Effects of formalin and ethanol preservation on otolith delta18O stable isotope signatures

The use of preserved otoliths for stable isotope analysis assumes handling and preservation procedures do not alter the isotopic composition of the otolith. Otoliths from wild and hatchery-reared salmonids (brook charr, Salvelinus fontinalis, and Atlantic salmon, Salmo salar) were used to test for possible delta(18)O preservation effects in ethanol and formalin preservation experiments at varying temperatures. Analysis of variance (ANOVA) demonstrated a significant interaction effect between species and preservative during preservation. Possible causes for the observed effect are discussed in relation to species-specific differences in otolith chemistry related to growth and environment including: (1) chemical mechanisms of dissolution-recrystallisation involving the precipitation of secondary minerals within and at the otolith surface; (2) adsorption of ions at available binding sites on the otolith surface; and (3) isotopic exchange during otolith surface dissolution and/or reprecipitation processes. Differential occurrence of vaterite and aragonite in otoliths is believed to account for some of the observed effects as a result of otolith density differences. Isotopic exchange is also argued to cause much of the observed variation in species-specific preservation effects. Biologically, study findings imply that preserved otoliths should not be used for baseline paleoclimatic or individual fish thermal reconstructions, or the development of delta(18)O-fractionation equations, without the prior use of pilot studies to determine preservation effects.     

A field-derived oxygen isotope fractionation equation for Salvelinus species

Field-collected otolith samples of young-of the-year Arctic charr (Salvelinus alpinus) and brook charr (Salvelinus fontinalis) and monitored water temperatures were used to estimate a delta(18)O fractionation equation for the genus Salvelinus. When compared to literature reported equations, the developed fractionation equation had a statistically similar slope but dissimilar intercept. Statistical similarities among fractionation equation slope estimates suggest a common otolith delta(18)O incorporation response among fish species that may be interpreted as widespread equilibrium otolith delta(18)O deposition. Statistical dissimilarities among intercept estimates question broad applicability of any single fractionation equation to all fish species and were interpreted here to have biological meaning as a result of known differences in standard metabolic rates among species. Attempts to statistically cross-validate fractionation equations by prediction of water temperatures used in other fractionation studies indicated significant biases in the range of -7.9 to 6.7 degrees C that preclude the broad use of any single fractionation equation for accurate thermal reconstructions. Differences in equation intercepts and the prevalence of predictive biases do not support the conclusion of previous studies concerning the wide applicability and/or general accuracy of fractionation equations and suggest fractionation equations are best developed at the species- or taxon-specific (e.g., genus) level.     

Identifying migratory Salmo trutta using carbon and nitrogen stable isotope ratios

Many Salmo trutta populations consist of non-anadromous (freshwater-resident) brown trout and anadromous (sea-run migratory) sea trout. Although adult brown trout and sea trout can usually be identified using differences in size and body colouration, it is not possible to easily identify eggs/alevins as the progeny of brown trout or sea trout. In this study we show that delta(13)C and delta(15)N, measured using a continuous flow isotope ratio mass spectrometer (CF-IRMS), can accurately identify fish eggs as the progeny of freshwater-resident (delta(13)C(egg) = -25.7 +/- 1.9 per thousand,delta(15)N(egg) = 9.2 +/- 1.8 per thousand) or migratory (delta(13)C(egg) = -19.9 +/- 1.1 per thousand, delta(15)N(egg) = 14. 3 +/- 1.5 per thousand) adult female Salmo trutta. Case studies show that stable isotope analysis is a more reliable technique for distinguishing anadromous adult fish than differentiation using morphological characteristics. For example, stable isotope analysis of brown trout from Loch Eck, Scotland, revealed that some individuals possessed delta(13)C and delta(15)N signatures indicative of marine feeding despite visual identification as freshwater-resident fish. It is most likely that these fish are misidentified sea trout although it possible that these fish may be brown trout that have adopted an estuarine feeding strategy to avoid interspecific competition for food within Loch Eck with salmon, powan and Arctic charr. Most stable isotope studies of fish ecology use terminal tissue sampling to provide sufficient biological material for isotopic analysis; however, our study suggests that adipose fin tissue could provide a comparable measure of delta(13)C and delta(15)N. Such a strategy would be invaluable when studying the trophic ecology or migration patterns of fish of high conservation value.     

