OCS Study 

MMS 2010-016 

 

Trophic Aspects of Sperm Whales 

(Physeter macrocephalus) in the 

Northern Gulf of Mexico Using 

Stable Isotopes of Carbon and Nitrogen 

U.S. Department of the Interior

Minerals Management Service 

Gulf of Mexico OCS Region 
OCS Study 

MMS 2010-016 

Trophic Aspects of Sperm Whales 

(Physeter macrocephalus) in the 

Northern Gulf of Mexico Using 

Stable Isotopes of Carbon and Nitrogen 

Authors 

R.I. Ruiz-Cooley 

D. Engelhaupt 

Prepared under MMS Contract 

1435-01-06-CT-39790 (M06PC00011) 

by 

New Mexico State University 

Arts & Science Research Center 

Box 3000, MSC RC 

Las Cruces, NM 88003 

Published by 

U.S. Department of the Interior

Minerals Management Service 

Gulf of Mexico OCS Region 

New Orleans

April 2010 
DISCLAIMER 

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

New Mexico State University (NMSU). 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 views 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 the 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 2010-016. 

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 

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Fax: (504) 736-2620 

CITATION 

Suggested citation: 

Ruiz-Cooley, R.I. and D.T. Engelhaupt. 2010. Trophic aspects of sperm whales (Physeter 

macrocephalus) in the northern Gulf of Mexico using stable isotopes of carbon and 

nitrogen. U.S. Dept. of the Interior, Minerals Management Service, Gulf of Mexico OCS 

Region, New Orleans, LA. OCS Study MMS 2010-016. 28 pp. 

ABOUT THE COVER 

Cover shows mother and calf sperm whales surfacing (upper photo) and a sperm whale fluking 

(lower photo) in the northwest Gulf of Mexico on the 2005 S-tag cruise (photos by Craig Hayslip, 

Oregon State University). 

ii
ACKNOWLEDGMENTS 

This work was made possible thanks to the significant support and collaborative work of many 

scientists from the Sperm Whale Seismic Study (SWSS). For their extensive support during field 

work, we thank Ann Jochens and Doug Biggs (Texas A&M University), Bruce Mate and Joel 

Ortega-Ortiz (Oregon State University), Deborah Epperson and Carol Roden (Minerals 

Management Service) and the scientific team and crew of the RV-Gyre (SWSS project). 

We are grateful to John Wormouth (TAMU), Kim Johnson, Andre Dubose, and Alonzo Hamilton 

(Department of Commerce/National Oceanic and Atmospheric Administration/ National Marine 

Fisheries Service) for the collection of squid specimens. 

iii
TABLE OF CONTENTS 

 Page

List of Figures............................................................................................. v 

List of Tables............................................................................................... vi 

1 Summary.......................................................................................... 1 

2 Introduction ....................................................................................... 3 

3 Methods............................................................................................. 4 

3.1 Sample Collection, Preservation and DNA Extraction............................... 4 

3.2 Squid Muscle Samples.................................................................. 4 

3.3 Genetic Analysis.......................................................................... 6 

3.4 Stable Isotope Analysis.................................................................. 6 

3.5 Statistical Analysis....................................................................... 6 

4 Results............................................................................................... 7 

4.1 Relative Trophic Position of Sperm Whales........................................... 7 

4.2 Comparison between Sperm Whales from the Gulf of Mexico and Gulf of 

California............................... ................................................... 8 

4.3 Isotopic Signatures between Sperm Whales and Squid from the Gulf of 

Mexico....................................................................................... 8 

5 Discussion........................................................................................... 9 

5.1 Relative Trophic Position of Sperm Whales.......................................... 9 

5.2 Temporal Variation ...................................................................... 11 

5.3 Spatial Variation.......................................................................... 12 

6 Recommendations................................................................................ 14 

7 References......................................................................................... 15 

Appendix A. Stable Isotopic Ratios from Skin Samples Collected in the Northern Gulf of 

 Mexico from 2002 to 2005.............................................................. 19 

iv
LIST OF FIGURES 

 Page

Figure 1. Sampling location of skin of sperm whales (X) and pelagic squid (, in red), 

 and inshore shallow water squid () from the northern Gulf of Mexico. 

Contours indicate the 200 m, 1,000 m, and 2,000 m isobaths..................... 5 

Figure 2. Temporal variation in carbon (A) and nitrogen (B) isotopic ratios (‰) of skin 

samples from immature male () and female () sperm whales from the 

northern Gulf of Mexico................................................................. 7 

Figure 3. Stable isotope ratios (‰; mean ± 95% confidence intervals) from skin samples 

of free-ranging sperm whales (SW; ), muscle tissue samples of mesopelagic 

and neritic squid (Loligo pealei, L. plei, Lolliguncula brevis), and a myctophid 

collected in the northern Gulf of Mexico ()......................................... 9 

 

v
vi

LIST OF TABLES 

 Page

Table 1. Stable isotope signatures (‰) from sperm whale (Physeter macrocephalus) 

skin tissues from the Mississippi Canyon (MC) and Rio Grande Slope 

(North West (NW)) Gulf of Mexico................................................... 8 
1. Summary 

In the northern Gulf of Mexico (GoM), seismic operations and oil exploration are increasing in 

deepwater areas which are inhabited by sperm whales (Physeter macrocephalus). Yet, little is 

known about the feeding ecology of sperm whales. This report provides initial information on 

the variability in the relative trophic position of sperm whales in the northern GoM. Stable 

isotope ratios of carbon (C) and nitrogen (N) were measured from skin samples of free-ranging 

whales. The isotopic signatures were different between females and immature males. However, 

no differences were detected between samples from the Mississippi Canyon and samples from 

the northwestern GoM, perhaps due to similar consumption of average diet. We compared the 

isotopic values between individuals from the GoM and Gulf of California (GC), and between 

squid from inshore and offshore areas of northern GoM. The isotopic signatures of sperm whales 

from the GoM were contrastingly lower than whales from the GC. This isotopic difference may 

be associated with a variation in diet and different trophic level. However, the variability in the 

amount of C and N and biochemical cycling seems to influence this isotopic difference. 

