Orientadores: Melissa Gurgel Adeodato Vieira, Ambrósio Florêncio de Almeida Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-21T04:22:05Z (GMT). No. of bitstreams: 1 GaonaGalindo_LuzStella_M.pdf: 4431618 bytes, checksum: c30f71e68b49dd362a3ed8f70bde5a0a (MD5) Previous issue date: 2012 / Resumo: Neste trabalho foi avaliada a capacidade de remoção de metais pesados comumente encontrados em efluentes e aterros sanitários da região metropolitana de Campinas, mais especificamente o chumbo e o cádmio, utilizando argila bentonítica sódica tipo Fluidgel extraída de jazias do nordeste brasileiro (Boa Vista - PB). A argila Fluidgel foi fornecida na forma comercial, composto de argila natural com a adição de carbonato de sódio. A argila Fluidgel comercial foi submetida a um tratamento ácido, uma vez que o pH natural desta ocasionava precipitação química do chumbo e do cádmio, sendo denominada Fluidgel TA. A argila Fluidgel comercial foi submetida ao tratamento térmico e químico por um período de 24 horas a temperatura de 750 ºC, com o objetivo de melhorar sua estabilidade física, sendo denominada Fluidgel TACal. As técnicas empregadas para a caracterização das argilas visando informações importantes para conhecer a viabilidade das suas propriedades na remoção de metais foram realizadas por termogravimetria (TG), calorimetria exploratória (DSC), difração de raios-X (DRX), espectroscopia de infravermelho (FTIR), área superficial, picnometria a gás hélio, microscopia eletrônica de varredura (MEV)/(EDS) e capacidade de troca catiônica (CTC). As técnicas de remoção estudadas neste trabalho foram a de banho finito e de leito fixo, permitindo avaliar os mecanismos e taxas de remoção dos íons de chumbo e cádmio, a partir da realização de curvas de equilíbrio e cinéticas de cada íon. A adsorção de Cd2+ e Pb2+ foi estudada em sistema estático e dinâmico para as argilas Fluidgel TA e Fluidgel TACal. A argila Fluidgel comercial não foi utilizada no processo de adsorção destes metais, já que seu alto teor de carbonatos ocasionou a precipitação química, além de não apresentar resistência mecânica adequada para aplicação posterior em sistema dinâmico de leito fixo. Os ensaios monocomponentes em sistema estático e dinâmico mostraram que o chumbo tem maior afinidade pelos sítios das argilas. Verificou-se a partir dos resultados obtidos em banho que as quantidades máximas adsorvidas pela argila Fluidgel TA foram de 0,038 e de 0,035 mmol de metal/g de argila, para o cádmio e o chumbo respectivamente. Utilizando argila Fluidgel TACal a quantidade máxima adsorvida de cádmio não foi significativa (0,005mmol/g), sendo para o chumbo de 0,033 mmol de metal/g de argila, podendo-se observar que a capacidade de remoção foi reduzida devido ao tratamento térmico. A partir das curvas de ruptura obtidas nas diferentes vazões verificou-se os diferentes comportamentos, indicando a forte influência da vazão nas resistências difusionais, mostrando que na vazão de 1 mL/min foi obtido um valor baixo de comprimento da ZTM (3,413), bem como uma baixa resistência à transferência de massa para a remoção de chumbo pela argila FluidgelTACal. Para regeneração dos leitos de argila FluidgelTACal foi utilizado o CaCl2 como eluente. A remoção do chumbo obtida até saturação do leito de argila FluidgelTACal, no primeiro ciclo foi de 0,03782 mmol/g, diminuindo no segundo e terceiro ciclos (0,02478 e 0,02060 mmol/g) / Abstract: This study aimed to evaluate the ability to remove heavy metals commonly found in industrial wastewater and landfills in the metropolitan region of Campinas, specifically lead and cadmium using as adsorbent sodic bentonite clay type Fluidgel, which was obtained from Boa Vista, Paraíba, located in the northeastem region of Brazil. Fluidgel was provided as a commercial product processed, being composed of natural clay with sodium carbonate addition. Fluidgel in nature was subjected to acid treatment because the pH of this clay occasioned chemical precipitation of lead and cadmium, it was named Fluidgel TA. In order to improve the physical stability, the Fluidgel clay was also carried out a chemical and thermal treatment for a period of 24 hours at 750 oC and it was named as Fluidgel TACal. The techniques employed for the characterization of clays seeking important information to identify the viability of their properties in the removal of metals and it was performed by thermogravimetry, scanning calorimetry, X-ray diffraction (XRD), Fourier transform infrared (FTIR), Thermal Analysis (TG and DSC), Scanning Electron Microscopy (SEM) with chemical analysis by Energy Dispersive X-ray (EDX), surface area, helium pycnometry, and cation exchange capacity. The removal techniques discussed in this work were realized in the finite bath and fixed bed with the purpose to evaluate the mechanisms and removal rates of lead and cadmium. The adsorption of Cd2+ and Pb2+ was studied in the finite bath and fixed bed systems using Fluidgel TA and Fluidgel TACal. Fluidgel was not used in the process of adsorption of these metals, due to their high content of carbonates caused the precipitation chemistry, besides it type of clay did not has mechanical strength suitable for application in dynamic system of fixed bed. According to obtained results, sorption affinity of clays towards lead is larger than their affinity towards cadmium. The results obtained in a finite bath showed that the maximum amount adsorbed by the clay Fluidgel TA was 0.038 and 0.035 mmol of metal/g of clay, for cadmium and lead, respectively. Using clay Fluidgel TACal the maximum amount of adsorbed cadmium was not significant (0.005 mmol / g), whereas for lead was of 0.033 mmol of metal / g of clay, can be observed that the removal capacity has been reduced due to heat treatment. Fixed bed experiments were developed evaluating the breakthrough curves obtained by different flow rates showed different behavior, indicating a strong influence on the flow rate of diffusional resistance, can observed that using the flow rate of 1 mL/min was obtained a low value of the length of the mass transfer zone (MTZ) of 3.413 and moreover, a low resistance to mass transfer to remove lead with Fluidgel TACal clay. The CaCl2 was used as eluent with the purpose to regeneration of the beds using Fluidgel TACal clay. The study conducted in fixed bed system for lead in cycles of adsorption/desorption showed that in the first cycle was 0.03782 mmol/g, reducing the second and third cycles (0.02478 and 0.02060 mmol /g) / Mestrado / Processos em Tecnologia Química / Mestra em Engenharia Química

