Polar Biology

, Volume 38, Issue 2, pp 131–143 | Cite as

Effects of lipid extraction and the utility of lipid normalization models on δ13C and δ15N values in Arctic marine mammal tissues

  • David J. Yurkowski
  • Nigel E. Hussey
  • Christina Semeniuk
  • Steven H. Ferguson
  • Aaron T. Fisk
Original Paper


Animals store lipids, which are 13C-depleted, in their tissues that often must be extracted to correctly interpret δ13C data. However, chemical lipid extraction (CLE) can alter δ15N values and lipid normalization (LN) models are not consistent across fauna. We determined whether lipids should be extracted by assessing effects of CLE and validating LN models for liver and muscle from seven and eight marine mammal species, respectively, and skin from one species. In liver, CLE significantly increased δ13C and δ15N values for all species, whereas only a significant increase in δ13C occurred in skin. For muscle, δ13C and δ15N values were generally greater after CLE, but this was not consistent across species. Extracted lipids were depleted by approximately 7 and 5 ‰ for δ13C and δ15N, respectively, in both muscle and liver compared with protein in all species. The reliability of LN models varied between tissues and species; thus, their use is largely dependent on the precision of stable isotope values needed to address the objectives of a study. A decision framework to decide whether CLE or LN models is required for ecological interpretation of stable isotopes based on species, tissue and study objectives is presented.


Carbon Lipids Marine mammals Nitrogen Stable isotopes 

Supplementary material

300_2014_1571_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 19 kb)
300_2014_1571_MOESM2_ESM.docx (21 kb)
Supplementary material 2 (DOCX 20 kb)


