Heavy metal pollution in water and soil is a serious concern to human health and the associated environment. Some heavy metals have bio-importance but the bio-toxic effects of many of them in human health are of great concern. Hence, there was a need for proper understanding of the concentration levels of these heavy metals in ground water and soil around the community residing in the vicinity of the defunct mine. Mining has become prominent in this area because of the existence of copper lodes, veins and veinlets. It was therefore necessary to assess these selected metals associated with copper mining as their concentration has a tendency to affect the environment and human health. The objective of this study was to establish the levels of lead (Pb)-zinc (Zn)-copper (Cu)-arsenic (As)-nickel (Ni) metals in ground water and soil associated with an old copper mine in the vicinity of the township and to compare them with the South African and international standards in order to safeguard the health of the community using such water for drinking purpose. Clean sampling plastic bottles were used to collect water from five water boreholes being used at present. Water samples were filtered using membrane filtration set LCW (0.45 μm). The samples were digested sequentially with different procedures for the total metal concentration. Concentrations of four metals commonly associated with Cu mining were examined at five different water boreholes which are used for drinking and industrial purposes. Flame Atomic Absorption Spectrophotometer (Perkin Elmar S/n 000003F6067A, Singapore) was used to analyze metals in water samples at Eskom Ga-Nala Laboratory: pH, electrical conductivity and turbidity were analyzed using an auto titrator meter (AT- 500,Japan), conductivity meter (Cole-parmer® YO-19601-00) and turbidity meter (AL 250TIR, Agua lytic, German) respectively. Soil samples were collected from the selected areas where human health is of a serious concern, and a hand held auger drill was used to recover samples, while shovels were used to prepare the sampling area. The samples were sieved up to 63.0 μm particle size and digested with aqua-regia. Flame Atomic Absorption Spectrophotometer (Model: AA400; Year: 2008; Manufacturer: Perkin Elmer; Germany; Serial no: 201S6101210) was used at the University of Venda Laboratory to analyze soil from the study area for possible heavy metal contamination due to the defunct Cu mine in the area. v The results showed variation of the investigated parameters in water samples as follows: pH, 6.0 to 7.51; EC, 70.0 to 96.40 μS/cm and turbidity, 1.05 to 4.56 NTU. The mean concentration of the metals increased in the followed order: Pb<Cu<As<Ni. Ni is the most abundant in the ground water determined with value of (6.49 μg/g). The observations have confirmed that most ground water contains an appreciable quantity of Ni. The mean value of As in water is (4.20 to 4.84 μg/g), Pb and Cu have (2.13 to 2.58 μg/g) and (1.52 to 2.52 μg/g) respectively. For soil samples, the mean concentration of the metals increased in the following order: Pb<Cu<Zn<As<Ni. Pb ranged from (0.023 to 0.036 μg/g) followed by Cu (0.28 to 0.45 μg/g) then Zn (0.026 to 0.053 μg/g), the mean range of As in soil ranged from (0.054 to 0.086 μg/g). However, some studies show much higher contamination of As from the natural sources and Ni with (0.057 to 0.144 μg/g) lastly. Accumulation of heavy metals in soil is of concern due to their toxic effects on human and animals. The quality of ground water from the five boreholes studied was satisfactory with turbidity (T), electrical conductivity (EC) and heavy metals (HM’s) below the WHO limit. The water therefore may, according to the WHO Standards be safely used as a drinking water. The concern lies on pH which was slightly (0.5) below the standard. There is a serious need to monitor the ground water which is now used for drinking purposes. This study revealed that heavy metal pollution in soil from the abandoned Cu mine in Musina is a threat to the health of the community. Although pollution was between medium and low in the contamination index, it is therefore important for the Musina Municipality or mine owner of Musina (TVL) Development Co Ltd copper mine to advocate possible remedial actions which will safeguard the environment and human health. The tailing at Musina’s old Cu mine have high pH and they lack normal soil stabilization