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The benthic biolayer is a shallow zone of reactive streambed sediments, widely believed to contribute disproportionately to whole‐stream reactions such as aerobic respiration and contaminant transformation. Quantifying the relative contribution of the biolayer to whole‐stream reactions remains challenging because it requires that hyporheic zone solute transport and reaction heterogeneity are explicitly captured within a single modeling framework. Here, we use field experiments and modeling to quantify the biolayer's aerobic reactivity relative to other stream compartments. We co‐injected and monitored several fluorescent tracers, including the reactive tracer resazurin, into a controlled experimental stream. We characterized reactive transport in the water column and at multiple depths in the hyporheic zone by fitting all data to a new mobile‐immobile model, using resazurin‐to‐resorufin conversion as an indicator of aerobic bioreactivity. Results show that the biolayer converted 8 times more resazurin to resorufin than all other stream compartments, and 80% of this conversion occurred within 2 reach advection times. This hotspot and hot moment behavior is attributed to the biolayer's ability to rapidly acquire, transiently retain, and rapidly degrade stream‐borne solutes. The model analysis shows that the majority of raz‐to‐rru conversion occurs in the biolayer across streams with a wide range of biolayer structural properties, including streams with a biolayer that is less reactive than deeper regions of the hyporheic zone. Together, our results show that the biolayer is a common feature of streams and rivers that should be considered in network‐scale models of aerobic reactivity. 

Access to and extensive use of fluorometric analyses is limited, despite its extensive utility in environmental transport and fate. Wide-spread application of fluorescent tracers has been limited by the prohibitive costs of research-grade equipment and logistical constraints of sampling, due to the need for high spatial resolutions and access to remote locations over long timescales. Recently, low-cost alternatives to research-grade equipment have been found to produce comparable data at a small fraction of the price for commercial equipment. Here, we prototyped and benchmarked performance of a variety of fluorometer components against commercial units, including performance as a function of tracer concentration, turbidity, and temperature, all of which are known to impact fluorometer performance. While component performance was found to be comparable to the commercial units tested, the best configuration tested obtained a functional resolution of 0.1 ppb, a working concentration range of 0.1 to >300 ppb, and a cost of USD 59.13.