In nitrogen-limited ecosystems, biological nitrogen (N) fixation can provide an important subsidy to ecosystem N budgets. In streams, however, N-fixation is most commonly measured at the scale of individual substrates or patches and has rarely been scaled to reaches or river segments. Here, we combined substrate-specific N-fixation rates with high-resolution spatial data to generate reach-scale estimates of N-fixation in the South Fork of the Eel River (SFER), California. We used a 15N₂ tracer approach to quantify mass-specific N-fixation rates and paired these measurements with areal standing stock estimates and percent cover derived from drone imagery. Five integrated reach-scale (~100 m) assessments were conducted across early, mid, and late summer during 2024–2025. A random, stratified sampling design was used to quantify N-fixation in (1) floating Cladophora glomerata mats and associated epiphytes, (2) benthic Cladophora turfs, (3) epilithic algae, and (4) moss.
Across both years, Cladophora and its epiphytes represented the largest biomass pool, whereas epilithic algae had the greatest spatial coverage. The diatom Epithemia spp., which hosts an N-fixing endosymbiont, dominated both epilithic and Cladophora epiphytic assemblages by mid- to late summer, comprising >80% of diatom abundance in Cladophora epiphytes and ~60% in epilithon. Preliminary 2024 estimates indicate high reach-scale N-fixation rates (~20 mg N m⁻² d⁻¹), with Cladophora epiphytes and epilithic assemblages contributing 45% and 54% of total activity, respectively. To place these rates in context, we estimated an annual dissolved N flux of 102.3 kg N yr⁻¹ into the study reach using discharge and repeated measurements of DIN and DON. Assuming N-fixation persists for 45 days annually, approximately 15 km of stream length would fix an amount of N equivalent to the annual upstream dissolved N input. Despite this, downstream increases in dissolved N were not observed, suggesting rapid biological uptake. If efficiently transferred through the food web, observed N-fixation rates could support secondary production of ~5.6–8.4 g dry mass m⁻² yr⁻¹. Combined, these measurements illustrate the ecosystem-scale importance of Epithemia spp. driven N-fixation in the SFER and the potential ecological consequences of a possible shift to N-fixing cyanobacteria if conditions change.