Effects of demineralization on the stable isotope analysis of bone samples

Rationale: The sampling of sequential, annually formed bone growth layers for stable carbon (δ¹³C values) and nitrogen (δ¹⁵N values) isotope analysis (SIA) can provide a time series of foraging ecology data. To date, no standard protocol exists for the pre-SIA treatment of cortical samples taken from fresh, modern, bones. Methods: Based on the SIA of historical bone, it is assumed that fresh bone samples must be pre-treated with acid prior to SIA. Using an elemental analyzer coupled to an isotope ratio mass spectrometer to measure stable carbon and nitrogen ratios, we tested the need to acidify cortical bone powder with 0.25 M HCl prior to SIA to isolate bone collagen for the determination of δ¹³C and δ¹⁵ values. We also examined the need for lipid extraction to remove potential biases related to δ¹³C analysis, based on a C:N ratio threshold of 3.5. Results: It was found that acidification of micromilled cortical bone samples from marine turtles does not affect their δ¹⁵N values, and the small effect acidification has on δ¹³C values can be mathematically corrected for, thus eliminating the need for pre-SIA acidification of cortical bone. The lipid content of the cortical bone samples was low, as measured by their C:N ratios, indicating that lipid extracting cortical bone samples from modern marine turtles is unnecessary. Conclusions: We present a standard protocol for testing fresh, modern cortical bone samples prior to SIA, facilitating direct comparison of future studies. Based on the results obtained from marine turtle bones, pre-acidification and lipid removal of cortical bone are not recommended. This is especially useful as there is frequently not enough bone material removed via micromilling of sequential growth layers to accommodate both acid treatment and SIA

Tracking turtles back in time: Linking stable isotope analysis with skeletochronology to determine life history patterns in endangered sea turtles

Of the five species of sea turtles that inhabit the east Pacific Ocean, two, the North Pacific loggerhead (Caretta caretta) and East Pacific green turtle (Chelonia mydas), experience some of the highest rates of globally documented mortality in a productive foraging hotspot near the Baja California Peninsula (BCP), Mexico. This area, the Gulf of Ulloa, overlaps with high levels of fishing, resulting in thousands of dead turtles, many that wash up on the beach of Playa San Lázaro on the BCP. It is unknown how long loggerheads inhabit distinct regions of the North Pacific, and to what degree juvenile individuals in the population demonstrate variation in their life history patterns. Similarly, it is unknown how long green turtles occupy oceanic regions of the eastern Pacific before settling into more nearshore habitats, or how they use the Gulf of Ulloa. My research determined the residency duration at different foraging grounds within the eastern Pacific, as well as basic demographic information such as age-at-settlement, age-at-maturation, and timing of ontogenetic shifts, for these two endangered sea turtle populations. By determining life history parameters and the length of time individuals in these populations spend in distinct high-risk habitats, managers can better understand exposure to spatially explicit threats and prioritize conservation approaches. I focused particularly on duration of time spent in the high-bycatch waters of the Gulf of Ulloa. To this end, I developed a novel technique that combined skeletochronology (the study of growth and age increments in bones) with sequential stable carbon (δ13C) and nitrogen (δ15N) isotope analysis of humerus bone growth layers. Naturally occurring stable isotope gradients exist in ocean systems such that habitats and foraging behaviors can be distinguished (i.e. nearshore vs. offshore habitats or high vs. low trophic levels). Given these isotope gradients, the combination of skeletochronology with stable isotope analysis of sequentially sampled growth layers provides a multi-year record of location, diet, size, age, and annual growth of individual turtles, allowing the reconstruction of life history and long-term habitat use patterns. The combination of these techniques allowed me to address questions that could not be answered using either technique alone

Methods for sampling sequential annual bone growth layers for stable isotope analysis