Ph.D. / This study consisted of a two-tiered approach to assessment of the effects of EDCs on aquatic organisms, and heavy metal accumulation in the aquatic environment, by integrating field work and laboratory-based experiments. In the last three decades a considerable portion of research in aquatic health and aquatic toxicology has been largely focused on endocrine disruptors, aiming to establish how certain chemicals discharged into the environment, especially organochlorine pesticides (OCs), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), surfactants and plasticisers, can mimic endogenous hormones and thereby induce reproductive abnormalities. The rationale behind the present study was the growing interest in the study of endocrine disrupting chemicals (EDCs) internationally and the aspects of this field of research that are relevant to South Africa’s aquatic environment and its endemic aquatic organisms. The field work was carried out in the Rietvlei Wetland System and consisted of a combination of plant root analysis and application of the South African Scoring System 5 (SASS5) macroinvertebrate index. Three characteristic wetland macrophytes used in the study were Typha capensis, Phragmites australis and Persicaria lapathifolia. Samples of plants, sediment and water were taken from predetermined locations along the wetland system in the Rietvlei Nature Reserve and analyzed for heavy metals by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The heavy metals analyzed in this study; lead, arsenic and cadmium, have also been implicated as endocrine disruptors. The results of heavy metal accumulation in the plant roots reflected a pollution trend along the wetland, suggesting that plant roots are useful bioindicators of contamination in freshwater systems.