Although no annual isotopic variation was detected during the years of study, this work 

recommends continued evaluation of how the relative trophic position (indicated by 13C and 

15N) in sperm whales’ skin may change through time. We documented significant spatial 

isotopic variability in squid between inshore and offshore sites of the northern Gulf; this 

variation has important implication for evaluating trophic relationships using stable isotope 

analysis. We recommend continued investigation of the feeding ecology of free-ranging whales 

by using stable isotope ratios in association with mesopelagic cephalopods. 

The objectives of the project were 

I. Analyze sperm whale skin samples collected in previous SWSS cruises 

(2002-2005) 

II. Conduct stable isotope analysis of different cephalopod species collected 

in 2003 from SWSS cruises where sperm whales were sighted 

III. Conduct analysis of cephalopods collected by NOAA/NMFS scientists in 

the summer and fall of 2005 in the northern Gulf of Mexico. 

Accomplished objectives include

I. Completed stable isotope analysis of sperm whale tissue samples from 

2002, 2003, 2004 and 2005. 

II. Completed stable isotope analysis from squid tissue samples collected in 

2003 in areas where sperm whales were distributed 

III. Completed stable isotope analysis from squid collected in 2005 by 

NOAA/NMFS scientists. 

 1
 2

Deliverables 

I. Final Report 

II. Presentation at the 2007 Information Transfer Meeting in Kenner, 

Louisiana 

III. Presentation at the 2007 17th Biennial Conference on the biology of 

Marine Mammals in South Africa 

IV. Reference values of isotopic ratios (13C and 15N) in sperm whale skin 

samples collected from 2002 to 2005 
Trophic Aspects of Sperm Whales (Physeter macrocephalus) in the Northern Gulf of 

Mexico Using Stable Isotopes of Carbon and Nitrogen 

R. I. Ruiz-Cooley 1, * and D. T. Engelhaupt 2 

1

 Department of Biology, New Mexico State University, MSC 3AF, Las Cruces, NM, 88003-

8001, USA 

* Current address: Protected Resources Division, Southwest Fisheries Science Center, NOAA 

Fisheries, 3333 North Torrey Pines Court, La Jolla, CA 92037, USA 

2 School of Biological and Biomedical Sciences, University of Durham, South Road, Durham, 

DH1 3LE, UK 

2. Introduction 

Optimal foraging theory predicts that animals select diets that maximize energy intake and 

minimize the cost to obtain nourishment (Schoener 1971). Optimal diet is crucial for endangered 

species as it may affect reproductive success. This is particularly important for those populations 

inhabiting areas where anthropogenic activities are increasing. Sperm whales inhabit the northern 

Gulf of Mexico (GoM) in water depths between 200 to 2000 m, near cyclone and confluence 

zones (Hansen et al. 1995, Davis and Fargion 1996, Davis et al. 2000). The distribution of sperm 

whales might be related to the occurrence of cephalopod species (Davis et al. 2000), but there is 

not much information about their diet composition and attributes of potential prey (e.g. 

abundance and diversity of cephalopods) in the GoM. 

The main used method to investigate the diet of sperm whales is analysis of stomach contents 

from carcasses obtained during whaling or from strandings (e.g. Okutani and Nemoto, 1964, 

Clarke et al. 1988). Measuring stable isotopes of carbon (C) and nitrogen (N) from animal tissues 

offers an alternative and non-invasive method to traditional analysis to investigate the trophic 

ecology of sperm whales (Ruiz-Cooley et al. 2004, Marcoux et al. 2007). The 13C/12C and 

15N/14N ratios from skin samples of sperm whales and muscle samples of cephalopods indicate a 

trophic position that depends on their assimilated diet. Isotopic ratios can be used to evaluate 

changes in trophic position at temporal and spatial scales, and also to infer trophic relationships 

between sperm whales and potential prey (Ruiz-Cooley et al. 2004, Marcoux et al. 2007). 

The goals of this study were: (1) to quantify 13C and 15N in skin samples of free-ranging sperm 

whales from the northern Gulf of Mexico, (2) to investigate temporal (interannual) variation in 

relative trophic position, and (3) to compare the relative trophic position between whales from 

two locations: Mississippi Canyon and the Northwestern Gulf of Mexico. We tested the null 

hypothesis of no differences in sperm whale trophic position between years and areas due to low 

changes in the average assimilated diet. Squid were collected in sites where sperm whales were 

observed and muscle tissue samples used to determine isotopic ratios. Potential trophic 

3
relationships between sperm whales and squid were inferred comparing their isotopic ratios. 

Since we lacked a representative sample size of mesopelagic squids (e.g.> 50 individuals by 

site), we also collected squid from inshore waters from the upper northern Gulf to understand 

spatial isotopic variation between inshore and offshore sites. This allowed us to recognize how 

13C and 15N in squid change as a function of location within the northern Gulf of Mexico. 

Efforts are needed to investigate mesopelagic squid and evaluate their relationship with sperm 

whales as potential prey simultaneously and at different temporal scales. 

3. Methods 

3.1 Sample Collection, Preservation and DNA Extraction 

Skin tissue from free-ranging sperm whales was collected under Marine Mammal Protection 

Act/Endangered Species Act permit #909-1465 and #909-1726. Samples included in this study 

were from adult female and immature whales. No samples were collected from physically mature 

adult males or calves. Tissue samples were collected with a biopsy dart or from sloughed skin 

(Lambertsen 1987, Palsbøll et al. 1991, Amos et al. 1992), including sloughed skin 

opportunistically collected from suction cups that had been attached to sperm whales (Miller et 

al. 2004a and b). Biopsy samples were collected with a sterilized corer tip attached to a dart 

(with float) fired from a crossbow or PAXARMS biopsy system. A total of 53 skin samples of 

sperm whales were collected from 2002 to 2004 and stored in an aqueous solution of 20 % (v/v) 

dimethyl sulfoxide (DMSO) saturated with sodium chloride (Amos and Hoelzel 1991). In 2005, 

each collected skin sample (n = 18) was divided into two pieces: one half was kept frozen at -20 

C for isotopic analysis and the other half in DMSO for gender determination. Whole-cell DNA 

was extracted for use in subsequent polymerase chain reactions (PCR) by a standard 

phenol/chloroform extraction technique (Hoelzel 1998). 

