Graduation date: 1983

No description available.

No description available.

The need to monitor fresh water ecosystems for pollution is increasing, as is the need to develop a biomarker sensitive to a range of environmental insults. Recently, heat shock proteins have been identified as possible biomarkers of environmental contamination. However, evaluation as to their use as a biomarker of metal contamination in fish species endemic to Southern Africa is limited. The purpose of this study was to identify what members of the 70 kDa family of heat shock proteins (Hsp70) were present in the liver of Oreochromis mossambicus, and if their accumulation was altered after short-term (96 hour) exposure to aqueous copper. In addition to copper exposure, the effect of acclimation media was also examined. Tissue-level analysis was done by means of histological examination so as to determine if alterations in the accumulation of the Hsp70 family had a marked effect on the structural integrity of the liver. Specimens of Oreochromis mossambicus acclimated in either aged tap water or borehole water were placed in flow-through systems and exposed to either 10% or 20% of the LC50 value of cupric chloride for a duration of 96 hours. Control groups were run in conjunction with the exposure groups so as to set control values by which to compare. Heat shock protein analysis was done by Western blotting after separation of hepatic proteins by SDS-PAGE. For the purpose of histological analysis, representative samples were randomly selected. Analysis of the hepatic heat shock protein 70 family identified the presence of three (3) members, each of a different molecular weight. These included members of 70 kDa (Hsp70), 74 kDa (Hsp74) and 76 kDa (Hsp76). In addition to these findings, it was found that Oreochromis mossambicus accumulated high levels of particular members of the heat shock protein 70 family under unstressed conditions, affording the fish adaptability to environmental extremes. Furthermore, individuals acclimated in aged tap water showed decreased Hsp76 accumulation after exposure to sub-lethal copper concentrations, whereas those individuals acclimated in borehole water retained relatively high levels of Hsp76. Additionally, it was shown that the hepatic structure deteriorated in those individuals acclimated to the aged tap water after copper exposure, with observed increases in vacuolation, number of macrophage centres present and the occurrence of intracellular golden-brown granules. However, there was little change from the already-altered hepatic structure of those individuals acclimated in borehole water, with conspicuous golden-brown granules the most obvious histopathological condition present. Histological examination therefore proved to supplement the heat shock protein results obtained. This study thus concluded that a decrease in the accumulation of the Hsp70 family resulted in a negative organismal response, initiating deleterious alterations in the hepatic structure. Additionally, this study concluded that past water quality has a marked effect on a given biomarker response, and should be taken into careful consideration when conducting biomarker studies. / Prof. J.H.J. Van Vuren

