Emissions of sulphur oxides, nitrogen oxides and other pollutants have increased in the Athabasca Oil Sands Region (AOSR) in Alberta, Canada. Atmospheric pollutants impact aquatic communities through a number of processes, but due to a lack of regional monitoring programs potential biological impacts have not been assessed. In this study, a bioassessment was conducted using approaches borrowed from a variety of protocols to establish a baseline dataset, determine appropriate methodologies, and to assess the current impact of emissions on benthic macroinvertebrate (BMI) communities in the AOSR. As a result, 32 lakes, including 5 test lakes located in a modelled high deposition region, were sampled for water chemistry and BMI. The Reference Condition Approach (RCA) was used because a baseline dataset does not exist and data were evaluated using three separate statistical techniques. All of the statistical methods used: One Sample T-Tests, Multivariate Analysis of Variance (MANOVA) and Test Site Analysis (TSA), showed that BMI assemblages in test lakes differed from BMI assemblages in reference lakes. Traditional statistics classified all 5 test lakes as "significantly impaired" whereas TSA identified 3 of the 5 test lakes as only potentially impaired and 2 lakes were in "reference condition". The variability in lake attributes present challenges in interpreting BMI data and establishing an accurate biomonitoring program in the AOSR which need to be addressed in future assessment studies.