Tissue and fixative dependent shifts of delta13C and delta15N in preserved ecological material

Carbon and nitrogen stable isotope analyses are routinely used to investigate aquatic food webs, and have potential application in retrospective investigations using archived materials. However, such analyses assume that storage does not alter isotopic signatures of materials preserved, or that changes in isotopic composition during storage are predictable. Here we examine preservation shifts on cod (Gadus morhua) muscle, roe and liver tissue over 21 months following preservation in 80% ethanol, in 4% formaldehyde, and by freezing. Preservation shifts were not consistent among tissues. High protein tissues exhibited greater delta(15)N shifts than low protein tissues in 4% formaldehyde, while greater delta(13)C shifts occurred in relatively higher fat tissues when preserved in alcohol. Freezing did not change isotopic signatures. Responses of delta(15)N and delta(13)C are explained by differences in the preservative's isotopic signature and the reaction properties and biochemical composition of the tissues preserved. The results clarify some of the processes that lead to isotopic change during preservation.     

Solid phase extraction for analysis of biogenic carbonates by electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS): an investigation of rare earth element signatures in otolith microchemistry

Uptake of trace elements into fish otoliths is governed by several factors such as life histories and environment in addition to stock and species differences. In an attempt to elucidate the elemental signatures of rare earth elements (REEs) in otoliths, a solid phase extraction (SPE) protocol was used in combination with electrothermal vaporization (ETV) as a sample introduction procedure for the determinations by inductively coupled plasma quadrupole mass spectrometry (ICP-MS). Effects of various parameters, such as carrier gas flow rate, atomization temperature and chemical modification, were examined for optimization of the conditions by ETV-ICP-MS. Atomization was achieved at 2800 °C. Lower temperatures (i.e. 2600 °C) resulted in severe memory problems due to incomplete atomization. Palladium was used as a chemical modifier. It was found that an increase in Pd concentration up to 0.5 μg in the injection volume (70 μl) led up to four-fold enhancement in the integrated signals. This phenomenon is attributed to the carrier effect of Pd rather than the stabilization since no significant losses were observed for high temperature drying around 700 °C even in the absence of Pd. Preconcentration was performed on-line at pH 5 by using a mini-column of Toyopearl AF-Chelate 650M chelating resin, which also eliminated the calcium matrix of otolith solutions. After preconcentration of 6.4 ml of solution, the concentrate was collected in 0.65 ml of 0.5% (v/v) HNO₃ in autosampler cups, and then analyzed by ETV-ICP-MS. The method was validated with the analysis of a fish otolith certified reference material (CRM) of emperor snapper, and then applied to samples. Results obtained from otoliths of fish captured in the same habitat indicated that otolith rare earth element concentrations are more dependent on environmental conditions of the habitat than on species differences.     

Preparation techniques alter the mineral and organic fractions of fish otoliths: insights using Raman micro-spectrometry

The high spatial resolution analysis of the mineral and organic composition of otoliths using Raman micro-spectrometry involves rigorous protocols for sample preparation previously established for microchemistry and trace elements analyses. These protocols often include otolith embedding in chemically neutral resin (i.e., resins which do not contain, in detectable concentration, elements usually sought in the otoliths). Such embedding may however induce organic contamination. In this paper, Raman micro-spectrometry reveals the presence of organic contamination onto the surface obtained from the use of epoxy resin, specifically Araldite. This contamination level varies depending on otolith structures. Core and checks, known as structural discontinuities, exhibit the most important level of contaminations. Our results suggest that otolith embedding with resin affects the organic matrix of the otolith, probably through an infiltration of the resin in the crystalline structure. The interpretation of chemical otolith signatures, especially Raman otolith signatures, and stable isotope analyses should then be revised in light of these results. In this respect, we propose a method for the correction of Raman otolith signatures for contamination effects.     

A comparison of muscle- and scale-derived delta13C and delta15N across three life-history stages of Atlantic salmon, Salmo salar

Stable isotope signatures were obtained from paired scale and muscle tissue samples from smolt, post-smolt and one-sea-winter adult Atlantic salmon (Salmo salar). Post-smolt and adult scales were separated into central and outer (marine) portions with analyses carried out on the marine growth section of both life-history stages and the central portion for the adult scales. Muscle and scale delta(13)C and delta(15)N signatures were assessed (1) to determine whether a linear relationship exists between tissue types, (2) to determine if a constant offset exists between tissue signatures across all life-history stages, and (3) to evaluate whether underplating imparts a significant bias to life-history scale segments that would preclude their use in retrospective analyses of any ontogenetic dietary changes between life-history stages. Significant correlations were found to exist between muscle and scale stable isotope signatures obtained from smolts (delta(13)C and delta(15)N) and adults (delta(15)N). Both the muscle and the scale signatures captured the dietary shift associated with the transition from freshwater to the marine environment. Post-smolt and adult scales were depleted relative to muscle tissue, which may be attributed to isotopic differences in amino acid composition between muscle and scale tissues. The results suggest that scales may better represent dietary carbon sources because they are not influenced by lipid dynamics. The scale, however, appears less responsive to short-term shifts in diet relative to muscle and, therefore, must be used only to infer seasonally integrated dietary patterns for slow-growing life-history stages. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Analysis of fish otoliths by electrothermal vaporization inductively coupled plasma mass spectrometry: aspects of precipitating otolith calcium with hydrofluoric acid for trace element determination

