Instrumental Analysis Conventional / Nutrients Emerging contaminants Assessing Metabolomic Response in Rainbow Trout Exposed to Sewage Effluents and Lake Water from Hamilton Harbour, Ontario Jonathan P. Benskin,1 Trudy Watson-Leung,2 Sonya Kleywegt,3 Bharat Chandramouli,1 Victoria Tkatcheva,4 Paul Helm5 jbenskin@AXYS.com 1. AXYS Analytical Services Ltd. 2045 Mills Road West, Sidney BC, Canada, V8L 5X2 2. Aquatic Toxicology Unit, Laboratory Services Branch, Ontario Ministry of the Environment and Climate Change, 125 Resources Rd Toronto ON M9P3V6 3. Standards Development Branch, Ontario Ministry of Environment and Climate Change, 40 St. Clair Avenue West, 7th Floor, Toronto, ON, Canada M4V 1M2 4. Natural Sciences and Engineering Research Council, Ottawa, ON, Canada. 5. Great Lakes Monitoring Unit, Ontario Ministry of Environment and Climate Change, 125 Resources Rd Toronto ON M9P3V6 Background and Objectives Results: Water Chemistry Municipal wastewater treatment plants (WWTPs) have been identified as point sources of a broad suite of endocrine-disrupting compounds, that have the potential to elicit an effect on aquatic organisms. Given the wide variety of chemicals present in municipal waste water, regulating discharges based on specific chemical monitoring may not be the most efficient way to ensure discharges do not impact downstream water use (e.g. aquatic life, drinking water sources). In the present work, targeted metabolomics was applied to rainbow trout which were exposed to sewage effluent and lake water from the Hamilton Harbour region. Comprehensive chemical characterization of the exposure media was also conducted. A B C D E F Figure 2. Selected water chemistry results from surface water (A) and effluents (B-F). Note higher BOD, CBOD, NH4+NH3 in Woodward effluents. Results: Targeted Metabolomics Experimental Methods * ‡ ∆ Sampling Surface water was collected from three locations in Hamilton Harbour (Figure 1). Effluents were collected from two wastewater treatment plants (WWTP) in Hamilton Harbour (Figure 1). A B C D * ∆ ‡ ∆ * Incubations All animals were acclimated prior to testing. 10 rainbow trout (5-10 g, 2 per bucket) were incubated at 15 ± 1 °C in test water for 48 h. Control incubations were conducted concurrently using dechlorinated City of Toronto tap water (culture water). At 48 h, all animals were euthanized and a section of liver was flash frozen in liquid nitrogen. Figure 3. Ions (A) and statistically significant organic metabolites (B-D) measured in rainbow trout plasma following exposure to surface water. Statstically significant differences (i.e. p<0.05; Wilcoxon rank-sum test) are indicated by: *(Arm and Control), ‡ (Index and Control), ∆ (West and Control). A B C D * ‡ * ‡ * ‡ Figure 1. Sampling locations. Water Chemistry Instrumental Analysis Targeted Metabolomics Chemical analysis of the surface water and wastewater was conducted by Ontario Ministry of Environment and Climate Change. Sample preparation and instrumental analysis procedures described in Benskin et al. 2014. Inorganic ions measured by ion chromatography. Figure 4. Ions (A) and statistically significant organic metabolites (B-D) measured in rainbow trout plasma following exposure to sewage effluent. Statistically significant differences (i.e. p<0.05; Wilcoxon rank-sum test) are indicated by: *(Woodward and Control), ‡ (Dundas and Control). Table 1. Structure and function of metabolites quantified in the present work. Table 2. Water chemistry measurements Summary Future Work Structure Target # of Analytes Glycerophospho lipids (PCs) 92 Sphingomyelines (SMs) 15 Acylcarnitines (carnitine shown) 41 Amino acids (alanine shown) 14 Biogenic amines (taurine shown) 19 ∑Hexose (glucose shown) 1 Bile acids (taurocholic acid shown) 13 Fatty acids (FAs; arachidonic acid shown) 17 Chemical grouping Surface Water    Conventional / Nutrients    Biological oxygen demand (BOD/BODC) Dissolved and suspended solids pH and alkalinity Dissolved nutrients Dissolved carbons (DIC/DOC) Legacy contaminants Metals PCBs PAHs Dioxins and Furans Dioxin-like PCBs Brominated diphenyl ethers (PBDEs) Emerging contaminants Perfluorinated compounds (PFCs) Pharmaceuticals and personal care products Nonyl phenols Exposure studies involving Hexagenia (lake water, effluents and sediments) and fathead minnow (lake water and effluent) have been conducted and are awaiting analysis. Transcriptomics to be conducted on all samples. Further contaminant chemistry to be conducted on effluent, sediments and lake water. Figure 5. Number of statistically different organic metabolites measured in trout livers exposed to surface water or effluents from Hamilton Harbour. References Benskin, J.P. et al. Environ Sci Technol 2014, 48, 11670-11678. Woodward WWTP effluent, followed by Dundas WWTP effluent, produced the greatest number of statistically different metabolites relative to controls. Woodward contained considerably higher concentrations of most contaminants relative to Dundas. Lake water produced a similar number of statistically different metabolites at all locations; however, exposure to Index Site water produced unique metabolomic changes primarily attributable to FAs. Changes in amino acids were only observed following exposure to Woodward WWTP effluents. Acknowledgements This project has received funding support from the Government of Ontario. Such support does not indicate endorsement by the Government of Ontario of the contents of this contribution.