Using clinical growth-based antimicrobial susceptibility tests as a sensitive indicator of oil sand process-affected water toxicity

Current approaches for petroleum extraction in northern Alberta oil sands use heat, agitation, chemicals, and water to separate useable bitumen from oil sand deposits. During this process, caustic chemicals and sequestered compounds of environmental concern become concentrated in these waters. These waters are referred to as oil sands process-affected waters (OSPW), and are alkaline, slightly brackish waters that contain several organic and inorganic compounds that have been shown to be detrimental to organism physiology and normal biological function using both in vivo and in vitro approaches. The deleterious effects of OSPW are often contributed to recalcitrant contaminants such as naphthenic acids (NAs), but there are no clear guidelines regarding acceptable limits for NAs in the environment. As a result, these waters are currently held under a zero-discharge policy, resulting in the accumulation 1.44 billion m3 of OSPW as of 2022. As required by provincial legislation, these waters must be returned to a pre-industrial state within 10 years of site closure, prompting significant efforts to remediate OSPW. A variety of treatment regimens that target NAs for removal are currently being tested and will require intensive toxicity monitoring to inform water quality status. As these waters are complex mixtures, the tools used to assess their toxicity and treatment efficacy must be equally varied and sensitive. While in vivo assays provide biologically relevant data for affected organisms, in vitro approaches allow for rapid, high-throughput screening of water samples to inform treatment decisions and further toxicity testing. Despite their apparent value in guiding both treatment and toxicity testing, relatively few approved in vitro methods exist for use in aquatic toxicity assessments. As government and industry seek new toxicity assessment tools to help achieve time-sensitive OSPW remediation targets, new approaches must be considered to fill these technical gaps. Bacterial toxicity assays are a candidate for this role, as these approaches are inexpensive, robust, reproduceable, and high throughput, making them a potentially valuable tool in informing rapid decisions on OSPW treatment, release, and monitoring. In clinical settings, growth inhibition as determined by microdilution minimum inhibitory concentration (MIC) assays have long stood as the gold standard for the assessment of antimicrobial activity against indicator organisms. These approaches are well standardized, widely accepted, and are supported by data showing their effectiveness and sensitivity. As such, I investigated the viability of adapting a microdilution MIC assay for determining the detrimental effects of OSPW and NAs on bacterial growth. Through preliminary optimization, a modified microdilution MIC assay was determined to be stable when exposed to physiochemical parameters seen in freshwater and OSPW. Additionally, this assay demonstrated dose-dependent inhibitory effects on growth as a result of commercial naphthenic acid (cNA) and OSPW exposures, with values comparable to what is observed in the most sensitive in vivo and in vitro aquatic toxicity assays. To further establish the sensitivity of this approach, a cNA standard and several OSPW samples were treated to remove organic contaminants. Removal of organic compounds was tracked using fluorescence spectroscopy, with chemical analysis data revealing efficient removal of potential contaminants. When assessed using a modified microdilution MIC approach, the reduction in organic compounds was associated with a decrease of inhibitory effects on growth. These approaches were then compared to a standardized bacterial luminescence inhibition assay currently approved for use in aquatic toxicity testing. My data showed that the modified microdilution MIC approach had greater sensitivity to untreated cNA and OSPW, with similar trends observed in terms of monitoring removal and persistence of toxicity in response to treatment. Furthermore, these approaches were resilient against optical interference and several other technical and substrate specific limitations that can perturb bioluminescence assays. Overall, my thesis data suggests that a modified microdilution MIC assay is a robust, high throughput, cost effective method for monitoring OSPW toxicity. These approaches display sensitivity equivalent to or greater than what is observed in bacterial luminesce inhibition assays but require less sample modification and have exposure times and toxicity endpoints of greater ecological and technical relevance.

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