16 
Fishery Bulletin 1 1 1 (1) 
Table 1 
Number of harbor seal samples, by location, sex, and season, used in our investigation of diet 
composition of harbor seals ( Phoca uitulina) in the Salish Sea through quantitative fatty acid 
signature analysis. 
Location 
Female 
Male 
Spring 
Fall 
Winter 
Spring 
Fall 
Winter 
Belle Chain 
4 
0 
0 
6 
0 
0 
Bird Rocks 
1 
0 
2 
5 
4 
2 
Padilla Bay 
14 
1 
0 
3 
0 
0 
Vendovi Island 
0 
2 
1 
0 
4 
0 
and December, 2008, with a variety of gear, including 
hook and line, longline, and trawl. Samples were ob- 
tained from 269 specimens representing these 20 spe- 
cies: Black (Sebastes melanops), Yellowtail (S. flauidus), 
Copper, and Puget Sound (S. emphaeus) Rockfish; Chi- 
nook, Chum ( Oncorhynchus keta), Coho (O. kisutch), 
Sockeye ( O . nerka ), and Pink (O. gorbuscha ) Salmon; 
Pacific Herring, Walleye Pollock; Pacific Sand Lance 
( Ammodytes hexapterus); Northern Anchovy ( Engrail - 
lis mordax ); Shiner Perch ( Cymatogaster aggregata ); 
Plainfin Midshipman ( Porichthys notatus ); Spiny Dog- 
fish ( Squalus acanthias ); Opalescent Inshore Squid 
( Loligo opalescens)-, Kelp Greenling ( Hexagrammos 
decagrammus ); Pacific Staghorn Sculpin ( Leptocottus 
armatus ); and Starry Flounder ( Platichthys stellatus). 
Specimens were identified with Hart (1973) for fish 
species and Roper et al. (1984) for squid. Because some 
species were represented by individuals with differenc- 
es in size and total fat content (for example, immature 
and mature species of salmon), 27 prey classes were 
defined (Table 2). 
Prey specimens were placed in airtight plastic bags 
and stored at -80°C as soon as possible after collec- 
tion. In the laboratory, each specimen was given a 
unique sample number, partially thawed, weighed and 
measured (standard, fork, and total lengths), and ho- 
mogenized with a medium or large mechanical blend- 
er, depending on fish size. The smallest prey animals 
were homogenized with a mortar and pestle because 
the blender was ineffective. Stomach contents were not 
removed from prey specimens, to mimic ingestion by 
predators (Budge et al., 2002). Approximately 5-10 g 
of homogenate was placed in labeled scintillation vials 
with Teflon lids and stored in a -80°C freezer. Samples 
were express shipped in a cooler on dry ice to the Ap- 
plied Sciences, Engineering, and Technology (ASET) 
Laboratory at the University of Alaska Anchorage. 
Fatty acid extraction and selection 
All samples were processed at the ASET Laboratory 
through the use of a method for microscale recovery 
of total lipids with the Dionex ASE 200 1 automated 
solvent extraction system (Thermo Fisher Scientific, 
Waltham, MA), which provides lipids for the determi- 
nation of 80 unique fatty acids (Dodds et al., 2005). The 
total body mass, percent fat composition, and fat mass 
of prey specimens were obtained for 27 prey classes 
(Table 2). Total mass data were not available for ma- 
ture Chinook, Sockeye, and Pink Salmon obtained from 
the Northwest Fisheries Science Center; therefore, an 
approximate mean mass for these prey classes (e.g., 
Quinn, 2005) was used in calculation of fat mass. Given 
the large range of mass among prey classes (Table 2), 
the results were insensitive to our use of these approxi- 
mate values. 
Extracted lipids were dissolved in hexane to a con- 
centration of 100 mg/mL, hydrolyzed by a base-cata- 
lyzed reaction with potassium hydroxide, and then 
esterified to form fatty acid methyl esters (FAMEs) 
by reaction with boron trifluoride in methanol. Each 
sample was spiked with a C21:0 internal standard (25 
pg/mL) and separated on a Hewlett-Packard 5890 gas 
chromatograph (GC) with a flame ionization detector 
(FID) (Hewlett-Packard Co., Palo Alto, California) by 
using a 60-m J&W DB-23 column (Agilent Technolo- 
gies, Inc., Santa Clara, CA) with a 0.25-mm inside di- 
ameter and 0.25-pm cyanopropyl polysiloxane film. Sig- 
nal data were collected and analyzed with Agilent GC 
Chemstation software. 
Supelco 37-Component FAME Mix (catalog no. 
47885-U; Sigma-Aldrich Co., St. Louis, MI) was used 
as a continuing calibration verification (CCV) to verify 
both the retention times and recovery values. This CCV 
also contained 25 pg/mL of a C21:0 internal standard, 
which is required to meet a tolerance of no greater 
than ±20% of actual value. Analyte identity was veri- 
fied further by mass spectrometry through the use 
of a Varian CP3800 GC (Agilent Technologies, Inc.) 
and a Varian Saturn 2200 ion trap mass spectrometer 
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