Length δ

15N δ13C δ34S %N %C %S

HSV 1.0000 0.0656 0.4818 0.1346 -0.2696 -0.1538 -0.0479

δ

15N 1.0000 0.5011 -0.7218 0.0294 -0.0194 -0.3308

δ

13C 1.0000 -0.3248 -0.2444 -0.0824 -0.2663

δ

34S 1.0000 0.0493 0.1331 0.0949

%N 1.0000 0.6936 0.3896

%C 1.0000 0.1434

%S 1.0000

(significant differences α=.05 highlighted in yellow).

Table 7. Bush Hill mussel parameter correlations 

Length δ

15N δ13C δ34S %N %C %S

HSV 1.0000 0.2461 0.0365 0.1518 -0.0649 -0.0732 -0.0461

δ

15N 1.0000 0.7713 -0.5468 0.1339 -0.0124 0.2994

δ

13C 1.0000 -0.7462 0.2560 0.0788 0.4086

δ

34S 1.0000 -0.2620 -0.0844 -0.3153

%N 1.0000 0.6507 0.6903

%C 1.0000 0.4594

%mgS 1.0000

(significant differences α=.05 highlighted in yellow).

4.1.2.1. Isotope Patterns Within Seeps

The presence of isotope spatial patterns within the simple design of sampling at the Brine Pool 

was examined using an unbalanced Factorial Analysis of Variance carried out for each isotope 

separately on pooled specimens from 1989 and 1991 (Table 8). For all three isotopes, the means 

were significantly different between the Northern and Southern end of the pool (position factor 1). 

There were also significant differences between means of all isotopes collected at the inner portion 

of the mussel ring and the outer edge (position factor 2) . Interactions between the two positional 

factors, however, were also significant and indicated that the effects of the main factors was 

inconsistent. Spatial differences exist, but in the form of simple spatial gradients. The 

inconsistency of the spatial pattern was confirmed with post-hoc Tukey Honest Significant 

Distance (TukeyHSD) analysis (Appendix II; Table 1).

The effects on isotope values from year of sampling and the pool end position (position factor 

1) along with the interaction of year of sampling and pool end position was also examined by 

unbalanced factorial analysis (Table 9). The isotopes varied different from one another. δ13C 

means were significantly different at different ends and at different years, but interactions were 
21

also significant indicating inconsistent year and position effects. δ15N also showed significant year 

effects, lacking position differences and also showed inconsistent results as indicated by significant 

interactions. Values of δ

34S show no significant year effects but did have different means between 

North and South ends of the pool. No significant interactions were found for δ34S. Post hoc 

TukeyHSD analyses reinforced the finding of interactions for δ13C and δ

15N (Appendix II; Table 

2).

Table 8. Within Brine Pool ANOVA for stable isotope spatial patterns

Response: δ

13C

Factor Sum Sq Df F value Pr(>F)

Pool End 216 1 10.238 0.00199 **

Edge:Inner 449 1 21.329 1.500e-05 ***

Interaction 443 1 21.055 1.679e-05 ***

Residuals 1643 78 Factor

Response: δ

15N

Factor Sum Sq Df F value Pr(>F)

Pool End 298.41 1 18.443 4.992e-05 ***

Edge:Inner 544.19 1 33.634 1.351e-07 ***

Interaction 248.28 1 115.345 0.0001907 ***

Residuals 1262.02 78

Response: δ

34S

Factor Sum Sq Df F value Pr(>F)

Pool End 434.57 1 104.302 4.928e-16 ***

Edge:Inner 510.48 1 122.522 < 2.2e-16 ***

Interaction 360.79 1 86.593 2.759e-14 ***

Residuals 324.99 78

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Spatial patterns at Bush Hill were sought using an unbalanced factorial analysis of variance 

pooling over the two years of sampling (Table 10). The spatial factors were which bed the samples 

came from and whether the samples came from the inner portion of the bed or the outer edge. All 

three isotopes had significantly different means at the three beds 1, 1A, and 2. δ15N showed the 

greatest significant difference between edge and inner locations. The interaction of the two 

position factors indicated an inconsistent effect. δ13C showed a significant inner:edge effect but 

only at a lower criteria of α=0.10; again significant interactions indicated an inconsistent spatial 

pattern. δ34S lacked a distinct inner:edge different. Due to the significant interaction for all three 

isotopes, post-hoc TukeyHSD analyses were carried out and confirmed the inconsistency of spatial 

patterns (Appendix II; Table 3).
22

Table 9. Within Brine Pool ANOVA for stable isotope pool-end and year effects

Response: δ

13C

Factor Sum Sq Df F value Pr(>F)

Year 600 2 16.7914 3.617e-07 ***

Pool End 147 1 8.2116 0.004903 ** 

Interaction 737 2 20.6418 1.899e-08 ***

Residuals 2178 122

Response: δ

15N

Factor Sum Sq Df F value Pr(>F)

Year 396.6 2 16.6642 3.996e-07 ***

Pool End 0.4 1 0.0348 0.852411

Interaction 120.0 2 5.0411 0.007878 ** 

Residuals 1451.9 122

Response: δ

34S

Factor Sum Sq Df F value Pr(>F)

Year 23.43 2 1.5172 0.223427 

Pool End 64.60 1 8.3677 0.004524 **

Interaction 23.77 2 1.5394 0.218649 

Residuals 941.82 122 

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

Temporal analysis at Bush Hill was limited to Bed 1 because it was the only bed sampled in 

both years (Table 11). δ13C and δ15N but not δ34S showed significant differences in the mean 

between the two years. δ13C and δ15N showed no differences between the inner and edge samples. 

δ

34S displayed a weak difference between the two positions but one that was inconsistent between 

the years. As with the previous analysis, post-hoc TukeyHSD analysis confirmed the position and 

year inconsistency (Appendix II; Table 4).

