Development of gene expression-based biomarkers of exposure to metals and pesticides in the freshwater amphipod Hyalella azteca

Ecological risk assessment (ERA) is a framework for monitoring risks of exposure and adverse effects of environmental stressors to populations or communities of interest. One tool of ERA is the biomarker, which is a characteristic of an organism that reliably indicates exposure to or effects of a stressor like chemical pollution. Traditional biomarkers which rely on characteristics at the tissue level and higher often detect only acute exposures to stressors. Sensitive molecular biomarkers may detect lower stressor levels than traditional biomarkers, which helps inform risk mitigation and restoration efforts before populations and communities are irreversibly affected. In this study I developed gene expression-based molecular biomarkers of exposure to metals and insecticides in the model toxicological freshwater amphipod Hyalella azteca. My goals were to not only create sensitive molecular biomarkers for these chemicals, but also to show the utility and versatility of H. azteca in molecular studies for toxicology and risk assessment. I sequenced and assembled the H. azteca transcriptome to identify reference and stress-response gene transcripts suitable for expression monitoring. I exposed H. azteca to sub-lethal concentrations of metals (cadmium and copper) and insecticides (DDT, permethrin, and imidacloprid). Reference genes used to create normalization factors were determined for each exposure using the programs BestKeeper, GeNorm, and NormFinder. Both metals increased expression of a nuclear transcription factor (Cnc), an ABC transporter (Mrp4), and a heat shock protein (Hsp90), giving evidence of general metal exposure signature. Cadmium uniquely increased expression of a DNA repair protein (Rad51) and increased Mrp4 expression more than copper (7-fold increase compared to 2-fold increase). Together these may be unique biomarkers distinguishing cadmium and copper exposures. DDT increased expression of Hsp90, Mrp4, and the immune response gene Lgbp. Permethrin increased expression of a cytochrome P450 (Cyp2j2) and decreased expression of the immune response gene Lectin-1. Imidacloprid did not affect gene expression. Unique biomarkers were seen for DDT and permethrin, but the genes studied were not sensitive enough to detect imidacloprid at the levels used here. I demonstrated that gene expression in H. azteca detects specific chemical exposures at sub-lethal concentrations, making expression monitoring using this amphipod a useful and sensitive biomarker for risk assessment of chemical exposure.

Improved Venting for Flammability Limit Testing Using ASTM E681 Apparatus

The literature on the determination of flammability limits was reviewed and experts on the ASTM E681 standard were interviewed to identify new means of improving the reproducibility of the ASTM E681 test. Venting was identified as a variable of flammability limits not yet addressed. Limitations of the current system for sealing and venting (a rubber stopper) were identified and addressed by the development of a custom burst disc. The burst disc was evaluated for its ability to hold and maintain a vacuum, its ability to vent at pressures of interest, and for its venting phenomena. The burst disc was deemed to be a satisfactory alternative to the rubber stopper and is recommended to be included in the ASTM E681 standard.