A method is developed for determination of trace elements, including Ag, As, Cd, Co, Cr, Cu, Mn, Ni, Se, Tl and Zn, in fish otoliths by electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS). Hydrofluoric acid was used to precipitate calcium resulting from acid dissolution of otolith calcium carbonate. Initial acidity of the sample solution influenced the precipitation efficiency of calcium fluoride. Up to 99.5% of Ca was precipitated in solutions that contained less than 2% (v/v) HNO₃. Recoveries of the elements obtained from spiked artificial otolith solutions were between 90 and 103%. Stabilization of the elements within the ETV cell was achieved with 0.3 μg Pd/0.2 μg Rh chemical modifier that also afforded optimum sensitivity for multielement determination. The method was validated by the analysis of a fish otolith reference material (CRM) of emperor snapper, and then applied to the determination of the trace elements in otoliths of several fish species captured in Raritan Bay, New Jersey. Results indicated that fish physiology and biological processes could influence the levels of Cu, Mn, Se and Zn in the otoliths of fish inhabiting a similar aqueous environment. Otolith concentrations of Cr and Ni did not show any significant differences among different species. Concentrations for Ag, As, Cd, Co and Tl were also not significantly different, but were very low indicating low affinity of otolith calcium carbonate to these elements.     

Use of compound-specific delta13C and deltaD stable isotope measurements as an aid in the source apportionment of polyaromatic hydrocarbons

The potential of using compound-specific stable carbon isotopic analysis for the source apportionment of environmental polycyclic aromatic hydrocarbons (PAHs) has already been demonstrated by the authors, and other researchers. PAHs arising from wood burning and vehicle emissions have been shown to exhibit different isotopic signatures, and the isotopic compositions of n-alkanes and PAHs produced from combustion of C3 and C4 plant species have been reported. 13C/12C isotopic ratios for PAHs derived from coal and wood pyrolysis and from diesel particulates have been noted to vary over a range by ca. 8 per thousand, which may provide a basis for source apportionment. In order to further improve the ability of stable isotope measurements to source apportion environmental PAHs, hydrogen stable isotopes (deltaD) of PAHs from a number of processes have been measured. The wide range of deltaD values, in conjunction with the delta13C values obtained, provide a much greater degree of differentiation between petrol and jet fuel derived PAHs, and between PAHs from different coal conversion processes, than the delta13C values alone.     

Using dual-bacterial denitrification to improve delta15N determinations of nitrates containing mass-independent 17O

The bacterial denitrification method for isotopic analysis of nitrate using N(2)O generated from Pseudomonas aureofaciens may overestimate delta(15)N values by as much as 1-2 per thousand for samples containing atmospheric nitrate because of mass-independent (17)O variations in such samples. By analyzing such samples for delta(15)N and delta(18)O using the denitrifier Pseudomonas chlororaphis, one obtains nearly correct delta(15)N values because oxygen in N(2)O generated by P. chlororaphis is primarily derived from H(2)O. The difference between the apparent delta(15)N value determined with P. aureofaciens and that determined with P. chlororaphis, assuming mass-dependent oxygen isotopic fractionation, reflects the amount of mass-independent (17)O in a nitrate sample. By interspersing nitrate isotopic reference materials having substantially different delta(18)O values with samples, one can normalize oxygen isotope ratios and determine the fractions of oxygen in N(2)O derived from the nitrate and from water with each denitrifier. This information can be used to improve delta(15)N values of nitrates having excess (17)O. The same analyses also yield estimates of the magnitude of (17)O excess in the nitrate (expressed as Delta(17)O) that may be useful in some environmental studies. The 1-sigma uncertainties of delta(15)N, delta(18)O and Delta(17)O measurements are +/-0.2, +/-0.3 and +/-5 per thousand, respectively.     

