Technical Abstract: Reduced plant growth in wood-based substrates, compared with traditional substrates, has been overcome with increased fertilizer rates and by storing the material for a period of time. A variety of factors have been attributed to reduced plant growth in whole pine tree substrates including nitrogen immobilization, particle size distribution, and reduced cation exchange capacity. Phytotoxicity, associated with certain organic compounds found in pine trees, could be another contributing factor. In this study, a Phytotoxkit™ was used to evaluate seed germination and initial root growth of cress, mustard, and sorghum in aged and fresh whole pine tree substrates, aged and fresh pine needles, a reference soil, and a saline pine bark. A traditional seedling growth test was used to evaluate seedling root growth of lettuce and tomato in aged and fresh whole pine tree substrates, a peat-lite substrate, and a pine bark substrate. In the Phytotoxkit™ experiment, cress and sorghum germination percentage was lowest in fresh pine needles, while mustard had 100% germination in all substrates except saline pine bark (43%). The greatest root length for cress and sorghum occurred in aged whole pine tree, yet root length was greatest in fresh whole pine tree for mustard. In the seedling growth experiment, total root length was greatest in peat-lite and least in aged whole pine tree for tomato and lettuce. The Phytotoxkit™ provided the most sensitive test for phytotoxicity, although the seedling growth test could be used for a more practical assessment of potential phytotoxicity for alternative substrates under typical production practices. Future research will include a more complete chemical analysis of substrate treatments to identify potential phytotoxic compounds, and methods for overcoming such issues will be evaluated.