OSPW After Ozonation with 80 mg O3/L Growth of Chironomus dilutus Exposed to Ozone-Treated and Untreated Oil Sands Process Water J. Anderson 1, S.B. Wiseman 1, E. Franz 1, M. Gamal El-Din 2, J. Martin 3, P. Jones 1,4, K. Liber 1,4, and J.P. Giesy 1, 5, 6, 7 1. Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada 2. Department of Civil and Environmental Engineering, University of Alberta, Edmonton, AB, Canada 3. Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Chemistry, University of Alberta, Edmonton, AB, Canada 4. School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada 5. Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada 6. State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, Peoples Republic of China 7. Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA BACKGROUND RESULTS DISCUSSION Ozonation reduced the growth inhibition observed in untreated OSPW and may be a treatment option to reduce OSPW toxicity towards aquatic invertebrates Need to identify an optimal dose of ozonation and investigate whether a dose effect of ozonation is present 1. Transcriptome responses to OSPW and ozonated OSPW Illumina RNAseq transcriptome sequencing of C. dilutus larvae exposed to freshwater, untreated OSPW, and ozone-treated OSPW to determine gene expression profiles Biomarkers of OSPW exposure Biomarkers of effect Molecular basis of toxicity Over 250 million sequence reads returned by Illumina Contigs (>300,000) created using CLC Workbench Scaffolds will be built and sequences will be annotated using BLAST2GO Gene expression profiles will be generated for the different treatments to determine exposure specific transcriptome responses 2. Behavioral Responses in C. dilutus exposed to OSPW and Ozonated OSPW Preliminary studies suggest OSPW exposure drastically alters larvae burrowing and tube-building behaviours (Fig. 4) Effects appear to be attenuated by ozonation of OSPW Canada is home to the second largest proven oil reserves, mostly in Alberta’s Athabasca region Extraction of usable material (bitumen) from oil sands requires vast quantities of fresh water and produces wastewater - “oil sands process water” (OSPW) Approximately 4 m3 of OSPW are produced per 1 m3 of oil sands processed, but more efficient water recycling has reduced freshwater needs OSPW has been shown to be toxic to a number of different species; naphthenic acids (NAs) are believed to be responsible for much of the toxicity Companies are currently held to a zero-discharge policy so treatment methods are required to target NAs and effectively eliminate toxicity of OSPW One potential treatment method is ozonation, a method commonly used in municipal water treatment and shown to target more persistent and chronically toxic NAs (Fig. 1) No significant differences in survival between any exposure group and the freshwater controls (Figs. 2a and 3a) Untreated (100%) OSPW significantly inhibited C. dilutus growth (Figs. 2b and 3b) Low ozonation study (50 mg/L) 100% OSPW-exposed larvae were 64% smaller than freshwater controls (Fig. 2b) Exposure to full strength ozone-treated OSPW  no difference in mean wet mass from controls High ozonation study (80 mg/L) 100% OSPW-exposed larvae were 77% smaller than freshwater controls (Fig. 3b) Exposure to full strength ozone-treated OSPW  only 40% smaller than controls STUDIES IN PROGRESS / FUTURE DIRECTIONS O3 OSPW NAs Before Ozonation OSPW After Ozonation with 80 mg O3/L Fig. 1 Naphthenic acid profile of OSPW prior to ozonation and after treatment with 80 mg/L O3. Survival Growth a b Fig. 2a and 2b Effects of low dose ozonation (50 mg/L) on survival and growth (individual wet mass) of C. dilutus larvae following a 10-day exposure. Differences from freshwater controls in survival were determined by Kruskal-Wallis one-way ANOVAs and differences in mass were determined by Student-Newman-Keuls comparisons across treatments (p=0.05). Significant differences designated by an asterisk (*). * Fig. 4 Burrowing behaviours observed in a) fresh water, b) OSPW, and c) ozonated OSPW. In the freshwater control, all individuals are within tubes, while the ozonated group has a few individuals not burrowing and the untreated OSPW group displays very little burrowing and tube-building activity. These preliminary results will be investigated further in upcoming studies. a b c Study Objectives: 1.To characterize the effects of exposure to OSPW in a model benthic invertebrate, Chironomus dilutus 2. To determine whether remediation of OSPW using ozonation effectively reduces its toxicity to aquatic invertebrates Survival Growth Figs. 3a and 3b Effects of high dose ozonation (80 mg/L) on survival and growth (individual wet mass) of C. dilutus larvae following a 10-day exposure. Differences from freshwater controls in survival were determined by Kruskal-Wallis one-way ANOVAs and differences in mass were determined by Student-Newman-Keuls comparisons across treatments (p=0.05). Significant differences designated by different letters. A B C a b METHODS Test organism – Chironomus dilutus larvae (formerly C. tentans), started 13-14 days post-oviposition Exposure waters – Freshwater control, saltwater control, ozonated OSPW, untreated OSPW Two degrees of ozonation – Low (50 mg/L) and high (80mg/L) Exposure conditions – Continuous aeration, 50% water change every 2 days, daily feeding, 10 larvae per rep, 4 reps per treatment, 10 day exposure period End points – Survival, growth (measured as wet mass) Parallel assay terminated at Day 4, total RNA collected from pooled samples, sequencing by Illumina RNASeq This research was supported by research grants from the Alberta Water Research Institute and Western Economic Diversification Canada to J.P.G. J.P.G. is supported by the Canada Research Chairs Program J.A. was supported by an NSERC Alexander Graham Bell Canada Graduate Scholarship Thanks to Syncrude Canada Ltd. for support and assistance ACKNOWLEDGEMENTS