OCS Study 

MMS 2010-002 

 

Coastal Marine Institute 

Proof of Concept for Platform Recruited 

Fish, Phase I: Do Platforms Provide 

Habitat for Subadult Red Snapper? 

Cooperative Agreement

Coastal Marine Institute 

Louisiana State University 

U.S. Department of the Interior

Minerals Management Service 

Gulf of Mexico OCS Region 
U.S. Department of the Interior Cooperative Agreement Minerals Management Service Coastal Marine Institute 

Gulf of Mexico OCS Region Louisiana State University 

OCS Study 

 MMS 2010-002 

Coastal Marine Institute 

Proof of Concept for Platform Recruited 

Fish, Phase I: Do Platforms Provide 

Habitat for Subadult Red Snapper? 

Authors 

Lauren K. Nowling 

James H. Cowan, Jr. 

Richard F. Shaw 

February 2010 

Prepared under MMS Cooperative Agreement 

1435-99-CA-30951-18181 

by 

Louisiana State University 

Coastal Fisheries Institute 

School of the Coast and Environment 

Baton Rouge, Louisiana 70803 

Published by 
iii 

DISCLAIMER 

This report was prepared under contract between the Minerals Management Service (MMS) and 

Louisiana State University. This report has been technically reviewed by the MMS, and it has 

been approved for publication. Approval does not signify that the contents necessarily reflect the 

view and policies of the MMS, nor does mention of trade names or commercial products 

constitute endorsement or recommendation for use. It is, however, exempt from review and 

compliance with MMS editorial standards. 

REPORT AVAILABILITY 

This report is available only in compact disc format from the Minerals Management Service, 

Gulf of Mexico OCS Region, at a charge of $15.00, by referencing OCS Study MMS 2010-002. 

The report may be downloaded from the MMS website through the Environmental Studies 

Program Information System (ESPIS). You will be able to obtain this report also from the 

National Technical Information Service in the near future. Here are the addresses. You may 

also inspect copies at selected Federal Depository Libraries. 

Minerals Management Service U.S. Department of Commerce 

Gulf of Mexico OCS Region National Technical Information Service 

Public Information Office (MS 5034) 5285 Port Royal Road 

1201 Elmwood Park Boulevard Springfield, Virginia 22161 

New Orleans, Louisiana 70123-2394 Phone: (703) 605-6040 

Telephone requests may be placed at Fax: (703) 605-6900 

(504) 736-2519, 1-800-200-GULF, or Email: bookstore@ntis.gov

Rush Orders: 1-800-553-6847 

Fax: (504) 736-2620 

CITATION 

Suggested citation: 

Nowling, L.K., J.H. Cowan, Jr., and R.F. Shaw. 2010. Proof of concept for platform recruited 

reef fish, Phase 1: Do platforms provide habitat for subadult red snapper? U.S. Dept. of 

the Interior, Minerals Management Service, Gulf of Mexico OCS Region, New Orleans, 

LA. OCS Study MMS 2010-002. xvii + 69 pp. 

ABOUT THE COVER 

Day and night images of the semisubmersible petroleum platform “Nakika” located in the Gulf 

of Mexico in Mississippi Canyon Block 474. Images by David L. Nieland, Louisiana Sea Grant 

College Program, Louisiana State University. 
v 

This report represents a Master's Thesis 

Submitted to the Graduate Faculty of the 

Louisiana State University and 

Agricultural and Mechanical College 

in partial fulfillment of the 

requirements for the degree of 

Master of Science 

in 

The Department of Oceanography and Coastal Sciences 

by 

Lauren Kay Nowling 

B.S., Auburn University, 2001 

December 2005 

Major Advisor: James H. Cowan, Jr. 

Committee Members: Richard F. Shaw 

 Sam Bentley 

 Barry Moser 
vii

ACKNOWLEDGMENTS

We would like to acknowledge and sincerely thank our colleagues at the University of Hawaii, 

Dr. Robert Gauldie, Dr. Eric DeCarlo, and Yvonne Perry, for analyzing our fish otolith samples 

for trace element isotope ratios using Inductivity Coupled Plasma-Mass Spectrometry. We 

would also like to thank Dr. Khal Spencer (Los Alamos National Laboratory, Los Alamos, NM) 

for his help originating the otolith digestion procedure for this study. We are indebted to the 

recreational charter boat captains, Mike Thierry of Dauphin Island, Alabama, and Steve Tomeny 

of Port Fourchon, Louisiana, that allowed the collection of red snapper. We would also like to 

acknowledge Dr. Will Patterson and Dr. Walter Ingram for their advice on this project. Finally, 

we would like to thank Drs. Ann S. Bull and James Sinclair, Mr. Gregory S. Boland, and an 

anonymous MMS reviewer for their constructive criticism of this manuscript. 
ix

TABLE OF CONTENTS 

Page 

FIGURES....................................................................................................................................... xi 

TABLES ....................................................................................................................................... xv 

ABSTRACT................................................................................................................................ xvii 

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

Red Snapper (Lutjanus campechanus)...................................................................................... 1 

Red Snapper Site Fidelity ......................................................................................................... 1 

Oil and Gas Platforms in the Gulf of Mexico........................................................................... 2 

Otoliths...................................................................................................................................... 3 

Otolith Microchemistry............................................................................................................. 3 

Incorporation of Elements into Otoliths ................................................................................... 5 

Otoliths as Natural Tracers ....................................................................................................... 6 

Objectives ................................................................................................................................. 7 

Hypotheses................................................................................................................................ 8 

MATERIALS AND METHODS.................................................................................................... 9 

Red Snapper Collection ............................................................................................................ 9 

Otolith Removal...................................................................................................................... 11 

Otolith Digestion Procedure of the University of Hawaii at Manoa ...................................... 12 

Inductively Coupled Plasma-Mass Spectrometry................................................................... 15 

Statistical Treatment of Data .................................................................................................. 17 

RESULTS ..................................................................................................................................... 19 

Simple Linear Regression....................................................................................................... 20 

Multivariate Analysis of Variance (MANOVA) .................................................................... 21 

Principal Components Analysis.............................................................................................. 26 

Linear and Stepwise Discriminant Analysis........................................................................... 28 

Canonical Discriminant Analysis (CDA) ............................................................................... 33 

Possible Influence of the Mississippi River............................................................................ 40 

Trends of Important Elements with Red Snapper Sex and Total Length ............................... 46 

DISCUSSION............................................................................................................................... 51 

CONCLUSIONS........................................................................................................................... 56 

REFERENCES ............................................................................................................................. 59 

APPENDIX: MORPHOMETRIC AND ICP-MS SUMMARY TABLES................................... 65 
xi

FIGURES 

Page 

Figure 1. Map of study area where red snapper were collected from artificial reefs in 

the Hugh Swingle Permit Area off Alabama (modified from Patterson, 

1999). ........................................................................................................................ 10 

Figure 2. Map of study area where red snapper were collected from oil and gas 

platforms off Louisiana in the South Timbalier Lease Block (taken from 

Peabody, 2004). ........................................................................................................ 10 

Figure 3. Study area south of Port Fourchon, Louisiana, for both artificial reef and oil 

and gas platform red snapper samples (taken from Stanley and Wilson, 

1990). ........................................................................................................................ 11 

Figure 4. Addition of DI water to beakers and otoliths for rinsing off surface 

contaminants (Yvonne Perry—University of Hawaii at Manoa). ........................... 13 

Figure 5. Heat lamps and units used to dry down otolith samples after addition of acid 

(University of Hawaii at Manoa).............................................................................. 13 

Figure 6. MDS 2100 Microwave Digestion System® used to dissolve otolith samples 

and blanks (University of Hawaii at Manoa)............................................................ 14 

Figure 7. ICP-MS diagram showing 6 main systems (taken from Thomas, 2001a)................ 15 

Figure 8. Fisons/VG PlasmaQuad® inductively coupled plasma-mass spectrometer 

used at the University of Hawaii at Manoa............................................................... 17 

Figure 9. Length frequency distribution for red snapper from all study sites combined 

(N = 98)..................................................................................................................... 19

Figure 10. Linear regression relating otolith weight (g) to fish weight (g) (Wt = weight)........ 20 

Figure 11. Comparison of means of 15 elements from otoliths analyzed for all three 

study sites.................................................................................................................. 21 

Figure 12. Enlarged view of comparison of means of 15 elements from otoliths 

analyzed for all three study sites to emphasize concentrations of trace 

elements found in lower concentrations in the previous figure................................ 22 

Figure 13. Further enlargement of the comparison of means of 15 elements from 

otoliths analyzed for all three study sites to emphasize concentrations of 

trace elements found in lower concentrations in the previous figure. ...................... 22 

Figure 14. Comparison of means of elemental concentration in otoliths from Louisiana 

oil and gas platforms and Louisiana artificial reefs.................................................. 23 

Figure 15. Enlarged view of comparison of means of elemental concentration in otoliths 

from Louisiana oil and gas platforms and Louisiana artificial reefs to 

emphasize concentrations of trace elements found in lower concentrations in 

the previous figure. ................................................................................................... 23 
xii

Figure 16. Further enlargement of the comparison of means of elemental concentration 

in otoliths from Louisiana oil and gas platforms and Louisiana artificial reefs 

to emphasize concentrations of trace elements found in lower concentrations 

in the previous figure................................................................................................ 24 

Figure 17. Comparison of means of elemental concentration in otoliths from Louisiana 

artificial reefs and Alabama artificial reefs. ............................................................. 25 

Figure 18. Enlarged view of comparison of means of elemental concentration in otoliths 

from Louisiana artificial reefs and Alabama artificial reefs to emphasize 

concentrations of trace elements found in lower concentrations in the 

previous figure. ......................................................................................................... 25 

Figure 19. Further enlargement of the comparison of means of elemental concentration 

in otoliths from Louisiana artificial reefs and Alabama artificial reefs to 

emphasize concentrations of trace elements found in lower concentrations in 

the previous figure. ................................................................................................... 26 

Figure 20. Scatter plot of principal component score 1 versus principal component score 

2 with observations labeled by site (site 1 = LA plat.; site 2 = LA reefs; site 3 

= AL reefs)................................................................................................................ 28 

Figure 21. Vertical bar graph of canonical variable 1, with observations separated by 

site, for a comparison of elemental concentration of otoliths collected from 

all three study sites.................................................................................................... 35 

Figure 22. A scatter plot of canonical variable 1 versus canonical variable 2, with 

observations separated by site, for a comparison of elemental concentration 

of otoliths collected from all three study sites. ......................................................... 36 

Figure 23. Vertical bar graph of canonical variable 1, with observations separated by 

site, for a comparison of elemental concentration of otoliths collected from 

Louisiana oil and gas platforms (site 1) and Louisiana artificial reefs (site 2)......... 38 

Figure 24. Vertical bar graph of canonical variable 1, with observations separated by 

site, for a comparison of elemental concentration of otoliths collected from 

Louisiana artificial reefs (site 2) and Alabama artificial reefs (site 3). .................... 40 

Figure 25. Cadmium 114 concentration in otoliths all three study sites showing a 

geographical distribution with higher concentrations west of the Mississippi 

River. ........................................................................................................................ 41 

Figure 26. Copper 65 concentration in otoliths at all three study sites showing a 

geographical distribution with higher concentrations west of the Mississippi 

River. ........................................................................................................................ 42 

Figure 27. Uranium 238 concentration in otoliths at all three study sites showing a 

geographical distribution with higher concentrations west of the Mississippi 

River. ........................................................................................................................ 42 

Figure 28. Silver 107 and 109 concentrations in otoliths at all three study sites showing 

a geographical distribution with higher concentrations west of the 

Mississippi River. ..................................................................................................... 43 
xiii

Figure 29. Cobalt 59 concentration in otoliths at all three study sites showing a 

geographical distribution with higher concentrations east of the Mississippi 

River. ........................................................................................................................ 43 

Figure 30. Nickel 62 concentration in otoliths at all three study sites showing a 

geographical distribution with higher concentrations east of the Mississippi 

River. ........................................................................................................................ 44 

Figure 31. Lead 206 concentration in otoliths at all three study sites showing no 

significant geographical distribution but having higher concentrations on 

Louisiana oil and gas platforms................................................................................ 44 

Figure 32. Lead 207 concentration in otoliths at all three study sites showing no 

significant geographical distribution but having higher concentrations on 

Louisiana oil and gas platforms................................................................................ 45 

Figure 33. Lead 208 concentration in otoliths at all three study sites showing no 

significant geographical distribution but having higher concentrations on 

Louisiana oil and gas platforms................................................................................ 45 

Figure 34. Vanadium 51 concentration in otoliths at all three study sites showing no 

significant geographical distribution but having higher concentrations on 

Louisiana oil and gas platforms................................................................................ 46 

Figure 35. Zinc 64 and Zinc 66 concentrations in otoliths at all three study sites 

showing no clear geographical distribution but having higher concentrations 

on both artificial reef sites. ....................................................................................... 46 

Figure 36. Vanadium 51 concentration in otoliths at all three study sites showing higher 

concentrations in male red snapper........................................................................... 47 

Figure 37. Uranium 238 concentration in otoliths at all three study sites showing higher 

concentrations in male red snapper........................................................................... 48 

Figure 38. Vanadium 51 concentration in otoliths at all three study sites showing no 

significant trend with red snapper total length. ........................................................ 48 

Figure 39. Lead 206 concentration in otoliths at all three study sites showing no 

significant trend with red snapper total length. ........................................................ 49 

Figure 40. Uranium 238 concentration in otoliths at all three study sites showing no 

significant trend with red snapper total length. ........................................................ 49 
xv

TABLES 

Page 

Table 1. Eigenvalues of the correlation matrix from the PCA showing cumulative 

percent of variation explained by each principal component score.......................... 27 

Table 2. Eigenvectors of principal components 1 and 2 for all 15 elements analyzed........... 27 

Table 3. Multivariate statistics and F approximations for linear discriminant analysis 

comparing the elemental concentration of otoliths among all study sites. ............... 29 

Table 4. Number of observations and percent of otoliths classified into each of 3 sites 

using linear discriminant analysis and cross-validation. .......................................... 29 

Table 5. Number of observations and percent of otoliths classified into each of 3 sites 

after rerunning the linear discriminant analysis using only the 9 most 

important elements.................................................................................................... 30 

Table 6. Multivariate statistics and exact F statistics for linear discriminant analysis 

comparing the elemental concentration of otoliths from Louisiana oil and gas 

platforms (site 1) and Louisiana artificial reefs (site 2)............................................ 31 

Table 7. Number of observations and percent of otoliths classified into Louisiana oil 

and gas platforms (site 1) and Louisiana artificial reefs (site 2) using linear 

discriminant analysis and cross-validation. .............................................................. 31 

Table 8. Number of observations and percent of otoliths classified into Louisiana oil 

and gas platforms (site 1) and Louisiana artificial reefs (site 2) after 

rerunning the linear discriminant analysis using only the 6 most important 

elements. ................................................................................................................... 32 

Table 9. Multivariate statistics and exact F statistics for linear discriminant analysis 

comparing the elemental concentration of otoliths from Louisiana (site 2) 

and Alabama artificial reefs (site 3).......................................................................... 32 

Table 10. Number of observations and percent of otoliths classified into Louisiana 

artificial reefs (site 2) and Alabama artificial reefs (site 3) using linear 

discriminant analysis and cross-validation. .............................................................. 33 

Table 11. Number of observations and percent of otoliths classified into Louisiana 

artificial reefs (site 2) and Alabama artificial reefs (site 3) after rerunning the 

original linear discriminant analysis using only the 6 most important 

elements. ................................................................................................................... 33 

Table 12. Pooled with-in class standardized canonical coefficients for canonical 

discriminant analysis comparing elemental concentration of otoliths from all 

3 sites. ....................................................................................................................... 34 

Table 13. Pooled with-in class standardized canonical coefficients for canonical 

discriminant analysis comparing elemental concentration of otoliths from 

Louisiana oil and gas platforms and Louisiana artificial reefs. ................................ 37 
xvi

Table 14. Pooled with-in class standardized canonical coefficients for canonical 

discriminant analysis comparing elemental concentration of otoliths from 

Louisiana artificial reefs and Alabama artificial reefs.............................................. 39 
xvii

ABSTRACT 

There are currently over 3,500 oil and gas structures in the northern Gulf of Mexico (Gulf). 

