Despite many studies of diluted bitumen (DB) behavior during spills in saltwater, limited information is available on DB behavior in fresh water. This study examined the collective weathering processes on changes of fresh DB spilled in the North Saskatchewan River water and sediment mixture in a mesoscale spill tank under average air/water temperatures of 14 °C/15 °C and 6 °C/2 °C. Temporal changes of the hydrocarbon and microbial community compositions in the water column were assessed during the two 35-day tests under intermittent wave action. The contents of total organic carbon (TOC), benzene/toluene/ethylbenzene/xylenes (BTEX) and polycyclic aromatic hydrocarbons (PAHs) in water decreased with time during both tests. The final contents remained at higher values in warm water (15 °C) than in cold water (2 °C) after the collective weathering processes. A quick response of the main phyla, Proteobacteria and Actinobacteria, was observed, where the members of Proteobacteria enriched during both DB spills. In contrast, the members of Actinobacteria reduced with time. The microbial shifts coincided with the changes of PAHs in the waters at both temperatures. A comparison of the physical properties and chemical compositions of fresh and weathered DBs at both temperatures showed that the oil had undergone weathering that increased oil density and viscosity due to losing the light oil fraction with boiling points < 204 °C and emulsifying with water. This corresponded to losses of 19.0 wt% and 17.2 wt% of the fresh DB at 15 °C and 2 °C tests, respectively. For organic compounds in the DB with boiling points > 204 °C, there were small losses of saturates and 2- & 3-ring PAH aromatics (more during the 15 °C test than the 2 °C test), and negligible losses in the subfractions of resins and asphaltenes by the ends of the tests. <1.0 wt% of the DB was recovered from the bottom sediment, regardless of the temperature.
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