Isotopic sampling and analysis

Gas samples were collected for CH4 isotopic analysis in the first week of August 2019. Additional samples from Cart Creek and Sawmill Brook from the first week of September 2018, collected in the same patches as 2019, were also included in analysis. An initial patch near a long-term monitoring location for water quality was installed at each stream, and subsequent patches were distributed approximately every 10 to 15 m upstream or downstream. Four patches were located at Cart Creek, Dube Brook, and Sawmill Brook, and three patches were chosen at College Brook due to limited access to this stream. Patch selection avoided rocky substrate, which makes installation of our trap design unfeasible. This limitation accounted for less than 10% of each stream reach and the larger stream network, a feature of these relatively low-gradient systems.  Traps were placed in established channels rather than in intermittently inundated portions of the sediments. No other preference for patch selection was made, and we assume the distribution of patches is representative of the stream reach apart from rocky substrates. Substrates ranged from fine silt and organic matter to sand, depending on stream and patch. Patches included runs and pools, but avoided riffles as these were typically rocky.

Dissolved CH­4 samples from the surface water were collected at each stream using 60-mL syringes fitted with three-way stopcocks. Syringes were rinsed with stream water prior to sample collection. To collect water samples, syringes were filled with approximately 60 mL of stream water from 5–10 cm depth below the stream water surface. Syringes were cleared of air bubbles by inverting and expelling bubbles and water until 30 mL of sample water remained. Samples were stored on ice until returned to the laboratory within 6 h. In the laboratory, 30 mL of ambient air was added to each syringe to achieve a 1:1 ratio of sample water to air. Syringes were then shaken for 2 min to equilibrate gases between water and headspace (Magen et al., 2014). The water was then dispelled from the syringe, and the remaining headspace gas was saved for analysis. If the gas samples were not analyzed immediately, they were stored in evacuated glass vials sealed with a rubber septum until analyzed. At Cart Creek, ten sediment gas samples and eight dissolved samples from the surface water were collected; 14 sediment and 12 surface water samples were collected at Sawmill Brook; six sediment and four surface water samples were collected at Dube Brook; and three sediment and three surface water samples were collected at College Brook. All samples were stored in evacuated glass vials sealed with a rubber septum for analysis.

Samples were analyzed for δ13C-CH4 and δD-CH4 using an Aerodyne dual tunable infrared laser direct absorption spectrometer (TILDAS; Aerodyne Research Inc., Massachusetts, USA) at the University of New Hampshire. These instruments use high resolution infrared spectrometry to quantify trace gases such as CH4 (Mcmanus et al., 2011; Nelson and Roscioli, 2015). The TILDAS used here is configured with two 8 µm quantum cascade lasers (QCLs, Alpes Lasers, Switzerland) and a 200 m multipass absorption cell to simultaneously monitor 12CH4, 13CH4, and CH3D. The instrument was regularly calibrated with three standard tanks with known isotopic mixing ratios for 13CH4/12CH4 and CH4/CH3D. The isotopic composition of the standards was determined using an Aerodyne calibration system. The spectroscopic isotope ratios of four Isometric (now Airgas) CH4 standards were measured at diluted concentrations ranging from < 1 to 12 ppmv. Keeling plot analysis was used to determine the relationship between the spectroscopic and standard isotope ratios of the Isometric standards. The linear relationship was made to the corresponding measured spectroscopic isotopic ratios of the UNH calibration tanks. The TILDAS is configured with an automated sampling system designed to measure small (≤ 5 mL) injections of high concentration samples by diluting them within the instrument to a target CH4 mixing ratio of 8 ppmv with ultra-zero air. The instrument precision was 0.1‰ for δ13C-CH4 and 3‰ for δD-CH3D at the target CH4 mixing ratio.