Platform operations and their prior drilling operations produce trace amounts of lead, barium, 

vanadium, and lanthanum residues that are leached into the surrounding waters and are deposited 

on the sea floor. These residues have isotopic ratios different from those typical of the Gulf 

seafloor and can be used as “markers” if they become incorporated into hard-parts, such as 

otoliths, of fishes associated with oil and gas platforms. These markers are harmless because 

they occur at extremely low concentrations and are bound to the inedible otolith, or ear bone, of 

the fish. From 2002 to 2004, 115 red snapper were collected from oil and gas platforms and 

artificial reefs off Louisiana and Alabama. Otoliths were removed and analyzed from 98 of 

those collected using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The objective 

of this study was to determine if a trace element isotope ratio fingerprint could be detected and 

described as unique to red snapper inhabiting the platforms. Stepwise and canonical 

discriminant function analyses were used to compare red snapper otolith fingerprints from on 

and off platforms, and from east and west of the Mississippi River. Classification accuracies 

based on the probability of an individual fish being correctly classified into the habitat from 

which it was sampled were over 90% for each of the two main comparisons. When comparing 

the elemental composition of red snapper otoliths from Louisiana oil and gas structures and 

Louisiana artificial reefs, the classification accuracy was 93.75%. When comparing the 

elemental composition of red snapper otoliths from Louisiana artificial reefs and Alabama 

artificial reefs, the classification accuracy was 91.06%. Vanadium 51, Lead 206, Lead 207, and 

Lead 208 all appear to be linked with oil and gas structures or their prior drilling operations, as 

the concentrations of these four elements or isotopes were significantly higher in otoliths 

sampled from platforms in Louisiana than in otoliths sampled from artificial reefs in either 

Louisiana or Alabama. These platform isotopic fingerprint results indicate that it may be 

possible to sub-sample from otoliths of adult fish in Phase II of our study to determine agespecific habitat affinity, and to determine if the new recruits now expanding into the eastern Gulf 

as the red snapper population rebuilds were associated with oil and gas platforms during some 

portion of their early life. We reason that if oil and gas platforms provide high quality habitat 

and refuge from shrimp trawls for juveniles, then high numbers of adult recruits should be 

derived from the pool of individuals who utilize said habitat, particularly off Louisiana and other 

areas where natural habitat is scarce. By focusing on recruitment to adult populations in Phase 

II, our quantitative approach will provide a more direct assessment of the relative contribution of 

different juvenile reef fish habitats than is possible via traditional habitat suitability approaches. 

Therefore it may be possible in future studies to determine if oil and gas platforms contribute 

disproportionately to the survival of juvenile and adult red snapper in other areas of the Gulf. If 

this contribution proves to be true, then platforms could be considered essential fish habitat and, 

therefore, a potentially viable management tool for stock rebuilding. 
1 

INTRODUCTION 

RED SNAPPER (LUTJANUS CAMPECHANUS) 

The Magnuson-Stevens Fishery Conservation and Management Act requires that fishery 

management plans identify and describe essential fish habitat (EFH), adverse impacts on EFH 

(including the effects from fishing), and actions to conserve and enhance EFH (Gallaway et al., 

1999). One of the most pressing federal fisheries management concerns in the Gulf of Mexico 

(Gulf) is the overfished status of red snapper Lutjanus campechanus (Schmitten, 1999). For 

example, National Marine Fisheries Service (NMFS) data collection provides evidence that the 

primary cause of overfishing on red snapper is bycatch of age-0 and age-1 (juvenile) red snapper 

by shrimp trawls (Schirripa and Legault, 1999). It is the hope of all concerned that the future 

identification, conservation, and enhancement of red snapper EFH may accomplish the 

following: 1) act to reduce the impacts of shrimp trawl mortality on juvenile red snapper 

(Schmitten, 1999); 2) enhance or protect forage and spawning areas; and 3) identify sources of 

recruits. The ongoing research reported here within begins to address several of these issues. 

Red snapper support the most economically valuable finfish fishery in the Gulf and are 

among the most targeted fishes by commercial and recreational fishers (Goodyear, 1995). The 

red snapper stock in the Gulf has been subjected to heavy fishing pressure since the 1800’s and is 

currently estimated to be severely overfished (Goodyear, 1995; Schirripa and Legault, 1999). 

Since 1991 both commercial and recreational fisheries have been constrained by size limits, creel 

or trip limits, and quotas as established by the Gulf of Mexico Fishery Management Council 

(Wilson and Nieland, 2001). 

Red snapper are demersal fish that are distributed along the continental shelf throughout the 

Gulf. Young red snapper spend most of their first year of life over sand and mud bottoms on the 

shrimping grounds in the northern Gulf, after which they move offshore to reef environments 

(Patterson et al., 1998). Adult red snapper are found in deeper offshore waters near coral, rocks, 

banks, outcrops, and manmade submerged objects such as oil and gas platforms and artificial 

reefs (Workman and Foster, 1994). They are reef-associated fish and only utilize reefs (natural 

or artificial) for certain parts of their life history. It has been noted that red snapper may use 

reefs for protection from predation but move off the reef to spawn and feed (Gallaway et al., 

1981; Bradley and Bryan, 1975). Once red snapper recruit to offshore reefs, such as oil and gas 

platforms, they have been found to feed primarily on benthic organisms in the surrounding 

sediment. As these fish mature, they tend to become more piscivorous, even feeding on juvenile 

red snapper (McCawley, 2003). 

The way in which different life stages of red snapper utilize these various habitats as they 

grow, e.g., for feeding, protection or both, is unknown. Information on age-0 red snapper is 

sparse, especially at first settlement (Szedlmayer and Conti, 1999). However, it has been 

demonstrated that as red snapper mature they show strong preferences for natural and artificial 

habitat with vertical relief (Gallaway et al., 1999; Szedlmayer and Conti, 1999; Workman and 

Foster, 1994). It is these areas that are considered by many to constitute red snapper EFH. 

RED SNAPPER SITE FIDELITY

Many studies have been performed to measure red snapper site fidelity and results have been 

variable. Some studies off of the Alabama coast have indicated that red snapper have high site 

fidelity to reefs and remain there for long periods of time. Szedlmayer and Shipp (1994) 
2 

performed a mark and recapture study off of coastal Alabama in 1994. They concluded that red 

snapper showed a high degree of residence to artificial reefs, with most fish recaptured within 2 

km of their release site, even after extended periods of time. In contrast, a more extensive study 

by Patterson et al. (2001) off of Alabama found that only 55% of recaptures not at liberty during 

hurricanes had remained at their release sites. They demonstrated that over time, even fish not 

exposed to hurricanes had a relatively low probability of remaining at their release sites 

(Patterson et al., 2001). 

Opinions of red snapper site fidelity have begun to change. It is now believed by some that 

red snapper site fidelity, considered to be a reef-associated rather than reef-dependant species, is 

much lower than previously suggested. Red snapper site fidelity on Louisiana oil and gas 

platform may be dependent upon the time scale being examined (Peabody, 2004; Peabody and 

Wilson, 2006). Results showed that red snapper displayed high short-term fidelity to a particular 

oil and gas platforms on the order of days, weeks, to even a month or two (Peabody, 2004; 

Peabody and Wilson, 2006). However, in the longer-term, over a period of a few or more 

months, red snapper exhibited low fidelity and it is unlikely that they will remain at any one 

particular location for more than a few consecutive months (Peabody, 2004; Peabody and 

Wilson, 2006). 

OIL AND GAS PLATFORMS IN THE GULF OF MEXICO

In the waters of the northern Gulf there are over 3,500 oil and gas structures (Stanley and 

Wilson, 1998). Since the first platform was installed, fishers and scientists have been aware of 

their associated nekton assemblages (Stanley and Wilson, 1997). They act as artificial reefs by: 

1) providing habitat that potentially increases the growth and survival of individuals; 2) affording 

shelter for protection from predation; 3) providing suitable spawning substrate; and, 4) acting as 

a visual attractant for organisms not otherwise dependent upon hard bottom (Gallaway et al., 

1981). Oil and gas platforms differ from most natural habitats and from traditional artificial 

reefs in that their vertical profile extends throughout the entire water column into the photic zone 

(Stanley and Wilson, 1991). Increased habitat quality on, or immediately around, oil and gas 

platforms is believed to be derived from increased in situ food production associated with 

encrustation by fouling organisms, but this concept has not been proven. Any amount of shell 

pad on the bottom for support of the structure or platform related debris is also believed to 

increase habitat quality on, or around, oil and gas platforms. 

Due to the noted presence of red snapper around oil and gas platforms, recreational and 

commercial fishing efforts have continually been concentrated in those areas. Gallaway et al., 

(1981) reported that of all species caught by bottom fishing in the Buccaneer gas and oil field 

area off Texas, red snapper comprised 80% of the catch (Gallaway et al., 1981). Later, Stanley 

and Wilson (1990) showed that a high diversity of fish (including red snapper) existed around oil 

and gas platforms off coastal Louisiana. They reported that catch rates of private vessel anglers 

and charter boat operators around oil and gas platforms in the northern Gulf were high, with their 

effort targeted towards red snapper while offshore bottom fishing (Stanley and Wilson, 1990). 

Artificial reefs, such as oil and gas platforms, may be useful tools for fishery managers if 

they increase fisheries production, but many researchers question whether or not they are a 

positive influence on reef stock dynamics (Bohnsack, 1989; Grossman et al., 1997). There have 

been doubts about whether or not they produce or attract fish and the resolution to this question 

is essential to the management of reef fish stocks (Seaman, 1997; Lindberg, 1997). If they 

indeed constitute EFH for reef fish, then they can be considered as viable management tools. If 
3 

they are simply attracting fish to the area, they may merely promote overfishing. Bohnsack 

(1989) offered a conceptual model which inferred that increased production is most likely at 

locations isolated from natural reefs, and for habitat-limited, demersal, philopatric, territorial, 

and obligatory reef species. Species that are recruitment-limited, pelagic, highly mobile, and 

either partially reef-dependant or opportunistic reef fish may be present on platforms simply due 

to attraction. This is particularly true in locations with abundant natural reef habitat and/or 

where exploitation rates are high. Therefore, the question of habitat limitation lies at the heart of 

the artificial reef controversy (Grossman et al., 1997). Bohnsack (1989) also stated that artificial 

reefs are unlikely to benefit heavily exploited or overfished populations without other 

management actions. Currently, the Gulf Management Council does not include oil and gas 

platforms, or any other artificial reef habitat, in their treatment of EFH. 

OTOLITHS

The teleost otolith is composed of calcium carbonate, mainly in the form of aragonite, and an 

organic matrix including proteins, carbohydrates and lipids, as well as trace elements that are 

deposited during otolith deposition (Takagi et al., 2000). Otoliths are located in the head of 

fishes in three pairs (sagittae, lapilli, and asteriscii) and function in the acoustico-lateralis system. 

They grow, or accrete, relative to somatic growth, forming concentric opaque and translucent 

rings; increments in otoliths can be deposited sub-daily, daily, and annually. 

Otoliths are bathed in endolymph within the inner ear sacs and the otolith grows without 

touching any cells (Takagi, 2002). It is generally believed that the organic matrix is first 

constructed, followed by aragonite crystallization. The cells of the membranous wall of the 

otolith organ synthesize components of the otolith matrix. The components are secreted into the 

endolymph, a framework is constructed and the aragonite crystallization occurs on that 

framework (Takagi et al., 2000). Therefore, the calcification process of otoliths is heavily 

dependent upon the composition of the endolymphatic fluid (Campana, 1999). The key 

regulating factors appear to be pH of the endolymph, which is determined by the concentration 

of bicarbonate ions in the endolymph (Romanek and Gauldie, 1996; Payan et al., 1997, 1998), 

and temperature. Calcium carbonate can crystallize as any one of three crystal morphs (calcite, 

aragonite or vaterite) and the rate and type of calcium carbonate crystals formed in otoliths is 

regulated by proteins (Campana, 1999). Aragonite is the norm for sagittae and lapilli otoliths, 

while most asteriscii are made of vaterite. Strontium carbonate is virtually isostructural with 

aragonite making substitution of Sr ions for Ca in aragonite very likely. Ions similar to Ca and 

Sr, such as other alkaline earth metals (Mg and Ba), can also be substituted for Ca in the 

aragonite matrix. 

OTOLITH MICROCHEMISTRY

In the field of fisheries biology and management, the analysis of otolith microstructure is a 

quickly expanding field of prime importance (Payan et al., 1997). Fish otoliths have 

traditionally been used as a hard part to age fish, but recent research indicates that they may also 

serve as ideal natural markers of individual fish or fish populations (Campana et al., 1994). 

Some goals of otolith research focus on transport, movement, and mixing hypotheses, as well as 

understanding the mechanisms by which minor and trace elements are incorporated into otoliths, 

and developing tools with which to measure the elements present. Secor et al. (1995b) stated 

that concerted efforts at the suborganismal and organismal level are required to determine the 

effect of the environment on otolith composition. 
4 

Early studies using otolith chemistry to differentiate between fish populations or stocks were 

carried out through the use of proton and electron microprobe analysis. Microprobe analysis can 

be useful to elucidate the elemental composition of calcified tissues of fish. The nondestructive 

nature and the multi-elemental sensitivity of these techniques make them useful. With this 

method samples are irradiated with photons or electrons or even protons, which results in the 

emission of x-rays. The wavelength and energy dispersion of the resultant x-rays are specific to 

each element and the amount of that element being analyzed (Radtke and Shafer, 1992). 

Another advantage of electron microprobe analysis is that the electron probe provides good 

resolution over small spatial scales (5 μm), as is needed to trace daily and sub-daily chemical 

histories in larval and juvenile fish otoliths. While this method is widely used, it does have a few 

drawbacks. This instrumentation is only capable of detecting a small number of elements, one to 

six. Recent studies indicate that a much broader suite of elements exist in otoliths, but at 

concentrations well below that detectable with the electron microprobe. There can be a great 

deal of variation in sample preparation, equipment and experimental design and there is a need to 

standardize techniques (Radtke and Shafer, 1992). 

One of the most recent techniques that is commonly used to assay otoliths is Inductively 

Coupled Plasma-Mass Spectrometry (ICP-MS). This technique has been extensively developed 

as a standard first approach for bulk analysis of micro-composition and has shown that trace 

elements are present in otoliths down to parts per trillion (ppt) (Thresher, 1999). It involves the 

use of argon gas plasma produced in a quartz torch at temperatures of greater than 5000o

 K. 

According to Thresher (1999), these high temperatures induce intense spectral lines from a wide 

range of elements, which facilitates multi-element analysis. The advantages of ICP-MS over 

other approaches include the ability to simultaneously assay numerous elements (and isotopes of 

elements) quickly and inexpensively, with a sensitivity that matches or exceeds that of x-ray 

microanalysis, the proton microprobe, neutron activation analysis, atomic absorption 

spectroscopy, inductively-coupled plasma atomic emission spectroscopy, and x-ray fluorescence 

(Campana and Gagne, 1995). 

Isotope dilution ICP-MS (ID-ICPMS), a variant of ICP-MS, is often used and is thought to 

be the most accurate of the otolith analytical techniques currently available. ID-ICPMS consists 

of dissolving whole otoliths into solution and analyzing them using the inductively coupled 

plasma-mass spectrometer. Advantages of whole-otolith assays include ease of preparation, 

absence of error associated with sampling or identifying growth increments, and the availability 

of accurate and precise assay protocols (Campana, 1999). The major disadvantage is associated 

with the inability to take advantage of the chronological growth sequence recorded in the otolith. 

