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National Water Quality Criteria intended to protect aquatic life and their uses from the adverse effects of pollutants may not be appropriate due to site-specific factors that alter chemical bioavailability. The Indicator Species Procedure may be used to derive site-specific criteria in order to account for differences in site-specific bioavailability. This procedure was implemented using zinc for three chemically different site (river) waters. The purpose of this study was to quantify the bioavailability of zinc in each site water and correlate results to water quality parameters and/or zinc speciation. Results demonstrated that national criteria for zinc accurately predicted the experimentally derived site-specific values within a factor of two when adjusted for water hardness. Particulate forms of zinc were shown to be biologically unavailable under conditions tested.

Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic network failure to develop in a concentration-dependent time window between 50% and 90% activity loss. Investigation of entry routes suggested the L-type but not N-type calcium channels to be the main entry pathway for zinc. Data are presented implicating the chloride channel to be an additional entry route.

M.Sc. / Heavy metals occur naturally in the environment and are found in varying levels in all ground and surface waters. Some heavy metals are essential elements for the normal metabolism of organisms, while others are non-essential and play no significant biological role. Anthropogenic activities do, however, cause an increased discharge of these metals into natural aquatic ecosystems. Aquatic organisms are exposed to unnaturally high levels of these metals. Fish are relatively sensitive to changes in their surrounding environment. Fish health may therefore reflect and give a good indication of the health status of a specific aquatic ecosystem. Early toxic effects of pollution may only be evident on cellular or tissue level before significant changes can be identified in fish behaviour or external appearance. Histological analysis appears to be a very sensitive parameter and is crucial in determining cellular changes that may occur in target organs, such as the liver. The liver is a detoxification organ and essential for both the metabolism and excretion of toxic substances in the body. Exposure to heavy metals may cause histological changes in the liver. Fish liver histology could therefore serve as a model for studying the interactions between environmental factors and hepatic structures and functions. In this study, the effect of two heavy metals, cadmium (Cd) and zinc (Zn), on the histology of the liver of the South African freshwater fish species, Oreochromis mossambicus, was investigated. The aim of this study was to determine the toxic effect of cadmium and zinc on the histology of the liver, by identifying significant histological changes in the liver tissue, after exposing the fish to two concentrations of a mixture of cadmium and zinc, over both short and long-term exposure periods. Seventy two, adult O. mossambicus specimens were selected for the study. Two experimental exposures were executed under controlled conditions by means of a flow-through system in an environmental room. For each of the two exposures, twenty-four fish were exposed to different concentrations of cadmium and zinc. The remaining twenty-four specimens were used as a control group. The two respective metal concentrations selected for each exposure were 5% and 10% concentrations of both cadmium and zinc calculated from known LC50 values for cadmium chloride and zinc chloride. Liver samples were fixated in 10% neutrally buffered formalin and prepared for light microscopy analysis using standard techniques for Haematoxylin and Eosin (H & E) and Periodic Acid Schiff (PAS) staining. The liver histology of all seventy two specimens - including the forty eight exposed specimens and twenty four control specimens - were analysed, compared and documented. Although histological analysis can provide a clear indication of the degree of damage caused in the tissue(s) or organ(s) of exposed specimens, the need arises to quantify the histological results in studies where the effects of the exposing substance(s) are compared, to illustrate the possible decrease or increase in histological changes over time or the effect of two different concentrations of the same exposure substance on the histology of the liver. The histological results in this study were quantified in terms of a histological index. An index value representing the specific histological characteristics of the liver was assigned to each individual specimen indicating either normal histological structure (index value of 0-2) or a possible pathological response (index value of 3-6). Histological changes were identified in specimens exposed for 12, 18, 24, and 96 hours to both the 5% and 10% concentrations of cadmium and zinc, indicating a toxic response after the short-term metal exposures. Similar histological changes were identified in both the 5% and 10% exposed livers. These histological changes included hyalnization, vacuolation, cellular swelling and congestion of blood vessels. The liver histology of fish exposed over a long-term period of 672 hours, did, however, appear relatively normal in both the 5% and 10% exposure groups, indicating an adaptative, regenerative response. According to the results obtained, it was clear that exposure period did influence the degree of histological changes identified. The two metal concentrations did however seem to have similar histological effects and no definite variation could be identified in terms of 5% and 10% metal concentrations used. It can therefore be concluded that low concentrations of cadmium and zinc exposure caused histological alterations in the livers of exposed specimens and therefore allows the liver of O. mossambicus to be used as a biomarker of prior exposure to cadmium and zinc. / Dr. G.M. Pieterse