Due to the many significant interaction terms, simple plots of data taking each pair of isotopes 

proved to be especially informative about within-seep spatial and temporal variation. At Bush Hill 

this variation is apparent in data plots for 1991 and 1992 and beds 1, 2, and 1A. Bed 1 was the 

only bed sampled in both years and tended to have tightly-clustered points in each of the three

graphs for both years (Figure 6). The greatest difference found between the years in the centroid 

of bed 1 data points was a shift of approximately 5‰ in δ34S (Figure 6 E and F). More conservative 

between years was δ13C with a shift of approximately 1.5‰ and a δ15N shift of approximately 1‰. 

Contrast between beds within each year was more dramatic. Beds 2 and 1 A had δ13C values 

overlapping bed 1, but with an expansion of range that consisted primarily of the addition of more 

depleted values. δ15N had a more modest expansion of range, again with more deleted values mixed 
23

into beds 2 and 1A. δ34S were shifted sufficient between bed 1A and 2 for there to be no overlap 

(Figure 6 E and F). Bed 2 had a similar shift compared to Bed 1, but a wider range of values caused 

the two data clusters to overlap (Figure 6 B and C).

Table 10. Bush Hill mussel tissue stable isotope factorial ANOVA for spatial effects

Response: δ

13C

Factor Sum Sq Df F value Pr(>F)

Bed 22 2 6.3274 0.002447 ** 

Inner:Edge 6 1 3.3124 0.071274 . 

Interaction 37 2 10.9366 4.363e-05 ***

Residuals 202 119 

Response: δ

15N

Factor Sum Sq Df F value Pr(>F)

Bed 8.00 2 4.6781 0.011072 * 

Inner:Edge 6.29 1 7.3544 0.007681 ** 

Interaction 7.10 2 4.1553 0.018013 * 

Residuals 101.73 119

Response: δ

34S

Factor Sum Sq Df F value Pr(>F)

Bed 491.23 2 42.8340 9.718e-15 ***

Inner:Edge 0.27 1 0.0472 0.828308

Interaction 137.94 2 12.0284 1.747e-05 ***

Residuals 682.36 119 

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

At Brine Pool NR-1 the range for δ13C and δ15N greatly exceed those found at all Bush Hill 

sites consistent with mixing of widely different sources (Figure 7). The patterns among bed 

locations and years (1989, 1992, and 2006) are complex and do not reflect a simple dependence 

on location with respect to the brine. With the exception of five outlier specimens from the 1991 

northern outer portion of the mussel mat, all values for δ13C and δ15N fell on a common curvilinear 

relationship (Figure 7 B, D, and F). In 1989 the curve of δ13C and δ15N points was quite wide;

northern outer and southern inner samples clustered at the enriched end of the range and those 

from the northern inner and southern outer samples were spread towards more depleted values

(Figure A, C, and E). The pattern of widely scattered points in 1989 and more tightly clustered

points in 1991 for δ

13C and δ15N also held true for δ34S (Figures 7 C, D, E, and F).

Examination of isotope plots for Brine Pool 2006 values compared with the pooled older 1989 

and 1992 values showed relative consistency over the long term (Figure 8; please note scale change 

between B and F). In some respects, samples from 2006 more closely resembled the widely 

scattered values found in 1989 than the tightly clustered values of 1991. 
24

Table 11. Inter-Year and With-In Bed ANOVA at Bed 1 mussel tissue stable isotope values

Response: δ

13C

Factor Sum Sq Df F value Pr(>F)

Year 63.0 1 137.2755 <2e-16 ***

Inner:Edge 0.4 1 0.9375 0.3363

Interaction 1.0 1 2.1242 0.1495 

Residuals 31.7 69

Response: δ

15N

Factor Sum Sq Df F value Pr(>F)

Year 7.30 1 14.8699 0.0002556 ***

Inner:Edge 1.47 1 3.0034 0.0875560 .

Interaction 0.00 1 0.0096 0.9223670

Residuals 33.86 69

Response: δ

34S

Factor Sum Sq Df F value Pr(>F)

Year

Inner:Edge 10.359 1 2.7969 0.09897 . 

Interaction 137.919 1 37 .2401 5.386e-08 ***

Residuals 255.543 69

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1
25

Figure 6. Bush Hill Bed 1 compared to Bed 2 in 1989 and Bed 1A in 1991.
26

Figure 7. Brine Pool Positions for 1989 and 1991 boundaries added for clarity. 
27

Figure 8. Brine Pool tissue values for1989, 1992, and 2006 with boundaries on 2006 for clarity.

4.1.3. Stable Isotope Estimation of Mixed Use of Four Trophic Sources

The stable isotope signatures of four probable trophic sources were identified for each seep. 

Phytoplankton detritus (DPHY) was the same at both sites. A total of four methane-pathways 

sources were identified and designated SM_ABH, SM_BBH, SM_CBP, and SM_CBP; the subscripts 

denotes the seep. Each seep also had a distinct thiotrophic-pathway source DSOMBH and DSOMBP. 

