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First-order half-lives for 26 trace organic compounds (TrOCs) were determined in the hyporheic zone (HZ) and along a 3 km reach of a first-order stream in South Australia during both dry and wet seasons. Two salt tracer experiments were conducted and evaluated using a transient storage model to characterize seasonal differences in stream residence time and transient storage. Lagrangian and time-integrated surface water sampling were conducted to calculate half-lives in the surface water. Half-lives in the HZ were calculated using porewater samples obtained from a modified mini-point sampler and hyporheic residence times measured via active heat-pulse sensing. Half of the investigated TrOCs (e.g., oxazepam, olmesartan, candesartan) were not significantly removed along both the investigated river stretch and the sampled hyporheic flow paths. The remaining TrOCs (e.g., metformin, guanylurea, valsartan) were found to be significantly removed in the HZ and along the river stretch with relative removals in the HZ correlating to reach-scale relative removals. Using the modeled transport parameters, it was estimated that wet season reach-scale removal of TrOCs was predominately caused by removal in the HZ when the intensity of hyporheic exchange was also higher. Factors that increase HZ exchange are thus likely to promote in-stream reactivity of TrOCs.