Effects of ethanol storage and lipid extraction on stable isotope compositions of twelve pelagic predators

Stable isotope analysis (SIA) has proven to be a powerful tool in reconstructing diets and characterizing trophic relationships for pelagic predators. Ethanol has been a common preservative solution for biopsy samples from remote areas and archived collections. It is still under debate whether the effects of ethanol (ET) would bias the trophic interpretation of the stable isotope values. Further, lipid extraction (LE) is becoming more popular as a general treatment for standardization prior to SIA, particularly for investigating intra and interspecific variation of sympatric species, because lipids have lower δ13C values. In this study, the long-term (up to 448 days) effects of treatment ET and combined treatments ET and LE (ET+LE) on stable carbon and nitrogen isotope values (δ13C and δ15N, respectively) of twelve pelagic predators from the open ocean were evaluated. Results showed that compared with control values, δ15N values displayed a positive change (δ15Nmean offset was 0.71 ± 0.56‰) but δ13C values had variable results (δ13Cmean offset was 0.42 ± 0.64‰) among all species following treatment with ET during the first 28 days and then remained stable throughout the experiment. Compared with treatment LE results, no difference was observed in δ13C, δ15N values, and C/N ratios through time following treatment ET+LE. These results indicated that treatment ET may have species-specific effects on stable isotope values, and the shifts from treatment LE could counter the changes caused by treatment ET. In addition, after 28 days of preservation, the values following treatment ET were similar to those following treatment LE in low C/N species (C/N<3.5), which suggested ethanol may also affect some of lipid contents from muscle tissues. Nevertheless, further research is needed to focus on the mechanisms that control changes in stable isotope composition in tissues stored in ethanol. Given the effects on pelagic predators, muscle tissue samples stored in ethanol from the open ocean or a museum after LE treatment could be used to develop SIA.

[1]  T. J. Willis,et al.  A Multi-Tissue, Multi-Species Assessment of Lipid and Urea Stable Isotope Biases in Mesopredator Elasmobranchs , 2022, Frontiers in Marine Science.

[2]  J. L. Varela,et al.  Trophic niche segregation between the sympatric tunas Thunnus alalunga and Katsuwonus pelamis in the Gulf of Cadiz (East Atlantic) , 2022, Marine Biodiversity.

[3]  G. Lepoint,et al.  Effects of preservation methodology on stable isotope compositions of sea stars. , 2020, Rapid communications in mass spectrometry : RCM.

[4]  M. Herbin,et al.  Noninvasive Fluid Identification: Potential of Micro-Raman Spectroscopy , 2020, Collection Forum.

[5]  K. Jerina,et al.  Effects of ethanol storage and lipids on stable isotope values in a large mammalian omnivore , 2019, Journal of Mammalogy.

[6]  Y. Yokoyama,et al.  Enhancing insights into foraging specialization in the world's largest fish using a multi-tissue, multi-isotope approach , 2019, Ecological Monographs.

[7]  Julien M. Claes,et al.  A global perspective on the trophic geography of sharks , 2018, Nature Ecology & Evolution.

[8]  A. Gallagher,et al.  Patterns of resource use and isotopic niche overlap among three species of sharks occurring within a protected subtropical estuary , 2017, Aquatic Ecology.

[9]  F. Ramírez,et al.  Lipid effects on isotopic values in bottlenose dolphins (Tursiops truncatus) and their prey with implications for diet assessment , 2017 .

[10]  X. Dai,et al.  Urea and lipid extraction treatment effects on δ(15)N and δ(13)C values in pelagic sharks. , 2016, Rapid communications in mass spectrometry : RCM.

[11]  K. Furuya,et al.  Effects of ethanol-preservation on stable carbon and nitrogen isotopic signatures in marine predators , 2015 .

[12]  V. Lesage,et al.  Effect of ethanol preservation on stable carbon and nitrogen isotope values in cetacean epidermis: Implication for using archived biopsy samples , 2014 .

[13]  Yu Liu,et al.  An Assessment of Sample Preservation Methods for the Determination of Stable Carbon and Nitrogen Isotope Ratios in Mollusks , 2013 .

[14]  Sora L. Kim,et al.  Methods to collect, preserve, and prepare elasmobranch tissues for stable isotope analysis , 2012, Environmental Biology of Fishes.

[15]  M. MacNeil,et al.  Stable isotopes and elasmobranchs: tissue types, methods, applications and assumptions. , 2012, Journal of fish biology.

[16]  A. C. James,et al.  On the Use of Stable Isotopes in Trophic Ecology , 2011 .

[17]  R. I. Ruiz‐Cooley,et al.  Effects of lipid removal and preservatives on carbon and nitrogen stable isotope ratios of squid tissues: Implications for ecological studies , 2011 .

[18]  V. Lesage,et al.  Stable isotopes and trace elements as indicators of diet and habitat use in cetaceans: predicting errors related to preservation, lipid extraction, and lipid normalization , 2010 .

[19]  F. Wessels,et al.  Carbon 13 discrimination during lipid biosynthesis varies with dietary concentration of stable isotopes: implications for stable isotope analyses , 2010 .

[20]  C. Somers,et al.  Effects of Lipid Extraction and Lipid Normalization on Stable Carbon and Nitrogen Isotope Ratios in Double-Crested Cormorants: Implications for Food Web Studies , 2010 .

[21]  Juan Freire,et al.  Effects of preservatives on stable isotope analyses of four marine species , 2009, Estuarine, Coastal and Shelf Science.

[22]  K. Bjorndal,et al.  Effects of Preservation Method on Stable Carbon and Nitrogen Isotope Values , 2008, Physiological and Biochemical Zoology.

[23]  D. Post,et al.  Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses , 2007, Oecologia.

[24]  M. Power,et al.  The effects of preservation on fish tissue stable isotope signatures , 2006 .

[25]  S. Jennings,et al.  Effects of chemical lipid extraction and arithmetic lipid correction on stable isotope ratios of fish tissues. , 2006, Rapid communications in mass spectrometry : RCM.

[26]  S. Jennings,et al.  Tissue and fixative dependent shifts of δ13C and δ15N in preserved ecological material , 2004 .

[27]  K. Becker,et al.  Effect of different lipid extraction methods on δ13C of lipid and lipid-free fractions of fish and different fish feeds , 2003, Isotopes in environmental and health studies.

[28]  Ransom A. Myers,et al.  Collapse and Conservation of Shark Populations in the Northwest Atlantic , 2003, Science.

[29]  D. Post.  USING STABLE ISOTOPES TO ESTIMATE TROPHIC POSITION: MODELS, METHODS, AND ASSUMPTIONS , 2002 .

[30]  H. Sarakinos,et al.  A synthesis of tissue-preservation effects on carbon and nitrogen stable isotope signatures , 2002 .

[31]  E. Pakhomov,et al.  Effects of storage and preservation on the δ13C and δ15N signatures of selected marine organisms , 2001 .

[32]  K. Hobson,et al.  Preservation of blood and tissue samples for stable-carbon and stable-nitrogen isotope analysis , 1997 .

[33]  K. Elliott,et al.  Lipid extraction techniques for stable isotope analysis of bird eggs: Chloroform–methanol leads to more enriched 13C values than extraction via petroleum ether , 2016 .

[34]  D. Kopp,et al.  Freezing and chemical preservatives alter the stable isotope values of carbon and nitrogen of the Asiatic clam (Corbicula fluminea) , 2010, Hydrobiologia.

[35]  B. Peterson,et al.  STABLE ISOTOPES IN ECOSYSTEM STUDIES , 1987 .