In addition, another disadvantage is if the otolith contains exceptionally high concentrations of 

the elements of interest, it can overload the detector as it passes through the machine and if there 

is not enough otolith sample left to rerun the analysis, then the specimen data are lost. 

Inductively Coupled Plasma-Mass Spectrometry has emerged as the instrument of choice for 

assays due largely to its capability for rapid and accurate isotopic and elemental assays over a 

wide range of elements and concentrations (Campana, 1999). The use of ID-ICPMS also 

minimizes (although not eliminates) the effects of instrumental drift. 

The elements useful for site-specific, chemical location markers in otoliths are: lead (Pb), 

strontium (Sr), barium (Ba), lanthanum (La), vanadium (V) and the elements of the thoriumuranium (Th-U) series. This group of elements forms carbonates whose crystal structure is 

isostructural with that of aragonite. By having carbonates that are similar in structure to 

aragonite, these elements are embedded in the crystal matrix of the aragonite itself, and therefore 
5 

cannot be mobilized by minor pH changes or by the effects of washing and handling (Proctor and 

Thresher, 1998). Lead is a desirable element to consider as a site-specific marker where there 

has been industrial activity, such as is the case for oil and gas platforms. Combustion engine 

operations, electrical motors and some kinds of metal priming paints result in lead residues, 

although in very small amounts. As early as 1976, higher levels of vanadium in fish tissues and 

higher levels of zinc in bottom-grab sediment samples were found at two platforms versus 

natural reef areas off California (Mearns and Moore, 1976). They speculated that the vanadium 

came from drilling/production operations and that the zinc came from the corrosion and leaching 

of submerged platform structures and resultant metal flakes or metal debris scattered under the 

platforms. The Mearns and Moore (1976) study did not find a significant difference in lead 

concentrations, but lead isotopes were not specifically examined. In the Buccaneer gas and oil 

field study, Gallaway et al. (1981) also stated that observed metal flakes were probably a major 

source of the trace metal contamination of sediments taken adjacent to the platforms. 

A further advantage for this study is that platform drilling operations usually involve the use 

of drilling muds that contain bentonite (mostly the mineral montmorillionite) that has traces of 

rare earths and barium. Drilling muds perform several functions integral to the drilling process. 

The most important of these include transport of drill cuttings to the surface, balance of 

subsurface and formation pressures thus preventing a blowout, and to cool, lubricate and support 

part of the weight of the drill bit and drill pipe (Neff et al., 1987). The industrial lead 

compounds mentioned previously, and montmorillionite, are derived from a single lead mine in 

Wisconsin and a single montmorillionite mine in Wyoming. Both industrial lead compounds and 

montmorillionite-containing bentonite will have mine-specific isotope ratios that reflect the 

unique geochemical histories of the mines involved. It is believed that existing oil and gas 

platform operations, and their prior history of drilling operations, are likely to produce trace 

amounts of residues of Pb, Ba, V, and La whose isotope ratio signatures will be different from 

those typical of the Gulf seafloor away from platforms. We hypothesize that this can be used as 

a “marker” in the otoliths of the sub adult fish that have spent time in close association with oil 

and gas platforms. By a marker, we mean that the elemental fingerprint, which will be at 

extremely low concentrations, is permanently bound to the otolith, a nonedible component of the 

fish. 

INCORPORATION OF ELEMENTS INTO OTOLITHS

Minor and trace elements may be incorporated into otoliths generally by incorporation into 

the otoliths’ protein structure or by replacing calcium, carbon, or phosphorus in the aragonite 

crystals of otoliths (Kalish, 1989). Certain elements may be more labile within otoliths than 

others (Rooker et al., 2001b). Those associated with the organic matrix rather than those that 

substitute for elements within the lattice may be more prone to contamination from, or loss to, 

surrounding solutions. Postmortem handling procedures can cause changes in elemental 

concentrations within both the endolymph and otolith (Proctor and Thresher, 1998). Therefore, 

special care has to be exercised when selecting the elements to be analyzed and when handling, 

storing, and processing the otoliths to minimize the risk of contamination. Rooker et al. (2001b) 

demonstrated that Thunnus albacares otoliths easily acquired trace impurities but acid washing 

was found to successfully remove surface contaminants. 

The pathway of a given element or ion from the environment into the calcium-carbonate 

matrix of the otolith is a multi-stage process and is characterized by a sequence of more or less 

independent barriers (Campana, 1999). The basic pathway of inorganic elements into the otolith 
6 

is from the water to the blood plasma via the gills or intestine, then into the endolymph, and 

finally as a molecular replacement of calcium into the crystallizing otolith (Campana, 1999). 

Elements entering the blood plasma via the gills is the primary method in freshwater fishes, 

while the continual drinking of seawater by marine fish is the main source of elements being 

incorporated in the intestine. It is believed that only a minimal amount of elements are gathered 

from food sources, but the specific portion is unknown (Campana, 1999). 

The intestine-water interface in saltwater fishes is likely one of the biggest barriers to 

elemental uptake. Osmoregulation regulates movement of water-bourne ions into the fish. The 

next barrier is the blood plasma-endolymph interface and cellular transport is responsible for 

moving the water-bourne elements there. Lastly, elements must pass through the endolymphotolith interface, which occurs during the otolith crystallization process. In general, the 

composition of the endolymph appears to be closer to the composition of the otolith than is the 

water or blood plasma (Campana, 1999). Payan et al. (1998) indicated that the elemental 

composition of the endolymph is less affected by starvation than is the plasma. 

OTOLITHS AS NATURAL TRACERS

There are two observations of otoliths that make otolith microchemistry successful: (1) 

otoliths grow throughout the life of the fish and, unlike bone, are metabolically inert; once 

deposited, otolith material is unlikely to be reabsorbed or altered; and, (2) the calcium carbonate 

and trace elements that make up 90% of the otolith appear to be mainly derived from the water 

(Casselman, 1987; Simkiss, 1974). The majority of elements are present at minor (> 100 ppm) 

and trace (< 100 ppm) levels, with many more trace elements presumably awaiting detection 

(Campana, 1999). The minor elements are represented by Na, Sr, K, S, N, Cl, and P, while the 

bulk of the trace elements are present at concentrations of less than about 10 ppm (Campana, 

1999; Rooker et al., 2001a). Broad spectrum elemental assays of otoliths have resulted in trace 

and minor element concentrations of less than 1% of the whole otolith and protein concentrations 

of about 3 to 4% (range of 1 to 8%) (Edmonds et al., 1992). 

The theory behind using otoliths as natural tracers of individual fish or stocks is a fairly 

simple concept. Since the otolith grows continuously throughout a fish’s life, the elemental 

fingerprint of the whole dissolved otolith integrates across the entire lifetime of the fish, and thus 

can be used to distinguish among fish which have experienced different overall environmental 

exposures (Edmonds et al., 1992; Campana and Gagne, 1995). It is thought that the 

environmental information stored upon the formation of fish otoliths is preserved internally, and 

that the otoliths contain not only information about age but also a large amount of other 

ecological data (Kakuta et al., 1997). The elemental composition of otoliths can reflect the 

elemental composition of the water body in which the fish lives, the elemental composition of 

the food of the fish, and the short and long term behavior of the pH of the fish inner ear resulting 

in changes in otolith chemistry in response to food, otolith growth rate, temperature, salinity and 

even stress induced by chasing (Mugiya and Satoh, 1995; Arai et al., 1996). 

The use of otolith elemental fingerprints as natural tags relies upon three central assumptions: 

(1) There are characteristic and reproducible markers for each group of interest. If the elemental 

fingerprints of the groups of interest do not differ significantly, little more can be done; (2) All 

possible groups of reference fish contributing to the population mixture being tested have been 

characterized. Otherwise, uncharacterized groups of fish present in the mixture could be 

mistakenly interpreted as one or more of the reference groups; and, (3) The marker remains 

stable over the temporal interval between characterization and mixing. The potential for drift in 
7 

elemental concentration of otoliths becomes greater as the interval length is extended (Campana, 

1999). 

Campana (1999) showed the extent to which otolith trace element concentration reflects (or 

fails to reflect) that of the ambient water from a comparison of water, blood and otolith 

composition from two very distinct environments: fresh water and salt water. The concentrations 

of many of the most common elements (Ca, Na, K, Mg, Cl) differed substantially between fresh 

and salt water, yet those differences did not appear to be reflected in the otolith when compared. 

Other elements such as P, Cu, and S that are physiologically important appeared to remain 

uninfluenced by the relative concentrations in the environment (Campana, 1999). When 

Campana (1999) examined the corresponding blood concentrations for most of the elements 

noted above, there was little or no difference in plasma concentrations between freshwater and 

saltwater. This stability in blood plasma composition is completely consistent with the strict 

osmoregulatory control required for the fish’s survival. Campana (1999) concluded that because 

the elements deposited in the otolith are derived penultimately from the blood plasma, it is 

clearly unrealistic to expect the otolith content of physiologically regulated elements to reflect 

environmental abundance. Through his comparisons Campana (1999) also concluded that the 

relative concentration of trace elements such as Sr, Zn, Pb, Mn, Ba, and Fe in freshwater and 

marine otoliths is consistent with an environmental effect. While it may be possible to use the 

concentration of any given element to distinguish between groups of fish, it is preferable to use 

all elements at once, e.g., a multivariate elemental fingerprint (Campana et al., 2000). 

OBJECTIVES

The main objective of this project (Phase I for ease of notation) is to test the hypothesis that 

association with oil and gas platforms during early life imparts a detectable “trace element 

isotope ratio fingerprint” in the otoliths of juvenile red snapper, i.e., prespawning adults (age 2 to 

4). If this phase of the project is successful, it will set the stage for future research (Phase II) that 

will test whether adult fishes containing the “platform fingerprint” in their otoliths contribute 

disproportionately to adult stocks on nearby natural and artificial reefs. The rationale for the 

choice of red snapper as a surrogate for reef-associated fish in Phase I of the project is due to 

their site fidelity as well as their recreational and commercial importance. Although there are 

differing conclusions on red snapper site fidelity, we believe that they exhibit the appropriate 

model life history to prove the concept of this study. Patterson et al. (2001) showed that red 

snapper have high site fidelity (70%) during their first years as recruits on offshore habitats, with 

site fidelity decreasing as fish get older. Based on the work by Patterson et al. (2001), we 

believe that red snapper remain at oil and gas platforms long enough to incorporate a signal into 

their otoliths, if one is available. Determination of oil and gas platforms as red snapper EFH 

would be an important tool for fisheries managers and may help to restore stock size. The 

determination of the impacts of artificial reefs (of any type) on the demographics of reefassociated fishes is also a necessary step in moving towards final resolution of the production vs. 

attraction debate. 

The specific objective is as follows: 

To test whether association with oil and gas platforms imparts a detectable “trace element 

isotope ratio fingerprint” in the otoliths of juvenile red snapper, making Phase II possible.
8 

HYPOTHESES

Ho1: Association with oil and gas platforms during early life does not impart a detectable 

“trace element isotope ratio fingerprint” in the otoliths of juvenile reef fishes, represented here 

by red snapper. 

HA1: Association with oil and gas platforms during early life imparts a detectable “trace 

element isotope ratio fingerprint” in the otoliths of juvenile reef fishes, represented here by red 

snapper. 
9 

MATERIALS AND METHODS 

RED SNAPPER COLLECTION

A total of 115 red snapper were collected from both artificial reefs (anything other than oil 

and gas platforms—controls) and oil and gas platforms in Alabama and Louisiana over a twoyear period from 2002 to 2004. The first sampling trip took place on October 18, 2002; fish 

were collected on artificial reefs off coastal Alabama. The red snapper sampled (N = 45) were 

caught aboard the recreational fishing vessel Lady Ann using hook and line. Exact location of 

capture of each fish is unknown but all were caught approximately 15 to 20 miles south of 

Dauphin Island in a grid of artificial reefs located within the Hugh Swingle Permit Area (Figure 

1). Most of the reefs in the Hugh Swingle are constructed from materials of opportunity; no 

reefs in this area are constructed from decommissioned oil and gas platforms. Upon collection, 

measurements of fork length (mm ± 1), total length (mm ± 1), dorsoventral height (distance from 

first dorsal spine to pectoral fin in mm ± 1), sex, and gonad development (if possible) were 

taken. Weight was calculated using a length-weight regression compiled by Patterson (1999) 

and is discussed in more detail below. Once these measurements were recorded, fish were 

cleaned and the head of each fish was removed using an electrical fillet knife. The heads were 

placed in individual plastic storage bags and then stored on ice until they were returned to 

Louisiana State University (LSU) for immediate otolith removal. For otolith processing at a later 

date, heads were kept frozen to minimize contamination. 

We used equations in this study to convert red snapper length (mm) to weight (kg) derived by 

Patterson (1999) based upon fish caught off Alabama because these charter boat-caught fish 

were filleted at the dock before being donated to us. He also fitted weight-length equations for 

each sex. Since only length measurements were taken as part of this study, the equations 

formulated by Patterson (1999) were used to calculate weights for male and female red snapper. 

The equations used are as follows: 

Male: Wt = 5.32 x 10-9*(TL)3.16

Female: Wt = 3.26 x 10-9*(TL)3.23

Although these equations were formulated using red snapper caught off Alabama, they were 

used to calculate weights for both Louisiana and Alabama samples in this study. 

The next sampling trip occurred on May 28, 2003. Thirty-five red snapper were collected on 

oil and gas platforms aboard a recreational charter boat in the South Timbalier Circle off the 

Louisiana coast, which is located in Minerals Management Service lease blocks 128, 134, 135, 

151 and 152 (Figure 2). These fish were caught using hook and line. Upon collection, 

measurements of fork length (mm), total length (mm), dorsoventral height (mm), sex, and gonad 

development (if possible) were taken. Once these measurements were obtained, the heads of the 

red snapper were then removed and stored in individual plastic storage bags on ice. The heads 

were returned to LSU for otolith removal. 
Figure 1. Map of study area where red snapper were 

collected from artificial reefs in the Hugh 

Swingle Permit Area off Alabama (modified 

from Patterson, 1999). 

Figure 2. Map of study area where red snapper were collected from oil and gas 

platforms off Louisiana in the South Timbalier Lease Block (taken from 

Peabody, 2004). 

In addition, thirty-five red snapper were collected on artificial reefs south of Port Fourchon, 

Louisiana, during two trips that took place on September 19, 2003, and October 17, 2003 (Figure 

3). The first ten red snapper were collected on the September trip using hook and line while 

fishing on sunken armored personnel carriers (APC’s), boats, and barges in the South Timbalier 

10 
Lease Block. At the time of capture none of the red snapper collected from artificial reefs off 

Louisiana were obtained from decommissioned oil and gas platforms. The other twenty-five 

specimens were collected using hook and line on the October trip while fishing in deeper water 

within the Grand Isle Lease Block. The same measurements of fork length (mm), total length 

(mm), dorsoventral height (mm), sex, and gonad development (if possible) were taken prior to 

cleaning. The heads were then removed using a fillet knife and were stored individually in 

plastic storage bags for transport to LSU. Although the sampling design called for collection of 

red snapper on oil and gas platforms east of the Mississippi River delta, we had difficulty 

catching them in the spring of 2004. Several attempts were made to collect these fish on 

recreational and private fishing trips once the red snapper season opened in April but all were 

unsuccessful. Therefore, that particular sample was excluded from the study. 

Figure 3. Study area south of Port Fourchon, Louisiana, for both artificial reef and oil 

and gas platform red snapper samples (taken from Stanley and Wilson, 

1990).

OTOLITH REMOVAL

For all 115 red snapper collected, the heads were thawed in the laboratory in sealed plastic 

bags inside collection containers. Dissection and handling tools were cleaned first with 18 

megohm water, followed by rinsing with quartz-distilled water (QED). Laboratory surfaces were 

cleaned with de-ionized water (DI) and covered with clean paper. All samples were dissected in 

a clean (Class-100) laboratory in a High Efficiency Particulate Air (HEPA) filtered air module. 

