Exposomics Based Methodologies for the Identification and Correlation of Organic Contaminants Contributing to Disease in Smallmouth Bass Populations
Open Access
- Author:
- Teehan, Paige
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 07, 2021
- Committee Members:
- Philip Bevilacqua, Major Field Member
Wendy Hanna-Rose, Program Head/Chair
Andrew Patterson, Chair & Dissertation Advisor
William Burgos, Outside Unit & Field Member
Ross Hardison, Major Field Member
Frank Dorman, Special Member & Co-Dissertation Advisor - Keywords:
- exposomics
multidimensional gas chromatography
liquid chromatography
mass spectrometry
smallmouth bass - Abstract:
- Since 2005, smallmouth bass (Micropterus dolomieu) throughout the Susquehanna River Basin have been observed exhibiting signs of disease; these observations include intersex adult males, high/chronic mortality in young-of-year fish, and generalized immunosuppression characterized as a single fish being co-infected with multiple pathogens. To date, no single factor or group of factors has been identified as a causative agent to explain the observed signs of disease, but environmental contaminants are suspected of being a contributing influence. Previous targeted studies have reported differing levels of contaminants, including polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (BDEs), legacy and current use pesticides, and pharmaceuticals and personal care products (PPCPs) in adult fish from sites throughout the river basin. Here, young-of-year fish are examined for both target and non-target compounds using an exposomics based approach to identify statistically significant features among populations experiencing disease traits. Previous investigations have focused largely on the identification of target compounds within adult fish or water samples, but this targeted investigative approach provides limited information about the samples. The relationship between environmental conditions, contaminants, pathogens, and the health of living organisms throughout their lifetime is complex and requires a more in-depth analysis than identifying target compounds in adults. By examining young-of-year fish, it is possible to identify exposures that are occurring during developmental life stages that may contribute to immunosuppression and intersex conditions later in life. And by expanding the analysis from a strict target list to any extractable non-target compound, it is possible to identify contaminants not included on the target list, new contaminants of emerging concern, and even biomarkers that are associated with disease profiles. Analyzing a complex matrix like whole young-of-year fish for both target and non-target compounds, required advancements in current sample preparation and data analysis techniques. Modifications to the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method have yielded a high-throughput sample preparation technique that is quicker, cheaper, and greener than current EPA methods for tissue analysis, while remaining suitable for both targeted and non-targeted analyses of fish tissue. This sample preparation has been combined with comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) as well as ultra-performance liquid chromatography coupled with high-resolution quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) to analyze young-of-year smallmouth bass from fourteen affected sites throughout the Susquehanna River Basin. Sample data sets were then processed to identify target and non-target compounds. Target compounds were quantified using internal standard normalized external calibration curves, while significant non-target compounds were identified using multivariate and univariate data analyses. The resulting compounds and features were then examined for correlations among disease characteristics or locations. A total of 146 young-of-year smallmouth bass were collected by electrofishing from 14 sampling sites throughout the Pennsylvania region of the Susquehanna River Basin, USA in 2015. The modified sample preparation methodology was used for the extraction and cleanup of all samples. Cleaned sample extracts were divided for analysis using both gas and liquid chromatography platforms. Targeted analyses using GC×GC-TOFMS and UPLC-QTOF-MS have revealed the presence of PCBs, BDEs, pesticides, plasticizers, polycyclic aromatic hydrocarbons, PPCPs, and perfluoroalkyl substances. Target compounds were analyzed for correlations with the disease characteristics observed at the time of young-of-year smallmouth bass collection. Several slight to moderate correlations were observed, but few were statistically significant. Non-targeted analyses were designed to elucidate differences between healthy fish and fish displaying outward signs of disease (lesions, parasitic infections, gill erosion, etc.) as well as site-specific features. The same data sets used for the targeted analyses, were mined for statistically significant non-targets. Chromatographic alignment was performed using commercially available software specific to either GC×GC or UPLC applications that produced aligned peak tables of normalized areas. For GC×GC-TOFMS data, Fisher ratio analysis was combined with principal component analyses or partial least squares discriminant analyses for feature reduction. This resulted in two final datasets, one for disease classification and one for collection sites, of tentatively identified, significant, non-target compounds. Comparisons between these non-target compounds revealed three chemical classes that were more abundant in fish displaying signs of disease and four compounds that were identified as significant in both datasets. For UPLC-QTOF-MS data, this optimized data reduction strategy was also applied, but with different results. No compounds or chemical classes were revealed to be significant to specific classifications of fish, either by disease state or collection site. Instead, the non-targeted analysis tentatively identified several pesticides, hormones, steroids, and pharmaceutical compounds not previously reported in smallmouth bass tissue. This work has produced a high-throughput sample preparation, analysis, and data processing methodology that can be applied to large environmental sample sets. While targeted analyses continue to be the default method of environmental monitoring efforts, the ongoing discovery of contaminants of emerging concern illustrates the need for robust non-targeted analysis methods as well. Chapter 2 has been published in Analytical Methods. Chapter 3 has been published in Science of the Total Environment. Data from Chapter 4 is currently in preparation for submission as a manuscript. I am first author on all these publications.