A gas chromatography/combustion/isotope ratio mass spectrometry system for high-precision delta13C measurements of atmospheric methane extracted from ice core samples

Past atmospheric composition can be reconstructed by the analysis of air enclosures in polar ice cores which archive ancient air in decadal to centennial resolution. Due to the different carbon isotopic signatures of different methane sources high-precision measurements of delta13CH4 in ice cores provide clues about the global methane cycle in the past. We developed a highly automated (continuous-flow) gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) technique for ice core samples of approximately 200 g. The methane is melt-extracted using a purge-and-trap method, then separated from the main air constituents, combusted and measured as CO2 by a conventional isotope ratio mass spectrometer. One CO2 working standard, one CH4 and two air reference gases are used to identify potential sources of isotope fractionation within the entire sample preparation process and to enhance the stability, reproducibility and accuracy of the measurement. After correction for gravitational fractionation, pre-industrial air samples from Greenland ice (1831 +/- 40 years) show a delta13C(VPDB) of -49.54 +/- 0.13 per thousand and Antarctic samples (1530 +/- 25 years) show a delta13C(VPDB) of -48.00 +/- 0.12 per thousand in good agreement with published data.     

δ13C and δ18O trends across overstory environments in whole foliage and cellulose of three Pinus species

Stable isotope ratios of carbon (delta(13)C) and oxygen (delta(18)O) are increasingly used to investigate environmental influences on plant physiology. Cellulose is often isolated for isotopic studies, but some authors have questioned the value of this process. We studied trends in delta(13)C and delta(18)O of whole foliage and holocellulose from seedlings of three Pinus species across three overstory environments to evaluate the benefits of holocellulose extraction in the context of a traditional ecological experiment. Both tissue types showed increasing delta(13)C from closed-canopy controls to thinned plots to 0.3 ha canopy gaps, and no change in delta(18)O between overstory environments. delta(13)C of P. resinosa and P. strobus was greater than delta(13)C of P. banksiana in whole foliage and holocellulose samples, and there were no differences in delta(18)O associated with species in either tissue type. Our results suggest whole foliage and holocellulose provide similar information about isotopic trends across broad environmental gradients and between species, but holocellulose may be better suited for studying differences in stable isotope composition between multiple species across several treatments.     

Methods to adjust for the interference of N2O on delta13C and delta18O measurements of CO2 from soil mineralization

In this paper we present an overview of the present knowledge relating to methods that avoid interference of N2O on delta13C and delta18O measurements of CO2. The main focus of research to date has been on atmospheric samples. However, N2O is predominantly generated by soil processes. Isotope analyses related to soil trace gas emissions are often performed with continuous flow isotope ratio mass spectrometers, which do not necessarily have the high precision needed for atmospheric research. However, it was shown by using laboratory and field samples that a correction to obtain reliable delta13C and delta18O values is also required for a commercial continuous flow isotope ratio mass spectrometer. The capillary gas chromatography column of the original equipment was changed to a packed Porapak Q column. This adaptation resulted in an improved accuracy and precision of delta13C (standard deviation(Ghent): from 0.2 to 0.08 per thousand; standard deviation(Lincoln): from 0.2 to 0.13 per thousand) of CO2 for N2O/CO2 ratios up to 0.1. For delta18O there was an improvement for the standard deviation measured at Ghent University (0.13 to 0.08 per thousand) but not for the measurements at Lincoln University (0.08 to 0.23 per thousand). The benefits of using the packed Porapak Q column compared with the theoretical correction method meant that samples were not limited to small N(2)O concentrations, they did not require an extra N2O concentration measurement, and measurements were independent of the variable isotopic composition of N2O from soil.     

Oxygen isotope corrections for online delta(34)S analysis

Elemental analyzers have been successfully coupled to stable-isotope-ratio mass spectrometers for online measurements of the delta(34)S isotopic composition of plants, animals and soils. We found that the online technology for automated delta(34)S isotopic determinations did not yield reproducible oxygen isotopic compositions in the SO(2) produced, and as a result calculated delta(34)S values were often 1-3 per thousand too high versus their correct values, particularly for plant and animal samples with high C/S ratio. Here we provide empirical and analytical methods for correcting the S isotope values for oxygen isotope variations, and further detail a new SO(2)-SiO(2) buffering method that minimizes detrimental oxygen isotope variations in SO(2).     

