Concentrations of copper, lead and zinc in sediments downstream of a metallurgical facility on the the Upper Columbia River (UCR) were significantly greater than those in regional reference locations. Sturgeons inhabit benthic habitats and there is concern that they might be at greater risk of exposure. A laboratory based, flow-through, experimental exposure system was used to characterize toxicity of metals in the different matrices associated with sediments collected from the UCR, and to assess risk to early life stages of white sturgeon (Acipenser transmontanus) under chronic bioassay conditions. Bioavailability and concentrations of metals in pore water, overlying water, and at the sediment-water interface were characterized. Several sampling methods including peepers, diffusive gradients in thin films (DGTs), and active sampling/suction techniques were employed. Results indicated that concentrations of metals in site sediments were significantly greater in comparison to upstream reference sediments. Of the four primary metals of concern, concentrations of copper, primarily in pore water, were significantly greater in exposure chambers containing site sediments compared to reference sediments. In addition, the different sampling techniques resulted in varying estimates of concentration depending on matrix, analyte, and method, and the present study highlights challenges in assessing the true risk of exposure. The analytical data reported herein is utilized in a parallel article to characterize risk and compare predictions to the bioassay results.