The ecotoxicological effects of hydrophobic organic compound (HOC) contamination in sediment are often assessed using laboratory exposures of cultured invertebrates to field-collected sediment. The use of a sediment holding time (storage at 4°C) between field sampling and the beginning of the bioassay is common practice, yet the effect of holding time on the reliability of bioassay results is largely unknown, especially for current-use HOCs, such as pyrethroid insecticides. Single-point Tenax extraction can be used to estimate HOC concentrations in the rapidly desorbing phase of the organic carbon fraction of sediment (i.e., bioaccessible concentrations), which relate to sediment toxicity and bioaccumulation in invertebrates. In this study, repeated measurements of bioaccessible concentrations (via Tenax), were made as a function of sediment holding time using pyrethroid-contaminated field sediment, and Hyalella azteca 10-d survival and growth was measured concurrently for comparison. Similarly, bioaccessible concentrations and 14-d bioaccumulation were measured in Lumbriculus variegatus as a comparison using the legacy HOCs, polychlorinated biphenyls (PCBs). While the bioaccessible and bioaccumulated PCB concentrations did not change significantly through 244d of holding time, the bioaccessible pyrethroid concentrations were more varied. Depending on when pyrethroid-contaminated sediments were sampled, the bioaccessible pyrethroid concentrations showed first-order loss with half-lives ranging from 3 to 45 d of holding, or slower, linear decreases in concentrations up to 14% decrease over 180 d. These findings suggest that at least for some contaminants in sediments, holding the sediments prior to bioassays can bias toxicity estimates