Table 12 presents the δ13C, δ15N and δ34S values for probable trophic sources (vertices) selected 

for the mixing model. They were chosen for their ecological feasibility in the upper continental 

slope seep setting and for the degree to which their values allowed for the tightest fit of the mixing 

tetrahedron around the sample data. The four sources included two different detrital sources and 

two different symbiotic-pathway sources with both site-specific differences and similarities. 
28

Table 12. Estimated sources (vertices) of a four-source mixing tetrahedron

Detrital Sources Symbiotic-Pathway Sources

δ

13C 

‰

δ

15N

‰

δ

34S

‰

δ

13C 

‰

δ

15N

‰

δ

34S

‰

δ

13C 

‰ 

δ

15N 

‰

δ

34S 

‰

δ

13C 

‰ 

δ

15N 

‰

δ

34S 

‰

Bush 

Hill

DPHY

Photosynthetic Carbon 

Seawater Nitrate,& 

Seawater Sulfate

DSOMBH

Chemosynthetic 

Carbon Seawater 

Nitrate & Seep Sulfide

SM_ABH

Methane Carbon, 

Seawater Nitrate

&Seawater Sulfate

SM_BBH

Methane Carbon, Seep 

Ammonium + Seawater 

Nitrate & Seep Sulfide + 

Seawater Sulfate 

-21.5 7 21 -31 7 -20 -50 7 21 -50 -5 0

Brine 

Pool

DPHY

Photosynthetic Carbon 

Seawater Nitrate 

&Seawater Sulfate

DSOMBP

Chemosynthetic 

Carbon Seep 

Ammonium &Seep 

Sulfide

SM_CBP

Methane Carbon, 

Seawater Nitrate & Seep 

Sulfide

SM_DBP

Methane Carbon, Seep 

Ammonium & Seawater 

Sulfate

-21.5 7 21 -31 -25 -20 -65 7 -5 -90 -25 21

Seawater and Photosynthesis Influenced Seep Influenced Depletions

Detritus influx originating from euphotic zone phytoplankton should be the same at the closelylocated seeps and relatively labile due to the 500m depth (Biggs et al 2008). Detritus Phytoplankton 

(DPHY) has a δ 13C value of -21.5‰ and a δ 15N value of 7‰ that is representative of surface 

particulate organic matter in the Northern Gulf (Rooker et al. 2006; Wells and Rooker 2009) and 

a δ 34S value of 21‰ representative of seawater sulfate (Rees et al. 1978). A second detrital source 

consists of free-living sulfur oxidizing microbes (DSOM) on or very close to bottom (Page et al. 

1990). The DSOM for both sites (DSOMBH and DSOMBP) have the same δ 13C value of -31‰,

obtained from thiotrophic Beggiotoa mats as a proxy for all free-living chemosynthetic microbes 

(Demopolous 2010). The DSOM values of δ 34S at both sites is the same extremely depleted -20‰ 

as the cumulative result of the fractionations during the reduction of seawater sulfate to interstitial 

seep sulfides (30–70‰ Canfield 2001) and the subsequent oxidation of those sulfides (10–18‰;

Dale et al. 2009). Fitting the tetrahedrons to the data requires that the DSOMBH and DSOMBP δ

15N values be different because the values for individuals at both sites have distinctly different 

ranges. For the Bush Hill DSOMBH, the δ 15N of 7‰ reflects a predominant usage of seawater 

nitrate (5–7‰) and at the Brine Pool DSOMBP δ

15N of -25‰ reflects the predominant utilization 

of brine-associated ammonium. 

The range and variance at both seeps indicate that two microbial methanotrophic-pathway 

sources were being used by the populations SM_ABH, SM_BBH, SM_CBP, and SM_CBP. The 

narrow range and symmetry of δ13C values (Figure 5B, 5D, &5F) and the fit of the tetrahedron 

(Figure 11) at Bush Hill are consistent with a single methane source with a δ13C value of -50‰ 

shared by two resources. The first resource SM_ABH combines that seep methane carbon with 

nitrogen from seawater nitrate δ 15N = 7‰ and sulfur from seawater sulfate δ34S = 20‰. The 

second resource SM_BBH incorporates seep methane carbon with seep-derived nitrogen and sulfur, 
29

or possibly a mixture of seep-derived ammonium and seawater-derived nitrate δ 15N = -5‰ and 

sulfate δ34S = 0‰. It is the relatively minor depletion of these two isotopes that suggests seep and

seawater mixing.

At the Brine Pool δ13C bimodality and asymmetry (Figure 5B, 5D, &5F) and tetrahedral fit (Figure 

9; the vertices represent possible trophic sources, and points within each tetrahedron are based on 

data from the mussels) are best explained by two seep methane-carbon sources, one highly 

depleted δ13C= -90‰ and another more moderately depleted δ13C= -65‰. The latter carbon 

combines in SM_BBH with nitrogen from seawater nitrate δ 15N = 7‰ and sulfur from seep sulfide 

δ

34S = -5‰. The highly depleted carbon combines in SM_CBP with highly depleted nitrogen from 

seep ammonium δ 15N = -25‰ and sulfur from seawater sulfate δ34S = 21‰.

Figure 9. Trophic mixing models as tetrahedrons surrounding Brine Pool (red) and Bush Hill (blue) data 

points in Cartesian space of the three stable isotopes data.

Because four distinct methane-pathway resources were indicated initial between Brine Pool 

and Bush Hill comparisons were carried out through separate t-tests for combined methane sources 

(SM_ABH + SM_BBH compared with SM_CBP + SM_DBP ), sulfur microbe sources (DSOMBH vs 

DSOMBP), and DPHY. Subsequent within-seep comparisons were carried out on each of the seepspecific four sources without combinations using an unbalanced Analysis of Variance (ANOVA)

and post hoc TukeyHSD analysis.
30

Paired t-tests between seeps indicated significant differences between the Brine Pool and Bush 

Hill in average use of the three source categories (Table 13). The mussel population at the Brine 

Pool made greater use of combined methane-pathway sources (61.1% ± 9.3) predominantly 

SM_DBP (49.4% ± 11.5) and lower and roughly equal particulate resources, DPHY (20.2% ± 7.0) 

and DSOBBP (18.7% ± 4.7). At Bush Hill the combined methane-pathways resources were 

significantly lower in the population (46.2% ± 11.1) and consisted of roughly equal amounts of 

SM_ABH (24.0% ± 6.6) and SM_BBH (22.2% ± 9.7). Offsetting the lower methane use was 

increased use of particulate sources; DSOBBH was a major resource (38.2% ± 13) and DPHY was

a minor resource (15.6% ± 4.6).

