Comparison of methods for stable isotope ratio (δ¹³C, δ¹⁵N, δ²H, δ¹⁸O) measurements of feathers

Abstract

Summary1. Natural variations in the stable isotope ratios of bioelements in bird feathers are being increasingly used by animal ecologists to investigate different aspects of bird life. However, to ensure reliability of the data, a critical and very delicate aspect is the preparatory phase (cleaning, drying and subsampling) and the proper analysis, mainly in relation to d 2 H and d 18O, respectively, for the presence of exchangeable Hs and of nitrogen and sulphur in keratin. 2. With respect to determination of the isotope ratios of C, N, O and H, in this work, we compare the cleaning mixture most commonly used in the literature (chloroform : methanol 2 : 1) with diethylether : methanol 2 : 1, which avoids the use of the carcinogenic solvent chloroform. We also compared oven-drying with airdrying of samples, as well as subsampling of feathers by cutting with surgical scissors or cryogenic pulverization. Finally, we investigated whether stable isotope ratios varied along the vane and between the rachis and vane. 3. The different methods compared in the three preparatory stages showed no differences performance-wise and can therefore be used interchangeably. Variability in stable isotope ratios can be considerable, both along the vane and between rachis and vane, which is because their compositions register changes in diet, area and climate. However, in this specific study, when the parts of the feather closest to the calamus were removed, the delta values were clearly more homogeneous. Finally, we demonstrate that a casein with a known d O, the use of a longer gas chromatography-GC column, its frequent change and the use of a linear equation built with matrix match equivalent reference materials seems to reduce the drift of GC column performance due to the presence of nitrogen and the accumulation of sulphur.

Stable isotope (C, N, O, and H) study of a comprehensive set of feathers from twoSetophaga citrina

“…Feather samples were collected and labeled from each bird (Fig 2): primaries P1-P9, secondaries S1-S9, right (RR) and left (LR) rectrices 1-6 (as seen from dorsal view), the primary, median, and greater coverts (PC, MC, and GC) and aggregate body feathers from the back, rump, throat, nape, breast, and belly. The feathers were cleaned in a solution of 2:1 diethyl ether:methanol and suspended in an ultrasonic bath for 2 three-minute cycles to ensure the removal of contaminants and organic detritus on the feathers' surface, following the technique of Bontempo et al 2 . The samples were then allowed to air dry for 48 hours in glass tubes before isotopic analysis.…”

Natal origins and timing of migration of two passerine species through the southern Alps: inferences from multiple stable isotopes (δ2H,δ13C,δ15N,δ34S) and ringing data

“…To obtain enough keratin to measure the four isotopes, we combined rectrix and wing feather vane material for each individual, as these feathers were grown in the nest contemporaneously. Feathers were washed in a solvent mixture (diethyl ether/methanol 2 : 1) prior to analyses (Bontempo et al 2014). For d 2 H f , about 0.2 mg for each sample was placed in 4.0 9 3.2 mm silver capsules and slightly crushed.…”

“…Isotope ratios were expressed in d notation against V-PDB (Vienna -Pee Dee Belemnite) for d 13 C, air for d 15 N, V-SMOW (Vienna -Standard Mean Ocean Water) for d 2 H, and V-CDT (Vienna -Canyon Diablo Troilite) for d 34 S. Accurate determination of d 2 H, d 13 C and d 15 N requires twopoint normalization, using at least two isotopic reference materials with different isotopic compositions to anchor the isotopic scale and to compensate for differences in instrumental responses, which commonly compress isotope-d scales (Schimmelmann et al 2016). The isotopic values of d 2 H were calculated as specified in Bontempo et al (2014). In particular, a regression line was built using the two reference materials Caribou Hoof Standard (CBS, USGS -U.S. Geological Survey, Reston Stable Isotope Laboratory, Reston, VA, USA) and Kudu Horn Standard (KHS, USGS).…”

Different prey resources suggest little competition between non-native frogs and insectivorous birds despite isotopic niche overlap

“…All samples, except bird feathers, were thoroughly rinsed with water to eliminate any contaminants before drying. We rinsed feathers with acetone to remove oils and then rinsed them thoroughly with water to remove the acetone before drying (Bontempo et al 2014). Once rinsed, we placed samples in a drying oven at 60°C for 48 h. We ground each sample into a very fine, evenly-sized powder using a mortar and pestle, but in the case of the feather samples, we cut feathers into very small (\1 mm in width and length) pieces with scissors (Bontempo et al 2014 …”