There are many types of chemicals/contaminants present in the environment, ranging from synthetic chemicals to trace metals that are required for life. When an organism is exposed to a toxicant, adverse effects are generally first at the sub-organismal level. There is thus a need for physiological and biochemical indicators of organismal health and sub-lethal toxicant effects. A bio-indicator, or biomarker, is a response of a living organism to environmental changes or because of exposure to contaminants. The main objectives of this study were to evaluate and investigate the possible use of biomarkers as indicators of sub-lethal (chronic) effects induced by certain chemicals/pollutants on the Mozambique tilapia, Oreochromis mossambicus. The biomarkers were evaluated as a possible sensitive and reliable measure of effects due to exposure to pollutants. This study was divided into laboratory experiments and field evaluations. The laboratory experiments consisted of the evaluation of biomarkers in exposure studies on 18-24 day old O. mossambicus juvenile fish, to determine the sub-lethal effects induced by chemicals. Acetylcholinestrase (AChE), Ethoxyresorufin-O-deethylase (EROD) and glucose levels acted as the battery of biomarkers evaluated. Thirty (30) juvenile fish were exposed for 24 and 96-hours to sub-lethal concentrations of cadmium, zinc, pentachlorophenol (PCP) and cyanide. For the field evaluation, mature Oreochromis mossambicus of both sexes, were caught during the summer and winter at the Rust de Winter Dam, Loskop Dam and Hartebeespoort Dam. Rust de Winter Dam acted as the reference site. The biomarkers evaluated during the field studies include: erythrocyte AChE, ƒx-Aminolevulinic acid dehydratase (ALAD) and plasma glucose levels, EROD and liver glycogen. The laboratory (exposure) test used during this study was not sensitive enough to obtain reliable, repeatable results. But standardised procedures were established for possible use in future studies. During the field studies, ALAD, EROD and plasma glucose showed significant results and differences between the reference and polluted sites. More accurate results could be obtained at impoundments with higher levels of pollution. Although, erythrocyte AChE and liver glycogen did not show significant results during this study, they might also show more accurate and reliable results at higher levels of pollution. In future, chemical water analysis should be carried out during field evaluation to determine what chemicals/toxicants are present in the aquatic environment. Biomarkers will show the level of effects of the toxicant on the organism, while chemical analysis will determine the specific pollutant present in the water. / Prof. J.H.J. Van Vuren

Growing interest in biofuel production from non-fossil fuel sources has resulted in several studies exploring different raw material sources as feedstock, including many algae species, for large-scale production of biofuel. Algae are promising feedstock due to advantages such as its short growth cycle, high biomass production, and lipid content. However, there are still challenges to overcome in order to use algae for commercial biofuel production. One of these challenges is the requirement for a large quantity of water and nutrients needed for growing large quantities of the algae. This work explores a potential solution to this challenge by studying the possibility of using industrial wastewater to grow algae for biofuel production. However, many industrial wastewaters, including effluents from semiconductor processing plants, are known to contain heavy metals that are toxic to humans and the environment. In this work, the effects of four of such metals ions, As(V), As(III), Ga(III), and In(III) on Chlorella sorokiniana and Scenedesmus obliquus strains were studied. In particular, the heavy metal toxicity on the strains, effects on its growth rate, biomass yield, lipid content and fatty acid methyl esters (FAME) were studied. Also, the effect of controlling pH on growth rate, biomass yield, lipid content, and FAME was studied for Chlorella sorokiniana in the presence of Ga(III). The results of the study confirmed the toxicity of these metals on both strains. However, Ga(III) and In(III) had the highest effect, while As(V) showed the least toxicity to the strains, with Chlorella sorokiniana withstanding concentrations of As(V) as high as 140mg/L. The heavy metals were slightly more toxic to Scenedesmus obliquus compared to Chlorella sorokiniana. In addition, the heavy metals reduced the growth rate of both strains. High percent changes in growth rate (more than 50%) were seen in cultures containing Ga(III) and In(III). Furthermore, concentration measurements with Inductively Coupled Plasma Optical Emission Spectrometer (ICP) before, during, and at the end of the growth period, showed that Scenedesmus obliquus adsorbed higher amounts of the heavy metals compared to Chlorella sorokiniana. Microalgae biosorption of heavy metals limits its end use, hence making Scenedesmus obliquus a less favorable option for this study, but may be a better choice for wastewater treatment applications. The effects of the four metals on the lipid content and FAME profile of Chlorella sorokiniana were studied. The result showed an increase in Chlorella sorokiniana lipid content in the presence of In(III), but a decrease in the presence of As(V) and As(III). The heavy metals had effects on the strain’s FAME compositions. The fatty acid composition included C16:0, C16:1, C16:2, C16:3, C18:0, C18:1, ω-6, C18:2, ω-6, and C18:3, ω-3 accounting for more than 97% of the total FAME composition. Furthermore, controlling the pH of the culture in the presence of Ga(III) at 6.5 led to higher adsorption of the heavy metal, increase in lipid content, but no significant change in FAME composition.