Table 13 Between-Seep t-Test comparisons with methane sources combined.

Site n Methane DPHY DSOB

Brine Pool 128 61.1 ± 9.3 20.2 ± 7 18.7 ± 4.7

Bush Hill 125 46.2 ± 11.1 15.6 ± 4.6 38.2 ± 13

t 11.6 6.2 -15.9

P_value <0.001 <0.001 <0.001

(significant values α=.05 highlighted in yellow)

At Bush Hill significant Bed differences were found for all four resources, but Position (Inner 

compared with Edge) differences were significant only for SM_BBH and DSOMBH (Table 14). Post

hoc analysis (Appendix II; Table 5) and box and whisker plots (Figure 10; based on Table 14) 

revealed that within-bed homogeneity was more prevalent than the ANOVA results might suggest. 

Beds 1, 1a, and 2 were not significantly different for use of SM_ABH and DPHY. When significant 

Position effects were found, they were restricted to only a single bed, bed 1 in the case of SM_BBH, 

bed 2 in the case of DSOMBH. Significant Bed:Position interaction for DPHY and DSOMBH 

indicated inconsistent factor effects.

Table 14. Bush Hill ANOVA for each resource and position factors both years pooled

BUSH HILL

Sum Sq Df F value Pr(>F) 

SM_ABH

Bed 0.0923 2 13.2810 6.218e-06 ***

Position 0.0000 1 0.383 0.9748 

Bed:Position 0.0105 2 1.5101 0.2251 

Residuals 0.4133 119 

SM_BBH

Bed 0.3791 2 31.619 9.71e-12 ***
31

Position 0.0862 1 14.383 0.00236 ** 

Bed:Position 0.0035 2 0.2878 0.75043 

Residuals 0.7135 119

DPHYBH

Bed 0.05720 2 19.450 4.91e-08 ***

Position 0.00525 1 3.574 0.0611 

Bed:Position 0.02966 2 10.0849 9.000e-05 ***

Residuals 0.17497 119

DSOMBH

Bed 1.0753 2 94.4439 < 2.2e-16 ***

Position 0.0977 1 17.1591 6.46e-05 ***

Bed:Position 0.1046 2 9.1906 0.0001943 ***

Residuals 0.6775 119

Unlike the relatively homogenous Bush Hill mussels, the Brine Pool sample contained five 

specimens from a single scoop sample taken in 1991 at the North Edge that were distinct outliers 

especially with respect to δ 15N values. ANOVA were carried out with the full data set (Table 15) 

and then with the outliers omitted (Appendix II; Table 6). For the full data set at the brine pool 

SM_CBP showed End (N vs S) effects and position effects (Inner vs Edge) but with significant 

interactions (Table 15). Post hoc analysis (Appendix II; Table 7) revealed that the significant 

results were caused primarily by high values encountered at the North Edge; the three other 

combinations of End and Position were homogenous. SM_B showed marginally significant End 

effects, Position effects and an interaction in the analysis of variance, but, in post hoc analysis, the 

End effect was not significant and differences were due primarily to the low values at the North

Edge. Use of DPHY showed significant End effects but insignificant Position effects and no 

significant interaction. Post hoc analysis revealed the same effects but pairwise comparisons 

indicated few major differences except for a variable range. DSOM showed no significant End or 

Position effects although a significant interaction was found. Post hoc analysis indicated a 

difference in means between Edge and Inner only at the South end. Otherwise, DSOM use was 

relatively uniform across the sampled locations. Interactions among factors are obvious in box and 

whisker plots (Figure 11; based on Table 15).

Omission of the outliers (Appendix II; Table 6) had little impact on the conclusion of the 

analyses; similar conclusions about Bed and Position effects were produced for SM_CBP ,SM_DBP 

and DPHY. The North End remained the location of significantly greater SM_CBP use, and the 

maximum found in the North Edge. The major change in results was for DSOM, which showed 

significant End, Position, and interaction effects. It had the greater use at the North End and the 

greatest overall at the North Edge.
32

Figure 10. Bush Hill between-bed and with-in bed proportional use of trophic resources presented as box 

and whisker plots with both sampling years pooled.

Temporal differences at Bush Hill could only be examined for Bed 1 because the other beds 

were sampled only in a single year (Table 16; Figure 12). DPHY was the only resource to be used

homogenously between years and between positions. SM_ABH use increased significantly in 1992 

and was consistently lower at the Edge in both years. SM_BBH was similarly higher in 1992 and 

within years was consistently higher at the Edge. DSOM was used significantly less in 1992 and 

there were no position effects within the years. Post hoc analyses (Appendix II; Table 7) supported 

the conclusions of the ANOVA.
33

Table 15. Brine Pool trophic resource ANOVA for position factors 2006 omitted

BRINE POOL

Sum Sq Df F value Pr(>F) 

SM_CBP

End 0.17956 1 11.22 0.0012450 ** 

Position 0.33114 1 20.703 1.941e-05 ***

End:Position 0.24444 1 15.283 0.0001961 ***

Residuals 1.24758 78

SM_DBP

End 0.03497 1 3.8889 0.05215 .

