The influence of diet on the distribution of nitrogen isotopes in animals was investigated by analyzing animals grown in the laboratory on diets of constant nitrogen isotopic composition. 
The isotopic composition of the nitrogen in an animal reflects the nitrogen isotopic composition of its diet. The δ^(15)N values of the whole bodies of animals are usually more positive than those of their diets. Different individuals of a species raised on the same diet can have significantly different δ^(15)N values. The variability of the relationship between the δ^(15)N values of animals and their diets is greater for different species raised on the same diet than for the same species raised on different diets. Different tissues of mice are also enriched in ^(15)N relative to the diet, with the difference between the δ^(15)N values of a tissue and the diet depending on both the kind of tissue and the diet involved. The δ^(15)N values of collagen and chitin, biochemical components that are often preserved in fossil animal remains, are also related to the δ^(15)N value of the diet. 
The dependence of the δ^(15)N values of whole animals and their tissues and biochemical components on the δ^(15)N value of diet indicates that the isotopic composition of animal nitrogen can be used to obtain information about an animal's diet if its potential food sources had different δ^(15)N values. The nitrogen isotopic method of dietary analysis probably can be used to estimate the relative use of legumes vs non-legumes or of aquatic vs terrestrial organisms as food sources for extant and fossil animals. However, the method probably will not be applicable in those modern ecosystems in which the use of chemical fertilizers has influenced the distribution of nitrogen isotopes in food sources. 
The isotopic method of dietary analysis was used to reconstruct changes in the diet of the human population that occupied the Tehuacan Valley of Mexico over a 7000 yr span. Variations in the δ^(15)C and δ^(15)N values of bone collagen suggest that C_4 and/or CAM plants (presumably mostly corn) and legumes (presumably mostly beans) were introduced into the diet much earlier than suggested by conventional archaeological analysis.

Influence of Diet On the Distribtion of Nitrogen Isotopes in Animals

Within an organism, lipids are depleted in (13)C relative to proteins and carbohydrates (more negative delta(13)C), and variation in lipid content among organisms or among tissue types has the potential to introduce considerable bias into stable isotope analyses that use delta(13)C. Despite the potential for introduced error, there is no consensus on the need to account for lipids in stable isotope analyses. Here we address two questions: (1) If and when is it important to account for the effects of variation in lipid content on delta(13)C? (2) If it is important, which method(s) are reliable and robust for dealing with lipid variation? We evaluated the reliability of direct chemical extraction, which physically removes lipids from samples, and mathematical normalization, which uses the carbon-to-nitrogen (C:N) ratio of a sample to normalize delta(13)C after analysis by measuring the lipid content, the C:N ratio, and the effect of lipid content on delta(13)C (Deltadelta(13)C) of plants and animals with a wide range of lipid contents. For animals, we found strong relationships between C:N and lipid content, between lipid content and Deltadelta(13)C, and between C:N and Deltadelta(13)C. For plants, C:N was not a good predictor of lipid content or Deltadelta(13)C, but we found a strong relationship between carbon content and lipid content, lipid content and Deltadelta(13)C, and between and carbon content and Deltadelta(13)C. Our results indicate that lipid extraction or normalization is most important when lipid content is variable among consumers of interest or between consumers and end members, and when differences in delta(13)C between end members is <10-12 per thousand. The vast majority of studies using natural variation in delta(13)C fall within these criteria. Both direct lipid extraction and mathematical normalization reduce biases in delta(13)C, but mathematical normalization simplifies sample preparation and better preserves the integrity of samples for delta(15)N analysis.

/pdf/getting-to-the-fat-of-the-matter-models-methods-and-1styosvrid.pdf

Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses

Stable isotope analysis (SIA) has proven to be a useful tool in reconstructing diets, characterizing trophic relationships, elucidating patterns of resource allocation, and constructing food webs. Consequently, the number of studies using SIA in trophic ecology has increased exponentially over the past decade. Several subdisciplines have developed, including isotope mixing models, incorporation dynamics models, lipid-extraction and correction methods, isotopic routing models, and compound-specific isotopic analysis. As with all tools, there are limitations to SIA. Chief among these are multiple sources of variation in isotopic signatures, unequal taxonomic and ecosystem coverage, over-reliance on literature values for key parameters, lack of canonical models, untested or unrealistic assumptions, low predictive power, and a paucity of experimental studies. We anticipate progress in SIA resulting from standardization of methods and models, calibration of model parameters through experimentation, and continu...

/pdf/on-the-use-of-stable-isotopes-in-trophic-ecology-lygvzayjij.pdf

On the Use of Stable Isotopes in Trophic Ecology

Summary 1. Lipids have more negative δ 13 C values relative to other major biochemical compounds in plant and animal tissues. Although variable lipid content in biological tissues alters results and conclusions of δ 13 C analyses in aquatic food web and migration studies, no standard correction protocol exists. 2. We compared chemical extraction and mathematical correction methods for freshwater and marine fishes and aquatic invertebrates to better understand impacts of correction approaches on carbon ( δ 13 C) and nitrogen ( δ 15 N) stable isotope data. 3. Fish and aquatic invertebrate tissue δ 13 C values increased significantly following extraction for almost all species and tissue types relative to nonextracted samples. In contrast, δ 15 N was affected for muscle and whole body samples from only a few freshwater and marine species and had a limited effect for the entire data set. 4. Lipid normalization models, using C : N as a proxy for lipid content, predicted lipid-corrected δ 13 C for paired data sets more closely with parameters specific to the tissue type and species to which they were applied. 5. We present species- and tissue-specific models based on bulk C : N as a reliable alternative to chemical extraction corrections. By analysing a subset of samples before and after lipid extraction, models can be applied to the species and tissues of interest that will improve estimates of dietary sources using stable isotopes.