3.2 Squid Muscle Samples 

C and N isotopic ratios in some squid species vary by size and location (Ruiz-Cooley et al. 

2006). Therefore, it is highly recommended to collect animal tissues from both predator and prey 

from the same area to evaluate trophic relationships properly. We obtained muscle tissue 

samples from mesopelagic cephalopods collected at night (Table 1) using 14.8 m2

 Isaacs-Kidd 

midwater trawl at different depths (0-400 m, 0-400-600 m and 0-600-800 m; Wormuth 2006).

These specimens were obtained in areas of the GoM where sperm whales were observed (Figure 

1) and presumed to be engaged in foraging activity (Miller et al. 2004a; Wormuth 2006). 

During the summer and autumn of 2005, we collected muscle tissues from inshore neritic species 

in areas where sperm whales do not occur. Our goal was to evaluate the spatial isotopic variation 

from the northern Gulf of Mexico and distinguish isotopic values from potential prey 

(mesopelagic squid) of sperm whales from those of a non-prey (inshore shallow squid). These 

inshore squid species were collected along the inshore northern Gulf of Mexico by personnel 

from the National Marine Fisheries Service, Pascagoula, Mississippi, aboard National Oceanic 

4
and Atmospheric Administration research vessels (Figure 1). For these specimens, we measured 

mantle lengths to the nearest mm using a digital caliper. We analyzed muscle samples from the 

arrow squid Loligo plei (Blainville 1823, n = 61), the brief squid Loligo pealei (Lesueur 1821, n 

= 29) and the long-finned inshore squid Lolliguncula brevis (Blainville 1823, n = 54). For these 

inshore species, L. brevis (mean and SD: 75.7 and 74.8 mm ML) had the smallest mantle length, 

while L. plei (mean and SD: 93.2 and 38 mm ML) and L. pealei (116 and 40 mm ML) were 

comparable in size.

We analyzed muscle tissue samples from the mesopelagic squid Ommastrephes bartrami (n = 1; 

10 cm mantle length (ML)), and from unidentified species from the family Ommastrephidae (n = 

1) and Histioteuthidae (n = 2) collected in 2003 in the same area where sperm whales were 

observed. 

-98 -96 -94 -92 -90 -88 -86 -84 -82 -80

19

21

23

25

27

29

Gulf of Mexico

Figure 1. Sampling location of skin of sperm whales (X) and pelagic squid (, in red), and 

 inshore shallow water squid () from the northern Gulf of Mexico. Contours indicate 

 the 200 m, 1,000 m, and 2,000 m isobaths. 

5
3.3 Genetic Analysis 

Sex was determined using the ZFX/ZFY technique described by Bérubé and Palsbøll (1996) and 

used to evaluate differences between females and males with respect to prey. Male and female 

strandings of known gender from the Gulf of Mexico and Northern Sea were included as a means 

of positive confirmation for PCR amplifications. To avoid biases associated with the 

unintentional inclusion of multiple samples from the same individual, genotype screening at a 

minimum of ten polymorphic microsatellite loci was performed on all samples, and any 

duplicates were eliminated as a result (Engelhaupt 2004). 

3.4 Stable Isotope Analysis 

Most of the tissue samples originated from biopsy darts rather than from sloughed skin. 

Therefore, we followed a lipid extraction of chloroform and methanol (2:1) modified from Bligh 

and Dyer (1959) to maximize removal of lipids. Ruiz-Cooley et al. (2004) analyzed mostly 

sloughed skin samples of sperm whales from the Gulf of California and followed a lipid 

extraction using chloroform and methanol with a volume ratio 1:1. To compare our result with 

the previous study, we obtained two subsamples of a total of five skin samples. For each, we 

extracted lipids following a mixture of chlorophorm: methanol with a volume 1:1 and the other 

one with a volume 2:1. Regarding potential effect from preservatives (i.e. DMSO) on the 

isotopic signature of skin samples, no effects of dimethyl sulfoxide (DMSO) were observed on 

lipid-extracted skin samples of cetaceans in short-term studies (Todd et al. 1997, Marcoux et al. 

2007). Thus, we assumed that presence of DMSO in our samples did not affect the results. 

Skin samples were analyzed by using an isotope ratio mass spectrometer (IRMS, Thermo 

Finnigan Delta Plus) at the New Mexico State University. The 13C and 15N were obtained for 

each sample. Isotope ratios are expressed in standard notation: 15N or 13C = [(R sample / R 

standard) - 1] X 1000, where R sample and R standard are the ratio of 15N/14N or 13C/ 12C in the sample 

and standards (PDB for carbon and atmospheric nitrogen, respectively). IAEA-N-1 and 

ammonium sulfate (IAEA-N-2) provided by NIST were used as a primary standard for nitrogen. 

For carbon, we used LSVEC (Li2CO3) and USGS24 (graphite). The typical long-term external 

precision obtained by repeated analyses of internal laboratory standards was lower than 0.2 ‰. 

3.5 Statistical Analysis 

 The resultant isotopic ratios of C and N from the northern Gulf of Mexico were categorized by 

location: Mississippi Canyon and northwest Gulf of Mexico. We tested the effect of location 

(using only isotopic data of females) and sex on 13C and 15N using a student t-test and 

assuming equal variances. Samples from females and males were pooled by year; we tested the 

effect of year on 13C and 15N values using ANOVA at  = 0.05. Ruiz-Castro (2002) performed 

a lipid extraction with chloroform and methanol with a volume 1:1, whereas we extracted lipids 

using the same mixture but with a volume 2:1. Therefore, we tested the effect of lipid extraction 

with these two methods using a paired t-test. Then, we used published isotopic ratios from 

females and immature sperm whales reported in Ruiz-Castro (2002) from the central Gulf of 

California to compare this area with our two sampling sites using 1-way ANOVA. 

