1. Compound specific stable isotope analysis (CSIA) of amino acids (AAs) has been rapidly incorporated in ecological studies to resolve consumer trophic position (TP). Differential ¹⁵N fractionation of ‘trophic’ AAs, which undergo ¹⁵N enrichment with each trophic step, and ‘source’ AAs, which undergo minimal trophic ¹⁵N enrichment and serve as a proxy for primary producer δ¹⁵N values, allows for internal calibration of TP. Recent studies, however, have shown the difference between source and trophic AA δ¹⁵N values in higher marine consumers is less than predicted from empirical studies of invertebrates and fish.
2. To evaluate CSIA-AA for estimating TP of cetaceans, we compared source and trophic AA δ¹⁵N values of multiple tissues (skin, baleen, and dentine collagen) from five species representing a range of TPs: bowhead whales, beluga whales, short-beaked common dolphins, sperm whales, and fish-eating (FE) and marine mammal-eating (MME) killer whale ecotypes.
3. TP estimates (TP_CSIA) using several empirically-derived equations and trophic discrimination factors (TDFs) were 1 to 2.5 trophic steps lower than stomach content-derived estimates (TP_SC) for all species. Although TP_CSIA estimates using dual TDF equations were in better agreement with TP_SC estimates for bowhead whales, belugas, and FE killer whales, our data do not support the application of a universal or currently available dual TDFs to estimate cetacean TPs. Discrepancies were not simply due to inaccurate TDFs, however, because the difference between consumer glutamic acid (Glu) and phenylalanine (Phe) δ¹⁵N values (δ¹⁵N_Glu-Phe) did not follow expected TP order, indicating it is not a reliable index of relative TP in these species.
4. In contrast with pioneering studies on invertebrates and fish, our data suggest trophic ¹⁵N enrichment of Phe is not negligible and should be examined among the potential mechanisms driving ‘compressed’ and variable δ¹⁵N_Glu-Phe values at high TPs. We emphasize the need for controlled diet studies to clearly understand mechanisms driving AA-specific isotopic fractionation before widespread application of CSIA-AA in ecological studies of cetaceans and other marine consumers.

Most of the isotope data presented here have been compiled from previously published studies where detailed sample preparation and analysis procedures can be found (Matthews and Ferguson 2014, 2015; Ruiz-Cooley et al. 2014, 2017; Pomerleau et al. 2017; Zupcic-Moore et al. 2017). Briefly, baleen samples were drilled from the proximal end of each plate where the most recent growth corresponds to foraging on the summer grounds (Matthews and Ferguson 2015), and no further sample preparation was carried out prior to isotope analysis. Bowhead whale skin samples were rinsed of DMSO using deionized water and were not lipid-extracted prior to analysis. Sperm whale skin samples were also rinsed of DMSO using deionized water, and then lipid-extracted using a 2:1 chloroform ethanol mixture (Lesage et al. 2010; Ruiz-Cooley et al. 2012). Dolphin skin was thawed and lipid-extracted with petroleum ether. Annual dentine growth layers of sperm whale teeth were sampled using a micromill and later combined, while a handheld rotary tool was used to collectively sample multiple dentine growth layers of beluga and killer whale teeth. All dentine was demineralized using repeated washes of 0.25 N HCl for 12-hr periods, and the remaining collagen was rinsed with distilled H₂O. All samples except baleen were freeze-dried and finely homogenized.

Compound specific stable isotope analysis

All bowhead, dolphin, beluga, and killer whale tissues were analysed at the University of California-Davis Stable Isotope Facility, while sperm whale tissues were analysed at University of California-Santa Cruz Stable Isotope Laboratory. Briefly, at UC Davis, approximately 3 mg of each freeze-dried, homogenized tissue sample was acid hydrolysed using 6 M HCl at 150 ^oC under a N₂ headspace for 70 min, and derivatized using methoxycarbonylation esterification (Walsh et al. 2014, Yarnes and Herszage 2017). Methods at UC Santa Cruz differed primarily in the use of trifluoroacyl-isopropyl ester as the derivatization agent. δ¹⁵N values of individual derivatized AAs were measured at both labs by gas chromatography-combustion isotope ratio mass spectrometry (GC-IRMS). At UC Davis, two AA mixtures, previously calibrated to the international reference scale for δ¹⁵N (atmospheric N₂), were used in calibration and scale-normalization procedures. Quality assurance of δ¹⁵N measurements followed Yarnes and Herzsage (2017). A third AA mixture served as the primary quality control reference material, while two well-known natural materials, baleen and fish muscle, were used as secondary quality control standards. At UC Santa Cruz, isotope values were calibrated to the isotopic composition of atmospheric N₂ through repeated measures of a co-injected reference compound of known isotopic composition (norleucine). Mean analytical precision assessed from duplicate or triplicate measures of samples and the reference compound was < 1 ‰ at both labs.

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