TNT (2,4,6-trinitrotoluene) is a persistent contaminant at many military installations and poses a threat to aquatic ecosystems. Data from environmental fate and toxicity studies with TNT revealed that sediment toxicity test procedures required modification to accurately assess sediment TNT toxicity. Key modifications included aging TNT-spiked sediments 8-14 d, basing lethal dose on measured sediment concentrations of the molar sum of TNT and its main nitroaromatic (NA) transformation products (SNA), basing sublethal dose on average sediment SNA concentrations obtained from integration of sediment SNA transformation models, avoiding overlying water exchanges, and minimizing toxicity test durations. Solid phase microextraction fibers (SPMEs) were investigated as a biomimetic chemical measure of toxicity and bioavailability. Both organism and SPME concentrations provided measures of lethal dose independent of exposure scenario (TNT-spiked sediment or TNT-spiked water) for Tubifex tubifex. Among all benthic organisms tested (Chironomus tentans, Ceriodaphnia dubia, T. tubifex) and matrixes, median lethal dose (LC50) estimates based on SPME and organism concentrations ranged from 12.6 to 55.3 mmol SNA/ml polyacrylate and 83.4 to 172.3 nmol SNA/g tissue, ww, respectively. For Tubifex, LC50s (95% CI) based on SNA concentrations in sediment and SPMEs were 223 (209-238) nmol SNA/g, dw and 27.8 (26.0-29.8) mmol SNA/ml, respectively. Reproductive effects occurred at slightly lower exposures. Median effective dose (EC50) estimates (95% CI) for Tubifex cocoon production, based on sediment and SPME concentrations, were 118 (114-122) nmol SNA/g, dw and 21.8 (21.2-22.4) mmol SNA/ml, respectively. Bioconcentration experiments with Tubifex revealed that compound hydrophobicity predicted the toxicokinetics and bioconcentration of these compounds from water, however, there was a large discrepancy between the toxicokinetics of absorbed versus metabolically-generated aminodinitrotoluenes. A large portion of bioconcentrated, radiolabeled TNT transformation products could not be identified. In addition to their ability to provide matrix-independent measures of dose, SPME concentrations were more accurate indicators of bioavailable NAs than were sediment concentrations.