Step 1 – From 2018, samples were gathered to assess algal biomass using specially designed floating devices known as "phytometers" deployed in the weir ponds in each monitored stream (HBWatER 2024, Thellman et al. 2024). The weir ponds are located at the base of each experimental watershed, where v-notch weirs are used to measure stream discharge with high precision. Phytometers were comprised of a 2 m2 Nitex mesh screen attached to a PVC frame, which was buoyed by flexible, expanded polyethylene foam. Mounted on each phytometer were strips of 1 cm2 scouring pads (Americo Manufacturing, #40031218). The scouring pads mimicked the physical structure of bryophytes due to their woven plastic fibers which increase interstitial space, offering habitats that accumulate stream particles and experience lower velocities (Thellman et al. 2024).
Step 2 – We processed the diatom samples following procedures described by Patrick et al. (1966) to remove extraneous material and create permanent slides for diatom identification. Each sample was sonicated on the lowest setting for 30 seconds to dislodge any diatoms from the scouring pad. The sonication time was determined based on preliminary trials, which ensured effective removal of diatoms while minimizing the risk of damage. Samples were boiled in 25 mL of nitric acid to oxidize organic matter for approximately one hour. After, the samples were thoroughly rinsed with 50mL of distilled water and allowed to settle until all residuals were eliminated and the rinse water achieved a neutral pH. Between rinses, the sample volume was reduced from 50 mL to about 5 mL through aspiration. Subsequently, 0.2 mL aliquots of the clean diatoms were dried onto coverslips and secured with Naphrax® mounting medium.
Step 3 – Diatoms were identified and species richness determined using brightfield microscopy with oil immersion at 1000x magnification (Leica DM2500 LED) . We did not estimate species abundance for these samples because, after preparing three additional slides from the same sample to check, over 60% of the prepared slides did not contain a sufficient number of diatoms for accurate abundance counts (>300 valves per slide) (Patrick et al. 1966). We instead estimated species richness, which refers to the number of different species within a given area, providing insight into the overall diversity of an ecosystem. Live/dead diatom counts were not conducted as part of this analysis, and only cleaned diatom valves were used for identification. Diatom identification was conducted by a team, including trained students, under close supervision. All diatom species present on each slide were counted, ensuring a comprehensive assessment of species richness.
References:
Hubbard Brook Watershed Ecosystem Record (HBWatER). 2024. The Hubbard Brook Stream Ecology Record: Light, 2018 - ongoing ver 1. Environmental Data Initiative. https://doi.org/10.6073/pasta/0f40b75b299494d736645d940fa2b5a4 (Accessed 2024-10-25).