Since many estuarine zones are impacted by copper contamination, there is an on-going effort to develop Biotic Ligand Models (BLMs) predicting copper toxicity in transitional environments. In the first stage of this project, a critical analysis of the BLM framework identified some aspects of the model that required further investigation. In particular, a BLM for estuaries needed (a) a better characterization of the dissolved organic matter (DOC) and its effect on copper availability, and (b) the inclusion in the model’s equation of a salinity-correction factor modulating the relationship between copper accumulation on the biotic ligand and toxicity. The first issue was addressed by modelling the data produced using a Chelex resin method to determine the labile fraction of copper in samples of mixed riverine and estuarine waters. A refined and simplified BLM equation was then presented, accounting for both the DOC characteristics and the relevance of the osmotic gradient in modulating the relationship between copper accumulation and toxicity. A critical analysis of the literature on copper toxicity and salinity led to the hypothesis that copper-exposed fish are more sensitive to osmotic stresses, as copper interferes with their osmoregulatory pathways. In particular, the cytosolic isoform-2 of the enzyme carbonic anhydrase (CA2) was identified as an osmotic effector protein targeted by copper and involved in osmotic stress response pathways, hence representing a mechanistic link between the combined effects of copper exposure and osmotic stress. To test this hypothesis, two in vivo studies were performed, using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) and applying different rates of salinity changes as a way of dosing osmotic stress. The results showed a disturbance in plasma ion homeostasis after the salinity transitions, but notably the magnitude of the disturbance was greater in the copper-exposed individuals, suggesting a sensitizing effect of copper on the responses of fish to osmotic stress. Gene expression data demonstrated that CA2 is targeted by copper and confirmed the role of the enzyme in osmoregulatory pathways, as further supported by a promoter analysis of the gene coding for zebrafish CA2, which revealed the presence of osmotic-stress related elements. Overall, these results suggest that CA2 is an osmotic effector protein whose response can be activated by a medium level of osmotic stress through a combination of transcriptional and post-translational control circuits.

<p> In this study the influence of several water chemistry parameters on the toxicity of Ni to Daphnia pulex in soft water were tested. A reconstituted soft water (pH 7.8, hardness 31.5 mg/L CaCO3) was used as the basis for culture and testing. Daphnia pulex was chosen as a typical cladoceran, one which can be acclimated to very soft water. An understanding of the influence of water chemistry on Ni toxicity in soft water is relevant because metals have higher bioavailability in soft water. The 48h EC50 in the reconstituted soft water (RSW) was 974 μg/L (16 μM) dissolved Ni (95%CI 830- 1081 μg/L). The following factors were examined for their potential for modifying Ni toxicity: Ca, Mg, Na, K, Cl, pH (3 different approaches used) and natural organic matter (NOM, 2 sources tested). Both Ca and Mg protected against Ni toxicity and the relative effect was greater for Ca. Varying the concentrations of Na, Cl or K did not alter the toxicity of Ni. Tests at different pH showed that as pH increased, Ni toxicity decreased. When the test solution pH was adjusted with the organic buffer 3-morpholinepropanesulfonic acid, there was a clear correlation between increasing pH and increasing EC50. The pH tests using bicarbonate to adjust pH did not show this relationship as clearly. Both types of NOM showed a protective effect on Ni toxicity with Nordic Reservoir NOM having a 4 fold greater effect than that of Suwannee River NOM. This research illustrated that the effect of alterations in water chemistry were generally as predicted within the context of the biotic ligand model (BLM) approach. The data provide the information required to develop a BLM for the acute effects of Ni in soft water.</p> / Thesis / Master of Science (MSc)