All tools and specimens were handled with clean-room grade vinyl gloves. 

Otoliths were removed in the HEPA-filtered air module using a chisel and Teflon forceps. 

The red snapper head was laid in a collection container within the air module. The operculum 

was pulled back and the gill rakers were cut and moved to the side using the chisel. The cavity 

containing the sagittal otolith was then exposed and pried open with the chisel. The otolith was 

11 
12 

removed using plastic forceps. This procedure was performed on each side of the head to obtain 

both of the sagittal otoliths. Once an otolith was removed it was wiped clean using sterile 

laboratory wipes and then placed in a clean, labeled glass vial. Vials were packed in boxes and 

made ready for shipment. Since all mass spectrometry of the otoliths was performed in 

laboratories at the University of Hawaii at Manoa, otoliths were immediately shipped to 

Honolulu. 

OTOLITH DIGESTION PROCEDURE OF THE UNIVERSITY OF HAWAII AT MANOA

Once otoliths arrived at the University of Hawaii, they were prepped for digestion. They 

were transferred from the glass vials used for shipping to acid washed petri-dishes. Otoliths 

from artificial reefs in Louisiana (fall 2003 sample) were the first to be digested. The first 15 

pairs of otoliths (AR-03-LA to AR-18-LA) from this sample were processed using the digestion 

procedure described below. This procedure was later modified and those modifications are 

described in full detail. 

Original Otolith Digestion Procedure 

One otolith from each pair was placed into a tared plastic beaker and weighed and the weight 

(g ± 0.00001) was recorded. A 1-ml volumetric pipette was then used to rinse the otolith with 

2.5 ml of DI water to remove surface contaminants (Figure 4). The water was then transferred 

out of the plastic beaker. The otolith was again covered with 3 ml of DI water and sonicated for 

1 minute. The 1-ml pipette was again used to remove the water from the beaker. For a final 

rinse, 3 ml of DI water were used to cover the otolith and then removed as before. These same 

steps were also performed on a “blank” beaker for comparison. The otolith was then transferred 

to an acid leached beaker. The tare weight of the beaker was recorded (g ± 0.00001). Four ml of 

DI water and a small amount of ~8-N HNO3 were added to the beaker to begin the otolith 

digestion. The beaker was covered loosely and put aside. Two-hundred and fifty μL of HNO3

were added every few minutes until the otoliths were completely dissolved. Once the otoliths 

were completely dissolved, an additional 250 μL of HNO3 was added to each beaker. The same 

procedure was performed on the “blank” as well. The otolith samples and blank were dried 

individually under heat lamps. Due to the limited number of dry down units and heat lamps 

(Figure 5), this procedure only allowed for 5 otolith samples and one blank to be processed at a 

time. 
Figure 4. Addition of DI water to beakers and otoliths for rinsing 

off surface contaminants (Yvonne Perry—University of 

Hawaii at Manoa). 

Figure 5. Heat lamps and units used to dry down otolith 

samples after addition of acid (University of 

Hawaii at Manoa). 

Once the samples were dry, 1.5 ml of 10.5-N HCl and 1 ml of 11.2-N HNO3 (a mixture 

known as aqua regia) were added to the beakers. The beakers were capped tightly and left over 

night on low heat under red lamps. The next day, samples were held in front of a light source 

and checked for residue remaining on the walls of the beakers. If no residue was present, 

beakers were again put into dry down units and dried slowly under heat lamps. Two ml of 7.5-N 

HNO3 were added to each sample and the blank to remove some of the chloride from the aqua 

regia stage. Once otolith samples were dried to a “puffy mass,” they were dissolved in 

approximately 6 ml of 1.5-N HNO3. The blank was dried to a small drop before adding 6 ml of 

13 
1.5-N HNO3. The samples were then completely digested and ready to be analyzed using ICPMS. 

Modified Otolith Digestion Procedure of the University of Hawaii at Manoa 

The original otolith digestion procedure using heat lamps to dry down samples was then 

modified to a procedure using microwave digestion. The new procedure allowed for 11 samples 

and one blank to be digested at a time and the digestion took less time than the previous method. 

Therefore, all other otoliths from artificial reefs in Louisiana, as well as all other otolith samples 

collected, were prepared using the microwave digestion procedure to improve the rate of sample 

processing. The new otolith digestion procedure required the use of Advanced Composite 

Vessels® (ACV) and a CEM Corporation MDS 2100 Microwave Digestion System® (Figure 6). 

Figure 6. MDS 2100 Microwave Digestion System® used to dissolve otolith 

samples and blanks (University of Hawaii at Manoa). 

Otoliths were placed into tared plastic beakers and weighed (g ± 0.00001). Weight was 

noted before washing. Otoliths were then rinsed with 2.5 ml of DI water. A 1-ml pipette was 

used to squirt water over the samples to loosen surface contaminants. The pipette was then used 

to transfer the water out of the beaker. The otolith samples were covered with 3 ml of DI water 

and sonicated for 1 minute. Water was again removed from the beaker using the pipette. 

Otoliths were transferred to a nitric cleaned ACV® made for the microwave digestion system. 

Two ml of quartz-distilled HNO3 was slowly added to the ACV® to dissolve the otolith. 

Samples were allowed to sit for at least two hours to allow for complete otolith dissolution. The 

same methods were also used on a blank ACV® for analysis. 

14 

Once otoliths had been completely dissolved, another 2 ml of HNO3 were added to the 

samples and the blank. A rupture membrane was placed into the caps of the ACV’s®, they were 

closed tightly, and loaded onto the microwave tray. Two programs were devised for the 

microwave digestion procedure, and were stored in the microwave. The first program called 

“OTOLITH” had two stages. Stage 1 lasted for 10 minutes at 80% power and a pressure of 60 

pounds per square inch (PSI). Stage 2 heated the otolith samples for a total of 15 minutes at 80% 
power. During this stage the pressure was increased from 60 PSI to 90 PSI, but this new 

pressure was only maintained for 10 of the 15 minutes. Once the OTOLITH program had been 

performed the samples were allowed to cool. The caps of the ACV’s® were removed and the 

“OTOLITH DRY DOWN” program was begun. This program was used repeatedly until the 

otolith samples were completely dry. The first time the “OTOLITH DRY DOWN” procedure 

was performed it lasted for 10 minutes at 10% power. The power was increased from 10 to 15 to 

20% with each run to obtain maximum dryness of each sample. Once the samples were 

completely dry they were transferred into vials using a total of 5 ml of 2% HNO3. The tare and 

final weight (g ± 0.00001) of the vials were noted to calculate the weight of the otolith sample. 

Once samples had been transferred to plastic vials using the HNO3 they were digested and ready 

for analysis using ICP-MS. 

INDUCTIVELY COUPLED PLASMA-MASS SPECTROMETRY

There are six major systems within an ICP-MS (Figure 7). The first system is the sample 

introduction area consisting of a spray chamber and a nebulizer. The job of the sample 

introduction area is to generate a fine aerosol of the sample so it can be efficiently ionized in the 

plasma discharge (Thomas, 2001b). This system has often been referred to as the Achilles heel 

of ICP-MS because only 1-2% of the sample actually makes it into the plasma. 

Figure 7. ICP-MS diagram showing 6 main systems (taken from Thomas, 2001a). 

The sample is transported from the sample introduction area to the plasma torch. The basic 

components used to generate the plasma source are a plasma torch, a radio frequency (RF) coil, 

and a RF power supply (Thomas, 2001c). The plasma torch is made up of three tubes, which are 

usually made from quartz. The process begins with a flow of argon gas through the quartz torch. 

The RF power supply is then administered to the coil and an electromagnetic field is formed. A 

high voltage spark produces free electrons, which are accelerated by the RF field, causing 

collisions and ionization of the argon gas (Thomas, 2001c). The inductively coupled plasma is 

formed at the open end of the quartz torch and the sample is introduced into the plasma. 

15 
16 

 

The sample is then moved to the interface region consisting of two cones, the sampler cone 

and the skimmer cone. The role of this system is to transport the ions efficiently to the mass 

analyzer region. After ions are generated in the plasma, they pass through the first cone, the 

sampler cone, which has an orifice diameter of 0.8-1.2 mm (Thomas, 2001d). They travel a 

short distance to the skimmer cone, which has a much smaller orifice of 0.4-0.8 mm in diameter. 

The interface region is probably the most critical area of the whole ICP-MS system because the 

most challenging problems to overcome are the movement of ions from the plasma to the mass 

spectrometer (Thomas, 2001a). 

Ions emerge from the skimmer cone and are directed through the ion focusing system. This 

system consists of one or more electrostatically controlled lens components that are made up of a 

series of metallic plates, barrels, or cylinders. The main role of this system is to transport the 

maximum number of analyte ions from the interface region to the mass separation device 

(Thomas, 2001e). This system rejects as many of the matrix components and non-analyte-based 

species as possible. These species cause signal instability and contribute to background levels, 

which ultimately affect the performance of the machine (Thomas, 2001e). 

Ions then move from the ion focusing system to the mass separation device where they are 

separated according to their mass-to-charge ratio (Thomas, 2001f). This device is made up of a 

number of metallic rods of the same length and diameter. A direct current (DC) field is placed 

on one pair of rods and a RF field is placed on the opposite pair of rods. By doing this, ions of a 

selected mass are allowed through the rods to the detector, while the others are ejected from the 

mass separation device (Thomas, 2001f). This scanning process is then repeated for another 

analyte at a different mass-to-charge ratio until all analytes in a multi-element analysis have been 

measured (Thomas, 2001a). 

Ions emerge from the mass separation device and are converted to an electrical pulse by the 

ion detector (Thomas, 2001a). The RF-DC voltage of the mass separation device is repeatedly 

scanned at different mass-to-charge ratios and the ions as electrical pulses are stored and counted 

by a multichannel analyzer. The multichannel data acquisition system typically has 20 channels 

per mass, and as the electrical pulses are counted in each channel, a profile of the mass is built up 

over the 20 channels, corresponding to the spectral peak of that ion (Thomas, 2001a). In a 

multielement analysis, repeated scans are made over the entire suite of analyte masses. 

Red snapper otolith samples were processed in a Fisons/VG PlasmaQuad® inductively 

coupled plasma-mass spectrometer (Figure 8) using flow injection analysis (FIA) for the 

presence of 15 different elements or isotopes. The flow injection system pulses a sample through 

the ICP-MS in a continuous flow of cleaning solution to reduce the amount of sample required 

and lessen the build up of deposits on the sampler and skimmer cones (Dove et al., 1996). The 

ICP-MS standard used for comparison of elemental recovery was comprised of red snapper 

otolith material gathered in the Gulf of Mexico and analyzed at the National Standards 

Laboratory (James H. Cowan, Jr.1—personal communication). 

1 Coastal Fisheries Institute, Louisiana State University, Baton Rouge, LA 70803
Figure 8. Fisons/VG PlasmaQuad® inductively coupled plasma-mass 

spectrometer used at the University of Hawaii at Manoa. 

The 15 elements or isotopes analyzed in this study were Vanadium 51, Cobalt 59, Nickel 62, 

Zinc 64, Copper 65, Zinc 66, Silver 107, Silver 109, Cadmium 110, Cadmium 111, Cadmium 

114, Lead 206, Lead 207, Lead 208, and Uranium 238. Although the elements and isotopes 

chosen to be analyzed were not the usual suite of characters used in otolith microchemistry work 

(Campana, 1999; Campana et al., 1994; Dove et al., 1996; Edmonds et al., 1992; Patterson et al., 

1998), they were picked because we felt they would be present due to platform operations and in 

the drilling muds used during drilling of the well (Continental Shelf Associates, Inc., 2004; 

Gallaway et al., 1981; Mearns and Moore, 1976; Neff, 1987; Neff et al., 1987). These elements 

are also heavy and replace calcium in the otoliths’ aragonite matrix. In addition, with the 

exception of zinc, it was felt that these elements would not be associated with a food web signal, 

but would be derived from the surrounding water mass (Campana, 1999; Ni et al., 2000). 

STATISTICAL TREATMENT OF DATA

All data obtained from mass spectrometry was standardized using the following equation: 

[X]sample = [Xsolution – Xblank](solution weight)(dilution factor) 

sample weight 

A number of different statistical methods were used to determine the elemental fingerprint of 

otoliths from each environment tested (platforms and reefs). A simple linear regression was used 

to determine the relationship between otolith weight (g) and fish weight (g). Differences 

between environments for individual elements or isotopes were tested using Multivariate 

Analysis of Variance (MANOVA) to determine which isotopes were most important in interenvironment fingerprint differences. An alpha (α) level of 0.05 was used to judge significance. 

17 
18 

A principal components analysis (PCA) was used to describe the variation in the ICP-MS 

data gathered for all the study sites combined. This analysis is used to describe the variation in a 

set of multivariate data in terms of a set of new, uncorrelated variables, each of which is a 

particular linear combination of the original variables (Der and Everitt, 2002). The main 

objective of this analysis was to determine whether the first few principal components accounted 

for a large part of the variation in the data, in which case they can be used to provide a suitable 

summary of the complete data set. The correlation matrix was used in this analysis as opposed to 

the covariance matrix due to the higher Zinc 64 and Zinc 66 concentrations than those of the 

other 14 elements analyzed. 

Unique signatures of each environment were determined using discriminant function 

analysis, with all isotopes as dependent variables. Using a set of observations containing one or 

more quantitative dependent variables and a classification variable defining groups of 

observations, discriminant function analysis computes linear (a linear function was used here = 

LDA) or quadratic discriminant functions classifying observations (red snapper otoliths) into two 

or more groups (platforms vs. other) on the basis of one or more of the quantitative variables 

(elements and isotopes) (SAS Institute, 1985). The discriminant functions (classification 

criterion) that are derived will be stored for later use to classify the adult snapper in Phase II of 

the project. 

A stepwise discriminant analysis (SDA) was performed to determine what elements were the 

most influential in discriminating between oil and gas platforms and artificial reefs in the Gulf of 

Mexico. Stepwise discriminant analysis finds a set of the original quantitative variables that best 

discriminate among the two sites or groups. A significance value of 0.05 was used in the 

analysis to determine when variables were chosen to enter and leave the discriminant function. 

To exclude variables that are highly correlated, the singular statement was set at a value of 0.2. 

Singularity is equal to 1 minus the tolerance, so setting the singularity to 0.2 infers an optimum 

tolerance value of 0.8. Tolerance values range from 0 to 1 and a small value indicates a high 

correlation between that variable with 1 or more of the other variables included in the 

discriminant function (Rooker et al., 2001a). 

A canonical discriminant analysis (CDA) was also used to compare the two environments. 

The canonical discriminant procedure determines the best linear combinations of the quantitative 

variables in which the means of the groups are most different. The canonical discriminant 

analysis was performed three times, first to compare the elemental concentration of otoliths from 

all sites, second to compare otoliths from both Louisiana sites, and third to compare otoliths from 

the two artificial reefs sites (Louisiana and Alabama) east and west of the Mississippi River. All 

statistical analyses were performed using SAS Version 9.0.
RESULTS 

Red snapper analyzed (N = 98 of 115 total collected) for this study ranged in size from 318 

mm to 776 mm in total length. These were divided into 5 size classes (mm TL): 301 – 400; 401 

– 500; 501 – 600; 601 – 700; and 701 – 800. Twenty red snapper were within the first size class, 

55 were in the second, 13 were in the third, 8 were in the fourth, and 2 red snapper were in the 

fifth and largest class (Figure 9). As expected, most of the red snapper were sub-adults (first two 

sized classes), although some adult fish were included in the analysis as well. For the most part, 

red snapper do not recruit to high vertical relief reefs until age-2, represented here by fish 

ranging in length from 301-400 mm TL. These fish were collected by permit, as they are below 

the legal size limit. Female red snapper in Louisiana waters are >50% mature at age-2, but do 

not reach 100% maturity until age-6. Fish greater than 405 mm TL (legal size) were collected at 

dockside and made up the majority of the fish used in this study. Of those, 45 were males and 53 

were females. When compared by site, the sex ratios were not evenly distributed. Of the thirtytwo red snapper analyzed from Louisiana oil and gas platforms, 11 were males and 21 were 

females. Similarly, of red snapper collected on artificial reefs in Louisiana, 12 were males and 

19 were females. Of red snapper collected on artificial reefs in Alabama, 22 were males and 

only 13 were females. By location, 32 otoliths came from Louisiana oil and gas platforms, 31 

from Louisiana artificial reefs, and 35 from Alabama artificial reefs. 

