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Abstract Canada has experienced a significant increase in the transport of diluted bitumen (dilbit), a predominant oil sands product that combines bitumen with diluents derived from oil-gas condensates and other proprietary compounds. The toxicity of dilbit to fish embryos, which are immobile and thus at a high risk of exposure to oil in the event of a spill, remains largely unknown for most species. This study assessed the toxicity of water accommodated fractions (WAF) and chemically enhanced water accommodated fractions (CEWAF) of two winter dilbit blends, Access Western Blend (AWB) and Cold Lake Blend (CLB), to fathead minnow (Pimephales promelas) embryos. The TPH-F EC50s for malformations were 834 and 1058 μg/L for AWB WAF and CEWAF, respectively, and 500 and 715 μg/L for CLB WAF and CEWAF, respectively. Levels of cyp1a mRNA increased up to 46- and 69-fold, respectively, reflecting increasing exposure to polycyclic aromatic compounds (PACs) in AWB and CLB. Similarly, levels of gst mRNA were elevated up to 3.8-fold and 2.7-fold with increasing total concentrations of PACs in AWB and CLB, respectively. However, there were no significant changes in mRNA levels of p53 , sod, cat , and gsr. These results suggest that the expression of cyp1a and gst may serve as biomarkers for dilbit exposure in fathead minnow, furthering our understanding of dilbit-responsive indicators of toxicity in fish species native to North America. This study is important as it utilizes the same exposure methodology to examine the toxicity of two commonly used Canadian dilbits, facilitating comparison of dilbit toxicity. [ABSTRACT FROM AUTHOR]