6
4 Results 

4.1 Relative Trophic Position of Sperm Whales 

A total of 71 sloughed skin samples and skin biopsies from free-ranging sperm whales were 

analyzed from 2002 to 2005 for stable isotope analysis. Based on molecular sexing analysis, we 

identified a total of 57 females and 14 immature males. 

Differences between genders were observed in 15N (t = 2.1, P = 0.04, DF = 69). N-isotopic 

values from females (mean, standard deviation (SD) = 12.31 (0.4) for 15N) were higher than 

males (12.05 (0.45) for 15N) by 0.26 ‰. However, variation in 13C (t = -0.47, p = 0.63, DF = 

69) was minimal between sexes (mean (SD) for females was -16.55 (0.43) and for males was -

16.5 (0.27)). No significant differences were found between years for either 15N (ANOVA, F3,67 

 = 1.04, P = 0.38) or 13C (F3,67 = 0.86, P = 0.46) (Figure 2, Table 1). Female isotopic signatures 

(mean (SD)) from the Mississippi Canyon (n = 41, -16.53 (0.47) for 13C and 12.36 (0.40) for 

15N) and Northwest Gulf (n = 16, -16.58 (0.29) for 13C and 12.17 (0.38) for 15N) were not 

detectably different between sites for either carbon (t = 0.41, P = 0.68, DF = 55) or nitrogen (t = 

1.64, P = 0.107, DF = 55) stable isotope ratios. 

Year

2001 2002 2003 2004 2005 2006

13C -17.5

-17.0

-16.5

-16.0

-15.5

-15.0

-14.5

-14.0

-13.5

A

Year

2001 2002 2003 2004 2005 2006

15N 11.0

11.5

12.0

12.5

13.0

13.5

14.0

B

Figure 2. Temporal variation in carbon (A) and nitrogen (B) isotopic ratios (‰) of skin samples 

 from immature male ( in blue) and female (▲) sperm whales from the northern Gulf 

 of Mexico. 

7
Table 1 

Stable isotope signatures (‰) from sperm whale (Physeter macrocephalus) skin tissues from the 

Mississippi Canyon (MC) and Rio Grande Slope (North West (NW)) Gulf of Mexico. 

 Gender 15N 13C 

Year n Area F:M Mean (SD) Min Max Mean (SD) Min Max 

2002 28 MC 19:09 12.26 (0.39) 11.22 12.97 -16.46 (0.53) -16.92 -13.97 

2003 22 MC 19:03 12.25 (0.43) 11.51 12.99 -16.62 (0.21) -17.06 -16.07 

2004 3 MC 3:00 12.65 (0.64) 12.19 13.56 -16.69 (0.15) -16.89 -16.51 

2005 18 NW 16:02 12.19 (0.37) 11.50 12.82 -16.52 (0.33) -16.9 -15.80 

 

4.2 Comparison between Sperm Whales from the Gulf of Mexico and Gulf of California 

The 13C (t = 1.56, p = 0.19, DF = 4) and 15N (t = 2.69, p = 0.054, DF = 4) values of lipid 

extracted skin samples with 1:1 and 2:1 chloroform: methanol ratios were not statistically 

different. The 13C (ANOVA, F1, 105 = 1097.45, P < 0.0001) and 15N (ANOVA, F1, 105 = 5489, 

P < 0.0001) from skin tissue samples from the Gulf of Mexico and Gulf of California were 

significantly different. Females and immature males from the GoM showed mean isotopic 

signatures that were lower by 7.42 ‰ for 15N and by 2.74 ‰ for 13C in comparison with mean 

values from the Gulf of California (Table 1). 

4.3 Isotopic Signatures between Sperm Whales and Squid from the Gulf of Mexico

Stable isotope differences between sperm whales and muscle samples from the family 

Histiotheutidae and Ommastrephidae were 1.6 ± 0.36 ‰ for 13C and 4.3 ± 0.58 ‰ for 15N 

(Figure 3). In contrast, the 15N between sperm whales from the Gulf of Mexico and small 

shallow water squid, L. plei and L. pealei, were similar; they varied by 0.45 ‰ for and 0.2 ‰, 

respectively. However, the 13C values between sperm whales and L. plei were distinct; they 

varied by 1.27 ‰, while the difference between whales and L. pealei was 0.9 ‰ (Figure 3). 

Remarkably, the 15N and 13C values from L. brevis were higher than those from sperm whales 

by 2.81 ‰ and 0.7 ‰, respectively (Figure 3). 

8


13C

-20 -19 -18 -17 -16 -15 -14

15

N

6

8

10

12

14

16

18

L. brevis

Female SW

Immature male SW

L. pealeii

L. pleii

Histioteuthidae

Histioteuthidae

unknown Ommastrephidae

squid sp.

Myctophid

Ommastrephes bartrami

Figure 3. Stable isotope ratios (‰; mean ± 95% confidence intervals) from skin samples of free 

 ranging sperm whales (SW; ), muscle tissue samples of mesopelagic and neritic 

 squid (Loligo pealei, L. plei, Lolliguncula brevis), and a myctophid collected in the 

 northern Gulf of Mexico () 

5. Discussion 

5.1 Relative Trophic Position of Sperm Whales 

Isotopic signatures of female sperm whales from Mississippi Canyon (MC) and the northwest 

Gulf (NW) were similar. Satellite-telemetry studies have been conducted on individuals from 

which skin samples were obtained. Telemetry data indicated that groups of adult females have 

large home ranges but spend most of their time in smaller ‘core’ areas in Mississippi Canyon and 

the Rio Grande Slope (Mate and Ortega-Ortiz 2008). Females remained at the edge of the upper 

continental slope with affinity for particular areas on monthly scales (Mate and Ortega-Ortiz 

2008). Moreover, although there was some overlap in the overall home range, the core areas 

were different for whales tagged (and biopsy sampled) in Mississippi Canyon than those tagged 

9
in the Western Gulf (Mate and Ortega-Ortiz 2008). In the Galapagos Islands, Marcoux et al. 