/pdf/lipid-corrections-in-carbon-and-nitrogen-stable-isotope-2xouwaiz2m.pdf

Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods

1. Stable isotope analyses coupled with mixing models are being used increasingly to evaluate ecological management issues and questions. Such applications of stable isotope analyses often require simultaneous carbon and nitrogen analyses from the same sample. Correction of the carbon isotope values to take account of the varying content of 13 C-depleted lipids is then frequently achieved by a lipid-normalization procedure using a model describing the relationship between change in δ 13 C following lipid removal and the original C:N ratio of a sample. 2. We evaluated the applicability of two widely used normalization models using empirical data for muscle tissue from a wide range of fish and for aquatic invertebrates. Neither normalization model proved satisfactory, and we present some modifications that greatly improve the fit of one of the models to the fish muscle data. For invertebrates we found no clear relationship between change in δ 13 C following lipid removal and the original C:N ratio. 3. We also examined the effect of lipid-normalization on the output of a mixing model designed to calculate the proportional contribution of prey items to the diet of a consumer. Mixing model output was greatly influenced by whether prey or consumer values alone or together were lipid-normalized and we urge caution in the interpretation of results from these models pending further experimental evidence. 4. Synthesis and applications . We describe a revised lipid-normalization model that should be applicable to a wide range of marine and freshwater fish species in studies applying stable isotope analyses to ecological management issues. However, we strongly advise against applying these kinds of lipid-normalization models to aquatic invertebrate data. The interpretation of outputs from mixing models is greatly influenced by whether the carbon isotope data have been lipid-normalized or not.

A revised model for lipid‐normalizing δ13C values from aquatic organisms, with implications for isotope mixing models

– We examined whether solvent-based lipid extractions, commonly used for stable isotope analysis (SIA) of biota, alters δ15N or δ13C values of fish muscle tissue or whole juvenile fish. Lipid extraction from muscle tissue led to only small (<1‰) isotope shifts in δ13C and δ15N values. By contrast, ecologically significant shifts (+3.4‰ for δ13C and +2.8‰ for δ15N) were observed for whole juvenile fish. Sample variance was not affected by lipid extraction. For tissue-specific SIA, two sample aliquots may be required: a lipid-extracted aliquot for stable carbon isotope analysis when differing lipid content among tissues is a concern, and a nonextracted aliquot for δ15N determination. Whole organism SIA is not recommended because of the mix of tissues having different turnover times; for very small fish, we recommend that fish be eviscerated, decapitated, and skinned to minimise differences with samples of muscle tissue.

Resumen

1. Cada vez con mayor frecuencia, los ecologos de peces utilizan analisis de isotopos estables. Por ello, se hace cada vez mas importante comprender las fuentes de variacion, - debido a diferencias inherentes entre muestreos biologicos o como resultado de tecnicas de procesamiento de muestreo - tanto como identificar estrategias para tratar tales fuentes. Examinamos si la extraccion de lipidos basada en disolventes, comunmente utilizada en analisis de isotopos de carbono estable, altera negativamente los valores de δ15N y δ13C de tejido muscular de tres peces de tamano pequeno y de peces juveniles completos.



2. La extraccion de lipidos de musculo de pez llevo a pequenos cambios isotopicos de + +0.4 a +1.0‰ y de +0.3 a +0.5‰ para δ13C y δ15N, respectivamente. Por el contrario, la extraccion de lipidos de peces juveniles completos vario marcadamente en +3.4‰ para δ13C y +2.8‰ para δ15N - ambos cambios ecologicamente importantes. La varianza de los valores de muestreos de δ13C y de δ15N tanto para tejido muscular como para los peces completos no difirieron entre los muestreos de lipidos extraidos y muestreos sin tratamiento.



3. Nuestros resultados recomiendan el analisis de isotopos estables de tejidos especificos. Cuando ello no es posible o deseable, dos alicuotas de muestreo pueden ser requeridas: una alicuota de lipidos extraidos para el analisis de isotopos de carbono estable cuando la varianza de δ13C, debida a diferencias en el contenido de lipidos de diferentes tejidos, y una alicuota de no-extraccion para determinaciones de δ15N.



4. Dada la mezcla de tejidos, el analisis de isotopos de un organismo completo no es recomendable – en el caso de peces muy pequenos, recomendamos que los peces sean eviscerados, decapitados, y despellejados para minimizar las diferencias de muestreos de tejido muscular.

M. A. Sotiropoulos

Papers

Effects of lipid extraction on stable carbon and nitrogen isotope analyses of fish tissues: potential consequences for food web studies