DOI: 10.5285/6aaa53e8-3d0a-48fe-838e-31c5b5f24ce7
title: ARISE project - Work package 3: Stable nitrogen isotopes of bulk tissue and amino-acids of ringed seals muscle and teeth's growth layer groups of harp seals from the Arctic and sub-Arctic

description: This dataset includes stable nitrogen isotopes of bulk tissue (δ15Nbulk) and compound specific stable nitrogen isotopes on amino acids (δ15NAA) measured in harp seal (Pagophilus groenlandicus) teeth from Southern Barents Sea, Greenland Sea, Northwest Atlantic, and ringed seal (Pusa hispida) muscles from Canadian Arctic Archipelago and Baffin Island, in the Arctic and sub-Arctic. Teeth of harp seals from the Northwest Atlantic (n=48) were taken from archives in Fisheries and Oceans Canada (DFO) St John's, Canada from 1979 to 2016. Teeth of harp seals from the Barents Sea (n=72) and Greenland Sea (n=55) were taken from archives of the Institute of Marine Research (IMR), Norway, from 1963 to 2018 and 1953 to 2014, respectively. Muscle tissue from ringed seals were opportunistically sampled as part of Inuit subsistence harvests. Samples from the CAA were collected in Resolute from 1992 to 2016 (n=66). Muscle samples from the Baffin Bay were collected in Pangirtung from 1990 to 2016 (n=39). The seal samples were collected as part of Norwegian commercial sealing and student field courses from the University of Tromso in Norway (Barents Sea and Greenland Sea) and the Inuit subsistence and commercial harvests in Canada (Northwest Atlantic, Baffin Island, Canadian Archipelago). Analyses of δ15Nbulk and δ15NAA of seal tissue were carried out at the Liverpool Isotopes for Environmental Research laboratory, University of Liverpool. Results are reported here in standard δnotation (per mille) relative to atmospheric N2. This work resulted from the ARISE project (NE/P006035/1 and NE/P006310/1), as part of the Changing Arctic Ocean programme, funded by the UKRI Natural Environment Research Council (NERC).
description: δ15Nbulk, δ13Cbulk and δ15NAA analyses were carried out at the Liverpool Isotopes for Environmental Research laboratory, University of Liverpool. Harp seal teeth preparation The roots of the teeth were sectioned along two planes: transverse and sagittal using a precision low speed diamond saw (Buehler, IsometTM). The transverse sections were used to determine the different growth layer groups (GLG) by the structure, width, and opacity of individual layers (Bowen et al. 1983). Each GLG corresponds to one year of life of the individual. A 700 µm sagittal section was cut as close as possible to the central plane of the tooth and de-mineralised with 0.25M HCl for between 12 and 24 hours. Once softened, any remaining gum tissue and cementum was cut away from the outer edge of the tooth. The pure dentine samples representing the individual GLGs for the second (GLG2) and third (GLG3) years of life were freeze-dried and stored in plastic vials until stable isotope analysis. Stable isotope analyses were conducted on GLG2 and GLG3 combined for each individual. GLGs covered years from 1976-1977 to 2011-2012 in the Northwest Atlantic, 1951-1952 to 2011-2012 in the Barents Sea, and from 1945-1946 to 2007-2008 in the Greenland Sea. The years indicated for the teeth in the data base are the years corresponding to GLG2 and GLG3, and not the sampling years. Sample preparation for bulk δ15N and δ13C of seal tissue ~ 0.5 mg of freeze-dried and homogenized muscle sample, and ~ 0.7 mg of dentine was precisely weighed (± 1 µg) and sealed in a tin capsule. For muscle tissue, samples were run for δ15Nbulk and δ13Cbulk separately. Lipids were removed from the samples using five repeated rinses with 2:1 DCM:methanol to avoid the bias due to the depletion in 13C in lipids relative to the diet, before δ13Cbulk analyses. Sample preparation for δ15N analyses on amino acids of seal muscle tissue ~ 5 mg of freeze-dried and homogenized muscle sample, and ~ 15 mg of dentine (GLG2 and GLG3 combined for each individual), was hydrolyzed in reaction vessels (6M, mL, 200 µl, 100 °C for 22h). L-Norleucine (Sigma-Aldrich) was added to each sample as an internal standard (80 µl of 5 mg mL-1). On cooling, the samples were transferred into a nanosep centrigal device (45 µm nylon filters) and centrifuged (10 000 rpm for 1 min). Samples were then transferred into clean micro-reaction vessels and lipids were extracted by addition of n-hexane:DCM (3:2 v/v, 0.5 mL). Each sample was shaken by hand for 10 seconds in order for the