Validating methods for measuring δ¹⁸O and δ¹³C in otoliths from freshwater fish

Abstract

The ability of the phosphoric acid digestion technique to extract carbon dioxide from biogenic carbonates and reliably reproduce delta(18)O and delta(13)C signatures from standard reference materials (NBS-18, NBS-19) was tested and shown to produce accurate, unbiased measurements of non-biologic materials. The effects of roasting preparation methods commonly reported when analyzing biogenic carbonates were also tested in a series of experiments using reference standards and otoliths obtained from aquacultured Arctic charr and rainbow trout. Roasting had no effect on the isotope measurement of reference standards. No significant differences between mean oxygen isotope signatures from paired experiments with roasted and non-roasted fish otoliths were found. However, otolith oxygen isotope measurements were significantly enriched in comparison to rearing water-based measurements for both species. Agreement between expected isotopic equilibrium and measured otolith delta(18)O values varied as a function of roasting temperature and between species. Criteria for the selection of appropriate roasting temperatures are suggested and favour 350 degrees C in freshwater fish where unbiased estimates of average rearing water temperatures and known differences in rearing temperatures were obtained. Carbon isotopic disequilibria were observed for both species. A mixing model analysis established differences in the percentage of metabolically derived carbon in studied otoliths, with Arctic charr deriving a greater proportion of otolith delta(13)C from metabolism as a result of higher metabolic rates.

Development of a species-specific fractionation equation for Arctic charr (Salvelinus alpinus (L.)): an experimental approach

“…They also noted that inappropriate use of fractionation equations not tuned to the physiology of a species would result in substantive inferential error about occupied thermal habitats. In the earlier study, Guiguer et al (2003) also noted the influence of differences in metabolic rate on the incorporation of CO 2 during otolith formation.…”

“…Jobling & Wandsvik, 1983;Jobling, 1985), and to avoid possible metabolic effects associated with differential growth rates (e.g. Guiguer et al, 2003;Høie et al, 2004), 10 fish from each chamber were selected for analysis from the middle of the size range of individuals raised in each chamber. In order to obtain sufficient sample material for analysis (0.05 mg), single or paired otoliths from individual fish were used.…”

“…In order to obtain sufficient sample material for analysis (0.05 mg), single or paired otoliths from individual fish were used. Prior to isotope analysis, otoliths were pre-treated following procedures described in Guiguer et al (2003) and Storm-Suke et al (2007), to remove organic impurities that might bias the isotopic signatures obtained. Otoliths were then reacted with 100% phosphoric acid under a constant flow of purified helium to obtain CO 2 from which oxygen isotope ratios could be measured.…”

“…Otoliths were then reacted with 100% phosphoric acid under a constant flow of purified helium to obtain CO 2 from which oxygen isotope ratios could be measured. Vials containing the reacted samples were heated to 40°C for at least 3 h, after which the evolved CO 2 gas was auto-injected into a Micromass IsoPrime (IRMS) stable isotope mass spectrometer with an analytical precision of *0.2% (Guiguer et al, 2003). The precision of the mass spectrometer was measured with a concurrent analysis of available international standards: NBS-18 (Carbonitite CaCO 3 : -15.68) and EIL-23 (Fisher CaCO 3 : d 13 C PDB = -3.42, d 13 O PDB = -13.45) with sets of n = 6 laboratory working standards being analysed before and after sample batches of 8-11 otoliths.…”

Spatial and temporal trends in stable carbon and oxygen isotope ratios of juvenile winter flounder otoliths

“…One sagittal otolith from each fish was weighed and analyzed for δ 13 C and δ 18 O using Continuous Flow-Isotope Ratio Mass Spectrometry (CF-IRMS) methodology similar to that of (Guiguer et al 2003). Whole otolith samples (0.4-0.8 mg) were transferred to 11-ml Labco Exetainer septum vials (Labco Limited) which were placed into a GasBench II sample tray (ThermoFisher Scientific).…”