(2007) reported isotopic differences between sympatric clans of sperm whales, but only one clan 

was identified in the northern Gulf of Mexico (Gordon et al. 2008). Because there is no evident 

oceanographic or biogeographic barrier between these areas, a comparable diversity and 

abundance of cephalopods and fish may exist at the isobathymetric boundary along the upper 

slope between depths of 200 and 1000 m. Therefore, it is possible that whales had similar diet 

between these two sites and fed at the same trophic level. Yet, the number of samples collected 

in the NW area represents only one year of collection compared with three years for the 

Mississippi Canyon (Table 1). Telemetry data showed that females did not spend much time in 

waters more than 2000 m deep (Mate and Ortega-Ortiz 2008) but groups of females with calves 

have been observed during surveys in that deep area of the lower continental slope (Mullin et al. 

2004). No skin samples from female sperm whales from the lower continental slope region were 

analyzed in the present study. Whales from that region may have different home ranges (Mate 

and Ortega-Ortiz, 2008), diet composition and consequently trophic position. Further studies are 

needed to test whether females from the lower continental slope region have different relative 

trophic position than the ones from the upper slope. 

The comparative variation in the relative trophic position between females and immature males 

from the northern GoM differed from previous findings in the Gulf of California (GC) (RuizCooley et al. 2004). In the GoM, immature males fed at a lower trophic position than females as 

indicated by the lower and significant differences in 15N between them, while in the GC 

immature males were higher in 15N than females, but no statistical differences were detected. 

This variation can be associated with length and age of immature males, range of foraging areas 

(horizontal and vertical) and prey size consumed. For example, in the northern GoM, many 

immature males tracked by satellite-telemetry moved to deeper waters and explored southern 

areas within the GoM, while females remained in the northern part (Mate and Ortega-Ortiz 

2008). Immature males are likely to forage during their journey within the Gulf and may prey 

upon other prey species that are not accessible to females. 

The isotopic differences between sperm whales and mesopelagic squid from the family 

Ommastrephidae, such as Ommastrephes bartramii, and squid from the family Histiotheutidae 

(Figure 3) suggest potential trophic-relationships between them, although a representative 

sample size of these squid species for each area (i.e. Mississippi Canyon and NW) is needed to 

evaluate this relationship. Our squid sample size is low, so caution should be taken with our 

interpretation. The overall predator-prey isotopic enrichment recognized in marine systems 

varied from 0.8 to 1.5‰ for 13C (DeNiro and Epstein 1978, Fry and Sherr 1984) and 2.5 to 

3.5‰ for 15N (Minagawa and Wada 1984, Wada et al. 1987, Fry 1988). For consumers raised 

on a high-protein diet, the average trophic enrichment is 3.3‰ for 15N (McCutchan et al. 2003). 

Currently, the isotopic difference between sperm whale skin and cephalopod muscle has not been 

rigorously tested. In this study, the 13C isotopic difference between sperm whales and these 

mesopelagic squid species (1.6 ± 0.36 ‰, Figure 3) may suggest a predator-prey relationship. 

However the difference in 15N between sperm whales and these squid was higher (4.3 ± 0.58 ‰ 

for 15N) than the overall predator-prey difference for 15N. Using archival tags or D-tags, Miller 

et al. (2004a) found that sperm whales from the northern Gulf of Mexico produce creaks 

(echolocation signals used for foraging) mainly at depths between 490 and 742 m. Because our 

mesopelagic cephalopod species were collected in areas and at depths (400 to 600 m) where 

10
sperm whales forage in the Gulf of Mexico (Miller et al. 2004a, Wormouth 2006), sperm whales 

may feed on these cephalopod species but of larger sizes than the ones that we analyzed. 

Stomach content analysis of four stranded sperm whales from the northern GoM revealed that 

Histioteuthis sp. had the highest percentage of occurrence (Barros 2003 cited by Wormouth 

2006). Our results recognized Histioteuthis sp. as a potential prey, but squid with mantle lengths 

larger than 20 cm ML are crucial to evaluate its role as a potential prey. Additionally, more 

efforts are needed to collect other mesopelagic cephalopod species from sperm whale foraging 

areas to evaluate the importance of these species in the sperm whale diet. 

5.2 Temporal Variation 

Comparable 13C and 15N values among years indicate that sperm whales maintained a similar 

diet composition throughout the period from 2002 to 2005. While each of these observations is 

the result of the diet over a period prior to sample collection, the length of that period is currently 

not well defined. Based on the turnover rate of sloughed skin from other odontocetes and the 

thickness of sperm whale skin, Ruiz-Cooley et al. (2004) suggested that stable isotope ratios 

from sloughed skin samples of sperm whale may reflect the diet assimilated over a period of at 

least 72 days. Seasonal changes in the C- and N-isotopic signatures in pilot whales were recorded 

in the Strait of Gibraltar (De Stephanis et al. 2008). These seasonal differences in isotopic ratios 

may suggest that stable isotope ratios from pilot whale skin change at rate of about four months. 

It is likely that adult sperm whales have slower metabolism rates than pilot whales. Hence, if 

sperm whales would have changed their diet composition significantly between years, we would 

have detected changes in their skin isotopic signatures. In the Gulf of California, no detectable 

differences were observed in the isotopic values of sperm whale skin over three years of study 

(1997 to 1999), including one year (1997) in which an El Niño event occurred (Ruiz-Cooley et 

al. 2004). In contrast, the skin isotopic values of sperm whales varied significantly in the 

Galapagos area in a longer-term study (i.e. in 1989, 1991 and 1995) and in Chile between 1993 

and 2000 (Marcoux et al. 2007). Our study analyzed skin samples of sperm whales collected 17 

years later, long after the oil industry began in deep areas in 1985 (French et al. 2006). A 

comparison of the isotopic signatures for sperm whales before and after oil industry expansion 

would have enabled us to evaluate potential changes in the sperm whale relative trophic position 

in association with the developing oil industry from the northern GoM. Unfortunately, we lacked 

samples from prior to oil development. 