Magister Scientiae - MSc / Male infertility in humans has increased in the last few decades and could be as high as 40%, while up to 50% of these men have ''unexplained'' (idiopathic) infertility. Although newly developed molecular techniques have great value in detecting subtle causes of male infertility, more detailed sperm functional tests are required to identify compromised fertility, especially in a clinical set-up. Since ethical constraints often preclude the pursuit of many basic research questions in humans, non-human primates (NHPs) have been identified as key models in human-related studies. NHPs are often used in studies on male fertility/infertility, IVF or assisted reproductive technology (ART) procedures, male contraception and reproductive toxicology. However, comparing results of NHP and human studies require that techniques used for assessment must be objective, standardized and sensitive to recognize compromised sperm function. The aim of this study was to evaluate standard sperm functional tests and develop new functional tests using NHP sperm, specifically from vervet monkeys (Chlorocebus aethiops), chacma baboons (Papio ursinus) and rhesus monkeys (Macaca mulatta), for application in human and NHP studies and to ultimately develop a basic primate model. The sperm functions investigated included sperm motility, longevity, vitality, DNA integrity, acrosome reaction, and hyperactivation. The sperm functional tests evaluated were: CASA motility analysis; Sperm Longevity test; Eosin-Nigrosin and Hoechst and Propidium Iodide staining, as well as the use of WST-1 cytotoxicity assay for vitality; the TUNEL assay for DNA integrity; Acrosome Intactness Test; and induction of hyperactivation via stimulants. The validity of each test was investigated by inhibiting sperm function through the use of copper sulphate and cadmium chloride. All functional tests were successfully performed across all three species, except the TUNEL assay for DNA integrity, and was further used for validation testing. Validation testing proved that all sperm functional parameters were significantly affected by the highest concentrations of the chemicals (250 µg/ml CuSO4 and 500 µg/ml CdCl2) and if not significant, trends of reduction were seen. The tests employed were therefore sensitive to the inhibitory effect of the metals. By evaluating these established sperm functional tests we found that primates would serve as good models for research study. Furthermore, we optimized and modified techniques for sperm and functional analysis in these three primate species and this study will standardize protocols for use in future studies on male infertility. Additionally, comparing human and NHP sperm function can possibly reveal or explain the high infertility rates in humans.

This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.

Abstract The thesis consists of regional forest condition studies, using different biomonitors. Heavy metal deposition was investigated in 1985–2000 on the basis of the heavy metal concentrations (As, Cd, Cr, Cu, Fe, Hg, Ni, Pb, V, Zn) in mosses in Finland. A comparison on the suitability of mosses, epiphytic lichens and pine bark as biomonitors of heavy metals was also carried. Bark was also used to study the dispersal of emissions from the Kola Peninsula into northern Finland. The occurrence of green algae on conifers in Finland was investigated in 1985 and 1995. Regional and temporal differences were found in the heavy metal concentrations of mosses in Finland. The concentrations of most metals were the highest in southern Finland, and they decreased towards the north. Some of the major emission source had a noticeable effect on the Cu, Ni and Cr concentrations of mosses in the surroundings of the emission sources. The Pb, Cd and V concentrations decreased the most during the study period. Mosses, lichens and bark gave a relatively similar result for heavy metal deposition in Finland. However, the comparisons indicated that mosses are better suited as biomonitors for regional surveys than epiphytic lichens, because the regional differences in heavy metal deposition were more readily reflected by concentrations in mosses than in lichens. Bark is relatively unsuitable for regional surveys due to the small range of variation in the concentrations. Emissions from the Kola Peninsula had a clear effect on the sulphur and heavy metal concentrations of pine bark. The concentrations in bark were at very high levels close to the smelters, but they rapidly decreased on moving towards the west. The effects of emissions were still clearly visible in north-eastern Lapland. There was strong increase in the abundance of green algae on conifers in southern and central Finland during the period 1985–1995. The increase is probably due to following factors: climate warming, and an increase in nitrogen and a decrease in sulphur in their habitats. Half of each biomonitor sample collected in the surveys has been stored in the specimen bank at Paljakka. The storage of samples offers advantages for monitoring purposes. The availability of long-term sample series makes it possible to construct retrospective time series of the pollutants. The specimen bank is to be further developed in the future by establishing a reputation as a storage facility for samples related to forest ecosystems.

Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.

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

M.Sc. (Zoology) / Please refer to full text to view abstract