EXAMINATION OF THE REPRODUCTIVE PERFORMANCE AND TISSUE-SPECIFIC METAL ACCUMULATION PROFILES IN FEMALE FATHEAD MINNOWS (PIMEPHALES PROMELAS) DURING CHRONIC WATERBORNE EXPOSURE TO INDIVIDUAL METALS AND BINARY METAL MIXTURES
The overall objective of the research presented in this thesis was to evaluate the effect of waterborne metals, alone and in binary combination on the reproductive capacity of fathead minnows (FHM; Pimephales promelas) during chronic exposure. A wide array of reproductive endpoints across multiple levels of biological organization (molecular, biochemical, sub-organismal and population) was evaluated to understand the reproductive effects of individual metals and metal-mixtures in fish. In addition, the thesis also focused on determining the interactive effects of binary metal mixture exposure on tissue-specific (gill, liver, gonad and carcass) metal accumulation patterns in fish. This thesis consists of two generalized methodological components - in vivo and in vitro experiments. The first component employed a 21-day FHM reproductive bioassay. Trios (1 male: 2 female) of FHMs were exposed to four different experimental treatments via water: control, two individual metals, and a binary metal-mixture. The individual metals used in this study were cadmium (Cd), copper (Cu), zinc (Zn) and nickel (Ni), alone and in binary combinations of Cd-Cu, Cd-Zn and Cu-Ni. In the reproductive bioassay, the effects of individual metals and binary metal-mixtures in female fish were assessed by evaluating serum estradiol level, hepatic mRNA expression of estrogen receptors (ER-α and ER-β), vitellogenin (Vtg) and metallothionein (MT), and ovarian histopathology, and a wide array of whole organismal reproductive endpoints including fish fecundity (cumulative egg production). The second methodological component (in vitro study) of my research was conceived based on the findings of first two reproductive bioassays (Cd-Cu and Cd-Zn), and focused on assessing the capacity of individual metals and metal-mixtures to directly affect estradiol production in FHM ovarian explants. The in vitro study also exhibited some first definitive evidence indicating that metals can directly impair steroidogenesis, further corroborating the findings of the in vivo reproductive bioassays. This thesis provided novel insights into the mechanisms of reproductive toxicity of individual metals, especially Zn and Ni. More importantly, my research demonstrated that metals in binary mixtures, irrespective of their apparent mode of toxic action, appear to act in a predominantly additive manner on fish fecundity, which was found to be the most sensitive reproductive endpoint in FHM bioassays. In general, the main findings of my thesis suggested that the interactions of metals elicit their reproductive effects in fish by altering estradiol production and/or hepatic vitellogenin synthesis, which possibly occur as an indirect consequence of metal-induced disruption of energy homeostasis in fish. The examination of tissue-specific metal accumulation revealed no notable interactions for any of the binary metal combinations examined, except an antagonistic effect of Cd on hepatic Cu burden, and Zn on hepatic Cd burden. However, none of these antagonistic interactions on tissue metal burden translated into amelioration of reproductive effects. On the other hand, the examination of ovarian histopathology revealed an additive effect of Cu and Ni, and Cd and Zn on follicular atresia. Collectively, these findings indicated that cumulative tissue burden of metals during chronic waterborne exposure to binary metal-mixtures contributes to the reproductive impairment in FHM by increasing the energetic cost of metal detoxification (increased metallothionein induction) and affecting the oocyte maturation process. Overall, this thesis demonstrated that waterborne exposure to metals in binary mixture impairs fish reproductive capacity essentially by disrupting the estrogen-mediated reproductive pathways.
metal, fish, fathead minnow, reproduction, mixture toxicity,
Doctor of Philosophy (Ph.D.)
Western College of Veterinary Medicine