Total Length Size Class (mm)

300 400 500 600 700 800 900

Frequency 

0

10

20

30

40

50

60

Figure 9. Length frequency distribution for red snapper from all study sites combined (N = 98).

19 
The discrepancy between the total number of otoliths collected and the number analyzed 

occurred because there were some failures while running the mass spectrometer due to 

exceptionally high concentrations of some elements. This caused the instrument detector to trip 

from pulse counting to analog mode. By the time the detector had settled into analog mode the 

entire otolith sample had already passed through the machine and was not properly analyzed. 

Unless there was enough otolith sample left to rerun that analysis, those samples could not be 

rerun and therefore were not included in the results of this study. 

SIMPLE LINEAR REGRESSION

Otolith weight (g) was related to fish weight (g) using a simple linear regression. 

The regression relationship is significant (α = 0.05) producing an R2 value = 0.95 (Figure 

10). It is expected that as fish weight increases, otolith weight also increases. While the 

relationship appears slightly non-linear, log transformation of the data only increases the R2

 by < 

1.0%. 

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2000 4000 6000 8000

Fish Weight (g)

Otolith Weight (

g Otolith Weight (g) 

R2 = 0.95 

Otolith Wt. = 0.291 + 1.766E-4(Fish Wt.) 

Figure 10. Linear regression relating otolith weight (g) to fish weight (g) (Wt = weight). 

20 
MULTIVARIATE ANALYSIS OF VARIANCE (MANOVA) 

A MANOVA was used to compare the means of all 15 elements in otoliths between each of 

the study sites. For all of the following analyses, site 1 represents Louisiana oil and gas 

platforms, site 2 represents Louisiana artificial reefs, and site 3 represents Alabama artificial 

reefs. Ten of the Louisiana oil and gas platforms’ 15 elements analyzed had significantly 

different means (α = 0.05 than those collected from Louisiana artificial reefs (site 2), and 

Alabama artificial reefs (site 3; Figures 11, 12, and 13). These 10 elements and their 

corresponding p-values, are as follows: Cobalt 59 (p=0.0070), Zinc 64 (p=0.0339), Copper 65 

(p=0.0255), Silver 107 (p<0.0001), Silver 109 (p<0.0001), Cadmium 110 (p=0.0091), Cadmium 

111 (p=0.0229), Cadmium 114 (p=0.0012), Lead 206 (p=0.0464), and Lead 207 (p=0.0331). 

Lead 208 (p=0.0958) and Uranium 238 (p=0.0888) are close to being significantly different 

between the three sites, with p-values greater than 0.05 but less than 0.10. 

0

50

100

150

200

250

300

350

400

V51

Co59

Ni62

Zn64Cu65Zn66

A g107A g109

Cd110Cd111Cd114

P b206Pb207Pb208

U238

Elements

Parts Per Billio

n

Louisiana Oil and Gas Platforms Louisiana Artificial Reefs

Alabama Artificial Reefs

Parts Per Billion 

Figure 11. Comparison of means of 15 elements from otoliths analyzed for all three study 

sites. 

21 
0

10

20

30

40

50

60

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Elements

Parts Per Billion

Louisiana Oil and Gas Platforms Louisiana Artificial Reefs

Alabama Artificial Reefs

Figure 12. Enlarged view of comparison of means of 15 elements from otoliths analyzed 

for all three study sites to emphasize concentrations of trace elements found 

in lower concentrations in the previous figure. 

0

0.5

1

1.5

2

2.5

3

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Elements

Parts Per Billion

Louisiana Oil and Gas Platforms Louisiana Artificial Reefs

Alabama Artificial Reefs

Figure 13. Further enlargement of the comparison of means of 15 elements from otoliths 

analyzed for all three study sites to emphasize concentrations of trace 

elements found in lower concentrations in the previous figure. 

22 

When comparing the means of each element in otoliths collected from Louisiana oil and gas 

platforms and Louisiana artificial reefs, the MANOVA results show that 8 of the 15 elements in 
question have significantly different means in otoliths from these two areas. The elements are 

Vanadium 51 (p=0.0001), Zinc 64 (p=0.0035), Zinc 66 (p=0.0003), Silver 107 (p=0.0015), 

Silver 109 (p=0.0001), Lead 206 (p=0.0075), Lead 207 (p=0.0092), and Lead 208 (p=0.0094) 

(Figures 14, 15, and 16). 

23 

0

100

200

300

400

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Element

Parts Per Billion

Louisiana Oil and Gas Platforms Louisiana Artificial Reefs

Figure 14. Comparison of means of elemental concentration in otoliths from Louisiana oil 

and gas platforms and Louisiana artificial reefs. 

0

5

10

15

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Element

Parts Per Billion

Louisiana Oil and Gas Platforms Louisiana Artificial Reefs

Figure 15. Enlarged view of comparison of means of elemental concentration in otoliths 

from Louisiana oil and gas platforms and Louisiana artificial reefs to emphasize 

concentrations of trace elements found in lower concentrations in the previous 

figure. 
0

0.5

1

1.5

2

2.5

3

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Element

Parts Per Billion

Louisiana Oil and Gas Platforms Louisiana Artificial Reefs

Figure 16. Further enlargement of the comparison of means of elemental concentration in 

otoliths from Louisiana oil and gas platforms and Louisiana artificial reefs to 

emphasize concentrations of trace elements found in lower concentrations in 

the previous figure. 

When comparing otoliths from the two artificial reef sites, one in Louisiana and one in 

Alabama (sites 2 and 3), 9 of the 15 elements analyzed are significantly different, and are 

believed to reflect water mass characteristics east and west of the Mississippi River. Those 

elements that have significantly different means are Vanadium 51 (p=0.0048), Copper 65 

(p<0.0001), Zinc 66 (p=0.0495), Silver 107 (p=0.0001), Silver 109 (p=0.0107), Cadmium 114 

(p=0.0004), Lead 206 (p=0.0218), Lead 208 (p=0.0103), and Uranium 238 (p=0.0374) (Figures 

17, 18, and 19). Cobalt 59 (p=0.0588), Cadmium 110 (p=0.0720), and Lead 207 (p=0.0529) are 

close to being significant, having p-values larger than 0.05 but less than 0.10. 

24 
0

50

100

150

200

250

300

350

400

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Element

Parts Per Billion

Louisiana Artificial Reefs Alabama Artificial Reefs

Figure 17. Comparison of means of elemental concentration in otoliths from Louisiana 

artificial reefs and Alabama artificial reefs. 

0

2

4

6

8

10

12

14

16

18

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Element

Parts Per Billion

Louisiana Artificial Reefs Alabama Artificial Reefs

Figure 18. Enlarged view of comparison of means of elemental concentration in otoliths 

from Louisiana artificial reefs and Alabama artificial reefs to emphasize 

concentrations of trace elements found in lower concentrations in the previous 

figure. 

25 
0.0

0.5

1.0

1.5

2.0

V51

Co59

Ni62

Zn64

Cu65

Zn66

Ag107Ag109

Cd110Cd111Cd114

Pb206Pb207Pb208

U238

Element

Parts Per Billion

Louisiana Artificial Reefs Alabama Artificial Reefs

Figure 19. Further enlargement of the comparison of means of elemental concentration in 

otoliths from Louisiana artificial reefs and Alabama artificial reefs to emphasize 

concentrations of trace elements found in lower concentrations in the previous 

figure. 

PRINCIPAL COMPONENTS ANALYSIS

Principal components analysis (PCA) was performed using all 98 observations to determine 

the variation in the data set. The 15 elements analyzed in this study were included in the analysis 

as variables. Based on the results of the PCA, and when referring to the eigenvalues of the 

correlation matrix, the first four principal component scores explain 70% of the variance in the 

ICP-MS data (Table 1). The first two principal component scores alone explain 47% of the 

variance suggesting that these two scores, for each of the elements analyzed, provide an adequate 

description of the data. When referring to the eigenvectors of principal components 1 and 2, a 

number of different elements account for the variation in the ICP-MS data. Silver 107, Silver 

109, Cadmium 110, Cadmium 111, Cadmium 114, Lead 206, Lead 207, and Lead 208 have 

some of the largest eigenvectors and appear to be explaining the greatest portion of the variance 

when looking at principal component 1 (Table 2). When referring to principal component 2, 

Zinc 64, Zinc 66, Vanadium 51, Cobalt 59, and Nickel 62 have the largest eigenvectors (Table 

2). Figure 20 shows a scatter plot of principal component 1 versus principal component 2 with 

observations labeled by site. Both principal component scores appear to be ranking observations 

collected from the same location closely together. 

26 
27 

Table 1. 

 

Eigenvalues of the correlation matrix from the PCA showing 

cumulative percent of variation explained by each principal 

component score. 

Eigenvalue Cumulative % 

1 5.02168453 0.3348 

2 2.02398796 0.4697 

3 1.74382553 0.5860 

4 1.67318932 0.6975 

Table 2. 

 

Eigenvectors of principal components 1 and 2 for all 15 elements 

analyzed. 

Principal Component 1 Principal Component 2 

V 51 0.023242 0.276143 

Co 59 -.092396 0.289524 

Ni 62 -.018790 0.296602 

Zn 64 0.184254 -.501276 

Cu 65 0.208920 -.115625 

Zn 66 0.194150 -.523766 

Ag 107 0.253426 -.104995 

Ag 109 0.250578 -.112389 

Cd 110 0.339336 0.139852 

Cd 111 0.342344 0.134835 

Cd 114 0.286382 -.107563 

Pb 206 0.380085 0.214434 

Pb 207 0.383314 0.200276 

Pb 208 0.375109 0.216806 

U 238 0.042430 -.083117 
-5

-4

-3

-2

-1

0

1

2

3

4

5

Pri nci pal Component 2

-5 -4 -3 -2 -1 0 1 2 3 4 5

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1 1

1 1

1

1 1

1

1

1

1

2

2

2

2

2 2

2

2

2

2

2

2

2

2

2

222 2

2

2

2

2

2

2

2

2

2

2

2

2

3

3

3 3

3

3

3

3

3

3

3

3

3

3

3

3

33

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

Figure 20. Scatter plot of principal component score 1 versus principal 

component score 2 with observations labeled by site (site 1 = LA 

plat.; site 2 = LA reefs; site 3 = AL reefs). 

LINEAR AND STEPWISE DISCRIMINANT ANALYSIS

Comparison Among All Study Sites 

Linear discriminant analysis (LDA) was performed using all 98 observations to determine if 

study sites could be discriminated based upon the elemental composition of otoliths. The 

variables used include the 15 elements analyzed, and the classes consist of the 3 sites previously 

designated. The F approximations for all of the multivariate statistics in this analysis are 

significant with p-values < 0.0001 (Table 3) indicating that it is possible to discriminate between 

the 3 sites based upon red snapper otolith elemental composition. It is also apparent that the two 

artificial reef sites (site 2 and site 3) cannot be combined as one large sample due to differences 

in the elemental composition of otoliths collected in these areas. Overall, the LDA resulted in a 

cross-validation misclassification rate of only 19.51%, and the number of observations and 

percent classified into each of the 3 sites are shown in Table 4. 

28 
Table 3. 

 

Multivariate statistics and F approximations for linear discriminant analysis comparing the 

elemental concentration of otoliths among all study sites. 

Statistic F-Value Pr > F 

Wilks’ Lambda 8.80 <0.0001 

Pillai’s Trace 8.81 <0.0001 

Hotelling-Lawley Trace 8.80 <0.0001 

Roy’s Greatest Root 10.66 <0.0001 

Table 4. 

 

Number of observations and percent of otoliths classified into each of 3 sites using linear 

discriminant analysis and cross-validation. 

From Site 1 2 3 Total 

1 23 

71.88 

5 

15.63 

4 

12.50 

32 

100.00 

2 3 

9.68 

26 

83.87 

2 

6.45 

31 

100.00 

3 3 

8.57 

2 

5.71 

30 

85.71 

35 

100.00 

Total 29 

29.59 

33 

33.67 

36 

36.73 

98 

100.00 

A stepwise discriminant analysis (SDA) was then used to determine which elements were 

most important in discriminating otoliths from fish collected among the sites. All 98 

observations were included in the analysis (PROC STEPDISC; SAS V 9.0), the 15 elements 

analyzed in the study were used as variables, and the 3 sites remained as class levels. Results of 

the SDA indicate that 9 of the 15 elements are useful to discriminate among otoliths collected 

from the 3 sites. These 9 elements are Silver 107, Zinc 66, Vanadium 51, Lead 208, Cadmium 

114, Cobalt 59, Copper 65, Uranium 238, and Nickel 62. The LDA was then rerun comparing 

otoliths from all 3 sites, but using only the 9 elements identified above; the number of 

observations and the classes remained the same. Rerunning the LDA using these 9 elements 

lowers the cross- validation misclassification rate from 19.51% to 15.03%. The number of 

observations and the percent of otoliths classified into each site are shown in Table 5. 

29 
30 

Table 5. 

 

Number of observations and percent of otoliths classified into each of 3 sites after rerunning the 

linear discriminant analysis using only the 9 most important elements. 

From Site 1 2 3 Total 

1 25 

78.13 

4 

12.50 

3 

9.38 

32 

100.00 

2 0 

0.00 

30 

96.77 

1 

3.23 

31 

100.00 

3 4 

11.43 

3 

8.57 

28 

80.00 

35 

100.00 

Total 29 

29.59 

37 

37.76 

32 

32.65 

98 

100.00 

Because it was determined that otoliths collected from all of the study sites were significantly 

different in elemental composition and we could not combine the artificial reef sites, two other 

discriminant analyses were performed. The first analysis compared the Louisiana sites to 

determine if we could successfully discriminate between red snapper otoliths from Louisiana oil 

and gas platforms (site 1) and Louisiana artificial reefs (site 2). Since no red snapper were 

collected on oil and gas platforms in Alabama, the Louisiana and Alabama artificial reef samples 

(sites 2 and 3) were compared to determine if we could discriminate between otoliths taken from 

east and west of the Mississippi River. 

Comparison of Louisiana Sites 

The elemental composition of otoliths from Louisiana oil and gas platforms (site 1) and 

Louisiana artificial reefs (site 2) were compared using LDA to determine if otoliths from the two 

sites could be successfully discriminated. The analysis included a total of 63 red snapper otoliths 

(32 from site 1 and 31 from site 2), 15 variables or elements, and 2 classes (site 1 and site 2). All 

four of the multivariate statistics and their exact F statistics are significant, with p-values < 

0.0001 (Table 6). Otoliths from site 1 and 2 could successfully be discriminated using this 

procedure, and the analysis resulted in a cross-validation misclassification rate of 15.83%. The 

number of observations and percent of otoliths classified into each of the two sites are shown in 

Table 7. 
31 

Table 6. 

 

Multivariate statistics and exact F statistics for linear discriminant analysis comparing the elemental 

concentration of otoliths from Louisiana oil and gas platforms (site 1) and Louisiana artificial reefs 

(site 2). 

Statistic F-Value Pr > F 

Wilks’ Lambda 8.23 <0.0001 

Pillai’s Trace 8.23 <0.0001 

Hotelling-Lawley Trace 8.23 <0.0001 

Roy’s Greatest Root 8.23 <0.0001 

Table 7. 

 

Number of observations and percent of otoliths classified into Louisiana oil and gas 

platforms (site 1) and Louisiana artificial reefs (site 2) using linear discriminant analysis 

and cross-validation. 