processes, as a result the tailing does not develop a good plant cover. Pollution of the ground water resources is also evident in the study area where there is seepage or ingress of polluted water to the underground aquifers. Small-scale mining in Musina is causing further degradation to the environment but it supports the South African Waste Hierarchy by promoting the reuse and recycling of the tailing and mine dumps for the production of bricks. Mine workers are exposed to the above mentioned toxic heavy metals daily. Medicine will not help stop the poisoning. The only way to stop the metal poisoning is to stop being exposed to the heavy metals. / Environmental Sciences / M. Sc. (Environmental Management)

Heavy metal pollution in water and soil is a serious concern to human health and the associated environment. Some heavy metals have bio-importance but the bio-toxic effects of many of them in human health are of great concern. Hence, there was a need for proper understanding of the concentration levels of these heavy metals in ground water and soil around the community residing in the vicinity of the defunct mine. Mining has become prominent in this area because of the existence of copper lodes, veins and veinlets. It was therefore necessary to assess these selected metals associated with copper mining as their concentration has a tendency to affect the environment and human health. The objective of this study was to establish the levels of lead (Pb)-zinc (Zn)-copper (Cu)-arsenic (As)-nickel (Ni) metals in ground water and soil associated with an old copper mine in the vicinity of the township and to compare them with the South African and international standards in order to safeguard the health of the community using such water for drinking purpose. Clean sampling plastic bottles were used to collect water from five water boreholes being used at present. Water samples were filtered using membrane filtration set LCW (0.45 μm). The samples were digested sequentially with different procedures for the total metal concentration. Concentrations of four metals commonly associated with Cu mining were examined at five different water boreholes which are used for drinking and industrial purposes. Flame Atomic Absorption Spectrophotometer (Perkin Elmar S/n 000003F6067A, Singapore) was used to analyze metals in water samples at Eskom Ga-Nala Laboratory: pH, electrical conductivity and turbidity were analyzed using an auto titrator meter (AT- 500,Japan), conductivity meter (Cole-parmer® YO-19601-00) and turbidity meter (AL 250TIR, Agua lytic, German) respectively. Soil samples were collected from the selected areas where human health is of a serious concern, and a hand held auger drill was used to recover samples, while shovels were used to prepare the sampling area. The samples were sieved up to 63.0 μm particle size and digested with aqua-regia. Flame Atomic Absorption Spectrophotometer (Model: AA400; Year: 2008; Manufacturer: Perkin Elmer; Germany; Serial no: 201S6101210) was used at the University of Venda Laboratory to analyze soil from the study area for possible heavy metal contamination due to the defunct Cu mine in the area. v The results showed variation of the investigated parameters in water samples as follows: pH, 6.0 to 7.51; EC, 70.0 to 96.40 μS/cm and turbidity, 1.05 to 4.56 NTU. The mean concentration of the metals increased in the followed order: Pb<Cu<As<Ni. Ni is the most abundant in the ground water determined with value of (6.49 μg/g). The observations have confirmed that most ground water contains an appreciable quantity of Ni. The mean value of As in water is (4.20 to 4.84 μg/g), Pb and Cu have (2.13 to 2.58 μg/g) and (1.52 to 2.52 μg/g) respectively. For soil samples, the mean concentration of the metals increased in the following order: Pb<Cu<Zn<As<Ni. Pb ranged from (0.023 to 0.036 μg/g) followed by Cu (0.28 to 0.45 μg/g) then Zn (0.026 to 0.053 μg/g), the mean range of As in soil ranged from (0.054 to 0.086 μg/g). However, some studies show much higher contamination of As from the natural sources and Ni with (0.057 to 0.144 μg/g) lastly. Accumulation of heavy metals in soil is of concern due to their toxic effects on human and animals. The quality of ground water from the five boreholes studied was satisfactory with turbidity (T), electrical conductivity (EC) and heavy metals (HM’s) below the WHO limit. The water therefore may, according to the WHO