Potential impacts from oil industry development on the sperm whale population are poorly 

understood. Modeling studies obtained from D-tagged whales exposed to controlled airgun 

emissions from seismic survey vessels revealed that these emissions may have an effect on 

sperm whales (Madsen et al. 2006), such as on their foraging effort (Miller et al. 2005, DeRuiter 

et al. 2008). Currently, it is unknown how seismic operations or activities of the oil industry 

influence mesopelagic cephalopod populations. Information on this aspect is of particular 

importance since mesopelagic squid are the main prey of sperm whales (Kawakami 1980, 

Wormouth 2006). As squid grow, some pelagic species occupy deeper waters (Rodhouse and 

Nigmatullin 1996), so larger sized squid are likely to occur in deeper waters of the mesopelagic 

zone. Miller et al. (2008) revealed that D-tagged sperm whales foraged both along the seafloor 

11
and at mid-depth in the water column in the northern Gulf, areas where higher quality prey 

patches are likely to occur. Since cephalopods change rapidly as a result of anthropogenic 

activities and environmental variables (Rodhouse and Nigmatullin 1996), airgun emissions or 

other anthropogenic activities may potentially affect the distribution of squid in the mid-water 

column or in the deep-ocean. For instance, Engås et al. (1996) demonstrated that fish populations 

were severely affected by seismic emissions from airguns on the North Cape Bank in the Barents 

Sea; the abundance and catch rates of cod and haddock stocks were reduced by almost 50%. 

Clearly, directed efforts are needed to understand the potential effects of the oil industry on 

meso- and bathypelagic squid communities (sperm whale prey). 

The resighting of individual sperm whales over periods of years, genetic findings indicating a 

unique GoM population, information on acoustic dialects, and satellite-telemetry results all 

support the hypothesis that at least some female sperm whales exhibit site-fidelity to the GoM 

coastal basins (Reeves and Notarbartolo di Sciara 2006, Mate and Ortega-Ortiz, 2008, Jochens et 

al. 2008, Engelhaupt et al., 2009). If so, changes in sperm whale diet composition (e.g. feeding 

on smaller sized squid or in prey patches with lower density) may result in a lower caloric gain 

per unit foraging effort and could reduce feeding success and consequently fitness. A reduction 

in feeding success of sperm whales would hinder allocation of energy to other biological 

activities such as reproduction and growth. Therefore, we recommend continuing monitoring the 

relative trophic position of sperm whales using stable isotope analysis and beginning of efforts to 

understand potential effects of the oil industry on mesopelagic cephalopod communities. A high 

concern would arise if sperm whales maintained their home ranges but the 15N in skin 

decreased through time.

5.3 Spatial Variation 

When comparing the isotopic signatures of sperm whales from the GoM and the Gulf of 

California (GC), we were expecting distinct isotopic values, since sperm whale prey composition 

varies by area (Kawakami 1980). In the Gulf of California, sperm whales prey on the jumbo 

Humboldt squid Dosidicus gigas (Ruiz-Cooley et al. 2004 and 2006), whereas in the GoM they 

seem to prey on squid of the families Ommastrephidae and Histiotheutidae (Barros 2003; Figure 

3). However, we observed contrasting differences in the 13C and 15N of sperm whales from 

the central Gulf of California and northern GoM. The isotopic ratios of sperm whales from the 

northern GoM were significantly lower (7.41 % for 15N and 2.74 % for 13C) compared to ratios 

from the GC. We found no differences in the13C and 15N values from skin lipid extracted with 

a volume of chloroform and methanol 1:1 versus 2:1 respectively. Therefore, we are confident 

that the small variation in this method did not have an effect on the resultant variation in isotopic 

ratios of sperm whale skin from these two oceanic basins. 

At first glance, the isotopic difference between GoM and GC may suggest that sperm whales 

from the GoM occupy a lower trophic position than those in the GC, since enrichment in 15N 

from prey to predators allows the δ15N value to serve as an indicator of trophic position, whereas 

the δ13C value traces origin of primary productivity (De Niro and Epstein 1978 and 1981, 

Minawaga and Wada 1984, Fry 1988, Rau et al. 1992). If we arbitrarily assumed that a 

12
difference of 3 % for 15N represents one trophic level, sperm whales from the GoM would be 

2.5 trophic levels lower than whales from the GC. Because sperm whales from the GC feed on 

larger jumbo Humboldt squid of approximately 60 to 70 cm mantle length (Ruiz-Cooley et al. 

2004, Davis et al. 2007), then sperm whales from the GoM would be feeding on tiny squid or 

fish that may be part of the zooplankton; this is very unlikely. 

Inferences about trophic position using stable isotopes in studies that lack samples at the base of 

the food web can be misleading. For instance, latitudinal variation in 13C values from plankton 

exist, e.g., a 13C decrease from tropical latitudes toward the poles, presumably due to gradients 

in temperature and CO2 concentration as a function of latitude (Rau et al. 1982). Using both C 

and N isotope ratios allows for discrimination between trophic positions within the same system 

but also between aquatic systems such as freshwater, estuaries and marine ecosystems (Owens 

1987). In marine systems, these biochemical tracers are also helpful to differentiate between 

offshore versus inshore marine animals (e.g. for 13C, Rau et al. 1983). A geographic isotopic 

variation in 15N between oceanic areas has been documented in squid (Takai et al. 2000, RuizCooley et al. in press), sea turtles (Wallace et al. 2006) and sperm whales (Marcoux et al. 2007). 

This geographic variability in 15N/14N from animal tissues suggest that N can be used to 

differentiate geographic subpopulations due to differences in the physical and biochemical 

characteristics among systems (Wallace et al. 2006, Ruiz-Cooley et al. in press). Hence, the 

distinct organic and isotopic pool derived from the variability in biochemical cycles of C and N 

between the GoM and GC may influence significantly isotopic differences between these 

populations. 

For instance, we also observed that 15N values from skin samples of sperm whales and muscle 

tissue samples from two small squid species (Loligo plei and L pealei) were remarkably similar, 

but the 13C values differed. The15 N from Lolliguncula brevis muscle tissues was even higher 

than sperm whale isotopic signatures, but there was an overlap in the 13C values (Figure 3).

