Determining the Impacts of Environmental Contaminants to Zebra Mussels Using Genetic Biomarkers

ABSTRACT DETERMINING THE IMPACTS OF ENVIRONMENTAL CONTAMINANTS TO ZEBRA MUSSELS USING GENETIC BIOMARKERS by Nicklaus James Neureuther The University of Wisconsin-Milwaukee, 2016 Under the Supervision of Professor Rebecca Klaper, PhD Persistent legacy contaminants and emerging chemicals of concern continue to be a threat to the function and health in the Great Lakes Areas of Concern (AOCs). While chemical monitoring programs traditionally sample water and sediment, these studies can only provide information of the type and level of contamination within an (AOC). This being said, information on the biological impacts to the biota are needed to measure impairments of chemical exposure, to support remediation efforts in their ability to eventually restore AOCs. To accomplish this, I proposed to measure chemical exposure using molecular biomarkers from D. polymorpha (Pallas, 1771), more commonly known as the zebra mussel. This species has been successfully used as a bioindicator of contamination in the Great Lakes and in Europe due to it being an invasive species and having the physiological qualities of being a sessile filter feeder. These unique physiological properties, in addition to the zebra mussel already having a library of gene expression biomarkers known to be critical in relation to stress and detoxification, including the genes: GST, AHR, P-gP and HSP70, made this organism an obvious choice. Working in conjunction with the already iii established chemical monitoring program, the NCCOS NOAA Mussel Watch Program (MWP), the goal was to test these genomic biomarkers to see how robust they could be as an indicator of exposure in conjunction with chemical data from the field. We demonstrated that in an aquatic environment that these genes of exposure revealed a significant relationship with the legacy contaminants polychlorinated bi-phenols (PCBs) and polycyclic aromatic hydrocarbons (PAHs) and the emerging contaminants, 4 nonylphenol (4NP) and triclocarban (TCC) over a gradient of contamination and that these results were affected by length of exposure. Likewise, AOCs are dynamic environments containing complex mixtures of contaminants, which tend to co-correlate, making it difficult to parse out effects of exposure from single contaminants. To investigate individual chemical and gene expression relationships further, zebra mussels were exposed to environmentally relevant levels of TCC under laboratory conditions. This study confirmed my field results that the gene GST could be a potential biomarker of TCC. As a whole, these two studies demonstrated that using the zebra mussel as not only a bioindicator of contamination but as a biomonitor of exposure using gene expression biomarkers could be an effective tool used by monitoring programs to help gauge restoration success

Transcriptome signatures of wastewater effluent exposure in larval zebrafish vary with seasonal mixture composition in an effluent-dominated stream

Wastewater treatment plant (WWTP) effluent-dominated streams provide critical habitat for aquatic and terrestrial organisms but also continually expose them to complex mixtures of pharmaceuticals that can potentially impair growth, behavior, and reproduction. Currently, few biomarkers are available that relate to pharmaceutical-specific mechanisms of action. In the experiment reported in this paper, zebrafish (Danio rerio) embryos at two developmental stages were exposed to water samples from three sampling sites (0.1 km upstream of the outfall, at the effluent outfall, and 0.1 km below the outfall) during base-flow conditions from two months (January and May) of a temperate-region effluent-dominated stream containing a complex mixture of pharmaceuticals and other contaminants of emerging concern. RNA-sequencing identified potential biological impacts and biomarkers of WWTP effluent exposure that extend past traditional markers of endocrine disruption. Transcriptomics revealed changes to a wide range of biological functions and pathways including cardiac, neurological, visual, metabolic, and signaling pathways. These transcriptomic changes varied by developmental stage and displayed sensitivity to variable chemical composition and concentration of effluent, thus indicating a need for stage-specific biomarkers. Some transcripts are known to be associated with genes related to pharmaceuticals that were present in the collected samples. Although traditional biomarkers of endocrine disruption were not enriched in either month, a high estrogenicity signal was detected upstream in May and implicates the presence of unidentified chemical inputs not captured by the targeted chemical analysis. This work reveals associations between bioeffects of exposure, stage of development, and the composition of chemical mixtures in effluent-dominated surface water. The work underscores the importance of measuring effects beyond the endocrine system when assessing the impact of bioactive chemicals in WWTP effluent and identifies a need for non-targeted chemical analysis when bioeffects are not explained by the targeted analysis