Influence of flooding on delta15N, delta18O, 1delta15N and 2delta15N signatures of N2O released from estuarine soils--a laboratory experiment using tidal flooding chambers

The influence of flooding on N2O fluxes, denitrification rates, dual isotope (delta18O and delta15N) and isotopomer (1delta15N and 2delta15N) ratios of emitted N2O from estuarine intertidal zones was examined in a laboratory study using tidal flooding incubation chambers. Five replicate soil cores were collected from two differently managed intertidal zones in the estuary of the River Torridge (North Devon, UK): (1) a natural salt marsh fringing the estuary, and 2 a managed retreat site, previous agricultural land to which flooding was restored in summer 2001. Gas samples from the incubated soil cores were collected from the tidal chamber headspaces over a range of flooding conditions, and analysed for the delta18O, delta15N, 1delta15N and 2delta15N values of the emitted N2O. Isotope signals did not differ between the two sites, and nitrate addition to the flooding water did not change the isotopic content of emitted N2O. Under non-flooded conditions, the isotopic composition of the emitted N2O displayed a moderate variability in delta18O and 2delta15N delta values that was expected for microbial activity associated with denitrification. However, under flooded conditions, half of the samples showed strong and simultaneous depletions in 1delta15N and delta18O values, but not in 2delta15N. Such an isotope signal has not been reported in the literature, and it could point towards an unidentified N2O production pathway. Its signature differed from denitrification, which was generally the N2O production pathway in the salt marsh and the managed retreat site.     

Evaluating high time-resolved changes in carbon isotope ratio of respired CO2 by a rapid in-tube incubation technique

Recent insights into fractionation during dark respiration and rapid dynamics in isotope signatures of leaf- and ecosystem-respired CO(2) indicate the need for new methods for high time-resolved measurements of the isotopic signature of respired CO(2) (delta(13)C(res)). We present a rapid and simple method to analyse delta(13)C(res) using an in-tube incubation technique and an autosampler for small septum-capped vials. The effect of storage on the delta(18)O and delta(13)C ratios of ambient CO(2) concentrations was tested with different humidity and temperatures. delta(13)C ratios remained stable over 72 h, whereas delta(18)O ratios decreased after 24 h. Storage at 4 degrees C improved the storage time for delta(18)O. Leaves or leaf discs were incubated in the vials, flushed with CO(2)-free air and respired CO(2) was automatically sampled within 5 min on a microGas autosampler interfaced to a GV-Isoprime isotope ratio mass spectrometer. Results were validated by simultaneous on-line gas-exchange measurements of delta(13)C(res) of attached leaves. This method was used to evaluate the short-term (5-60 min) and diurnal dynamics of delta(13)C(res) in an evergreen oak (Quercus ilex) and a herb (Tolpis barbata). An immediate depletion of 2-4 per thousand from the initial delta(13)C(res) value occurred during the first 30 min of darkening. Q. ilex exhibited further a substantial diurnal enrichment in delta(13)C(res) of 8 per thousand, followed by a progressive depletion during the night. In contrast, T. barbata did not exhibit a distinct diurnal pattern. This is in accordance with recent theory on fractionation in metabolic pathways and may be related to the different utilisation of the respiratory substrate in the fast-growing herb and the evergreen oak. These data indicate substantial and rapid dynamics (within minutes to hours) in delta(13)C(res), which differed between species and probably the growth status of the plant. The in-tube incubation method enables both high time-resolved analysis and extensive sampling across different organs, species and functional types.     

Treatment methods for the determination of delta2H and delta18O of hair keratin by continuous-flow isotope-ratio mass spectrometry

The structural proteins that comprise approximately 90% of animal hair have the potential to record environmentally and physiologically determined variation in delta2H and delta18O values of body water. Broad, systematic, geospatial variation in stable hydrogen and oxygen isotopes of environmental water and the capacity for rapid, precise measurement via methods such as high-temperature conversion elemental analyzer/isotope ratio mass spectrometry (TC/EA-IRMS) make these isotope systems particularly well suited for applications requiring the geolocation of hair samples. In order for such applications to be successful, however, methods must exist for the accurate determination of hair delta2H and delta18O values reflecting the primary products of biosynthesis. Here, we present the results of experiments designed to examine two potential inaccuracies affecting delta2H and delta18O measurements of hair: the contribution of non-biologic hydrogen and oxygen to samples in the form of sorbed molecular water, and the exchange of hydroxyl-bound hydrogen between hair keratin and ambient water vapor. We show that rapid sorption of molecular water from the atmosphere can have a substantial effect on measured delta2H and delta18O values of hair (comprising approximately 7.7% of the measured isotopic signal for H and up to approximately 10.6% for O), but that this contribution can be effectively removed through vacuum-drying of samples for 6 days. Hydrogen exchange between hair keratin and ambient vapor is also rapid (reaching equilibrium within 3-4 days), with 9-16% of the total hydrogen available for exchange at room temperature. Based on the results of these experiments, we outline a recommended sample treatment procedure for routine measurement of delta2H and delta18O in mammal hair.     