  1. Balter V, Simon L, Fouillet H, Lécuyer C (2006) Box-modeling of 15N/14N in mammals. Oecologia 147:212–222PubMedCrossRefGoogle Scholar
  2. Barrow LM, Bjorndal KA, Reich KJ (2008) Effects of preservation method on stable carbon and nitrogen isotope values. Physiol Biochem Zool 81:688–693PubMedCrossRefGoogle Scholar
  3. Bearhop S, Teece MA, Waldron S, Furness RW (2000) Influence of lipid and uric acid on δ13C and δ15N values of avian blood: implications for trophic studies. Auk 117:504–507Google Scholar
  4. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917PubMedCrossRefGoogle Scholar
  5. Bodin N, Le Loc’h F, Hily C (2007) Effect of lipid removal on carbon and nitrogen stable isotope ratios in crustacean tissues. J Exp Mar Biol Ecol 341:168–175CrossRefGoogle Scholar
  6. Bond AL, Diamond AW (2011) Recent Bayesian stable-isotope mixing models are highly sensitive to variation in discrimination factors. Ecol Appl 21:1017–1023PubMedCrossRefGoogle Scholar
  7. Bosley K, Wainright S (1999) Effects of preservatives and acidification on the stable isotope ratios (15N:14N, 13C:12C) of two species of marine animals. Can J Fish Aquat Sci 56:2181–2185CrossRefGoogle Scholar
  8. Bowen WD, Boness DJ, Oftedal OT (1987) Mass transfer from mother to pup and subsequent mass loss by the weaned pup in the hooded seal, Cystophora cristata. Can J Zool 65:1–8CrossRefGoogle Scholar
  9. Burnham KP, Anderson DR (2002) Model selection and multimodel inference: a practical information-theoretic approach. Springer, New YorkGoogle Scholar
  10. Caut S, Angulo E, Courchamp F (2009) Variation in discrimination factors (Δ15N and Δ13C): the effect of diet isotopic values and applications for diet reconstruction. J Appl Ecol 46:443–453CrossRefGoogle Scholar
  11. Caut S, Laran S, Garcia-Hartmann E, Das K (2011) Stable isotopes of captive cetaceans (killer whales and bottlenose dolphins). J Exp Biol 214:538–545PubMedCrossRefGoogle Scholar
  12. DeNiro MJ, Epstein S (1977) Mechanism of carbon isotope fractionation associated with lipid synthesis. Science 197:261–263PubMedCrossRefGoogle Scholar
  13. DeNiro MJ, Epstein S (1978) Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmochim Acta 42:495–506CrossRefGoogle Scholar
  14. DeNiro MJ, Epstein S (1981) Influence of diet on the distribution of nitrogen isotopes in animals. Geochim Cosmochim Acta 45:341–351CrossRefGoogle Scholar
  15. Ehrich D, Tarroux A, Stien J, Lecomte N, Killengreen S, Berteaux D, Yoccoz NG (2011) Stable isotope analysis: modelling lipid normalization for muscle and eggs from arctic mammals and birds. Methods Ecol Evol 2:66–76CrossRefGoogle Scholar
  16. Elliott KH, Davis M, Elliott JE (2014) Equations for lipid normalization of carbon stable isotope ratios in aquatic bird eggs. PLoS ONE 9:e83597PubMedCentralPubMedCrossRefGoogle Scholar
  17. Fagan K-A, Koops MA, Arts MT, Power M (2011) Assessing the utility of C:N ratios for predicting lipid content in fishes. Can J Fish Aquat Sci 68:374–385CrossRefGoogle Scholar
  18. Falk-Petersen S, Hagen W, Kattner G, Clarke A, Sargent J (2000) Lipids, trophic relationships, and biodiversity in Arctic and Antarctic krill. Can J Fish Aquat Sci 57:178–191CrossRefGoogle Scholar
  19. Fisk AT, Tittlemier SA, Pranschke JL, Norstrom RJ (2002) Using anthropogenic contaminants and stable isotopes to assess the feeding ecology of Greenland sharks. Ecology 83:2162–2172CrossRefGoogle Scholar
  20. Focken U, Becker K (1998) Metabolic fractionation of stable isotopes: implications of different proximate compositions for studies of the aquatic food webs using δ13C data. Oecologia 115:337–343CrossRefGoogle Scholar
  21. Fry B, Baltz DM, Benfield MC, Fleeger JW, Gace A, Haas HL, Quiñones-Rivera ZJ (2003) Stable isotope indicators of movement and residency for brown shrimp (Farfantepenaeus aztecus) in coastal Louisiana marshscapes. Estuaries 26:82–97CrossRefGoogle Scholar
  22. Gaye-Siesseggar J, Focken U, Abel H, Becker K (2003) Feeding level and diet quality influence trophic shift of C and N isotopes in Nile tilapia (Oreochromis niloticus (L.)). Isotopes Environ Health Stud 39:125–134CrossRefGoogle Scholar
  23. Hendrixson HA, Sterner RW, Kay AD (2007) Elemental stoichiometry of freshwater fishes in relation to phylogeny, allometry and ecology. J Fish Biol 70:121–140CrossRefGoogle Scholar