From Site 1 2 Total 

1 26 

81.25 

6 

18.75 

32 

100.00 

2 4 

12.90 

27 

87.10 

31 

100.00 

Total 30 

47.62 

33 

52.38 

63 

100.00 

An SDA was then performed to determine the most important elements in discriminating 

otoliths from site 1 and 2. This procedure identifies 6 elements as being important in 

differentiating between elemental composition of otoliths; these are Vanadium 51, Zinc 66, 

Silver 109, Lead 206, Uranium 238, and Nickel 62. Using only these 6 elements to rerun the 

LDA lowers the original cross-validation misclassification rate from 15.83% to 6.25%. The 

number of observations and percent of otoliths classified into site 1 and site 2 after rerunning the 

LDA are shown in Table 8. 
32 

Table 8. 

 

Number of observations and percent of otoliths classified into Louisiana oil and gas 

platforms (site 1) and Louisiana artificial reefs (site 2) after rerunning the linear 

discriminant analysis using only the 6 most important elements. 

From Site 1 2 Total 

1 28 

87.50 

4 

12.50 

32 

100.00 

2 0 

0.00 

31 

100.00 

31 

100.00 

Total 28 

44.44 

35 

55.56 

63 

100.00 

Comparison Between Artificial Reef Sites 

Because no red snapper were collected on oil and gas platforms in Alabama (east of the 

Mississippi River), otoliths from fishes collected at the two artificial reef sites were compared to 

determine if otoliths collected east and west of the Mississippi River could be successfully 

discriminated. The LDA consisted of 66 red snapper otoliths (31 from site 2 in Louisiana and 35 

from site 3 in Alabama), the 15 elements analyzed, and 2 classes (site 2 and site 3). All of the 

multivariate statistics and exact F statistics from this analysis are significant with p-values < 

0.0001 (Table 9). Otoliths collected on Louisiana and Alabama artificial reefs can successfully 

be discriminated, with a cross-validation misclassification rate of 12.35%. The number of 

observations and percent of otoliths classified into site 2 and site 3 are shown in Table 10. 

Table 9. 

 

Multivariate statistics and exact F statistics for linear discriminant analysis comparing the elemental 

concentration of otoliths from Louisiana (site 2) and Alabama artificial reefs (site 3). 

Statistic F-Value Pr > F 

Wilks’ Lambda 9.90 <0.0001 

Pillai’s Trace 9.90 <0.0001 

Hotelling-Lawley Trace 9.90 <0.0001 

Roy’s Greatest Root 9.90 <0.0001 
33 

Table 10. 

 

Number of observations and percent of otoliths classified into Louisiana artificial reefs 

(site 2) and Alabama artificial reefs (site 3) using linear discriminant analysis and crossvalidation. 

From Site 2 3 Total 

2 26 

83.87 

5 

16.13 

31 

100.00 

3 3 

8.57 

32 

91.43 

35 

100.00 

Total 29 

43.94 

37 

56.06 

66 

100.00 

An SDA was performed to determine which elements are the most influential in 

discriminating otoliths from Louisiana and Alabama artificial reefs (sites 2 and 3). This analysis 

identifies 6 of the 15 elements as being the most important for discrimination. 

The 6 elements are Copper 65, Silver 107, Lead 208, Cobalt 59, Vanadium 51, and Uranium 

238. The LDA was then rerun using only these 6 elements. Rerunning the analysis in this 

manner lowers the original cross-validation misclassification rate of 12.35% to 8.94%. The 

number of observations and the percent of otoliths classified into site 2 (Louisiana) and site 3 

(Alabama) after rerunning the LDA are shown in Table 11. 

Table 11. 

 

Number of observations and percent of otoliths classified into Louisiana artificial reefs 

(site 2) and Alabama artificial reefs (site 3) after rerunning the original linear 

discriminant analysis using only the 6 most important elements. 

From Site 2 3 Total 

2 29 

93.55 

2 

6.45 

31 

100.00 

3 4 

11.43 

31 

88.57 

35 

100.00 

Total 33 

50.00 

33 

50.00 

66 

100.00 

CANONICAL DISCRIMINANT ANALYSIS (CDA) 

Comparison Among All Three Study Sites 

Although results from LDA and SDA are commonly reported in otolith microchemistry 

research, a CDA was also used to compare the red snapper otolith samples. Although these 

statistical techniques are similar to one another, the LDA will later allow an otolith of unknown 

origin to be classified into one of the three habitats mentioned above. The use of CDA allows 
34 

for better graphical visualization of the discrimination of otolith elemental concentrations from 

the three study areas. 

The first CDA included all study sites to determine what quantitative variables were 

contributing most to the discrimination of red snapper otoliths from among the sites. This 

procedure included all 98 otolith samples, the 15 elements used in the previous analyses, and 3 

classes (site 1, site 2, and site 3) (PROC CANDISC; SAS V 9.0). Based upon results from the 

CDA, and referring to the pooled with-in class standardized canonical coefficients, a number of 

different elements are identified as important for discriminating otoliths from all sites (i.e., have 

high coefficients). The CDA identifies Vanadium 51, Zinc 64, Zinc 66, Lead 207 and Lead 208 

as the most important variables in discriminating between otolith composition among these 3 

areas (Table 12). Nickel 62, Silver 107, and Cadmium 114 may also play a role in the 

discrimination between red snapper otoliths from the three sites, but these have smaller 

coefficients than the elements mentioned above. A vertical bar chart of canonical variable 1, 

with observations separated by site, shows a clear distinction between site 1 (LA oil and gas 

platforms), site 2 (LA artificial reefs), and site 3 (AL artificial reefs) (Figure 21). A scatter plot 

of canonical variable 1 versus canonical variable 2, with observations separated by site, also 

shows the discrimination of these three study areas (Figure 22). 

Table 12. 

 

Pooled with-in class standardized canonical coefficients for canonical discriminant 

analysis comparing elemental concentration of otoliths from all 3 sites. 

Variable Can 1 Can 2 

Van 51 -0.326287286 1.271772749 

Co 59 -0.364269086 -0.111424522 

Ni 62 0.164067374 -0.830804671 

Zi 64 -2.614762035 0.455984967 

Cu 65 0.226111971 -0.051381164 

Zi 66 2.124772164 -1.061174832 

Ag 107 0.727485480 0.043888748 

Ag 109 -0.232535143 0.423190943 

Cd 110 0.165261904 0.004690588 

Cd 111 -0.550891827 0.138192167 

Cd 114 0.711935361 -0.182457948 

Pb 206 -0.261392235 0.668427423 

Pb 207 3.391593583 0.770820882 

Pb 208 -3.253403361 -0.991748720 

U 238 0.395437128 -0.665813787 
si t e 1 2 3

FREQUENCY

0

10

20

30

Can1 MIDPOINT

-3.6 -2.4 -1.2 0.0 1.2 2.4 3.6

Can 1 midpoint 

Site 1 = Louisiana Oil and Gas Platforms; 

Site 2 = Louisiana Artificial Reefs; 

Site 3 = Alabama Artificial Reefs 

Figure 21. Vertical bar graph of canonical variable 1, with observations separated 

by site, for a comparison of elemental concentration of otoliths 

collected from all three study sites. 

35 
36 

Figure 22. A scatter plot of canonical variable 1 versus canonical variable 2, with observations separated 

comparison of elemental concentration of otoliths collected from all three study sites. 
37

Comparison Between Louisiana Sites 

A CDA was also performed comparing the elemental composition of otoliths from Louisiana 

oil and gas platforms (site 1) and Louisiana artificial reefs (site 2). This procedure contained a 

total of 63 red snapper otoliths, all 15 elements, and 2 classes (site 1 and site 2). Based upon 

results from the CDA, and referring to the pooled with-in class standardized canonical 

coefficients, 7 elements are identified to be most important (i.e., have high coefficients); these 

are Vanadium 51, Silver 109, Lead 206, and Lead 207 (Table 13). Silver 107, Lead 208, and 

Uranium 238 may also play a minor role in the discrimination of site 1 from 2, but their 

coefficients are smaller. A vertical bar graph of canonical variable 1, with the 63 otolith samples 

separated up by site, shows a clear separation of elemental composition of otoliths from 

Louisiana oil and gas platforms (site 1) and Louisiana artificial reefs (site 2) (Figure 23). 

Table 13. 

 

Pooled with-in class standardized canonical coefficients for canonical 

discriminant analysis comparing elemental concentration of otoliths 

from Louisiana oil and gas platforms and Louisiana artificial reefs. 

Variable Can 1 

Van 51 1.150511481 

Co 59 -0.315485812 

Ni 62 -0.386722369 

Zi 64 -0.510542370 

Cu 65 0.075796429 

Zi 66 -0.407428082 

Ag 107 -0.828019113 

Ag 109 1.288229683 

Cd 110 0.266889436 

Cd 111 -0.200465328 

Cd 114 0.067606075 

Pb 206 1.319984482 

Pb 207 -1.428899832 

Pb 208 0.741607422 

U 238 -0.758786655 
38

Figure 23. Vertical bar graph of canonical variable 1, with observations 

separated by site, for a comparison of elemental concentration of 

otoliths collected from Louisiana oil and gas platforms (site 1) and 

Louisiana artificial reefs (site 2). 

Comparison Between Artificial Reef Sites 

Elemental composition of otoliths collected from Louisiana artificial reefs (site 2) and 

Alabama artificial reefs (site 3) were compared using CDA. This comparison contained 66 red 

snapper otoliths, 15 elements, and 2 classes (site 2 and site 3). Based upon results from the 
39

CDA, and referring to the pooled with-in class standardized canonical coefficients, 7 of the 15 

elements included are identified as being important in discriminating between otoliths from sites 

2 and 3 (i.e., have high coefficients); these elements are Vanadium 51, Nickel 62, Zinc 64, Zinc 

66, Lead 207, Lead 208, and Uranium 238 (Table 14). A vertical bar graph of canonical variable 

1, with all 66 otoliths separated by site, shows a clear distinction between the composition of 

otoliths from Louisiana and Alabama artificial reefs (Figure 24). 

Table 14. 

 

Pooled with-in class standardized canonical coefficients for canonical 

discriminant analysis comparing elemental concentration of otoliths 

from Louisiana artificial reefs and Alabama artificial reefs. 

Variable Can 1 

Van 51 -1.599721728 

Co 59 -0.219237785 

Ni 62 1.344612829 

Zi 64 -2.600825746 

Cu 65 0.123169822 

Zi 66 2.264223297 

Ag 107 0.307630655 

Ag 109 0.104474403 

Cd 110 0.224670174 

Cd 111 -0.306991225 

Cd 114 0.575792743 

Pb 206 -0.326919757 

Pb 207 1.042553437 

Pb 208 -0.910109476 

U 238 0.942455608 
40

Figure 24. Vertical bar graph of canonical variable 1, with 

observations separated by site, for a comparison of 

elemental concentration of otoliths collected from Louisiana 

artificial reefs (site 2) and Alabama artificial reefs (site 3). 

POSSIBLE INFLUENCE OF THE MISSISSIPPI RIVER

A number of the same elements are identified as being important in both the stepwise and 

canonical discriminant analyses. Of the 15 elements analyzed, 13 reoccur frequently in the 

results. The 13 most important elements identified in discrimination of red snapper otoliths 

using the two statistical techniques are Vanadium 51, Cobalt 59, Nickel 62, Copper 65, Zinc 64, 

Zinc 66, Silver 107, Silver 109, Cadmium 114, Lead 206, Lead 207, Lead 208, and Uranium 
41

238. These elements are essential in differentiating between red snapper otoliths collected from 

Louisiana oil and gas platforms and artificial reefs, as well as between otoliths from artificial 

reefs east and west of the Mississippi River. To determine if the concentrations of these 

important elements are being influenced by the discharge of the Mississippi River, we plotted 

and compared concentrations of the elements in red snapper otoliths collected at all three study 

sites. 

Elements attributable to Mississippi River discharge should have mean concentrations in 

otoliths from both Louisiana sites that were significantly higher than in otoliths collected at site 3 

in Alabama. The elements that meet this criteria are Cadmium 114 (Figure 25), Copper 65 

(Figure 26), Uranium 238 (Figure 27), Silver 107 (Figure 28), and Silver 109 (Figure 28), all 

having means significantly higher in red snapper otoliths from coastal Louisiana, west of the 

Mississippi River. Conversely, Cobalt 59 (Figure 29) and Nickel 62 (Figure 30) have 

significantly higher means in otoliths from site 3, east of the Mississippi River and perhaps 

reflect discharge from Mobile Bay. However, otolith concentrations of Lead 206 (Figure 31), 

Lead 207 (Figure 32), Lead 208 (Figure 33), and Vanadium 51 (Figure 34) do not vary 

geographically, yet remain significantly higher in otoliths from Louisiana oil and gas platforms 

than from either artificial reef site. Otolith Zinc 64 and Zinc 66 concentrations (Figure 35) also 

show no significant geographical distribution, but are higher in otoliths collected on Louisiana 

and Alabama artificial reefs than in those from Louisiana oil and gas platforms. 

Figure 25. Cadmium 114 concentration in otoliths all three study sites showing 

a geographical distribution with higher concentrations west of the 

Mississippi River. 
42

Figure 26. Copper 65 concentration in otoliths at all three study sites showing a 

geographical distribution with higher concentrations west of the 

Mississippi River. 

Figure 27. Uranium 238 concentration in otoliths at all three study sites showing 

a geographical distribution with higher concentrations west of the 

Mississippi River. 
43

Figure 28. Silver 107 and 109 concentrations in otoliths at all three study sites 

showing a geographical distribution with higher concentrations west 

of the Mississippi River. 

Figure 29. Cobalt 59 concentration in otoliths at all three study sites showing a 

geographical distribution with higher concentrations east of the 

Mississippi River. 
44

Figure 30. Nickel 62 concentration in otoliths at all three study sites showing a 

geographical distribution with higher concentrations east of the 

Mississippi River. 

Figure 31. Lead 206 concentration in otoliths at all three study sites showing no 

significant geographical distribution but having higher concentrations 

on Louisiana oil and gas platforms. 
45

Figure 32. Lead 207 concentration in otoliths at all three study sites showing no 

significant geographical distribution but having higher concentrations 

on Louisiana oil and gas platforms. 

Figure 33. Lead 208 concentration in otoliths at all three study sites showing no 

significant geographical distribution but having higher concentrations 

on Louisiana oil and gas platforms. 
46

Figure 34. Vanadium 51 concentration in otoliths at all three study sites showing 

no significant geographical distribution but having higher 

concentrations on Louisiana oil and gas platforms. 

Figure 35. Zinc 64 and Zinc 66 concentrations in otoliths at all three study sites 

showing no clear geographical distribution but having higher 

concentrations on both artificial reef sites. 

TRENDS OF IMPORTANT ELEMENTS WITH RED SNAPPER SEX AND TOTAL LENGTH

An important potential influence on otolith elemental site-specific markers detected in this 

study are the sex and total length of red snapper collected for analysis. To investigate these 

trends, the concentrations of the 13 most important elements identified in both of the 

discriminant techniques for all sites combined were plotted against sex and total length. The 13 

elements are Vanadium 51, Cobalt 59, Nickel 62, Copper 65, Zinc 64, Zinc 66, Silver 107, Silver 
47

109, Cadmium 114, Lead 206, Lead 207, Lead 208, and Uranium 238. Any significant 

relationship between otolith concentration and red snapper sex or total length could reduce 

confidence in the concept of an “oil and gas platform” site-specific marker. 

When compared with red snapper sex, only 2 of the 13 elements show a significant 

relationship. Vanadium 51 (Figure 36) and Uranium 238 (Figure 37) both have higher mean 

concentrations in otoliths of male red snapper than females at all three study areas. None of the 

elements show any significant trends when compared to red snapper total length. Figures 38, 39, 

and 40 are a few examples of the graphic results obtained when comparing otolith elemental 

concentration at all three study sites to red snapper total length. Although a number of elements 

appear to have higher concentrations in otoliths of smaller (younger) red snapper, i.e., those less 

than 500 mm in total length, This result is believed to be a spurious correlation attributable to 

unequal and small sample sizes, i.e., high number of individuals collected in the smaller size 

classes relative to fish greater than 500 mm TL. 