Comparison of secondary ion mass spectrometry and micromilling/continuous flow isotope ratio mass spectrometry techniques used to acquire intra‐otolith δ18O values of wild Atlantic salmon (Salmo salar)

The chemical signals in the sequential layers of fish otoliths have the potential to provide fisheries biologists with temporal and spatial details of migration which are difficult to obtain without expensive tracking methods. Signal resolution depends, however, on the extraction technique used. We compared the use of mechanical micromilling and continuous flow isotope ratio mass spectrometry (CF‐IRMS) methods with secondary ion mass spectrometry (SIMS) to obtain δ¹⁸O profiles from otoliths of wild Atlantic salmon (Salmo salar) and used these to corroborate the time of freshwater emigration of the juvenile with macroscopic patterns within the otolith. Both techniques showed the transition occurring at the same visible feature on the otolith, allowing future analyses to easily identify the juvenile (freshwater) versus adult (marine) life‐stages. However, SIMS showed a rapid and abrupt transition whereas micromilling provided a less distinct signal. The number of samples that could be obtained per unit area sampled using SIMS was 2 to 3 times greater than that when using micromilling/CF‐IRMS although the δ¹⁸O values and analytical precisions (～0.2‰) of the two methods were comparable. In addition, SIMS δ¹⁸O results were used to compare otolith aragonite values with predicted values calculated using various isotope fractionation equations. Copyright © 2010 John Wiley & Sons, Ltd.     

Stable isotope ratio analysis as a tool to discriminate between rainbow trout (O. mykiss) fed diets based on plant or fish-meal proteins

The use of stable isotope ratio analysis (SIRA) as a rapid analytical tool to characterize and discriminate farmed fish on the basis of the feedstuffs included in the diet formulation is discussed. Two isoproteic (44.8%) and isolipidic (19.6%) extruded diets were formulated: a fish-meal-based diet (FM diet), containing fish meal as the sole protein source; a plant-protein-based diet (PP diet), where pea protein concentrate and wheat gluten meal replaced 80% of fish meal protein. The diets were fed to eight groups of rainbow trout (initial body weight: 106.6g) for 103 days in two daily meals under controlled rearing conditions. Growth performance (final body weight: 318.5 g; specific growth rate: 1.06%) and feed-to-gain ratio (0.79) were not affected by the dietary treatment. The differences in isotopic values of the two diets were clearly reflected in the different carbon and nitrogen isotopic values in rainbow trout fillets. The delta(13)C and delta(15)N values of muscle of farmed rainbow trout showed differences between farmed fish fed a fish-protein-based diet (-20.47 +/- 0.34 and 12.38 +/- 0.57 for delta(13)C and delta(15)N, respectively) and those fed a plant-protein-based diet (-23.96 +/- 0.38 and 7.15 +/- 0.51 for delta(13)C and delta(15)N, respectively). The results suggest that SIRA provides a robust and verifiable analytical tool to discriminate between fish fed on a plant or a fish protein diet.     

Effects of preservation methods on stable isotope signatures in bird tissues

Increasing use is being made of stable isotopes as indicators of habitat use and trophic ecology of animals. Preservation of tissues can alter stable isotope signatures. We investigated the effects of addition of ethanol and NaCl solution (hereafter 'salt'), and of freezing and drying, on carbon and nitrogen isotopic values in blood of the spectacled petrel Procellaria conspicillata, and compared these with those from simultaneously growing feathers. The mean delta(13)C values of blood preserved in ethanol was significantly higher, and of blood preserved in salt was significantly lower than that of dried or frozen samples. delta(13)C values in ethanol showed high variation according to brand and batch and could account for the differences found in delta(13)C ratios in ethanol-preserved blood samples. Mean delta(13)C and delta(15)N values in growing feathers were higher than in blood, suggesting tissue-specific fractionation. We conclude that different methods of preserving tissues such as blood may bias stable isotope values, and urge researchers to consider this issue. Air drying is proposed as a practical and unbiased method for blood preservation in field situations where freezing is not a practical option, and a mathematical approach is suggested to permit comparison between studies using different preservation methods or tissues.