Metal mines release treated effluents that contain a variety of metals, metalloids, and organics into the aquatic environment. A number of metal mine effluents (MMEs) have been found to contribute to adverse effects in fish and benthic invertebrates, such as decreased diversity and density, however the specific causal factors of toxic responses during chronic exposures to the MMEs are often unknown. Therefore, the overall objective of this dissertation was to explore causative and modifying factors of MME toxicity to a resident fish species, the fathead minnow (Pimephales promelas), during chronic, multi-trophic exposures. The representative MME used in this study was the process water effluent (PWE) of a Canadian metal mine, which is released into Junction Creek in Sudbury, Ontario, Canada. Chronic exposure to the MME has been a source of decreased reproductive output in fathead minnows in several previous studies, however, these same studies were not able to determine the potential causal factors of the reproductive impairment. In order to address the overall objective, several laboratory mesocosm studies were conducted, which consisted of three separate components. The first component included exploring several metals (Cu, Ni, and Se; alone and in mixture) that are consistently present in the MME and are known to cause toxicity at fairly low concentrations as potential causes for decreased egg production in fathead minnows. The second component included evaluating the role of decreased food availability (a possible indirect effect of MME in the receiving environment) as a potential cause of decreased egg production in fathead minnows. The third and final component included examining the role of water chemistry [(increased alkalinity and dissolved organic carbon (DOC)] as potential modifying factors of chronic MME toxicity to fathead minnows. In general, my results suggest that the metals present in the MME likely do not contribute directly to decreased reproductive performance in fathead minnows during chronic exposures, under the conditions examined. Instead, the MME appears to decrease food availability, therefore indirectly influence fathead minnow egg production. Furthermore, water chemistry modifications tested in this thesis were not able to entirely mitigate the reproductive effects in fish induced by the MME, although they did improve egg production relative to unmodified MME. Metal concentrations in fish tissues were not influenced by increases to alkalinity or DOC level in the exposure water, suggesting that bioavailability of metals during chronic exposure to metal-mixtures cannot be fully explained based on our understanding of metal complexation with abiotic ligands (inorganic and organic) during single metal or acute exposures. From a regulatory perspective, water chemistry modifications may somewhat improve fathead minnow reproductive performance during chronic exposure to the MME, however the MME would still not be entirely free of effects relative to the uncontaminated water. Future studies should focus on understanding the factors responsible for decreased food availability in MME-impacted aquatic ecosystems, and further explore potential approaches for ameliorating effluent quality.

abstract: Due to analytical limitations, thermodynamic modeling is a lucrative alternative for obtaining metal speciation in chemically complex systems like life. However, such modeling is limited by the lack of equilibrium constant data for metal-complexation reactions, particularly for metal-organic species. These problems were ameliorated estimating these properties from 0-125°C for ~18,000 metal complexes of small molecules, proteins and peptides. The estimates of metal-ligand equilibrium constants at 25°C and 1 bar were made using multiple linear free energy relationships in accordance with the metal-coordinating properties of ligands such as denticity, identity of electron donor group, inductive effects and steric hindrance. Analogous relationships were made to estimated metal-ligand complexation entropy that facilitated calculation of equilibrium constants up to 125°C using the van’t Hoff equation. These estimates were made for over 250 ligands that include carboxylic acids, phenols, inorganic acids, amino acids, peptides and proteins. The stability constants mentioned above were used to obtain metal speciation in several microbial growth media including past bioavailability studies and compositions listed on the DSMZ website. Speciation calculations were also carried out for several metals in blood plasma and cerebrospinal fluid that include metals present at over micromolar abundance (sodium, potassium, calcium, magnesium, iron, copper and zinc) and metals of therapeutic or toxic potential (like gallium, rhodium and bismuth). Metal speciation was found to be considerably dependent on pH and chelator concentration that can help in the selection of appropriate ligands for gallium & rhodium based anticancer drugs and zinc-based antidiabetics. It was found that methanobactin can considerably alter copper speciation and is therefore a suitable agent for the treatment of Wilson Disease. Additionally, bismuth neurotoxicity was attributed to the low transferrin concentration in cerebrospinal fluid and the predominance of aqueous bismuth trihydroxide. These results demonstrate that metal speciation calculations using thermodynamic modeling can be extremely useful for understanding metal bioavailability in microbes and human bodily fluids. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

The goal of this study was to characterize the binding sites on the surface of wheat roots, Triticum turgidum, involved in the adsorption of protons and metals, and quantify the thermodynamic constants needed for a surface complexation model to predict metal binding. The adsorption of protons, Cd(II), Cu(II), and Ni(II) to the root surface as a function of pH and ionic strength in single metal exposure scenarios was quantitatively described using potentiometric titrations, batch metal adsorption experiments, and the least squares fitting program FITEQL. Model predictions from single metal exposures were compared to measured metal adsorption concentrations when roots were exposed to binary and ternary combinations of the metals. Proton dissociation was a function of three discrete monoprotic acid sites on the root surface with log proton dissociation constants of -4.50, -6.23, and -7.37 respectively, upon which varied ionic strength had no effect. The total proton binding capacities for the three sites were 2.58 x 10-4, 1.29 x 10-4, and 2.58 x 10-4 M, respectively. Metal complexation was best described by a two-site model having conditional stability constant log values of 3.04 and 3.30 for Cd(II), 3.21 and 3.25 for Cu(II), and 2.83 and 2.84 for Ni(II) at ionic strength 0.01M. At ionic strength 0.1 M the conditional stability constants log values were 2.37 and 3.36 for Cd(II), 3.11 and 2.56 for Cu(II), and 2.18 and 3.00 for Ni(II). When roots were exposed to binary or ternary mixtures of the metals, the two monoprotic acid single metal model did not provide ideal fits to the data indicating that adsorption in a metal mixture scenario cannot be considered additive and is dependent on the combination of metals present in the exposure environment. The experimentally determined proton dissociation constants and metal stability constants could be used in commercial geochemical speciation programs such as Visual MINTEQ to predict metal adsorption to plants. / Natural Sciences and Engineering Research Council of Canada, The Mining Association of Canada, Ontario Power Generation, Environment Canada.