A protocol for distilling animal body water from biological samples and measuring oxygen and hydrogen stable isotopes via cavity ring-down spectroscopy

ABSTRACT

The application of stable isotope analysis (SIA) to the fields of ecology and animal biology has rapidly expanded over the past three decades, particularly with regards to water analysis. SIA now provides the opportunity to monitor migration patterns, examine food webs, and assess habitat changes in current and past study systems. While carbon and nitrogen SIA of biological samples have become common, analyses of oxygen or hydrogen are used more sparingly despite their promising utility for tracing water sources and animal metabolism. Common ecological applications of oxygen or hydrogen SIA require injecting enriched isotope tracers. As such, methods for processing and analyzing biological samples are tailored for enriched tracer techniques, which require lower precision than other techniques given the large signal-to-noise ratio of the data. However, instrumentation advancements are creating new opportunities to expand the applications of high-throughput oxygen and hydrogen SIA. To support these applications, we update methods to distill and measure water derived from biological samples with consistent precision equal to, or better than, ± 0.1 ‰ for δ¹⁷O, ± 0.3 ‰ for δ¹⁸O, ± 1 ‰ for δ²H, ± 2 ‰ for d-excess, and ± 15 per meg for Δ¹⁷O.

KEYWORDS:

• Cryogenic
• doubly labeled water
• hydrogen-2
• isotope ecology
• metabolic water
• oxygen-17 excess
• oxygen-18
• Picarro
• plasma
• triple oxygen isotopes

Acknowledgements

We thank J. Ritter, C. Penix, W. Jensen, and A. Gaines for their data processing and contributions to the distillation process, and A.R. Gerson for contributing methodological support. We thank S. Newsome and Z. Sharp for insights regarding Δ¹⁷O applications. We also thank S. Clayton for contributing data management support. Samples for this research were contributed via collaborations with: (1) D.P. Costa and R.R. Holser at the University of California, Santa Cruz (northern elephant seal samples were collected under protocols NMFS #19108 & 23188 and UCSC IACUC protocol Costd2009-1); (2) M. Ditmer at the Rocky Mountain Research Station (U.S. Forest Service – U.S. Department of Agriculture; Rocky Mountain mule deer samples were collected as part of routine sampling by the Utah Division of Wildlife Resources [UDWR] and all animal handling and sample collection were done under the direct supervision of a veterinarian within the UDWR). (3) G. Villegas and P. Morrow at the Environmental Division of the U.S. Army Garrison White Sands Missile Range, New Mexico (gemsbok samples were voluntarily collected post-mortem by hunters from culled carcasses, after regulated, routine harvest that is managed by White Sands Missile Range and the New Mexico Department of Game and Fish).

Data deposition

https://github.com/ZacharyTSteele/Automated_Templates

https://github.com/ZacharyTSteele/IEHS_VideoS1

https://github.com/ZacharyTSteele/IEHS_Example_Dataset

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data related to post-processing of isotope analyses in this manuscript is included in the listed Github repository. An example dataset is provided as well in the listed Github repository to demonstrate achievable precision with this method. All other data is available upon request from the corresponding author (ZTS).

Additional information

Funding

This work was supported by the National Science Foundation under grant IOS-1941853 (JPW); T & E, Inc. Conservation Grant (ZTS); a Virginia Space Grant Consortium Graduate Research STEM Fellowship (ZTS); and Old Dominion University.