  24. Hobson KA, Clark RG (1992) Assessing avian diets using stable isotopes II: factors influencing diet-tissue fractionation. Condor 94:189–197CrossRefGoogle Scholar
  25. Hobson KA, Schell DM, Renouf D, Noseworthy E (1996) Stable carbon and nitrogen isotopic fractionation between diet and tissues of captive seals: implications for dietary reconstructions of marine mammals. Can J Fish Aquat Sci 53:528–533CrossRefGoogle Scholar
  26. Hobson KA, Gloutney ML, Gibbs HL (1997a) Preservation of blood and tissue samples for stable-carbon and stable-nitrogen isotope analysis. Can J Zool 75:1720–1723CrossRefGoogle Scholar
  27. Hobson KA, Sease JL, Merrick RL, Piatt JF (1997b) Investigating trophic relationships of pinnipeds in Alaska and Washington using stable isotope ratios of nitrogen and carbon. Mar Mamm Sci 13:114–132CrossRefGoogle Scholar
  28. Hobson KA, Fisk AT, Karnovsky N, Holst M, Gagnon J-M, Fortier M (2002) A stable isotope (δ13C, δ15N) model for the North Water food web: implications for evaluating trophodynamics and the flow of energy and contaminants. Deep-Sea Res II 49:5131–5150CrossRefGoogle Scholar
  29. Horstmann-Dehn L, Follmann EH, Rosa C, Zelensky G, George C (2012) Stable carbon and nitrogen isotope ratios in muscle and epidermis of arctic whales. Mar Mamm Sci 28:E173–E190. doi:10.1111/j.1748-7692.2011.00503.x CrossRefGoogle Scholar
  30. Hussey NE, Brush J, McCarthy ID, Fisk AT (2010) δ15N and δ13C diet-tissue discrimination factors for large sharks under semi-controlled conditions. Comp Biochem Phys A 155:445–453CrossRefGoogle Scholar
  31. Hussey NE, MacNeil AM, Olin JA, McMeans BC, Kinney MJ, Chapman DD, Fisk AT (2012a) Stable isotopes and elasmobranchs: tissue types, methods, applications and assumptions. J Fish Biol 80:1449–1484PubMedCrossRefGoogle Scholar
  32. Hussey NE, Olin JA, Kinney M, McMeans BC, Fisk AT (2012b) Lipid extraction effects on stable isotope values (δ13C and δ15N) of elasmobranch muscle tissue. J Exp Mar Biol Ecol 434–435:7–15CrossRefGoogle Scholar
  33. Hussey NE, MacNeil MA, McMeans BC, Olin JA, Dudley SJF, Cliff G, Wintner SP, Fennessey ST, Fisk AT (2014) Rescaling the trophic structure of marine food webs. Ecol Lett 17:239–250PubMedCentralPubMedCrossRefGoogle Scholar
  34. Ingram T, Matthews B, Harrod C, Stephens T, Grey J, Markel R (2007) Lipid extraction has little effect on the δ15N of aquatic consumers. Limnol Oceanogr-Methods 5:338–343CrossRefGoogle Scholar
  35. Jacob U, Mintenbeck K, Brey T, Knust R, Beyer K (2005) Stable isotope food web studies: a case for standardized sample treatment. Mar Ecol Prog Ser 287:251–253CrossRefGoogle Scholar
  36. Kelly JF (2000) Stable isotopes of carbon and nitrogen in the study of avian and mammalian trophic ecology. Can J Zool 78:1–27 CrossRefGoogle Scholar
  37. Kiljunen M, Grey J, Sinisalo T, Harrod C, Immonen H, Jones RI (2006) A revised model for lipid-normalizing δ13C values from aquatic organisms, with implications for isotope mixing models. J Appl Ecol 43:1213–1222CrossRefGoogle Scholar
  38. Layman CA, Arrington DA, Montana CG, Post DM (2007) Can stable isotope ratios provide for community-wide measures of trophic structure? Ecology 88:42–48PubMedCrossRefGoogle Scholar
  39. Lee RF (1974) Lipid composition of the copepod Calanus hyperboreus from the Arctic Ocean. Changes with depth and season. Mar Biol 26:313–318CrossRefGoogle Scholar
  40. Lesage V, Morin Y, Rioux È, Pomerleau C, Ferguson S, Pelletier É (2010) Stable isotopes and trace elements as indicators of diet and habitat use in cetaceans: predicting errors related to preservation, lipid extraction, and lipid normalization. Mar Ecol Prog Ser 419:249–265CrossRefGoogle Scholar
  41. Logan JM, Jardine TD, Miller TJ, Bunn SE, Cunjak RA, Lutcavage ME (2008) Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods. J Anim Ecol 77:838–846PubMedCrossRefGoogle Scholar
  42. Martínez del Rio C, Wolf N, Carleton SA, Gannes LZ (2009) Isotopic ecology ten years after a call for more laboratory experiments. Biol Rev 84:91–111CrossRefGoogle Scholar
  43. McConnaughey T (1978) Ecosystems naturally labeled with carbon-13: applications to the study of consumer food webs. MSc Thesis, University of Alaska, Fairbanks, AKGoogle Scholar
  44. McConnaughey T, McRoy CP (1979) Food-web structure and the fractionation of carbon isotopes in the Bering Sea. Mar Biol 53:257–262CrossRefGoogle Scholar
