** The title, authors, and abstract for this completion report are provided below.  For a copy of the completion report, please contact the author at mssepulv@purdue.edu or via telephone at 765-496-3428. Questions? Contact the GLFC via e-mail or via telephone at 734-662-3209 **



Effects of lampricides on target and non-target species: from protein expression to ecological consequences


Maria S. Seplveda and Tomas O. Hk


Department of Forestry and Natural Resources, Purdue University, 195 Marsteller St., West Lafayette, Indiana 47907




June 2013




Although the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) has been used for over 60 years to control sea lamprey (Petromyzon marinus) in the Great Lakes, the mode of toxic action of TFM is not completely understood and impacts (including sub-lethal effects) of TFM on non-target species have not been fully evaluated. We explored these knowledge gaps by quantifying subcellular (metabolomics and protein expression), cellular (histology) and whole organism (growth and behavior) changes in several fish species acutely (12 hours) exposed to TFM. We replicated TFM stream treatments by exposing fish to increasing concentrations of TFM until the ~ 10th hour of treatment, decreasing concentrations to baseline levels in the last two hours of exposure. We exposed juveniles of four species of fish (sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss) and adult fathead minnows (Pimpehales promelas) to various concentrations of TFM (0.6-10 mg/L). Following exposure, tissues (livers and gills) from a subset of sea lamprey, lake sturgeon and rainbow trout, were collected and immediately preserved for metabolomics analysis (livers) using liquid chromatography-mass spectrometry; for semi-quantification of cytochrome p450 (CYP1A, livers) using western blots; or for histopathological (gills) evaluation using standard hematoxylin and eosin staining. Growth (for up to a month) was monitored for the remaining rainbow trout and lake sturgeon. In addition, behavioral studies that involved testing avoidance to TFM by rainbow trout and predatory avoidance by fathead minnows to a predator (largemouth bass, Micropterus salmoides) were also conducted. Exposure to TFM induced significant changes in metabolite profiles for all three species tested, with most of the significant metabolites observed being involved in glycerophospholipid and protein metabolism. These results are in agreement with a recent study reporting TFM as an uncoupler of mitochondrial oxidative phosphorylation (Birceanu et al. 2011) in rainbow trout and sea lamprey. Importantly, metabolite changes were similar across species. Although our results with CYP1A expression are preliminary at the time of this report, due to high variation between individuals, it appears that levels of expression of this protein were highest in the most sensitive species, the sea lamprey. This could be explained by having tested TFM at the lower end of the toxicity curve for sea lampreys (which was our intention in order to better understand mechanisms of toxicity in this species). Higher TFM levels would have likely induced overt toxicity and cell death pathways resulting in a decrease in the expression of this protein. The only lesion observed in gills was detected in rainbow trout exposed to 10 mg/L TFM which showed severe epithelial lifting. No effects on growth or behavior were observed. We conclude that our metabolomics work supports TFMs mode of toxic action as an uncoupler of oxidative phosphorylation and that differences in species sensitivity are not likely due to lower metabolizing ability by sea lampreys; that TFM is unlikely to cause mortality due to gill damage, although this could be a secondary effect at high doses; and that TFM exposure in the conditions tested in the present study are unlikely to lead to meaningful physiological effects such as decreased growth or impairment in avoidance behaviors.