Figure 36. Vanadium 51 concentration in otoliths at all three study sites showing 

higher concentrations in male red snapper. 
48

Figure 37. Uranium 238 concentration in otoliths at all three study sites showing 

higher concentrations in male red snapper. 

Figure 38. Vanadium 51 concentration in otoliths at all three study sites showing 

no significant trend with red snapper total length. 
49

Figure 39. Lead 206 concentration in otoliths at all three study sites showing no 

significant trend with red snapper total length. 

Figure 40. Uranium 238 concentration in otoliths at all three study sites showing 

no significant trend with red snapper total length. 
51

DISCUSSION 

Red snapper (our surrogate reef-associated species) from the north-central Gulf which were 

collected in close proximity to oil and gas platforms had a statistically recognizable or 

characteristic otolith microchemical “fingerprint.” Results also show that otoliths from red 

snapper collected over artificial reefs located east and west of the Mississippi River had a 

statistically different and characteristic fingerprint, as well. Once these differences were 

discovered, further analyses proved much more revealing and interesting. The discriminant 

function classification accuracies based on the probability of an individual fish being correctly 

classified into the habitat from which it was sampled are over 90% for each of the two main 

comparisons. If these results prove to be temporally stable (Campana, 1999), it may be possible 

to take any adult red snapper, analyze the otoliths using ICP-MS, and determine if that individual 

spent any portion of its earlier life associated with oil and gas platforms. This will, therefore, 

help to determine if red snapper that have the “oil and gas platform fingerprint” occur in high 

numbers on other habitat types or in different locations. For example, if large numbers of red 

snapper collected in the eastern Gulf of Mexico, where oil and gas platforms are relatively few, 

exhibit the “oil and gas platform signature,” this may indicate that platforms act as sources that 

supply recruits to other locations, in this case from Louisiana to Florida. This hypothesis is as of 

yet untested, but a proof of concept was necessary to determine if the oil and gas platform 

signature could be detected. As such, elaboration of this argument will be the basis of our next 

proposal (Phase II). In Phase II, if it is funded, we will examine a much larger sample size of 

fish collected on and off of platforms Gulf-wide over multiple years to examine the geographic 

and temporal stability of the “platform fingerprint.” If the fingerprint is stable, we then will be 

able to use it to determine if older fish collected in areas that do not have platforms contain the 

platform marker. 

If these findings hold true, the core of red snapper otoliths collected from any location could 

be analyzed to determine if the red snapper had at one time been resident on a platform, and 

single annual increments could be isolated to determine temporal patterns of residency. Such 

knowledge would be valuable for fisheries managers. If platforms are acting like refugia for the 

young because of their surrounding no trawl zones, or as source locations, they may be acting in 

much the way that marine protected areas (MPA’s) are thought to behave, and perhaps should be 

considered for designation as MPA’s. The determination of the impacts of artificial reefs (of any 

type) on the demographics of reef-associated fishes such as red snapper is also a necessary step 

in moving towards final resolution of the production vs. attraction debate. In Phase I, our fish 

were obtained mostly from dockside samples of opportunity, and were thus constrained both in 

number and size distribution (via legal size limits). If the second phase of our study is funded, 

we plan to include a much larger sample size, and to collect smaller fish by permit, to fill out the 

study design. This will include collecting fish from oil and gas platforms located east of the 

Mississippi River mouth, which we were unable to do previously despite several tries. In study 

Phase II, if funded, we plan to sample at several fishing rodeos, including spear fishing 

tournaments, east of the river to insure that we are able to collect red snapper from platforms in 

this location. 

Vanadium 51, Lead 206, Lead 207, and Lead 208 may be dissolution products linked with oil 

and gas platform operations or their prior drilling operations (Continental Shelf Associates, Inc., 

2004; Gallaway et al., 1981; Mearns and Moore, 1976; Neff, 1987; Neff et al., 1987) and the 
52

presence of these elements or isotopes in the otoliths of reef-associated fishes could indicate their 

association with these structures. The concentrations of these four isotopes are significantly 

higher in red snapper otoliths sampled in close proximity to oil and gas platforms in Louisiana 

than in otoliths sampled from artificial reefs in either Louisiana or Alabama. The feeding of red 

snapper on benthic organisms in the sediment surrounding oil and gas platforms may also be a 

method of incorporating these elements into otoliths, although the specific portion of elements 

gathered from food sources is thought to be minimal (Campana, 1999), since the main source of 

inorganic elements into the intestine and then the otolith is through drinking the water. 

In contrast, concentrations of Copper 65, Silver 107, Silver 109, Cadmium 114, and Uranium 

238 in otoliths of red snapper may be markers attributable to Mississippi River discharge. These 

five isotopes have significantly higher concentrations in otoliths collected west of the Mississippi 

River both on Louisiana oil and gas platforms and Louisiana artificial reefs. Cobalt 59 and 

Nickel 62 concentrations are both higher in otoliths sampled east of the Mississippi River off 

Alabama. Due to the close proximity of the sampling area to Mobile Bay, this fingerprint may 

be derived from Mobile Bay discharge, or from materials used to construct artificial reefs from 

which red snapper were sampled in Alabama. It must be noted, however, that the inferences 

above are speculative and will require more study before firm conclusions can be drawn. 

When comparing otolith elemental concentration with red snapper sex, Vanadium 51 and 

Uranium 238 both have higher mean concentrations in otoliths of male red snapper than females 

at all three study areas. One possible explanation of these results may be that these elements are 

lipophilic. Female red snapper may be distributing these elements into egg yolk and “dumping” 

them during spawning, whereas male red snapper do not have this opportunity. These findings 

suggest the need for more research into how these particular elements and reproduction interact. 

The ideas mentioned are speculative and require more research before firm conclusions can be 

drawn. 

The main statistical techniques utilized to discriminate red snapper otolith elemental 

concentration in those fish sampled on oil and gas platforms and on artificial reefs were LDA, 

SDA, and CDA. These analyses were chosen based upon their wide use in otolith 

microchemistry research. For example, Campana et al. (1994), Campana and Gagne (1995), and 

Patterson et al. (1998) all used SDA while Edmonds et al. (1992), Edmonds et al. (1995), and 

Milton et al. (1997) utilized CDA instead to interpret their otolith microchemical data. 

Although SDA and CDA are very similar techniques and both successfully discriminated 

between otoliths collected on oil and gas platforms and artificial reefs, elements or isotopes 

identified by these methods were not entirely consistent. Canonical discriminant analysis 

employed a larger number of isotopes than did SDA. For example, when discriminating between 

Louisiana oil and gas platforms and Louisiana artificial reefs, both techniques identify Vanadium 

51, Silver 109, Lead 206, and Uranium 238 as key isotopes. However, SDA also identifies Zinc 

66 and Nickel 62 as important for discrimination between the two habitats. In contrast, CDA did 

not indicate Zinc or Nickel as being important, but included Lead 207, Lead 208, and Silver 107. 

Although differences exist, both statistical techniques identify the main elements we felt were 

associated with oil and gas platforms in coastal Louisiana (Continental Shelf Associates, Inc., 

2004; Neff, 1987; Neff et al., 1987), namely Vanadium and the 3 isotopes of Lead previously 

mentioned. 

When comparing the results of the SDA and CDA for the discrimination of otoliths collected 

east and west of the Mississippi River, Lead 208, Vanadium 51, and Uranium 238 are chosen as 

important for discrimination by both techniques. SDA identifies Copper 65, Silver 107, and 
53

Cobalt 59 as important isotopes for discrimination as well. The CDA did not identify any of 

these isotopes as important, and instead recognizes Nickel 62, Zinc 64, Zinc 66, and Lead 207. 

Most importantly, while the two techniques use different elements to discriminate between 

otoliths collected on artificial reefs east and west of the Mississippi River, neither employs 

elements associated with the platform signature. All of the elements identified by SDA and 

CDA as being important for discrimination in any of the three comparisons were also identified 

by the principal components analysis as explaining a large portion of the variance within the 

ICP-MS data. 

Care needs to be taken with using ID-ICPMS and the dissolution of whole otoliths if 

concentrations of some of the elements are too high. During this study, 17 otoliths were lost to 

the analysis due to the detector limits being set at a concentration range outside of the values 

encountered with the sample, and all of the sample being used up during the instrument run. 

Although the results of this study are promising, there are some identifiable weaknesses that 

should be mentioned. The inability to collect red snapper on oil and gas platforms east of the 

Mississippi River limited the ability to determine if the microchemical signature in otoliths 

collected on Louisiana oil and gas platforms are evident and similar to those in fishes collected 

from platforms east of the River. Future studies should address this issue by sampling red 

snapper east of the Mississippi River and rerunning the analyses described here. Also, Zinc 

anodes that are attached to boat hulls and oil and gas platforms to prevent electrolysis may have 

contributed to the high Zinc 64 and Zinc 66 concentrations that were seen in otoliths collected in 

all three of the study areas. 

The red snapper collected on Louisiana oil and gas platforms were smaller in total length 

(mm) than the specimens collected both on Louisiana artificial reefs (by 100 mm on average) 

and Alabama artificial reefs (by 50 mm on average). Therefore, the otoliths from the Louisiana 

sample were smaller also. Because it has been determined that otolith growth rates of larval and 

juvenile fish may affect otolith elemental microchemistry (Thorrold et al., 1997; Patterson et al., 

1998, we assumed that otolith growth rates were similar despite differences in size of red snapper 

collected. This assumption was based on the results of Fischer et al. (2004) who examined 

growth rates, size-at-age, and length and weight information of red snapper collected from the 

recreational harvests off Alabama, Louisiana, and Texas from 1999 to 2001. Fischer et al. 

(2004) collected red snapper from Dauphin Island, Alabama, Port Fourchon, Louisiana, and Port 

Aransas, Texas and recorded pertinent morphometric measurements. Red snapper growth was 

modeled from weighted mean fork length at age and mean total weight at age by using the von 

Bertalanffy growth equation (Fischer et al., 2004). Results found that models of mean red 

snapper fork length at age for Alabama and Louisiana were similar, with likelihood ratio tests 

showing no significant differences between growth rates of fish collected in Louisiana and 

Alabama (Fischer et al., 2004). 

It is important to reemphasize that the 15 elements analyzed in this study are not the usual 

suite of elements analyzed in most otolith microchemistry research (Campana, 1999; Campana et 

al., 1994; Dove et al., 1996; Edmonds et al., 1992; Patterson et al., 1998). The elements chosen 

for analysis were those that other data suggested would be present as a unique “oil and gas 

platform signature.” We expected this signature to be attributable to both drilling muds used 

during the initial drilling operations of the oil and gas platforms, as well as from the natural 

weathering processes that occur on these structures over time (Continental Shelf Associates, Inc., 

2004; Gallaway et al., 1981; Mearns and Moore, 1976; Neff, 1987; Neff et al., 1987). Some 

examples of these weathering processes include natural corrosion and degradation of the 
54

platform gratings, decks, and “legs” into the surrounding water column. The elements chosen 

for analysis are also heavy metals and replace calcium in the aragonite crystal matrix. In 

addition, with the exception of zinc, We felt that these elements would not be associated with a 

food web signal, but would be incorporated from the surrounding water mass and sediments 

(Campana, 1999; Ni et al., 2000). Elements that are associated with a food web signal tend to be 

light and get incorporated into the protein matrix rather than the aragonite crystal matrix (James 

H. Cowan, Jr.—personal communication). 

Otolith microchemistry research has been a widely expanding field, yet such work involving 

red snapper in the Gulf of Mexico has been limited. Most otolith microchemistry studies, 

particularly in the early years, were aimed primarily at questions of stock discrimination. Now 

there are more studies that are attempting to use this technique to answer questions about habitat 

value and differential sources of recruitment to adult habitats, especially for fishes like red 

snapper where early life history stages occupy different habitats than adults. 

For example, a previous study by Patterson et al. (1998) aimed to differentiate between three 

important nursery areas of age-0 red snapper in the Gulf of Mexico using otolith microchemical 

fingerprints. Patterson et al. (1998) collected age-0 red snapper by shrimp trawls at randomly 

located stations (NMFS Fall Groundfish Survey) from the near-shore waters of south Texas, 

western Louisiana, and Alabama/Mississippi, which are thought to be three of the main nursery 

areas for red snapper in the Northern Gulf. Otoliths collected were dissolved and analyzed using 

inductively coupled plasma-atomic emissions spectrometry (ICP-AES). The presence of twelve 

elements was detected in the red snapper otolith solutions with the ICP-AES and those elements 

were Al, As, Ba, Ca, Fe, K, Mg, Mn, Na, Se, Sr, and Zn. Otolith elemental data was analyzed 

statistically to determine the microchemical fingerprints from each nursery area (Patterson et al., 

1998). Statistical analyses included an analysis of covariance (ANCOVA), an analysis of 

variance (ANOVA), and a multivariate analysis of variance (MANOVA). 

The results of the study by Patterson et al. (1998) found 10 of the 12 elements analyzed to be 

significantly different among the three nursery areas; Al, As, Ca, Fe, Mg, Mn, Se, Zn, K, and Na. 

However, only Al, As, Ca, Fe, Mg, Mn, Se, and Zn were used in subsequent multivariate 

analyses to determine otolith microchemical fingerprints of the three nursery areas (Patterson et 

al., 1998). K and Na were excluded because incorporation of these elements into otoliths is most 

likely under physiological control and thus may not result from true differences in water 

chemistry (Campana and Gagne, 1995; Kalish, 1989). Those eight elements were then entered 

into a stepwise discriminant function analysis that was significant, with the best model including 

Mg, Se, As, Fe, and Al entered in that order (Patterson et al., 1998). Classification accuracies 

based upon the probability of an individual fish being correctly classified into the nursery area 

from which it came were over 90% for each nursery area (Patterson et al., 1998). The results of 

the chemical and statistical analyses showed that 1995 age-0 red snapper in the northern Gulf of 

Mexico had otolith microchemical fingerprints unique to their nursery area (Patterson et al., 

1998). Once Patterson et al. (1998) determined that they could successfully use otolith 

microchemistry to determine the nursery area signature of juvenile red snapper in the Gulf of 

Mexico, they inferred that they could extend this hypothesis to adult red snapper, allowing them 

to sample adults on offshore habitats and determine whether fish were recruiting from nearby or 

distant nursery areas. Their results showed that most recruits to offshore reefs were derived from 

nearby nursery grounds, but the movement between reefs was more frequent as fish got older. 

More importantly, Patterson et al. (1998) clearly demonstrated that otolith microchemical 
55

fingerprints were useful for estimating patterns of long-term movement of reef-associated 

species. 

Despite some obvious differences, this experiment is an extension of the “nursery area 

hypothesis” explored by Patterson et al. (1998). Instead of determining the signatures of 

different red snapper nursery areas, or natural habitats, this study’s goal was to determine if it 

was possible to identify the signature of oil and gas platforms, or man-made red snapper habitat, 

which are much smaller in spatial extent despite the large number of individual platforms. 

Similar to the work by Patterson et al. (1998), a number of other studies have attempted to 

use otolith microchemistry to link fishes to specific habitats. Gillanders and Kingsford (1996), 

on the east coast of Australia, aimed to determine if populations of blue grouper (Achoerodus 

viridis) on rocky reefs were sustained by (1) recruitment to estuarine seagrass habitat followed 

by movement to rocky reefs, (2) direct recruitment to rocky reefs, or (3) a combination of the 

two. Recruits were collected from estuarine seagrass and rocky reef habitats and elements in 

their otoliths were analyzed by ICP-MS to determine if different habitat specific elemental 

fingerprints could be found (Gillanders and Kingsford, 1996). With further analysis, the larger 

goal was to determine the possible source (i.e., seagrass or rocky reef) of adult reef fish by 

analyzing the core of otoliths from adults (i.e., deposited during the juvenile recruitment phase) 

and relating this to the composition of otoliths from recruits from each environment. 