These three squid species generally occupy warm, shallow, and inshore waters of the northern 

GoM and occur on the edge of the continental shelf at depths between 40 and 250 m (Hanlon et 

al. 1983). L. plei is often found at depths greater than 75 m, while L. brevis often occurs in 

shallower waters, for example in Galveston Bay at depths from 1 to 20 m (Hanlon et al. 1983). In 

contrast, sperm whales are distributed in the northern Gulf of Mexico in areas with depths 

between 200 to 2000 m (Hansen et al. 1995, Davis and Fargion 1996, Davis et al. 2000) while 

their prey would occupy the mesopelagic ocean at depths greater than 400 m (Miller et al. 

2004a). There is no overlap in range between these three inshore squid species and sperm 

whales. Therefore, the stable isotopic signatures of these cephalopod species reflect the 

metabolism of N and C from inshore shallow waters of the northern Gulf, whereas isotopic ratios 

of sperm whales reflect their diet in offshore deep waters. The resultant 15N and 13C from 

specimens from inshore and shallow water systems (Loligo species) and offshore pelagic systems 

(sperm whales and mesopelagic squid) (Figure 3) illustrate the importance of collecting tissues 

samples from predator and prey from the same area and time; accurate inferences of trophic 

relationships using stable isotope analysis depend on that information. 

13
6. Recommendations 

This study provides baseline information regarding the relative trophic position of sperm 

whales in the northern Gulf of Mexico. Stable isotope analysis is a useful tool to infer aspects of 

the feeding ecology of sperm whales, but more effort is needed to evaluate temporal shifts in 

their trophic position. This is particularly important due to the fact that sperm whales from the 

northern Gulf of Mexico appear to exhibit unique characteristics. Recent findings based on 

mtDNA and nuclear DNA suggest that this ‘stock’ is genetically distinct from those whales 

comprising the neighboring North Atlantic Ocean stock (Engelhaupt et al. 2009), they belong to 

a unique acoustic clan (Gordon et al. 2008), and they have a smaller body length compared with 

other areas (Jochens et al. 2008). Seismic airguns used in energy exploration may have a 

negative impact on sperm whale feeding ecology (Miller et al. 2005, DeRuiter et al. 2008). 

Therefore, it is of great importance to establish a continual evaluation of the relative trophic 

position of sperm whales from the northern GoM and to investigate the relative trophic position 

of the offshore groups. Stable isotope analysis provides a feasible method to accomplish this 

goal. However, we need to invest more effort to investigate the diversity, distribution and 

abundance of mesopelagic cephalopods, especially in the core areas recognized by Mate and 

Ortega-Ortiz (2008). We need to understand the function of these areas in the biology of sperm 

whales. In particular, a large sample of the large sized mesopelagic squid (e.g. > 30 cm mantle 

length) or fish concurrent with skin samples of sperm whales would allow us to evaluate trophic 

relationships. Additionally, more effort is needed to collect and analyze samples from mid-water 

cephalopods as well as from particulate organic material or plankton concurrent with tissue 

samples of sperm whales and squid to determine trophic level and their changes through time. 

This is crucial to distinguish (1) if sperm whales from the northern Gulf have a lower trophic 

level than whales from the Gulf of California or (2) if the variability in the biochemical cycle of 

N and C between areas is responsible for the observed spatial isotopic variation. 

14
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18
Appendix A. Stable Isotopic Ratios from Skin Samples Collected in the Northern Gulf of 

Mexico from 2002 to 2005 (tag # corresponds to Oregon State University S-tag codes)

Year Date Latitude Longitude 15N 13C Tag # Gender 

2002 24-Jun-02 28.056 -89.669 11.84039 -16.5913 Female 

2002 24-Jun-02 28.012 -89.696 12.97605 -13.9777 2505660 Female 

2002 02-Jul-02 28.866 -88.496 12.28513 -16.5473 2505678 Female 

2002 02-Jul-02 28.884 -88.478 12.66605 -16.2659 Female 

2002 03-Jul-02 28.794 -88.808 12.00501 -16.2022 Female 

2002 03-Jul-02 28.705 -88.764 12.54042 -16.6176 2505709 Female 

2002 03-Jul-02 28.84 -88.692 12.24767 -16.5741 2505670 Female 

2002 03-Jul-02 28.85 -88.673 12.36314 -16.7803 2505720 Female 

2002 03-Jul-02 28.799 -88.699 12.15242 -16.5428 2505670 Female 

2002 03-Jul-02 28.808 -88.684 12.39353 -16.4660 2505669 Female 

2002 03-Jul-02 28.805 -88.698 12.20104 -16.7156 2505701 Female 

2002 06-Jul-02 28.426 -89.05 12.01514 -16.9087 Female 

2002 07-Jul-02 28.701 -88.665 12.68226 -16.7197 Female 

2002 07-Jul-02 28.742 -88.875 12.42291 -15.9152 Female 

2002 23-Aug-02 28.301 -89.519 12.41075 -16.6135 Female 

2002 23-Aug-02 28.301 -89.519 12.74912 -16.7662 Female 

2002 25-Aug-02 28.78 -88.693 12.49078 -16.7833 Female 

2002 26-Aug-02 28.686 -88.944 12.3368 -16.0506 Female 

2002 28-Aug-02 28.651 -89.005 12.65186 -16.5463 Female 

2002 28-Jun-02 29.208 -87.178 11.22545 -16.2477 2505648 Male 

2002 01-Jul-02 28.958 -88.109 11.79328 -16.4635 2505685 Male 

2002 01-Jul-02 28.956 -88.109 11.67627 -16.5388 2505650 Male 

2002 01-Jul-02 28.951 -88.113 12.16255 -16.7065 2505726 Male 

2002 01-Jul-02 28.903 -88.099 11.67627 -16.7126 2505725 Male 

2002 06-Jul-02 28.445 -88.991 12.04604 -16.9258 Male 

2002 08-Jul-02 28.992 -88.239 12.43101 -16.6388 Male 

2002 23-Aug-02 28.239 -89.562 12.04098 -16.6226 Male 

2002 29-Aug-02 28.832 -88.6 12.87525 -16.3791 Male 

2003 02-Jun-03 27.265 -93.825 12.391 -16.6711 Female 

2003 02-Jun-03 27.399 -93.885 12.90716 -16.3666 Female 

2003 02-Jun-03 27.399 -93.885 11.77355 -16.8785 Female 

2003 03-Jun-03 27.387 -93.811 12.47052 -16.0769 Female 

2003 04-Jun-03 27.423 -93.842 12.1943 -16.6445 Female 

2003 05-Jun-03 27.455 -93.003 12.174 -16.6671 Female 

2003 08-Jun-03 27.527 -90.736 11.5395 -17.0643 Female 

19
Appendix A. Stable Isotopic Ratios from Skin Samples Collected in the Northern Gulf of 

Mexico from 2002 to 2005 (tag # corresponds to Oregon State University S-tag codes) 