The decline in mussel fauna of the Powell River has been attributed to extensive mining activities in headwater streams of Wise and Lee counties, Virginia. Surface mining causes erosion, sedimentation, and contamination of water with toxic substances from coal washing and waste piles. Historical water quality data of the Powell River have documented concentrations of metals at levels determined to be toxic to molluscs, specifically zinc (Zn) and copper (Cu). Acute toxicity tests with Zn, Cu, and mixtures of these two metals were conducted with glochidia and juvenile freshwater mussels. Effects of varying conditions such as water source, temperature, length of exposure, species, and lifestage were determined. Additionally, the effects of Powell River sediment on survival and growth of juvenile mussels were evaluated. The Cumberland moccasin shell (Wedionidus conradicus) was the most sensitive species tested, with 24-hr and 48-hr LC50 values for glochidia ranging from 423 to 725 μg Zn/L. Glochidia of the pheasantshell (Actinonaias pectorosa) exhibited LC50 values from 274 to 2886 μg Zn/L, depending on test conditions. Similar results were obtained for glochidia of the rainbow (Villosa iris), with LC50 values ranging from 577 to 4123 μg Zn/L. Juveniles were more sensitive, with 48-hr LC50 values ranging from 360 to 1885 μg Zn/L for A. pectorosa, and 339 to 1122 μg Zn/L for V. iris, depending on test conditions. Juvenile mussels were affected by Zn at lower concentrations as evidenced by valve gaping and a swollen and nonresponsive foot. Copper was 5 to 15 times more toxic than Zn, with 48-hr LC50 values ranging from 52 to 156 μg Cu/L, and ECS5O values ranging from 25 to 115 μg Cu/L for juveniles of A. pectorosa. Copper appeared to exert a different toxic mode of action, as evidenced by closed valves and reduced siphoning. In general, sensitivities of early life stages of mussels to Zn and Cu increased with higher temperature, soft water, and length of exposure. At certain concentrations, Zn seemingly had an antagonistic effect (less than additive) when mixed with Cu. This effect was evidenced by reduced mortality of juveniles in Cu solutions when Zn was added at concentrations of about 400 to 800 μg/L. However, this antagonistic effect was not reflected in the percent of juveniles affected, which increased continuously with increasing metal concentrations. Glass beads were found unsuitable as a control substratum for use in sediment testing with juvenile mussels. Results of sediment tests indicated that sediment in some areas of the Powell River may be toxic to juvenile mussels, and that toxicity may be linked to water quality. After 10 days, survival of juveniles in sediment collected downstream of a coal processing plant was significantly lower than survival in sediment upstream of the plant (p = 0.01). Further, survival in sediments with dechlorinated tap water was significantly higher than survival in sediment with river water (p = 0.0002). After 20 and 30 days, survival was similar among sites and water types. High metal concentrations in the river water appeared to contribute to toxicity, because juveniles in tap water displayed consistently better growth, and initially better survival than juveniles in river water and sediment. This toxicity was not apparent in sediments collected from the same sites less than two months later, suggesting the character of the sediments may change as new suspended sediment is deposited. The USEPA water quality criteria for Zn (adjusting for water hardness) are 174 μg/L (acute) and 158 μg/L (chronic), whereas copper criteria are set at 28 μg/L (acute) and 18 μg/L (chronic). Powell River water samples collected during 1991 contained concentrations of Zn and Cu exceeding these criteria, as well as concentrations shown to have adverse effects on mussel populations. Results suggest that some metals are introduced into the river system in runoff, whereas Cu is being introduced as an episodic event. Intensive monitoring of water quality is needed to identify specific sources of metal pollution. If levels of heavy metal concentrations remain high, then the declining mussel populations of the Powell River will not recover, and endangered species may be extirpated from Virginia. / Master of Science

Chronic toxicity of zinc to Pimephales promelas was estimated by conducting replicate static and static-renewal short-term embryo-larval tests and static-renewal seven-day larval tests. The two test methods were highly reproducible. Daily renewal of test solutions had little effect on the toxicity of zinc, however, the stage of development at which exposure was initiated affected the sensitivity of the toxic endpoints measured. The most sensitive and reproducible endpoint in the embryo-larval tests was survival of viable (non-deformed) larvae and in the seven-day larval test was growth of the larvae, which was slightly more sensitive than the embryo-larval test endpoint. The estimated MATC of 0.18 and 0.15 mg/L mean total and mean soluble zinc, respectively, compared well with published results. Because of its advantages and similar sensitivity, the short-term embryo-larval test was recommended for estimating chronic toxicity.

Splenic and lymph node lymphocytes from Balb/C mice were activated in vitro by the heavy metal cations, Zn⁺⁺ and Hg⁺⁺, as noted by the several-fold increases in ³H-thymidine incorporation at 144 hours of culture. Optimal mitogenic concentrations of Zn⁺⁺ and Hg⁺⁺ were 200 μM and 10 μM, respectively. Data from experiments in which three different methods were used to enrich for either T or B splenic lymphocytes, i.e. cell passage over nylon wool columns, use of athymic Nu/Nu mouse spleen cells, or cell lysis with monoclonal anti-Thy-1 antibody plus complement, suggested that Zn⁺⁺ and Hg⁺⁺ were mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron followed by cell passage through nylon wool eliminated the lymphocyte responses to Zn⁺⁺ and to Hg⁺⁺. Moreover, addition of these macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn⁺⁺ and Hg⁺⁺ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce gamma interferon. Furthermore, Zn⁺⁺ induced low levels of natural killer activity in spleen cells. In contrast to spleen and lymph node cells, thymocytes and bone marrow lymphocytes did not respond to either cation under standard culture conditions. However, when cultured in the presence of E. coli-derived lipopolysaccharide (LPS) and 2-mercaptoethanol for 144 hours, thymocytes were activated by Zn⁺⁺ (200 μM) but not by Hg⁺⁺. Quantities of LPS as low as 1.0 ng/ml satisfied this culture requirement. Purified interleukin 1 could not replace the helping activity mediated by LPS. Thymocyte subpopulation studies showed that Zn⁺⁺ activated enriched peanut lectin receptor-negative mature thymocytes, but LPS was required for the response. Spleen cells from mice, intraperitoneally injected with ZnCl₂ for 7 to 14 days, were not activated in vivo as assessed by ³H-thymidine incorporation in vitro, nor did they display enhanced responses to T-cell or B-cell mitogens. However, zinc administration had negative effects by decreasing spleen cell numbers by 31% and thymic weight by 59%. A theoretical model is presented in which Zn⁺⁺ and Hg⁺⁺ may mediate their stimulating effects in vitro by altering histocompatibility "self" structures on the surface of lymphocytes and macrophages via interactions with sulfhydryl groups on these structures to which T lymphocytes with receptors for "altered self" structures respond with proliferation or cytotoxicity.