Standards be safely used as a drinking water. The concern lies on pH which was slightly (0.5) below the standard. There is a serious need to monitor the ground water which is now used for drinking purposes. This study revealed that heavy metal pollution in soil from the abandoned Cu mine in Musina is a threat to the health of the community. Although pollution was between medium and low in the contamination index, it is therefore important for the Musina Municipality or mine owner of Musina (TVL) Development Co Ltd copper mine to advocate possible remedial actions which will safeguard the environment and human health. The tailing at Musina’s old Cu mine have high pH and they lack normal soil stabilization processes, as a result the tailing does not develop a good plant cover. Pollution of the ground water resources is also evident in the study area where there is seepage or ingress of polluted water to the underground aquifers. Small-scale mining in Musina is causing further degradation to the environment but it supports the South African Waste Hierarchy by promoting the reuse and recycling of the tailing and mine dumps for the production of bricks. Mine workers are exposed to the above mentioned toxic heavy metals daily. Medicine will not help stop the poisoning. The only way to stop the metal poisoning is to stop being exposed to the heavy metals. / Environmental Sciences / M. Sc. (Environmental Management)

Thesis submitted in fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology 2008 / River systems can become contaminated with micro-organisms and metals and the routine monitoring of these rivers is essential to control the occurrence of these contaminants in water bodies. This study was aimed at investigating the metal contamination levels in the Berg-, Plankenburg- and Diep Rivers in the Western Cape, South Africa, followed by the remediation of these rivers, using bioreactor systems. Sampling sites were identified and samples [water, sediment and biofilm (leaves, rocks and glass, etc.)] were collected along the Berg- and Plankenburg Rivers from May 2004 to May 2005 and for the Diep River, from February 2005 to November 2005. The concentrations of aluminium (Al), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb) and zinc (Zn) were determined using the nitric acid digestion method and analysed by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). For the Berg River, the highest concentrations in water samples were recorded for Al, Mn and Fe at the agricultural area (Site A – chapter 2). In the sediment and biofilm samples, the highest metal concentrations were once again recorded for Al and Fe. The concentrations of Al and Fe were significantly higher (p < 0.05) than than Cu, Zn, Pb, Ni and Mn in water, sediment and biofilm samples, and were mostly higher than the quality guidelines recommended by the Department of Water Affairs and Forestry (DWAF, 1996) and the Canadian Council for the Ministers of the Environment (CCME, 2001). Possible sources of contamination in the Berg River could be due to the leaching or improper discarding of household waste from the informal- and established residential areas, as well as the improper discarding of pesticides at the agricultural area. For both the Plankenburg and Diep Rivers the Al and Fe concentrations were higher than all the other metals analysed for in sediment and water samples. The highest concentrations recorded in the Plankenburg River was 13.6 mg.l-1 (water - Week 18, Site B) and 15 018 mg.kg-1 (sediment - Week 1, Site C) for Al and 48 mg.l-1 (water - Week 43, Site A) and 14 363.8 mg.kg-1 (sediment - Week 1, Site A) for Fe. The highest concentrations recorded in the Diep River was 4 mg.l-1 (water - Week 1, Site A) and 19 179 mg.kg-1 (sediment - Week 1, Site C) for Al and 513 mg.l-1 (water - Week 27, Site A) and 106 379.5 mg.kg-1 (sediment - Week 9, Site C) for Fe. For most of the metals analysed the concentrations were higher than the recommended water quality guidelines as stipulated by the Department of Water Affairs and Forestry (DWAF, 1996b), the Canadian Council for the Ministers of the Environment (CCME, 2001) and the ‘World average’ (Martin and Windom, 1991). Point sources of pollution could not conclusively be identified, but the industrial and residential areas could have influenced the increased concentrations. Metal concentrations should be routinely monitored and the guidelines should