Position 0.26860 1 29.8679 5.358e-07 ***

End:Position 0.41237 1 45.8540 2.136e-09 ***

Residuals 0.70146 78 

DPHY

End 0.03589 1 8.2427 0.005265 **

Position 0.00113 1 0.2584 0.612662 

End:Position 0.00000 1 0.0009 0.975885 

Residuals 0.33965 78 

DSOMBP

End 0.00022 1 0.0747 0.78540

Position 0.00416 1 1.4599 0.23060

End:Position 0.02243 1 7.8711 0.00634 **

Residuals 0.22223 78

Temporal differences at the Brine Pool were examined with an unbalanced ANOVA with two 

factors: Year and Pool End North or South (Table 17). The predominant methane-pathway 

resource SM_DBP and showed no significant differences among the three years and between North 

and South ends (Figure 13; based on Table 17). In 2006, however, the North and South End 

mussels had significantly different means; the South used the greater proportion. Thiotrophic 
34

particulate material was similarly homogenous among Years and Ends, except in 2006 the North 

end. SM_CBP was lowest in 1991 and homogenous between Ends. Use in 1989 and 2006 were not 

significantly different but Ends differed inconsistently. DPHY showed an inverse pattern with 

highest use and similar Ends in 1991. Means in 1989 and 2006 were similar and Ends differed 

inconsistently. Post hoc analyses (Appendix II; Table 8)supported the conclusions of the ANOVA.

 

Figure 11. Brine Pool trophic resource proportional use at pool ends and positions displayed as whisker 

and box plots by position 1991 and 1992 years pooled and 2006 omitted. 
35

Table 16. Bush Hill Bed 1 ANOVA of trophic use between years

Bush Hill Year and Pool-End Effects Bed 1 Only

Sum Sq Df F value Pr(>F) 

SM_ABH

Year 0.0362 1 15.2349 0.0002181 ***

Position Bed 1 0.0156 1 6.5690 0.0125615 *

Year:Position 0.0028 1 1.1634 0.2845209 

Residuals 0.1642 69

SM_BBH

Year 0.08858 1 25.9948 2.864e-06 ***

Position Bed 1 0.01672 1 4.9072 0.03005 * 

Year:Position 0.00003 1 0.0096 0.92237 

Residuals 0.23514 69

DPHY

Year 0.00001 1 0.0115 0.9151 

Position Bed 1 0.00001 1 0.0081 0.9287

Year:Position 0.00010 1 0.0828 0.7744 

Residuals 0.08601 69

DSOMBP

Year 0.2345 1 77.8162 6.311e-13 ***

Position Bed 1 0.0000 1 0.0004 0.9836 

Year:Position 0.0013 1 0.4480 0.5055 

Residuals 0.2079 69

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1
36

Figure 12. Bush Hill between-year and position within Bed 1 only in trophic resource use displayed as box 

and whisker plots.
37

Table 17. Brine Pool among-year and pool-end trophic resource use ANOVA

BRINE POOL

Sum Sq Df F value Pr(>F) 

SM_CBP

Year 0.02758 2 8.8690 0.0002592 ***

End 0.00031 1 0.2001 0.6554899 

Year:End 0.10165 2 32.6879 5.276e-12 ***

Residuals 0.18192 117 

SM_DBP

Year 0.0061 2 0.7003 0.498488 

End 0.0285 1 6.5408 0.011821 * 

Year:End 0.0490 2 5.6207 0.004669 ** 

Residuals 0.5104 117 

DPHY

Year 0.0479 2 8.9247 0.000247 ***

End 0.0139 1 5.1909 0.024520 * 

Year:End 0.2016 2 37.5927 2.462e-13 ***

Residuals 0.3137 117 

DSOMBP

Year 0.0001 2 0.0388 0.96198

End 0.0047 1 3.3771 0.06864 .

Year:End 0.0076 2 2.7166 0.07027 .

Residuals 0.1629

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1
38

Figure 13. Brine Pool position and year proportional use of trophic resources for all three years presented 

as box and whisker plots. 

4.2. MUSSEL SHELL INCORPORATION OF SEEP RESPIRATORY METHANE

Though mussel shell was found to be composed of predominantly carbon from the seawater 

inorganic carbon pool, respired carbon from the methane-based tissue was detectable. That amount 

was distinctly different at the two seeps and indicated a greater incorporation rate at the Brine Pool. 

Additionally, a size effect was found when HSV was taken as the measure of animal size. Separate 

regression of Cm compared to HSV at Brine Pool and Bush Hill revealed significant (p<0.0086, 

p<0.0001), but relatively small, linear positive relationships between Ln(HSV) and %Cm at each 

site that are not significantly different from one another (p=0.1977). The intercepts (13.9, 8.1) at 

the two sites were significantly different (p<0.0001). An ANOVA with a Tukey’s post hoc test 

(α= 0.05) was performed on the Brine Pool site and on %Cm for groupings of HSV from 0-10 cm3, 

10–20 cm3, and >20 cm3. This resulted in significant differences between the 0-10 cm3(14.4%) 

and the >20 cm3(16.4%) groupings. It also resulted in a significant difference between the 10–20 

cm3(17.8%) and >20 cm3 (16.4%). A second ANOVA performed on the Bush Hill site resulted in 

significant differences between 0–10 cm3(9.8%) and 10-20 cm3(11.6%) groupings and between 

10–20 cm3(11.6%) and >20 cm3(13.1%) groupings.
39

When these samples were grouped on ranges of HSV and compared between the Brine Pool 

and Bush Hill sites, there was a significant difference (p<0.0001) between the Bush Hill and Brine 

Pool sites (Figure 12). That is, a mussel with a HSV of 0–10 cm3and a mean of 14.4 % ± 3.12 at 

Brine Pool (n=36) but at Bush Hill (n=40) for a mussel with an HSV of 0–10 cm3 there is a mean 

of 9.8 % ± 1.5. For a mussel with a HSV of 10-20 cm3, there was also a significant difference 

(p<0.0001) between the two sites. The mean at the Brine Pool (n=18) site was 17.8 % ± 2.2 and 

the mean at the Bush Hill (n=18) site was 11.6% ± 2.4. For a mussel HSV of 20+ cm3, there was 

a significant difference (p<0.004) between the two sites the mean at the Brine Pool (n=38) site was 

16.4 % ± 3 and the mean at the Bush Hill (n=6) site was 12.2 % ± 2.3. There is a statistically 

significant difference in the incorporation of metabolic carbon between the sites for all size ranges;

Brine Pool had significantly increased incorporation of respiratory carbon across the entire range 

of mussel HSV.

