| dc.description.abstract | While fish toxicity testing methods have been vital in predicting toxicity of pollutants dissolved in water, advancements with in vitro ecotoxicological techniques have continued to gained momentum. In vitro alternatives allow identification of a toxicant’s mode of action, high throughput approaches, reduced test duration and the number of animals used. This dissertation investigated novel animal alternative methods (i.e., fish embryo and cytotoxicity assays) and their ability to predict acute toxicity of chemicals dissolved in water or of actual effluents in fish in vivo. Fathead minnow (Pimephales promelas) embryos and the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, were successful in predicting toxicity of a broad list of legacy pollutants, chemicals of emerging concern (CECs) and wastewater samples similarly to fish. Moreover, the 24 hours cytotoxicity assay using RTgill-W1 cells was able to predict toxicity in all samples deemed toxic to fish using the 96- hour larvae assays. Additionally, to facilitate direct water sample exposures in fish gill cell monolayers, RTgill-W1 cells were cultured on transwell inserts. When cells are cultured on transwell inserts for 14-day in symmetrical culture media a monolayer is formed. In this culturing condition, RTgill-W1 cells were able to tolerate freshwater but not seawater exposure on the apical side. Furthermore, mixing between apical and basolateral compartments and lack of the formation of tight junction proteins reinforced the fact that RTgill-W1 cells do not form a truly tight epithelium. Finally, incorporation of additional rainbow trout cell lines from the liver, RTL-W1, and the intestine, RTgutGC, along with RTgill-W1 cells were used for cytotoxicity assays and identification of biomarkers to determine tissue specific mechanisms of toxic action and sensitivity to different classes of pollutants. RTgill-W1 cells were found to be the most sensitive to metal toxicity through cytotoxicity and gene expression of metallothionein. Our findings have highlighted embryo and cell lines applicability and reliability as advanced alternatives to traditional acute fish assays, and a more sensitive and specific toxicity forensic tool. | |
