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High-resolution Mass Spectrometry for Monitoring Physiological Impacts and Biotransformation Products in Fish Exposed to Wastewater Effluent
Citation:
Mosley, J., M. Evich, I. Ntai, D. Ekman, J. Cavallin, D. Villeneuve, G. Ankley, AND T. Collette. High-resolution Mass Spectrometry for Monitoring Physiological Impacts and Biotransformation Products in Fish Exposed to Wastewater Effluent. 2019 ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, June 02 - 06, 2019.
Impact/Purpose:
Mass spectrometry-based untargeted metabolomics led to the discovery of contaminant biotransformation products in the skin mucus and bile of fish.
Description:
Introduction High-resolution mass spectrometry is advantageous for both metabolomics analyses and the monitoring of contaminant biotransformation products in fish exposed to wastewater effluent. We evaluated liver tissue and skin mucus from male and female fathead minnows exposed to control water or control water mixed with treated wastewater effluent at levels of 5%, 20% or 100% effluent. Exposures were conducted for 21 days, using an onsite, flow-through system providing real-time exposure at the Western Lake Superior Sanitary District (WLSSD) wastewater treatment plant in Duluth, MN, USA. We also developed a novel untargeted method to confirm the presence of glucuronide-based contaminant biotransformation products in bile from these same fish as a potential means of screening for relevant contaminants found in complex environmental mixtures. Methods We used a Q-Exactive coupled with liquid chromatography to analyze liver, bile and skin mucus samples from control and exposed fish. Untargeted metabolomics data were processed using an in-house R-based workflow. Endogenous metabolites for liver and skin mucus were annotated using mummichog and Compound Discoverer. Bile samples were split into two portions, and one was treated with glucuronidase. Both portions were analyzed with the Q-Exactive followed by data analysis using Compound Discoverer to identify biotransformation products. Untreated bile samples were analyzed in parallel with a structure-specific neutral loss-dependent MS3 fragmentation method using an Orbitrap ID-X Tribrid mass spectrometer, both to confirm the presence of a glucuronide moiety and to elucidate the structures of the parent chemical contaminants. Preliminary Data Both sex-specific and non-sex-specific responses were observed in the liver and skin mucus metabolomes, the latter suggesting the induction of general compensatory pathways for xenobiotic exposures. The mummichog software package was used to elucidate impacted biochemical pathways and to enhance metabolite annotation. Partial least squares regression models revealed relationships between the liver and skin mucus metabolomes and upregulated hepatic mRNA transcripts reported previously for these same fish [1]. Furthermore, these regression models suggest that skin mucus metabolomic changes reflected, in part, processes by which the fish biotransformed xenobiotics in the WLSSD effluent. Regression models built with liver data provide additional evidence that xenobiotic transformation processes are induced, while also shedding light on the sex-specific nature of these responses. The detection of sex-specific phase II transformation products (e.g., glucuronidated bisphenol A) of chemicals from the effluent in the fish skin mucus led to the development of a novel untargeted method to identify glucuronidated biotransformation products in fish bile. In brief, characteristic neutral loss and product ions during fragmentation are used as handles to further elucidate the glucuronidated contaminants. Preliminary results of this method will be presented here.