Figure 14. Percent of metabolic carbon in shell compared with size (LnHSV used).

A paired T-test between Bush Hill and Brine Pool mussel %Cm yields a statistically significant 

difference (p<0.0001) between the two sites (Figure 14). The Brine Pool (n=98) site had a mean 

of 15.8 % ± 3.1 and Bush Hill (n=64) had a mean of 10.5 % ± 2.1. The T-tests performed on within 

site differences (North and South) revealed a statistically significant difference (p< 0.0357) 

between the Brine Pool and Bush Hill sites with all sample years pooled. In individual year 

samplings for North versus South, the 1989 and 1991 samplings were not significant, but the 2006 

sampling was significant (p<0.0048). There is a statistically significant difference between the two 

sites for %Cm, but the further detailed sampling design yields few significant results and as such 

is not useful for the spatial analysis of %Cm.
40

4.3 BATHYNERITA NATICOIDEA

The dominant seep snail, Bathynerita naticoidea, on the Louisiana upper slope is a 

heterotrophic consumer. Figure 15 displays three plots of each pair of isotopes with both mussels 

and snails presented. At both seeps the snails were enriched in 13C and 15N relative to the substrateforming mussel population. The extent of that enrichment, however, is greater at the Brine Pool 

site than at the Bush Hill site. At both seeps the enrichment is not the simple 3 δ15N and 1 δ13C 

associated with source:consumer pairs (Phillips 2012). From this information an alternate 

interpretation is that the snail populations at each seep are using a differ suite of detrital resources. 

For δ34S, the snails showed more depleted values indicating that their detrital resource was 

primarily generated by sulfide microbes of the seep system rather than phytoplankton-based 

detritus. At Bush Hill this pattern of δ13C and δ15N enrichment is consistent with consumption of 

the same DSOM as consumed by that mussel population. At the Brine Pool it is inconsistent with 

the estimated detritus resources. The snails are highly mobile and demonstrated no within-seep 

sample effects.

Figure 15. Plots of each pair of isotope values for all mussels (_M) and snails (_S) at Bush Hill (BH) and 

Brine Pool (BP).
41

5. DISCUSSION

5.1 ANSWERING THE INITIAL THREE QUESTIONS

In the following subsections the original three questions that this study set out to answer will 

be considered in light of the above results. Following that, the management relevance of the new 

findings are considered.

 Does the mass percent of carbon, nitrogen, and sulfur in tissue of the seep mussel

Bathymodiolus childressi provide a useful tool for assessing the overall condition of

populations?

 Does the stable isotope composition of carbon, nitrogen, and sulfur in mussel tissue

provide a useful indication of trophic condition of a population that is capable of both

chemosynthesis and heterotrophic feeding?

 When trophic condition of a foundation species is determined, is that condition also

reflected in a consumer organism (the snail Bathynerita naticoidea) in the same

system?

Does analysis of mussel shells have the potential of providing information on the trophic 

condition that might be used to assess past seepage condition?

5.1.1. C, N, & S Mass Proportions in Mussel Populations as a Condition Index

The purpose of a condition index is to provide a simple indication of an organism’s overall 

health which can be extended to a population with appropriately designed sampling. Populations 

with lower condition can be suspected as being under stress. Methods applied to molluscs were 

reviewed in section 2.5. Previous finding of higher condition at the Brine Pool and lower condition 

at Bush Hill were presented in Table 1. The most common methods are best suited for fresh 

specimens and cannot be applied to archival material.

The finding that mass percent of C, N, and S are significantly different at the Brine Pool and 

at Bush Hill is an indication that the element mass method may have utility. Based on random 

subsamples of the data, significant differences may be tested on samples of as few as 10 specimens 

per seep site. The full utility of the method, however, will require experimental work to link C, N,

and S mass to one of the accepted physiological method. Such experimentation can be carried out 

on an easily-obtained proxy for seep mussels and then confirmed based upon new sampling of 

seep specimens.

5.1.2. Tissue Isotopes and Trophic Mixing

Although often included in comprehensive trophic analyses, seep mussels are usually 

considered a proxy for a chemosynthetic food source to the larger system and their own trophic 

structure left unanalyzed (MacAvoy et al. 2008, Becker et al. 2010, Becker et al. 2013). The work 

reported herein approached the task of trophic assessment in two ways. The traditional approach 

using stable isotope values provided very strong indication that Bathymodiolus childressi 

population are mixotrophic rather than simply chemosynthetic. The ranges and variance of the 

stable isotope values established from large sample sizes at both seeps are simply two great to be 

explained by a single trophic pathway. A four-source mixing model was developed taking 

advantage of the fact that four distinct sources can be resolved given three isotope values. 
42

The most important result of the four-source analysis comes from the estimation of trophic 

mixing. The data indicate that the mussels are experiencing two symbiotic pathways at each seep 

based on the local source of methane but different sources of nitrogen and sulfur (SM_ABH, 

SM_BBH, SM_CBP, and SM_DBP). Previously, a single path for seep methane has been assumed. 

Also important is the apparent two sources of detrital food at each seep; one source reflects

phytoplankton (DPHY) and the other reflects microbial chemosynthesis through thiotrophy 

(DSOMBH and DSOMBP).

Figure 16 provides a schematic representation of the sources and their relationship to the local 

geochemistry. Detrital sources as circles reflect combinations of seep and water column influences. 

