This study was conducted in the Shambley Creek research watershed (outlet location: 32.98410915, -88.01334337) on privately owned property in Greene County (AL, USA) as part of the Aquatic Intermittency effects on Microbiomes in Streams (AIMS) project, an NSF EPSCoR funded project (OIA 2019603). The project sought to explore the impacts of stream drying on downstream water quality across Kansas, Oklahoma, Alabama, and Idaho, integrating datasets on hydrology, microbiomes, macroinvertebrates, and biogeochemistry. The watershed drains a non-perennial unnamed tributary to Shambley Creek, and contains 0.70 km^2 of coniferous forest managed for silviculture in the East Gulf Coastal Plain physiographic section. Located near Eutaw, AL, the watershed spans an elevation range from 63 to 94 m above sea level, and is a tributary to the Sipsey River (within the larger Mobile-Tombigbee basin). The region has a humid subtropical climate, with mean daily January and July air temperatures of 7.3°C and 27.4°C respectively, and mean annual precipitation of 1,350 mm/yr.

We collected samples every ~3 weeks from October 14, 2021, to March 1, 2023, at the outlet of our watershed (WHM01, approach 1), and seasonally at seven distributed sites (approach 2). We collected samples for water isotopes by triple-rinsing and filling clear borosilicate sample bottles with conical caps underwater until no headspace remained in the sample to reduce potential evaporative fractionation. We then sealed caps of each water isotope sample with parafilm and stored at room temperature until analysis. The first sample from each duplicate pair was designated for analysis, while the second was archived. Oxygen and hydrogen isotope ratios (δ18O and δD) were measured using a cavity ring-down spectroscopic isotope water analyzer (Picarro L2130-i, Picarro Inc., CA). For each sample, six sequential sub-samples were analyzed to account for memory effects. The first three sub-samples equilibrated the measurement cavity, while the final three sub-samples were used to calculate δ18O and δD ratios. To correct for instrument drift and ensure measurement precision, all samples were calibrated against internal secondary standards. The internal secondary standards were previously calibrated against International Atomic Energy Agency (IAEA) primary standards referenced to Vienna Standard Mean Ocean Water. All isotope values are reported as per mil (‰) deviations relative to VSMOW.