Ph.D. / Heavy metals like copper are toxicants commonly found in mining, industrial, agricultural and waste effluents in South Africa¡¯s riverine systems. In high concentrations these metals could have a negative effect not only on river systems, but also on fish populations. It is therefore important to determine the effect of these heavy metals on the fish at an early stage, before the fish show any sign of being exposed to the pollutants and before any bodily functions are affected. It is known that polluted water, especially heavy metal pollution like copper, can cause physiological and biochemical alterations in fish. Histopathology is a mechanism, which can provide an indication of fish health by determining early injury to cells and can therefore be considered an important tool to determine the effect of pollutants like copper on fish tissue. At present very little is known about the effect of toxicants on the histology of fish tissue of freshwater fishes in South Africa. This is important in gonads, where many variables not only in different fish species, but also in the number of developing germ cells in the testes exist. The large number of fish species and the variation in their anatomy and reproductive patterns make knowledge of the anatomy of all fish species difficult. In order to perform a histopathological analysis, it is important to be familiar with the normal histology of the fish organs that are being investigated. It was therefore necessary to describe the histology of selected organs in South Africa¡¯s freshwater fish species. The Mozambique tilapia, O. mossambicus was chosen as test organism and copper was selected as toxicant. The normal morphology and histology of the testes of O. mossambicus were described in order to serve as a baseline study and to provide histological parameters for further toxicological work on the gonadal histopathology of fish. Fish were exposed to sub-lethal concentrations of copper (CuCl2) for a short-term (96 hours) in experimental flow-through systems under controlled laboratory conditions (23 ¡¾ 1¨¬C). An external investigation of each of the 30 control and 40 copper exposed fish was performed. Standard length, body mass and gonadal mass were recorded and the maturity of each fish was obtained histologically. A light and electron microscopic examination was performed in order to describe the histology and histopathological changes. The histopathological changes were obtained by comparing the histology and ultrastructure of the testis of the control group with the information obtained for the testes that were exposed to copper. Fish obtained from the control group that were run simultaneously with every exposure group were used to describe the normal histology of the testes of O. mossambicus. The aim was to describe the normal morphology and histology of the testes of O. mossambicus so that the information obtained during this investigation could serve as a baseline study when dealing with toxicity studies. The different germ cells of the spermatogenesis process were also described with a view to assess not only the reproductive pattern, but also to provide histological parameters which might form a basis for any toxicological work on the gonadal histopathology of fish. Identification of reproductive biomarkers requires an understanding of all the principal cellular components of the testes and their normal range of variation. The cellular components that were described are the Sertoli and Leydig cells as well as the primary and secondary spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa. The information on the GSI, gonad reproductive stages as well as the histological examination provided the baseline which was needed for comparison of the testes of fish that were exposed to copper. The cellular and sub-cellular responses at light and electron microscopic level were examined to determine early cell changes and damage in the testes which were caused by different sublethal exposures of copper. There were no differences in the gross structure of the testes of the control group and the group that were exposed to copper. The testes of the fish that were exposed to 0.16, 0.40 and 2.0 mg Cu/§¤ all revealed disorganisation of the lobules, disintegration of the spermatogonia as well as disintegration of the interstitial tissue. The exposure of fish to sublethal concentrations of copper induced histopathological changes in the testes of all three groups of fish that were exposed to copper. The results also showed that the intensity of the histopathological changes increased with an increase in the concentration of copper. As a biomarker of exposure to toxicants, histopathology represents a useful tool to asses the degree of pollution. The results from this investigation showed a decrease in the GSI after being exposed to copper, when compared with the control groups, which were not exposed to copper. The information on the effect of copper on the GSI, gonad reproductive stages as well as the histological examination also provides information on gonadal health and maturational stage. This information however provides structural, rather than functional information regarding gonadal health. The results showed that the GSI and gonadal staging should be used together with the gonadal histopathology in order to provide a baseline for comparison when dealing with other toxicological studies. The information obtained during this investigation showed that fish histology and histopathology could be used as a biomarker to provide information on the effect of heavy metals on fish health. It is however suggested that the information obtained during this investigation should be extended to field investigation in order to be validated as a reliable histopathological biomarker. / Prof. J.H.J. van Vuren Prof. G.J. Steyn

Thesis. 1975. Ph.D.--Massachusetts Institute of Technology. Dept. of Earth and Planetary Sciences. / Bibliography: leaves 138-143. / by William Sunda. / Ph.D.

Copper toxicity was investigated using the green alga, Selenastrum capricornutum. Two major areas were investigated: the forms of copper which are toxic to growth of the alga and factors that can change the concentration of free copper, Cu2+ (aq), in the growth medium during the logarithmic growth of the organism. This research has shown that S. capricornutum is sensitive to the free Copper ion, Cu 2+ (aq), irrespective of the total copper concentration.

