Delta 5 plus irms

Each dry sample (both modern macrophytes, Daphnia, fish and subfossil Daphnia), weighing 100–200 µg, was packed into a small tin cup for isotope analysis. The subfossil Daphnia ephippia samples were oxidised and the resultant CO₂ and N₂ analysed with a Finnigan Delta Plus mass spectrometer interfaced via a Conflo II to a NC2500 Elemental Analyzer. The δ¹³C, δ¹⁵N isotopic values, were determined using a Thermo Finnigan Flash EA 112 interfaced via a Thermo Conflo III with a Thermo Delta V Plus IRMS^{47 (link),48 (link)}. Samples of acetanilide of known isotope composition were analyzed within each run to verify isotope values. Reproducibility for both δ¹³C and δ¹⁵N was 0.2‰ while that for both %C and %N was 1%^{47 (link),48 (link)}. Ratios of ¹³C/¹²C and ¹⁵N/¹⁴N were expressed as the relative per mil (‰) difference between the sample and conventional standards (PDB carbonate and air N₂, respectively). These ratios were expressed as: δX = [R (sample)/R (standard) − 1] × 1,000 (‰), where X = ¹³C or ^l5N and R = ¹³C/^l2C or ^l5N/¹⁴N^{32 (link)}.
The modern Daphnia isotopic values of nitrogen were derived from the samples collected in 2014, while the subfossil Daphnia isotopic values of nitrogen were from the top sections (0–10 cm) of the core that dated back to c. 2006–2011.

We formulated an amino acid working standard to facilitate conversion of raw measurements to intercomparable isotope values. Pure (>99%) amino acid powders, corresponding to each amino acid measured, were acquired from Sigma-Aldrich and characterized via continuous flow isotope ratio mass spectrometry (IRMS) at UC Riverside to determine their carbon and hydrogen isotope compositions. δ²H, relative to VSMOW, of amino acid powders was measured via the comparative equilibration method (Wassenaar and Hobson, 2003 (link)) and a high temperature conversion elemental analyzer (TCEA, Thermo Scientific) interfaced to a Thermo Scientific Delta V Plus IRMS. δ¹³C values, relative to VPDB, of amino acid powders were measured with a Costech 4010 Elemental Analyzer (Valencia, CA) interfaced to a Delta V Plus IRMS. The carbon and hydrogen isotope compositions of amino acid powders varied from −42.6 to −10.0‰, and −236.9 to 99.4‰ for δ¹³C and δ²H, respectively. 0.25 M stocks of individual amino acids were brought up in 30 mM Pierce™ HCl [formulated free of nitrogenous compounds, e.g., amines]. Amino acid stocks were mixed prior to the initial drying step of our derivatization procedure so each working standard compound was able to be derivatized simultaneously, and analyzed over the course of a single GC injection.

Concentrations and carbon isotope composition of acetate and ethanol were analyzed in the slurries’ supernatants of incubations amended with cellulose. Prior to analysis, 50 µl phosphoric acid were added to 200 µl of each supernatant retrieved from the incubations and incubated overnight to remove the high amount of ¹³C dissolved inorganic carbon. The δ¹³C values of acetate and ethanol were determined by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) according to approaches described previously by Heuer et al. [48 (link)]. The δ¹³C values of TOC were measured on a Flash 2000 elemental analyzer coupled with DELTA V Plus IRMS via a ConFlow II interface (EA-IRMS, Thermo Scientific, Bremen, Germany) [49 ]. Prior to analysis, dried sediment from 0.5-ml slurry was acidified using 1 ml HCl (37%) overnight to remove inorganic carbon and followed by evaporation for several days until HCl acid was fully evaporated.