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Development of Discovery-based Analytical Methodology for the Identification and Quantification of Legacy and Emerging Contaminants that Contribute to Current Disease in Smallmouth Bass Micropterus Dolomieu from the Susquehanna River Basin

The focus of this study was to develop methodologies to discover, identify, and quantify legacy persistent organic pollutants (POPs) and contaminants of emerging concern (CECs) that contribute to the negative health impairments of smallmouth bass (Micropterus dolomieu, SMB) of the Susquehanna River Basin. Many potential risk factors have been investigated in this complex issue, but there is still a lack of data surrounding organic contaminants and their relation to the observed signs of disease in fish. This is likely due to the reliance of strictly targeted analytical methods previously. Unfortunately, this approach negates the discovery of CECs or even known POPs that were not included on the selected target list for analysis. Developing inclusive, discovery-based analytical approaches will allow for the collection of far more data and increase the probability of finding correlations between contaminant exposure and organism health.

For this study, new sample preparation methodology was tailored to accommodate young-of-year (YOY) SMB based on the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction. QuEChERS was optimized to extract a broad range of chemical classes in a high-throughput manner to accommodate large numbers of samples needed to identify correlations among populations. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) was chosen to analyze samples for target and non-target compounds of interest. The powerful analytical tool provides large data sets that required the development of data reduction techniques to identify statistically significant non-target compounds. A data reduction pipeline utilizing multiple software platforms was implemented and proved successful on a small data set of 21 samples.

The sample preparation, analysis, and data reduction methodologies developed in this study are capable of quantitatively identifying known target contaminants and discovering unknown compounds of interest that warrant further investigation and molecular identification efforts using high resolution mass spectrometry. 147 YOY SMB were subjected to a targeted analysis to quantify the presence of 127 target compounds. 41 were detected in at least one sample at a concentration above the method detection limit. No correlations could be determined between any of the target compounds detected and the observed signs of disease in the YOY SMB. A non-targeted analysis of 21 YOY SMB identified 10 compounds as statistically significant and warranting further study. It is expected that similar results will be found at the completion of the non-targeted analysis of all 147 YOY SMB.

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