Thiotrophic detritus at the Brine Pool is greatly influenced by the very high seep ammonium levels 

found in the pool. Lacking that chemical resource at Bush Hill, thiotrophic microbes use seawater 

nitrate. Symbiotic pathways as octagons also show a distinct difference between seeps. At Bush 

Hill, SM_A combines seep methane with water-column sulfur and nitrogen. SM_B uses seep 

carbon, sulfur, and nitrogen. The difference may simply reflect the distance from the bottom where 

the mussel siphons. Those very near the sediment surface get the most seep products. Those a few 

centimeters off bottom receive a mixture. Siphon level may also be a factor at the Brine Pool but 

a substrate effect may be more important. The SM_A may represent the effects of feeding where 

the substrate is brine-saturated mud. The SM_B may be feeding in portions of the mat which 

extend out over brine. The over-brine source reflects the very high ammonium but only modest 

sulfide in the pool itself. Sulfide emitted by the mud and ammonium emitted by the brine may 

have such a high flux rate that siphon position has minimal effects.

The greater seepage rate at the Brine Pool is reflected in the 61% of trophic resources coming 

through methane pathways; detrital input is less important. At Bush Hill, only 36% comes through 

a methane path, and detritus is more important. Thiotrophic detritus is more important that 

phytoplankton. These proportions cannot be directly related to seepage rates, but they are 

consistent with previous conclusions that seepage is higher at the Brine Pool. High dependence on 

detritus may be a useful Condition Index. Phytoplankton detritus alone, however, may not be able 

to sustain a methane-limited population. There must be adequate microbial production of detritus 

via sulfide-based chemosynthesis. The data obtained from the seep snail Bathynerita naticoidea

confirms that the trophic status of the foundation species is reflected in the food web.

It had been anticipated that simple spatial patterns would be found both within the mussel beds 

at Bush Hill and the ring of mussels at the Brine Pool. This expectation was based on the 

assumption that methane seepage must be variable across these areas. One would expect at the 

minimum that the outer boundaries of mussel populations might reflect the limit of a seepage 

feature. It was surprising that no such simple relationships were found. Resource use was definitely 

variable and that variation had a spatial component, but not a spatial effect that could be found,

given the scale of sampling.
43

Figure 16. Trophic resources for Brine Pool and Bush Hill representing detrital (circles) and methane 

resources (hexagons) used by seep mussels.

The temporal analyses were the most tenuous given the limitations of using archival 

sampling, but the results were informative. There was significant variation over the two years 

(1991 and 1992) at Bush Hill Bed-1 for δ

13C, δ15N, and δ34S and at the Brine Pool (1989 and1991) 

for δ

13C, δ15N, and δ34S (2006 showed no significant variation for δ34S). Though the temporal 

variation was statistically significant, it is noteworthy that in 2006 the Brine Pool mussel 

population was still quite similar to 1991. The Brine Pool appears to be a relatively constant 

feature. A similar long-term comparison at Bush Hill was not possible based on the archive at 

Louisiana State University. 

5.1.3. Shell as a Historical Record of Metabolic Stress 

Assessment of status in seep systems is complicated by the fact that seepage passes through a 

geologically controlled sequence from initiation to termination (Roberts and Carney 1997). Seep 

communities also pass through stages and end in exposed or sediment-covered authigenic 

carbonate with no active seepage (Bergquist et al. 2004, 2005). In the search for seeps, it is very 

common to encounter dead systems; these often consist of a mixture of bivalve shells and 

authigenic carbonate gravel and there is no obvious evidence of the cause of death (Callender and 

Powell 1997). Is it possible that the isotopic composition of such shell material contains a record 

of seepage failure? This study did not carry out such a determination, but it did execute a “proof 

of method” study to see if respiratory methane could be detected in shells.

The significant between-seeps, between-site, and between-years incorporation of metabolic 

carbon into the mussel shells indicates that shells can preserve a temporal record of mussel 

exposure to methane flux. Sampling across shell age on both live and dead specimens may afford 

good indication of both the history of flux and the flux conditions at time of depth. The current 

study using only an edge piece of shell proved the feasibility of such an approach.
44

The observed variation can be interpreted as an indicator of stress. At the Bush Hill site, there 

is a smaller range of %Cm across shell volume, and overall mussel size is smaller than at the Brine 

Pool site. This indicates resource limitation at the site. This limitation and adverse effect of 

increased volume is observed in the mussels that range from 4–15 cm3, where a substantial portion 

of the %Cm values are in the range of 7–10% and fall well below the regression for this site. The 

Brine Pool site mussels had a steady availability of seepage resources from 1989 to 1991; there 

was no change in %Cm for this time period, but in the 2006 sampling a significantly increased 

%Cm indicates an increase in available seep resources for that time period. The Bush Hill site has 

a significant increase in %Cm from 1991 to 1992, which indicates a quicker increase in resources 

in a more resource limited setting. These differences on both a decadal and yearly time scales 

demonstrate the sporadic nature of methane availability at cold seep sites. Increases and decreases 

of methane seepage greatly affect the productivity in populations that are reliant on it as a basal 

resource.

6. CONCLUSIONS AND RECOMMENDATIONS

The primary conclusion is that populations of the foundation species Bathymodiolus childressi

are mixotrophic rather than purely methanotrophic. Its populations and the community that it 

supports may be resilient in the face of methane flux decrease. The mussel’s varied diet is reflected 

in the consumers of its system. There is enough respired carbon originating from the mixed diet 

found in the shells for the cause of dead-mollusc assemblages to be investigated.

Working at only two contrasting seeps, we have reinforced with larger sample sizes that most 

mussels at a particular seep reflect local average conditions. Contrary to previous interpretation 

seep-to-seep differences are reflective of different tropic systems and not just different methane 

sources. On a smaller scale it has also been shown that some distinctly different mixotrophic

conditions may exist at spatially restricted places as in the case of the Brine Pool outliers at the 

Northern edge.