hydrolysate and solvents to mix. The organic solvents were removed. This was repeated 3 times. Hydrolysates were blown down under N2 for 2 min in order to ensure that all organic solvents were removed and were frozen at -80 °C prior to lyophilization. The amino acids were propylated in 0.25 mL of acidified isopropanol solution (prepared by addition of acetyl chloride to anhydrous isopropanol (1:4 v/v) in an ice bath) at 100 °C for 1 h. The reaction was quenched in a freezer and reagents were evaporated under a gentle stream of N2, DCM was added (3 x 0.25 mL) and evaporated to remove excess reagents. Amino acid methyl esters were then treated with 1 mL of a mixture of acetone:triethylamine:acetic anhydride (5:2:1, v/v), which was added to each sample, and heated at 60 °C for 10 min. Following acetylation, the reagents were evaporated under a gentle stream of N2 and were dissolved in 2 mL of ethyl acetate, to which 1 mL of saturated NaCl solution was added. Phase separation was enabled via mixing and the organic phase was collected; separation was repeated 3 times with addition of 2 mL ethyl acetate. Residual water was removed from the combined organic phases by passing through a Pasteur pipette plugged with glass wool and filled with MgSO4.Finally, samples were evaporated under N2 and the derivatized amino acids were dissolved in DCM and stored at -20 °C until analysis.
description: Instrumental analysis for bulk δ15N and δ13C Samples were analysed using an elemental analyser (Costech) coupled to Delta V isotope ratio mass spectrometer (IRMS; Thermo-Scientific). Isotope values are reported in standard δnotation (per mille) to Vienna PeeDee Belemnite and atmospheric N2 for δ13Cbulk and δ15Nbulk, respectively. Instrumental analysis for δ15N of amino acids (δ15NAA) δ15NAA values were determined using a Trace Ultra gas chromatograph (GC) coupled to a Delta V Advantage IRMS with a ConFlo IV interface (Cu/Ni combustion reactor held at 1000 °C, Thermo Fisher). A liquid nitrogen trap was added after the reduction oven to remove CO2 from the sample stream. The separation of amino acids was achieved using an HP Innowax capillary column (30 m x 0.25 mm i.d. x 0.5 µmeter film thickness, Agilent). Each sample was introduced to the column using a split/splitless injector set at 260 °C. The GC was programmed as follows: held at 50 °C for 2 min, 10 °C min-1 to 180 °C and 6 °C min-1 to 260 °C, and held for 16.7 min. The carrier gas was ultra-high purity helium (flow 1.4 mL.min-1). The ion intensities of m/z 28, 29 and 30 were monitored and the δ15N of each amino acid peak were automatically computed (Isodat version 3.0; Themo fisher) by comparison with a standard reference N2 gas, which was repeatedly measured (x4) at the beginning and the end of each sample analysis.
description: Analyses for bulk δ15N and δ13C: To ensure accuracy, international reference standards, USGS40 and USGS41a, were analysed at the beginning, middle and end of each run. An internal standard of ground prawn Penaeus vannamei with well characterized δ13C and δ15N values (-22.6 and 6.8 per mille, respectively) was analysed every 10 samples to monitor precision, which was &lt;0.2 per mille for both δ13C and δ15N. Analyses for δ15N of amino acids (δ15NAA) Each sample was analyzed in duplicate and a triplicate measurement was made if the mean δ15NAA values fell outside the expected measurement error (&lt;1.0 per mille). Precision and accuracy were determined using a mixed amino acid standard prepared from 7 amino acids (alanine, valine, leucine, aspartic acid, glutamic acid, glycine and phenylalanine) with known δ15N values (University of Indiana, USA and SI Science Japan). The mixed standard was analyzed every 4 injections. The mean precisions and accuracies were ± 0.9 per mille and ± 0.7 per mille (1 sigma, n = 29), respectively. Raw δ15NAA sample values were corrected following the methods of McCarthy et al. (2013). This method takes into consideration the response of individual amino acids to the stationary phase of the column and is based on the offset between the measured δ15NAA values in the nearest mixed standard and their known δ15NAA values (Eq. 1). Eq. 1: δ15N-Sample-reported= Avg δ15N-Sample-measured (δ15N-Standard-measured - δ15N-known), where Avg δ15N-Sample-measured is the average δ15N for an amino acid in a sample (n = 2), δ15N-Standard-measured is the δ15N for the AA in the nearest mixed standard and δ15Nknown the known elemental analysed offline value for the same standard.