Significant differences in the elemental composition of otoliths were found between recruits 

collected from seagrass and rocky reef environments (Gillanders and Kingsford, 1996). 

Strontium occurred in significantly higher concentrations in otoliths from fish collected on rocky 

reefs, whereas Ba, Mn, and Co occurred in significantly greater concentrations in otoliths of fish 

collected from seagrass habitats. Strontium, Mn, and Ba were used as variables in a 2 group 

(seagrass and rocky reef) LDA that resulted in a significant separation of recruits between rocky 

reef and seagrass environments. There was a misclassification error rate of 5.5% when 

classifying recruits using the cross-validation method. The algorithms generated from recruits 

classified 59% of adult fish as having settled directly on rocky reefs while 41% of the adults had 

first recruited to seagrass environments before moving to the reefs (Gillanders and Kingsford, 

1996). 

In another example, Thorrold et al. (1997) used laser ablation ICP-MS to assay sectioned 

otoliths from juvenile Atlantic croaker (Micropogonias undulatus) collected from the Neuse 

River, North Carolina, and the Elizabeth River, Virginia. They hoped to identify factors that 

potentially influenced larval survival and transport from spawning sites to juvenile nursery areas 

and to gain insight into the geographic extent of self-recruiting populations of Atlantic croaker in 

the Mid-Atlantic and South Atlantic Bights. Yet, they found little evidence for differences in the 

trace element composition between the cores of otoliths from croakers collected in the Neuse and 

Elizabeth Rivers. More variation between locations was seen in assays from the otolith edge 

with Mg:Ca and Ba:Ca being significantly different. Both elements were found to be 

significantly higher in otoliths from the Neuse River than in those from the Elizabeth River 

(Thorrold et al., 1997). Despite this variability, Thorrold et al. (1997) were unable to reject the 

hypothesis that croaker larvae form north and south of Cape Hatteras originated from different 

spawning sites, which might indicate that the larvae were spawned in close geographic 

proximity. 

Finally, Gillanders and Kingsford (2000) collected juvenile trumpeter Pelates sexlineatus

over a 2-year period from two sites in seven estuaries along the east coast of Australia. The main 

objective of the study was to determine variation in elemental fingerprints among and within 
56

estuaries. Otoliths were removed and analyzed by solution-based ICP-MS; preliminary analyses 

suggested that seven elements (Mg, Mn, Cu, Zn, Sr, Ba, and Pb) were detectable in the otoliths 

(Gillanders and Kingsford, 2000). However, their results showed that significant differences 

were found in the otolith chemistry of juvenile trumpeter from different estuaries, but that 

success rates of classification to recruited estuaries ranged from 50 to 100% (Gillanders and 

Kingsford, 2000). While their results were promising, Gillanders and Kingsford (2000) 

suggested that the addition of further elements to the discriminant function and the use of stable 

isotopes might improve their classification accuracies. 

There are several such examples in the literature similar to the studies described above (Secor 

et al., 1995a; Jessop et al., 2002; Sanchez-Jerez et al., 2002; Swan et al., 2003; Arai et al., 2004; 

Brazner et al., 2004; Chittaro et al., 2004; and Arslan and Secor, 2005, to name but a few). 

Although the study by Patterson et al. (1998) was the only other from the Gulf of Mexico using 

red snapper, it is plausible to compare this study to those discussed above, as they represent 

direct comparisons to this research and others in the current literature, with one notable 

exception. This study is unique because it deals with an elemental signature of oil and gas 

platforms, i.e., a fingerprint derived from a man-made habitat. 

A study by Spencer et al. (2000) that utilized distinct anthropogenic sources of lead in fish 

otoliths as a potential nursery ground stock marker in Hawaii is the “bridge” that closes the gap 

between this study and those in the previous literature. Spencer et al. (2000) collected three 

species of juvenile tropical reef fish (parrotfish, sergeant major, and domino damselfish) at 5 

locations in Kaneohe Bay, Oahu, and used ICP-MS for otolith analysis. Variations measured in 

the lead stable isotope ratios in the otoliths reflected mixing of anthropogenic lead from the 

Kaneohe Bay watershed and “background” lead characteristic of the adjacent ocean (Spencer et 

al., 2000). They found that the lead isotopic composition of the watershed has a low 206Pb/204Pb 

signature primarily reflecting past combustion of tetra-ethyl Pb additives in fuels, while the 

ocean water has a high 206Pb/204Pb isotopic composition (Spencer et al., 2000). The key issue 

was that the characteristic anthropogenic Pb isotope ratios are a qualitative rather than 

quantitative marker, so that the reliable detection of the presence of distinct Pb isotopes is all that 

is required for nursery ground discrimination (Spencer et al., 2000). The use of an 

anthropogenic otolith signature instead of naturally occurring markers inspired the expansion of 

the “nursery area hypothesis” to manmade rather than natural nursery habitats. 

The overall objectives of all of the studies mentioned involve determination of the origin of 

adult recruits in coastal and offshore environments. This is very similar to the main goal of the 

next phase of this research; namely to determine if adult red snapper now recruiting to habitats in 

the eastern Gulf and elsewhere have spent any portion of their lives on oil and gas platforms. 

The real distinction between this research and the studies performed earlier in the literature is 

that it is the first to deal with determining the otolith “elemental fingerprint” of reef fishes 

attributable to their association with man-made habitats rather than natural habitats. If findings 

hold true, it may provide a new direction in which this type of research may expand. 

CONCLUSIONS 

Despite the fact that the main goal of this study was to prove the concept that otolith 

microchemistry could be used to determine association of red snapper with oil and gas platforms 

in the Gulf of Mexico, a number of other important conclusions can be drawn. They are as 

follows: 
57

• This method was successful; otolith microchemistry can be used to determine 

the trace element signature of oil and gas platforms in otoliths of red snapper. 

• Vanadium 51, Lead 206, Lead 207, and Lead 208 may be dissolution products 

incorporated into red snapper otoliths from oil and gas platform operations 

and their prior drilling operations. 

• It is plausible to move forward with microdrill sub-sampling of either the 

otolith cores or progressively out from the core of adult fish, to determine if 

the new recruits that are now expanding into the eastern Gulf were associated 

with oil and gas platforms during some portion of their early life, and to 

determine age-specific habitat affinity. 

• It may be possible to determine if there are a disproportionate number of adult 

red snapper in the eastern Gulf and elsewhere that have acquired this “oil and 

gas platform signature” in their otoliths, during sometime in their early life 

history. 

• If the above statement is true, oil and gas platforms may constitute red snapper 

essential fish habitat and, as such, should be considered as viable tools in 

management of red snapper.
59

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temperature and strontium calcium concentration ratios in otoliths of the Red Sea Bream 

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65

APPENDIX: MORPHOMETRIC AND ICP-MS SUMMARY TABLES 

Morphometric data for the 32 red snapper collected on oil and gas platforms off of Louisiana. 

ID # Site # Total 

Length 

(mm) 

Otolith Wt 

(g) 

Fish Wt (g) Sex 

GP01-LA 1 375 0.36634 724.19 M 

GP02-LA 1 500 0.63084 1797.45 M 

GP03-LA 1 351 0.37334 542.7 F 

GP04-LA 1 360 0.35163 636.54 M 

GP05-LA 1 366 0.36236 670.67 M 

GPO6-LA 1 318 0.27575 394.51 F 

GPO7-LA 1 346 0.3904 518.12 F 

GPO9-LA 1 386 0.43072 737.73 F 

GP10-LA 1 321 0.29512 406.66 F 

GP11-LA 1 342 0.35701 541.3 M 

GP12-LA 1 346 0.3163 561.55 M 

GP13-LA 1 410 0.44523 960.08 M 

GP14-LA 1 332 0.27466 453.41 F 

GP15-LA 1 335 0.33814 466.78 F 

GP16-LA 1 324 0.31931 419.06 F 

GP17-LA 1 357 0.32612 573.24 F 

GP18-LA 1 321 0.3267 443.07 M 

GP20-LA 1 347 0.34578 522.98 F 

GP21-LA 1 400 0.46436 827.7 F 

GP23-LA 1 491 0.642 1697.18 M 

GP24-LA 1 408 0.4376 945.36 M 

GP25-LA 1 615 0.9057 3321.12 F 

GP26-LA 1 402 0.442 841.14 F 

GP27-LA 1 442 0.53 1142.7 F 

GP28-LA 1 422 0.4918 1051.72 M 

GP29-LA 1 405 0.4392 861.58 F 

GP30-LA 1 419 0.4941 961.54 F 

GP31-LA 1 422 0.4693 983.96 F 

GP32-LA 1 421 0.4601 976.45 F 

GP33-LA 1 404 0.4131 854.73 F 

GP34-LA 1 776 1.4123 7038.35 F 

GP35-LA 1 730 1.39556 5777.64 F 
66

Morphometric data for the 31 red snapper collected on artificial reefs off of Louisiana. 

ID # Site # Total 

Length 

(mm) 

Otolith Wt 

(g) 

Fish Wt (g) Sex 

ARO3-LA 2 416 0.44801 939.48 F 

ARO4-LA 2 415 0.44238 997.57 M 

ARO5-LA 2 411 0.41423 967.5 M 

ARO7-LA 2 454 0.51818 1245.97 F 

AR08-LA 2 428 0.51677 1099.7 M 

AR10-LA 2 423 0.52221 1059.61 M 

AR11-LA 2 415 0.4452 932.21 F 

AR13-LA 2 416 0.52625 939.48 F 

AR14-LA 2 573 0.75479 2642.76 F 

AR17-LA 2 461 0.54027 1390.61 M 

AR18-LA 2 460 0.59004 1299.95 F 

AR19-LA 2 535 0.7277 2117.39 F 

AR21-LA 2 411 0.42337 967.502 M 

AR15-LA 2 470 0.57196 1478.23 M 

AR16-LA 2 450 0.4869 1288.44 M 

AR23-LA 2 615 0.85701 3321.12 F 

AR24-LA 2 659 0.96513 4151.62 F 

AR25-LA 2 648 0.89098 3931.92 F 

AR26-LA 2 600 0.79487 3066.52 F 

AR27-LA 2 502 0.68821 1820.27 M 

AR28-LA 2 424 0.42704 999.1 F 

AR29-LA 2 607 0.90285 3183.59 F 

AR30-LA 2 421 0.44649 1043.86 M 

AR31-LA 2 641 0.88207 3796.37 F 

AR32-LA 2 655 1.03172 4070.77 F 

AR33-LA 2 490 0.61123 1594.23 F 

AR34-LA 2 678 1.03883 4550.82 F 

AR35-LA 2 561 0.76898 2468.13 F 

AR36-LA 2 480 0.62642 1491.51 F 

AR37-LA 2 570 0.84107 2719.42 M 

AR38-LA 2 581 0.83424 2888.74 M 
67

Morphometric data for the 35 red snapper collected on artificial reefs off of Alabama. 

ID # Site # Total 

Length 

(mm) 

Otolith Wt 

(g) 

Fish Wt (g) Sex 

AR01-AL 3 500 0.5507 1797.45 M 

AR02-AL 3 470 0.6021 1478.23 M 

AR03-AL 3 435 0.4406 1157.54 M 

AR04-AL 3 438 0.5579 1182.96 M 

AR05-AL 3 450 0.5582 1210.86 F 

AR06-AL 3 483 0.5557 1611.33 M 

AR07-AL 3 457 0.5546 1272.76 F 

AR08-AL 3 447 0.5291 1261.49 M 

AR09-AL 3 491 0.6259 1697.18 M 

AR10-AL 3 539 0.6972 2278.94 M 

AR11-AL 3 467 0.5313 1448.61 M 

AR12-AL 3 510 0.688 1913.52 M 

AR13-AL 3 472 0.5819 1498.2 M 

AR14-AL 3 408 0.4527 945.36 M 

AR15-AL 3 544 0.6514 2346.41 M 

AR16-AL 3 439 0.5072 1117.84 F 

AR17-AL 3 430 0.4999 1045.49 F 

AR18-AL 3 402 0.4501 902.12 M 

AR19-AL 3 426 0.4738 1014.4 F 

AR20-AL 3 392 0.3647 833.1 M 

AR21-AL 3 448 0.5001 1193.56 F 

AR22-AL 3 402 0.4518 902.12 M 

AR23-AL 3 390 0.4786 819.74 M 

AR24-AL 3 426 0.5526 1014.4 F 

AR25-AL 3 596 0.8786 3131.05 M 

AR26-AL 3 447 0.5305 1184.98 F 

AR27-AL 3 495 0.636 1741.26 M 

AR28-AL 3 571 0.8468 2734.52 M 

AR29-AL 3 400 0.4541 888.02 M 

AR30-AL 3 465 0.5437 1429.1 M 

AR31-AL 3 401 0.4459 834.4 F 

AR32-AL 3 408 0.4961 882.37 F 

AR33-AL 3 505 0.558 1757.31 F 

AR34-AL 3 474 0.57 1432.13 F 

AR35-AL 3 419 0.4783 961.54 F 
68 

Summary table of the ICP-MS data (concentration in ppb) for all three study sites. 

SAMPLE: Gas Platform LA samples (n = 32 ) Artificial Reef LA samples (n = 31) Artificial Reef A

AVERAGE STD DEV MAX MIN AVERAGE STD DEV MAX MIN AVERAGE ST

Vanadium V 51 1.51 1.11 5.52 0.28 0.57 0.64 3.31 0.06 1.19 

Cobalt Co 59 1.56 1.17 6.86 0.53 1.94 1.02 3.78 0.51 4.08 

Nickel Ni 62 14.2 11.1 42.1 3.6 13.8 9.9 39.1 4.0 25.1 5

Zinc Zn 64 247 84 532 120 341 153 783 129 317 

Copper Cu 65 53.4 21.8 157.5 30.1 57.2 13.0 87.8 18.3 44.9 

Zinc Zn 66 263 78 539 145 369 135 681 151 302 

Silver Ag 107 0.27 0.10 0.48 0.08 0.20 0.08 0.39 0.05 0.11 Silver Ag 109 0.27 0.09 0.44 0.10 0.18 0.08 0.41 0.05 0.13 

Cadmium Cd 110 0.24 0.30 1.15 -0.03 0.16 0.08 0.41 0.05 0.12 Cadmium Cd 111 0.22 0.29 1.20 -0.04 0.15 0.08 0.35 0.03 0.11 Cadmium Cd 114 0.37 0.31 1.37 0.07 0.40 0.45 2.47 0.02 0.11 

Lead Pb 206 2.46 2.63 10.47 0.15 1.10 0.68 4.07 0.28 1.58 Lead Pb 207 2.34 2.51 10.01 0.14 1.10 0.61 3.81 0.34 1.46 Lead Pb 208 2.41 2.55 10.03 0.07 1.15 0.63 3.85 0.37 1.69 

Uranium U 238 0.07 0.19 1.05 -0.02 0.08 0.20 1.13 0.00 0.01 
69

Summary table of the elements identified as important by each of the statistical analyses 

performed on the ICP-MS data. (site 1 = LA oil and gas platforms; site 2 = LA artificial reefs; 

site 3 = AL artificial reefs) 

Analysis: MANOVA PCA LDA & SDA CDA 

Site: All Sites 1 vs 2 2 vs 3 All Sites All Sites 1 vs 2 2 vs 3 All Sites 1 vs 2 2 vs 3

V 51 X X X X X X X X X 

Co 59 X X X X 

Ni 62 X X X X X 

Zn 64 X X X X X 

Cu 65 X X X X 

Zn 66 X X X X X X X 

Ag 107 X X X X X X X X 

Ag 109 X X X X X X 

Cd 110 X X 

Cd 111 X X 

Cd 114 X X X X X 

Pb 206 X X X X X X 

Pb 207 X X X X X X 

Pb 208 X X X X X X X X 

U 238 X X X X X X 
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