1. Stable carbon (§13C) and nitrogen (§15N) isotope analysis (SIA) has proven useful in addressing fundamental questions in ecology such as reconstructing trophic interactions, habitat connections and climate regime shifts. The temporal scales over which SIA can be used to address ecological problems vary depending on the protein turnover times of the analysed tissue. Hard, inert tissues, such as teeth, bones and mollusc shells, grow in regular intervals (i.e. daily or annually), and sequential sampling of these growth layers provides a time series of isotopic patterns. As a result, SIA on these tissues is useful for elucidating behaviour and ecology of animals over time, especially those with cryptic life-history stages, such as marine turtles that retain growth layers in their humerus bones. To date, there exists no standard protocol for the sequential sampling of cortical bone samples taken from fresh, modern samples for SIA. 2. We tested two different methods,micromilling untreated bone cross sections and biopsy coring bone cross sections processed for skeletochronology, for sequentially sampling individual growth layers from marine turtle humerus bones. 3. We present a standard protocol for sequential bone growth layer sampling for SIA, facilitating direct comparison of future studies. We recommend using the micromilling sampling technique on untreated bone cross sections, as it facilitated higher precision sampling of growth layers that were not affected by chemical processing, and minimized sample handling, thereby reducing chances for contamination. 4. This is the first study to present a standardized method to sequentially sample annual bone growth layers for stable isotope analysis and facilitates direct comparison among future studies

Effects of demineralization on the stable isotope analysis of bone samples.

RationaleThe sampling of sequential, annually formed bone growth layers for stable carbon (δ(13)C values) and nitrogen (δ(15)N values) isotope analysis (SIA) can provide a time series of foraging ecology data. To date, no standard protocol exists for the pre-SIA treatment of cortical samples taken from fresh, modern, bones.MethodsBased on the SIA of historical bone, it is assumed that fresh bone samples must be pre-treated with acid prior to SIA. Using an elemental analyzer coupled to an isotope ratio mass spectrometer to measure stable carbon and nitrogen ratios, we tested the need to acidify cortical bone powder with 0.25 M HCl prior to SIA to isolate bone collagen for the determination of δ(13)C and δ(15)N values. We also examined the need for lipid extraction to remove potential biases related to δ(13)C analysis, based on a C:N ratio threshold of 3.5.ResultsIt was found that acidification of micromilled cortical bone samples from marine turtles does not affect their δ(15)N values, and the small effect acidification has on δ(13)C values can be mathematically corrected for, thus eliminating the need for pre-SIA acidification of cortical bone. The lipid content of the cortical bone samples was low, as measured by their C:N ratios, indicating that lipid extracting cortical bone samples from modern marine turtles is unnecessary.ConclusionsWe present a standard protocol for testing fresh, modern cortical bone samples prior to SIA, facilitating direct comparison of future studies. Based on the results obtained from marine turtle bones, pre-acidification and lipid removal of cortical bone are not recommended. This is especially useful as there is frequently not enough bone material removed via micromilling of sequential growth layers to accommodate both acid treatment and SIA

RamirezMatthewFishWildlifeEffectsDemineralizationStable.pdf

Rationale: The sampling of sequential, annually formed bone growth layers for stable carbon (δ¹³C values) and nitrogen (δ¹⁵N values) isotope analysis (SIA) can provide a time series of foraging ecology data. To date, no standard protocol exists for the pre-SIA treatment of cortical samples taken from fresh, modern, bones. Methods: Based on the SIA of historical bone, it is assumed that fresh bone samples must be pre-treated with acid prior to SIA. Using an elemental analyzer coupled to an isotope ratio mass spectrometer to measure stable carbon and nitrogen ratios, we tested the need to acidify cortical bone powder with 0.25 M HCl prior to SIA to isolate bone collagen for the determination of δ¹³C and δ¹⁵ values. We also examined the need for lipid extraction to remove potential biases related to δ¹³C analysis, based on a C:N ratio threshold of 3.5. Results: It was found that acidification of micromilled cortical bone samples from marine turtles does not affect their δ¹⁵N values, and the small effect acidification has on δ¹³C values can be mathematically corrected for, thus eliminating the need for pre-SIA acidification of cortical bone. The lipid content of the cortical bone samples was low, as measured by their C:N ratios, indicating that lipid extracting cortical bone samples from modern marine turtles is unnecessary. Conclusions: We present a standard protocol for testing fresh, modern cortical bone samples prior to SIA, facilitating direct comparison of future studies. Based on the results obtained from marine turtle bones, pre-acidification and lipid removal of cortical bone are not recommended. This is especially useful as there is frequently not enough bone material removed via micromilling of sequential growth layers to accommodate both acid treatment and SIA