  45. McMeans BC, Olins JA, Benz GW (2009) Stable-isotope comparisons between embryos and mothers of a placentatrophic shark species. J Fish Biol 75:2464–2474PubMedCrossRefGoogle Scholar
  46. Mintenbeck K, Brey T, Jacob U, Knust R, Struck U (2008) How to account for the lipid effect on carbon stable-isotope ratio (δ13C): sample treatment effects and model bias. J Fish Biol 72:815–830CrossRefGoogle Scholar
  47. Murry BA, Farrell JM, Teece MA, Smyntek PM (2006) Effect of lipid extraction on the interpretation of fish community trophic relationships determined by stable carbon and nitrogen isotopes. Can J Fish Aquat Sci 63:2167–2172CrossRefGoogle Scholar
  48. Newsome SD, Martínez del Rio C, Bearhop S, Phillips DL (2007) A niche for isotopic ecology. Front Ecol Environ 5:429–436CrossRefGoogle Scholar
  49. Newsome SD, Tinker MT, Monson DH, Oftedal OT, Ralls K, Staedler MM, Fogel M, Estes J (2009) Using stable isotopes to investigate individual diet specialization in California sea otters (Enhydralutris nereis). Ecology 90:961–974PubMedCrossRefGoogle Scholar
  50. Peterson BJ, Fry B (1987) Stable isotopes in ecosystem studies. Annu Rev Ecol Syst 18:293–320CrossRefGoogle Scholar
  51. Pinnegar JK, Polunin NVC (1999) Differential fractionation of δ13C and δ15N among fish tissues: implications for the study of trophic interactions. Funct Ecol 13:225–231CrossRefGoogle Scholar
  52. Post DM, Layman CA, Arrington DA, Takimoto G, Quattrochi J, Montaña CG (2007) Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses. Oecologia 152:179–189PubMedCrossRefGoogle Scholar
  53. Ricca MA, Miles AK, Anthony RG, Deng X, Hung SSO (2007) Effect of lipid extraction on analyses of stable carbon and stable nitrogen isotopes in coastal organisms of the Aleutian archipelago. Can J Zool 85:40–48CrossRefGoogle Scholar
  54. Rubenstein DR, Hobson KA (2004) From birds to butterflies: animal movement patterns and stable isotopes. Trends Ecol Evol 19:256–263PubMedCrossRefGoogle Scholar
  55. Ryan C, McHugh B, Trueman C, Harro C, Berrow SD, O’Connor I (2012) Accounting for the effects of lipids in stable isotope (δ13C and δ15N values) analysis of skin and blubber of balaenopterid whales. Rapid Commun Mass Spectrom 26:2745–2754PubMedCrossRefGoogle Scholar
  56. Ryg G, Smith TG, Øritsland NA (1990) Seasonal changes in body mass and body composition of ringed seals (Phoca hispida) on Svalbard. Can J Zool 68:470–475CrossRefGoogle Scholar
  57. Schlechtriem CH, Focken U, Becker K (2003) Effect of different lipid extraction methods on δ13C of lipid and lipid-free fractions of fish and different fish feeds. Isotopes Environ Health Stud 39:135–140PubMedCrossRefGoogle Scholar
  58. Sotiropoulos MA, Tonn WM, Wassenaar LI (2004) Effects of lipid extraction on stable carbon and nitrogen isotope analyses of fish tissues: potential consequences for food web studies. Ecol Freshw Fish 13:155–160CrossRefGoogle Scholar
  59. Sweeting CJ, Polunin NVC, Jennings S (2004) Tissue and fixative dependent shifts of delta13C and delta15 N in preserved ecological material. Rapid Commun Mass Spectrom 18:2587–2592PubMedCrossRefGoogle Scholar
  60. Sweeting CJ, Polunin NVC, Jennings S (2006) Effects of chemical lipid extraction and arithmetic lipid correction on stable isotope ratios of fish tissues. Rapid Commun Mass Spectrom 20:595–601PubMedCrossRefGoogle Scholar
  61. Tarroux A, Ehrich D, Lecomte N, Jardine TD, Bêty J, Berteaux D (2010) Sensitivity of stable isotope mixing models to variation in isotopic ratios: evaluating consequences of lipid extraction. Methods Ecol Evol 1:231–241. doi:10.1111/j.2041-210X.2010.00033.x Google Scholar
  62. Thompson DR, Phillips RA, Stewart FM, Waldron S (2000) Low δ13C signatures in pelagic seabirds: lipid ingestion as a potential source of 13C-depleted carbon in the Procellariiformes. Mar Ecol Prog Ser 208:265–271CrossRefGoogle Scholar
  63. Varela JL, Larrañaga A, Medina A (2011) Prey-muscle carbon and nitrogen stable-isotope discrimination factors in Atlantic Bluefin tuna (Thunnus thynnus). J Exp Mar Biol Ecol 406:21–28CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • David J. Yurkowski
    • 1
  • Nigel E. Hussey
    • 1
  • Christina Semeniuk
    • 1
  • Steven H. Ferguson
    • 2
  • Aaron T. Fisk
    • 1
  1. 1.Great Lakes Institute for Environmental ResearchUniversity of WindsorWindsorCanada
  2. 2.Freshwater InstituteFisheries and Oceans CanadaWinnipegCanada

Personalised recommendations