Relative to other metals, little is known about lead toxicity in fish. The use of predictive models such as the biotic ligand model (BLM) has been limited, a situation that is at least partially due to the lack of understanding of lead's acute toxic mechanism and characterization of key binding sites involved in this toxicity. Using the rainbow trout as a model species, the acute toxic mechanism for lead was found to be ionoregulatory disruption. While having no apparent respiratory or acid/base effects, Pb exposure resulted in significant ionoregulatory impacts that affected Ca2+ homeostasis, as well as Na+ and Cl- balance. Active Ca2+ uptake by the gills obeyed typical Michaelis-Menten kinetics, and Pb interacted in a competitive fashion with the uptake process. Exposure to increasing waterborne Pb concentrations resulted in significant increases in Km value while Jmax showed little or no change. A slower, non-competitive interaction occurred after prolonged Pb-exposure, evidenced by a significant reduction of high-affinity Ca2+ -ATPase activity that correlated well with branchial Pb accumulation. Conversely, calcium had a protective effect against branchial Pb accumulation, this relationship being predominately competitive in nature. Voltage-independent calcium channel blockers La3+, Cd, and Zn significantly reduced gill Pb burden while the voltage-dependent, L-type calcium channel blockers, nifedipine and verapamil, did not, suggesting Pb enters fish by a similar mechanism to that of Ca2+. Stimulated stanniocalcin release by CaCl2 injection also significantly reduced branchial Pb accumulation. Based on the evidence presented in this thesis, it is apparent that acute Pb toxicity occurs by ionoregulatory disruption. It is likely that Pb shares a similar uptake pathway as that for Ca2+ and that resulting accumulation results in disruption of Ca2+ influx as well as Na+ and Cl- balance. This study has provided data essential to the characterization of key binding sites involved in Pb toxicity, and ultimately, validates the development and application of predictive models such as the BLM. / Thesis / Master of Science (MSc)

<p>Dissolved organic matter (DOM) in natural waters is a heterogeneous mixture of organic molecules with direct and indirect influences on aquatic organisms. Although the influences are usually attributed to DOM quantity (quantified as Dissolved Organic Carbon, DOC), the role of quality (optical and binding characteristics obtained by absorbance and fluorescence spectroscopy and potentiometric titration, respectively) is not well-understood. Through an initial critical review of the literature, followed by experimental geochemical, toxicological, and physiological investigations, a number of conclusions were reached that improve our knowledge in this area. Freshwater DOM sources exhibit source-dependent protection against metal toxicity, in particular copper (Cu). Generally, for this indirect effect, optically-dark terrestrially-derived or allochthonous DOMs offer better protection than microbially-derived or autochthonous sources. Linear regressions revealed that the better ameliorative effect is principally related to a higher aromatic composition (specific absorption coefficient, SAC₃₄₀) and a greater humic-like fluorescent component as quantified by parallel factor analysis (PARAFAC). In addition, the allochthonous DOMs were shown to have relatively higher magnitudes of titration index (TI), a new summary of chemical reactivity of DOM molecules obtained by titration analysis, and closely related to optical properties. TI was strongly correlated with SAC₃₄₀, suggesting greater binding capacities for DOM molecules with higher SAC₃₄₀. Consequently, a method for incorporation of SAC₃₄₀ as a DOM quality measure into the Biotic Ligand Model (BLM) was developed which improved Cu toxicity predictions in experimental tests with natural DOMs. For direct effects, two basic physiological functions (Na⁺ metabolism and nitrogen excretion) of the adult water flea (<em>Daphnia magna</em>, a cladoceran crustacean) and the zebrafish (<em>Danio </em><em>rerio</em>, a teleost fish) were investigated at circumneutral and acidic pH (≥ 7 and ~ 5, respectively). Three previously characterized, chemically-distinct natural DOM sources as well as a commercial humic acid (AHA) were examined. Regardless of the pH conditions, while Na⁺ regulation of <em>D</em>. <em>magna </em>remained unaffected by the presence of all DOMs, the passive diffusive efflux of Na⁺ in zebrafish was attenuated, indicating ameliorative action against unidirectional Na⁺ loss. In addition, only a distinct allochthonous-autochthonous DOM source stimulated the Na⁺ uptake rate of zebrafish at low pH. Ammonia excretion rates of <em>D</em>. <em>magna </em>were reduced at circumneutral pH by the most highly coloured, allochthonous DOM, and at low pH by all three natural DOMs. Both in <em>D. magna </em>and in <em>D. rerio</em>, urea excretion rates at both pH conditions were not influenced by the presence of the various DOMs, and the same was true for ammonia excretion in the zebrafish. A commercially prepared humic acid (Aldrich humic acid, AHA) exerted anomalous actions relative to those of natural DOMs, and does not appear to be representative of their normal effects. In contrast to the actions of DOM in detoxifying metals, these direct effects of DOMs on freshwater organisms appeared highly unpredictable with variable dependencies on the source, pH and species. This thesis has advanced our understanding of the relationships between DOM quality and its indirect and direct effects on aquatic organisms, and points to new directions for future work.</p> / Doctor of Philosophy (PhD)

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.