Contamination of aquatic ecosystems (for example, dams, lakes, rivers, streams) with heavy metals (according to McDonald and Wood (1993), the metals that are of greatest concern in fresh waters are Hg, Pb, Cd, Cu, Zn, AI, Mn and Cr (approximately in order of decreasing toxicity)) has been receiving increased worldwide attention, and the literature has many publications on the topic. These amplified levels of contamination have become a global problem in both developed and developing countries and the impact of them on aquatic environments is severe due to the inability of water to disperse contaminants. To meet this threat, ecotoxicologists face a difficult challenge on how to assess the long-term impacts of metals which have already been released into the environment and which persist in sediment, water and biota. In recent years, the field of biomarker study has evolved and expanded rapidly to address this void. Biomarkers examine molecular and biochemical responses in exposed individuals in an effort to assess the status of an impacted environment and they can also serve as a basis for a Rapid Risk Assessment of Fish Health (RRAFH) employed by water quality managers. This effort to incorporate biomarkers into RRAFH research will eventually be worthwhile. Moreover, with this multidisciplinary approach, cause-effect relationships may be examined. It was in this context that the present study evaluated three biomarkers (metallothionein (MT), Na+K+ ATPase and heat shock/stress protein 70 (HSP 70)) to determine the integrated physiological responses in Oreochromis mossambicus to one metal (copper (Cu)) at a sub lethal level over the short term (96, 48, 24, 16, 12, 6, 4 and 2 h respectively). The information gained from these tests was valuable in suggesting what the baseline levels of biomarkers in O. mossambicus would be, as studies of metabolic adjustments to stress in fish have been focused on salmonids. The demand for sensitive, specific and rapid biological assays was also addressed by the development of an HSP 70 ELISA. This assay with, with additional refinement, will also prove to be a valuable tool in the management of Cu pollution in this species. Evaluation of the biomarkers in the gills of O. mossambicus indicated that this species has relatively high levels of MT in their gills compared with other species of fish. Exposure to sub lethal Cu concentrations [Cu] caused fluctuating, but not significant, levels in Na+K+ ATPase and HSP 70 during the exposure time, and the actual [Cu] in the gill seems to be the biggest factor when interpreting results. Significant decreases were found in MT levels in the gill tissue at 2 h and 12 h of exposure. HSP 70 results also indicated non-significant decreases to Cu exposure. Data from the gills analysed in the current study suggest that down-regulation in the biomarker response might be a better indicator of Cu exposure or effect in O. mossambicus. These data also suggest that further studies should rethink the amount of Cu that the fish under investigation would find sub-lethal as this metal seems to be very well regulated in this species of fish and seems frequently to non-significantly effect the chosen biomarkers. Evaluation of the biomarkers in the liver of O. mossambicus indicated that MT levels present in the liver most likely fall within the in range of normal baselevels for tilapia living in a Cu-rich environment and that HSP 70 in the liver only responds significantly to increased aqueous [Cu] at 16 h of exposure. This study also confirms current knowledge that the toxicity assessment of any pollutant, including Cu, cannot be exclusively based on one biomarker and that the true value of these biomarkers only becomes evident when they are used not as individual indicators but as integrated responses that influence one another. All these results furnish useful data for future research into the sub lethal effects of a pollutant such as Cu. The next step would be to test the usefulness of these physiological methods for detection of similar responses in natural fish populations inhabiting Cu polluted waters. This will provide a database relating the presence of Cu to biological effects at a molecular level. / Prof. J.H.J. Van Vuuren

Copper exposures of 400 μg/L for 5,10 and 15 days resulted in no significant differences in antibody titers of largemouth bass, Micropterus salmoides injected with Aeromonas hydrophila compared to control-injected bass. Twenty days of exposure did significantly increase titers. The control group had significantly lower antibody titers than either control-injected or copper-exposed.

The chemistry of copper sulfate dosing for effective algal kill was investigated in laboratory studies with Selenastrum capricornutum and Microcystis aeruginosa. The goal was to give practical information for water treatment plants operators who often must apply copper sulfate for algae control in impoundments. Research focused on the theoretical relationships involved in copper dosing, with emphasis on the applicability of these relationships to Virginia freshwater. Concentrations of alkalinity and humic acid were varied to study their effect on percentages of ionic copper available. Long-term (2 weeks) growth-rate studies were performed to study the effect of humic acid on the toxicity of ionic copper to algae. Some protection of algae from copper by humic acid was observed. As the humic acid concentration was increased, more ionic copper was complexed and, thus, was unavailable for algal kill. The only case in which humic acid was not demonstrably protective was at a low concentration (4 mg/L TOC). It is possible that in a long-term test, low humic acid levels may not protect algae, because the copper may be released from the humic acid complex. Alkalinity and the humic acid concentration (TOC) both complex ionic copper, however alkalinity appears to be more important in determining available ionic copper in freshwater. At alkalinities greater than 10 mg/L (as CaCO₃) and humic acid less than 8.0 mg/L, alkalinity predominated in determining available ionic copper. A short term (4-hour) procedure involving measurement of oxygen production by algae exposed to copper was also used to determine the effect of humic acid on ionic copper toxicity to algae. The data showed that short-term tests were best used to predict trends shown in the long-term tests. Both Selenastrum capricornutum (a green alga) and Microcystis aeruginosa (a blue-green alga) were protected from ionic copper by humic acid. Oxygen production was found to be best used to approximate toxic concentrations of a suspect compound, as opposed to being used to precisely determine toxic concentrations. / Master of Science