Conflo 3

Samples were milled and exposed to HCl-vapor for 4 h at room temperature in an extraction chamber to remove inorganic C and then milled again. Concentration measurements of nitrogen and carbon were performed simultaneously with a Thermo/Finnigan MAT V isotope ratio mass spectrometer, coupled to a Thermo Flash EA 1112 elemental analyzer via a Thermo/Finnigan Conflo III interface.

Carbon and nitrogen concentrations (total, organic, and inorganic) and isotopic signatures (δ¹³C and δ¹⁵N) were measured by grinding dry sediment in a mortar and pestle to create a fine powder. To determine organic content, sediment was subsampled into unacidified sediment and acidified sediment. Acidified sediment was treated with 1 mL of 1 N HCl per 0.3 g sediment. 20–25 mg of dry sediment in tin capsules was measured using a Costech ECS4010 Elemental Analyzer paired to a Thermo-Finnigan Delta + XL mass spectrometer via a Thermo-Finnigan Conflo III. The setup used helium gas and a high temperature, >1,000°C, to analyze the sediment. Organic concentrations of carbon and nitrogen were calculated by the difference between inorganic and total carbon and nitrogen concentrations, respectively.

0.6-0.8 mg samples for leaves and 1 mg samples for stems and roots was weighed using a high-precision analytical balance (XSE105 DualRange, Metler Toledo, Greifensee, Switzerland) into into 4x6 mm silver capsules (Costech Analystycal Technologies, Inc., Valencia, CA, USA). Prepared capsules were shipped for analysis to the Stable Isotope Core Laboratory at Washington State University. The samples were analyzed with continuous-flow pyrolysis using TC/EA interfaced with an IRMS (Delta Plus, ThermoFinnigan, Bremen, Germany) through a continuous flow device (Conflo-III, ThermoFinnigan,Bremen, Germany). Oxygen isotope ratios of each sample were then determined, and the values reported in “delta” notation as δ values in parts per thousand (‰):
Equation 2
δ18O was calculated as the ratio of the heavy isotope (18O) over the light oxygen isotope (16O) in the samples divided by the heavy isotope (18O) over the light oxygen isotope (16O) of the standard. The δ18O was compared with the standard Vienna-Standard Mean Ocean Water (VSMOW), the international standard commonly used for oxygen isotope analysis.

The unpolar lipid fraction was separated by silica column chromatography (SiO₂). Neutral lipids were eluted with two column volumes DCM, followed by glycolipids with two column volumes acetone and phospholipids with four column volumes MeOH. Only the phospholipid containing fraction was further analyzed in this study. Samples (0.01 mg–2.45 mg) were transferred to tin capsules, dried (40 °C, 1 h) and submitted for elemental analysis and δ¹³C/δ¹²C measurements. Elemental analysis and carbon isotope ratio analysis was performed using an elemental analysis isotope ratio mass spectrometer (EA-IRMS) fitted with an elemental analysator A (NA 1110, CE Instruments, Mailand) coupled with a ConFlo III and a Delta XL-IRMS (Thermo-Finnigan, Bremen). Acetylanilid (Ali-j3) and caffeine (caf-j3) were used as analytical standards (Table S15).

The dried collagen was placed in tin foil capsules and analyzed for carbon and nitrogen isotope ratios at the Alaska Stable Isotope Facility (ASIF) in the University of Alaska Fairbanks by using continuous-flow isotope ratio mass spectrometers. A Costech ECS4010 Elemental Analyzer (Costech Scientific Inc, Valencia, CA) combusted samples to carbon dioxide and nitrogen gas, which were carried in a constant flow of helium to a Finnigan Delta Plus XP isotope ratio mass spectrometer via the Conflo III interface (Thermo-Finnigan Inc, Bremen, Germany). Data are presented in the accepted delta notation as δX = (R sample − R standard)/(R standard) × 1000‰, where R is the ratio of heavy to light isotope (for both nitrogen and carbon) and the internationally recognized standards are atmospheric nitrogen and Vienna Pee Dee Belemnite for carbon. Multiple peptone standards (δ¹³C = − 15.8‰, δ¹⁵N = 7.0‰) were concurrently run to assess analytical accuracy and precision. The carbon and nitrogen isotope values were calculated relative to the Vienna Pee Dee Belemnite (VPDB) for δ¹³C and atmospheric N₂ (AIR) for δ¹⁵N, respectively. Replicate measurement errors on known standards were less than ± 0.2‰ for both δ¹³C and δ¹⁵N.

The copepods collected at the start and the termination of the feeding experiment were transferred to tin capsules, dried at 60°C for 48 h, weighed (∼0.2–0.3 mg dry mass sample^–1), and stored in a desiccator until shipped to the analytical facilities. The samples were analyzed at the Center for Physical Science and Technology, Vilnius, Lithuania. The relative abundance of stable carbon and nitrogen isotopes were measured using a Flash EA 1112 Series Elemental Analyzer connected via a Conflo III to a DeltaV Advantage isotope ratio mass spectrometer (all Thermo Finnigan, Bremen, Germany). Ratios of ¹⁴N:¹⁵N and ¹²C:¹³C are expressed relative to the international standards, atmospheric air (N) and Pee Dee Belemnite (C). Internal reference (pike muscle tissue) was analyzed every 10 samples. Overall analytical precision was 0.15 ‰ for δ¹⁵N and 0.10 ‰ for δ¹³C.

Xylem water in root crowns and soil water were extracted for isotopic analyses using a cryogenic water extraction line [30 (link)] and measured with a TC/EA high-temperature conversion/elemental analyzer coupled with a DeltaplusXP isotope ratio mass spectrometer via a ConFlo III interface (Thermo-Finnigan, Bremen, Germany; see Werner et al. [36 (link)] for further information). Oxygen and hydrogen isotopic compositions of the water samples are given in δ notation measured as (R_Sample/R_Standard)—1, and expressed in ‰. R is the ratio of ¹⁸O to ¹⁶O or ²H to ¹H of the sample or the standard. Our standard was a working control standard, regularly calibrated against international standards (V-SMOW, SLAP, GISP). The overall precision of the measurements was ± 0.09 ‰ for δ¹⁸O and ± 0.37 ‰ for δ²H.