Bathymodiolus childressi is mixotrophic with the trophic plasticity to draw form methane and 

detrital sources. For the short term, this may provide a degree of resilience should methane seepage 

decline or become highly variable. Mixotrophy also means that populations may become stressed 

by natural or manmade alteration of the four sources identified. In a hypothetical case of a 

manmade impact, offshore development may impact mussel populations via impacts on 

combinations of trophic sources. A population highly dependent on detritus feeding such as at 

Bush Hill might be more sensitive to drilling-caused turbidity. It must be noted that no industry 

impacts to seep systems has been identified or fully explored.

Though lacking a long time range, our observations suggest the need for some refinement of 

the existing model for seep development. There must be a progression from the fluid-prone 

initiation phase to mineral-prone methane seepage that provides a window of suitability for B. 

childressi colonization. Suitability for a mixotrophic foundation species requires that there must 

also be a progression in microbial sulfur geochemistry which provides an adequate thiotrophic 

detritus food source.

We have also shown that mussel shell retains a record of methane use. This opens up the 

possibility of documenting methane usage through the lifetime of individuals and reconstructing 

the final stages of seepage that produced large areas of shell hash in restricted areas of the Gulf. 
45

Living mussel populations and extensive areas of dead shell, including mussels, are stages in a 

very poorly understood natural succession. 

A valuable goal for the management of offshore resources and protection of seep systems at 

any stage of succession is to continue locating those systems as is now done and then provide an 

assessment of the status of the major foundation species. Such a status assessment is now only 

feasible for tubeworms. The work presented herein is a start at assessment of mussel populations 

and that the cause of death assemblages might be recorded in the shell material.

That succession and its indicators must be known before we can assess the potential impacts 

of offshore development. In order to gain better understanding, seven recommendations can be 

made.

1. Confirm the existence and method of feeding on thiotrophic detritus. The existence of an 

important chemosynthetic detritus as a food source must be tested rather than inferred from 

the consumers. 

2. If confirmed, multiple seeps should be examined to determine the apparent stages of 

detritus generation.

3. Long-term monitoring of Bush Hill, the Brine Pool. or other suitable sites should be 

formally organized, initiated. and coordinated. Monitoring or condition and trophic 

resources should be done without excessive collecting of live material.

4. Both Bathymodiolus childressi and Bathynerita naticoidea survive collection and 

rudimentary aquarium conditions. Therefore, use of detritus by chemosynthetic mussels 

should be carried out under fully-controlled laboratory conditions.

5. The study of death assemblages of chemosynthetic bivalves should be extended and 

include reconstruction of methane incorporation from shell material.

6. Multi-site surveys should adopt protocols for within-seep spatial sampling as well as 

species and specimen replication so as to optimize future inter-comparisons.

7. Specimen archives should be maintained for analysis of replicates rather than the more 

limited archiving of taxonomic voucher specimens.
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APPENDIXES
52

APPENDIX A: MUSSEL AND SNAIL DATA TABLES

The appendix files consist of Microsoft® Excel® spreadsheets that may be accessed by means of 

the attachment menu of the final report pdf file.

Appendix A Table 1. Stable Isotope Values for Brine Pool Mussels

Appendix A Table 2. Proportions of four trophic resources for Brine Pool Mussels

Appendix A Table 3. Brine Pool Elemental Mass of Nitrogen, Carbon and Sulfur as Milligrams 

and Mass Percent

Appendix A Table 4. Stable Isotope Values for Bush Hill Mussels

Appendix A Table 5. Proportions of Four Trophic Resources for Bush Hill Mussels

Appendix A Table 6. Bush Hill Nitrogen, Carbon and Sulfur Mass Percent

Appendix A Table 7. Mussel Shell Half Shell Volume, δ13C Shell, δ13C Tissue (‰) and 

Percent Metabolic Carbon for Bush Hill and Brine Pool

Appendix A Table 8. Brine Pool Bathynerites naticoidia Stable Isotope Data

Appendix A Table 9. Bush Hill Bathynerites naticoidia Stable Isotope Data
53

APPENDIX B: SUPPLEMENTAL DATA ANALYSIS TABLES

The appendix files consist of Microsoft® Excel® spreadsheets that may be accessed by means of 

the attachment menu of the final report pdf file.

Appendix B Table 1. Brine Pool TukeyHSD post hoc Analyses of Position Effects on Stable 

Isotope Content of Mussel Tissue

Appendix B Table 2. Brine Pool TukeyHSD post hoc Analyses with Year, Position and 

Interaction Effects on Stable Isotope Content of Mussel Tissue

Appendix B Table 3. Bush Hill TukeyHSD post hoc Analyses with Position Effects on Stable 

Isotope Content of Mussel Tissue

Appendix B Table 4. Bush Hill TukeyHSD post hoc Analyses with Position, year and interaction 

Effects on Stable Isotope Content of Mussel Tissue

Appendix B Table 5. Bush Hill TukeyHSD post hoc Analyses of Four Mussel Tissue Trophic 

Source Positions with Position Effects

Appendix B Table 6 Brine Pool ANOVA with Position Effects in Stable Isotopes of Mussel 

Tissue with Five Outliers Excluded

Appendix B Table 7. Brine Pool TukeyHSD post hoc Analyses of four Mussel Tissue Trophic 

Source Positions with Position Effects

Appendix B Table 8. Brine Pool TukeyHSD post hoc Analyses of four Mussel Tissue Trophic 

Sources with Year, Position and Interaction Effects

Appendix B Table 9. Bush Hill TukeyHSD post hoc Analyses of four Mussel Tissue Trophic 

Sources for Bed 1 with Year, Position and Interactions Effects
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