European Union Water Framework Directive (WFD) (2000/60/EC) waterbody statuses are often derived from the assemblage of mixed-taxon organisms found within bed sediments. Yet no routine water chemistry samples are taken from riverbed substrate, despite many interstitial species being dependant on specific physicochemical conditions. This paper examines water and nutrient exchanges between stream and substrate, the hyporheic zone, and the consequent alteration to the chemistry of interstitial and in-stream waters, which in turn leads to small-scale but significant changes in habitat. Bed topography, principally a pool-riffle sequence, was surveyed and hydraulically sampled to examine the hyporheic flow pathways generated between the stream, substrate, and groundwater—creating an ecotone. Hyporheic zone, in-stream and groundwater hydrochemistry, and hydraulic measures were assessed at a braided woodland river reach using a dense monitoring approach. The findings demonstrate that through a 23-m pool-riffle sequence, where water infiltrates at the riffle-head and subsequently exfiltrates at the riffle-tail, there is a 5% reduction in mean in-stream nitrate-N and a 73% reduction in hyporheic zone concentration. When calcium-enriched riffle-tail exfiltrate meets riffle-runoff water that is turbulently oxygenated from riffle-flow, an interstitial redoxcline is created. Dissolved oxygen nocturnally drops, with photosynthetic rate reduction, which causes hyporheic nitrate-N to double its daytime concentration. The results are related to the Yorkshire River Esk freshwater pearl mussels, Margaritifera margaritifera Linnaeus (1758), a declining endangered species. At present, scant monitoring of interstitial hydrochemistry and diurnal change across stream and substrate occurs.