(continued)

Year Date Latitude Longitude 15N 13C Tag# Gender 

2003 08-Jun-03 27.535 -90.713 11.87179 -16.9582 Female 

2003 08-Jun-03 27.592 -90.755 12.20915 -16.8953 Female 

2003 05-Jul-03 28.637 -88.7452 12.69543 -16.5195 2800843 Female 

2003 05-Jul-03 28.698 -88.7648 12.9318 -16.6446 2805719 Female 

2003 08-Jul-03 28.61 -88.938 11.9668 -16.6629 2505669 Female 

2003 09-Jul-03 28.5083 -89.034 11.8805 -16.6133 2505719 Female 

2003 09-Jul-03 28.5487 -88.9961 12.121 -16.6371 2810820 Female 

2003 09-Jul-03 28.4822 -89.0432 12.9945 -16.6533 2800829 Female 

2003 11-Jul-03 28.6016 -88.8991 12.83219 -16.4296 2800833 Female 

2003 11-Jul-03 28.7873 -88.2583 12.29019 -16.6530 Female 

2003 11-Jul-03 28.7873 -88.2583 11.74 -16.4300 2805678 Female 

2003 11-Jul-03 28.805 -88.7221 12.518 -16.7100 2800826 Female 

2003 02-Jun-03 27.399 -93.885 12.393 -16.3414 Male 

2003 05-Jun-03 27.411 -92.995 12.18686 -16.5893 Male 

2003 11-Jul-03 28.7965 -88.763 11.5143 -16.6595 2800827 Male 

2004 06-Jun-04 28.2664 -89.4285 12.215 -16.6705 3205660 Female 

2004 06-Jun-04 28.2467 -89.6693 13.562 -16.5114 3202083 Female 

2004 07-Jun-04 28.0311 -89.9294 12.18939 -16.8889 3205670 Female 

2005 11-Jun-05 26.567 -95.732 12.36516 -16.1567 3700836 Female 

2005 15-Jun-05 26.954 -95.137 12.09 -16.8100 Female 

2005 18-Jun-05 26.976 -95.408 12.00096 -16.7995 Female 

2005 18-Jun-05 26.96 -95.398 11.50252 -16.8586 Female 

2005 18-Jun-05 26.974 -95.361 11.93866 -16.9036 Female 

2005 21-Jun-05 26.922 -95.451 12.63059 -15.9920 3705644 Female 

2005 21-Jun-05 26.931 -95.484 11.82722 -16.4862 3700846 Female 

2005 21-Jun-05 26.928 -95.488 12.81902 -16.0829 3701386 Female 

2005 21-Jun-05 26.909 -95.485 11.82 -16.6040 3705726 Female 

2005 24-Jun-05 27.274 -93.845 11.95486 -16.7227 3705701 Female 

2005 24-Jun-05 27.311 -93.858 12.28715 -16.8945 Female 

2005 26-Jun-05 27.299 -93.768 12.2213 -16.7500 3700847 Female 

2005 27-Jun-05 27.502 -94.554 12.48369 -16.4064 Female 

2005 27-Jun-05 27.537 -94.511 12.28006 -16.6479 Female 

2005 28-Jun-05 27.084 -95.074 11.70463 -16.4943 Female 

2005 28-Jun-05 27.066 -95.124 12.8018 -16.7480 Female 

2005 11-Jun-05 26.545 -95.799 12.05212 -16.2340 Male 

2005 15-Jun-05 26.974 -95.141 12.62856 -15.8020 Male 

20
The Department of the Interior Mission

As the Nation's principal conservation agency, the Department of the Interior has responsibility 

for most of our nationally owned public lands and natural resources. This includes fostering 

sound use of our land and water resources; protecting our fish, wildlife, and biological diversity; 

preserving the environmental and cultural values of our national parks and historical places; 

and providing for the enjoyment of life through outdoor recreation. The Department assesses 

our energy and mineral resources and works to ensure that their development is in the best 

interests of all our people by encouraging stewardship and citizen participation in their care. 

The Department also has a major responsibility for American Indian reservation communities 

and for people who live in island territories under U.S. administration. 

The Minerals Management Service Mission 

As a bureau of the Department of the Interior, the Minerals Management Service's (MMS) 

primary responsibilities are to manage the mineral resources located on the Nation's Outer 

Continental Shelf (OCS), collect revenue from the Federal OCS and onshore Federal and Indian 

lands, and distribute those revenues. 

Moreover, in working to meet its responsibilities, the Offshore Minerals Management Program 

administers the OCS competitive leasing program and oversees the safe and environmentally 

sound exploration and production of our Nation's offshore natural gas, oil and other mineral 

resources. The MMS Minerals Revenue Management meets its responsibilities by ensuring the 

efficient, timely and accurate collection and disbursement of revenue from mineral leasing and 

production due to Indian tribes and allottees, States and the U.S. Treasury. 

The MMS strives to fulfill its responsibilities through the general guiding principles of: (1) being 

responsive to the public's concerns and interests by maintaining a dialogue with all potentially 

affected parties and (2) carrying out its programs with an emphasis on working to enhance the 

quality of life for all Americans by lending MMS assistance and expertise to economic 

development and environmental protection.