be updated and revised based on the current state of the rivers and pollution influences. Micro-organisms isolated from flow cells after exposure to varying metal concentrations were investigated for possible metal-tolerance. A site where high metal concentrations were recorded along the Plankenburg River was investigated. The micro-organisms isolated from the flow cells were cultured and identified using the Polymerase Chain Reaction (PCR) technique, in conjunction with universal 16SrRNA primers. The phylogeny of the representative organisms in GenBank, were analysed using the Neighbour-joining algorithm in Clustal X. After exposure, the channels were stained with the LIVE/DEAD BacLightTM viability probe and visualised using Epifluorescence Microscopy. The results revealed that when exposed to the highest concentrations of Al (900 mg.l-1), Fe (1000 mg.l-1), Cu (10 mg.l-1) and Mn (80 mg.l-1), the percentage of dead cells increased, and when exposed to the lowest concentrations of Al (10 mg.l-1), Cu (0.5 mg.l-1), Mn (1.5 mg.l-1) and Zn (0.5 mg.l-1), no significant differences could be distinguished between live an dead cells. When exposed to the highest concentrations of Zn (40 mg.l-1) and Ni (20 mg.l-1), no significant differences between the live and dead cell percentages, were observed. The phylogenetic tree showed that a diverse group of organisms were isolated from the flow cells and that some of the isolates exhibited multiple metal resistance (Stenotrophomonas maltophilia strain 776, Bacillus sp. ZH6, Staphylococcus sp. MOLA:313, Pseudomonas sp. and Delftia tsuruhatensis strain A90 exhibited tolerance to Zn, Ni, Cu, Al, Fe), while other isolates were resistant to specific metals (Comamonas testosteroni WDL7, Microbacterium sp. PAO-12 and Sphingomonas sp. 8b-1 exhibited tolerance to Cu, Ni and Zn, respectively, while Kocuria kristinae strain 6J-5b and Micrococcus sp. TPR14 exhibited tolerance to Mn). The efficiency of two laboratory-scale and one on-site bioreactor system was evaluated to determine their ability to reduce metal concentrations in river water samples. The laboratory-scale bioreactors were run for a two-week and a three-week period and the on-site bioreactor for a period of ten weeks. Water (all three bioreactors) and bioballs (bioreactor two and on-site bioreactor) were collected, digested with 55% nitric acid and analysed using ICP-AES. The final concentrations for Al, Ni and Zn (bioreactor one) and Mn (bioreactor two), decreased to below their recommended concentrations in water samples. In the on-site, six-tank bioreactor system, the concentrations for Fe, Cu, Mn and Ni decreased, but still exceeded the recommended concentrations. The concentrations recorded in the biofilm suspensions removed from the bioballs collected from bioreactor two and the on-site bioreactor, revealed concentrations higher than those recorded in the corresponding water samples for all the metals analysed, except Fe. The bioballs were shown to be efficient for biofilm attachment and subsequent metal accumulation. The species diversity of the organisms isolated from the bioreactor (bioreactor two) experiment after three days (initial) differed from the organisms isolated after 15 days (final). Hydrogenophaga sp., Ochrobactrum sp, Corynebacterium sp., Chelatobater sp. and Brevundimonas sp. were present only at the start of the bioreactor experiment. The surviving populations present both in the beginning and at the end of the bioreactor experiment belonged predominantly to the genera, Pseudomonas and Bacillus. Metal-tolerant organisms, such as Bacillus, Pseudomonas, Micrococcus and Stenotrophomonas, amongst others, could possibly be utilised to increase the efficiency of the bioreactors. The bioreactor system should however, be optimised further to improve its efficacy.

MSc (Chemistry) / Department of Chemistry / Increasing health and environmental concern about the effects of most toxic heavy metals emitted from cement plants in developing countries, which are going through rapid development, has led to this study. Cement industry in South Africa has been the primary industry over the years contributing immensely to infrastructure development and economic growth. Cement has been used to build many large cities, industries, homes, bridges and shopping malls around the country and still continue to be used by constructors. At this point, there has been no other substitute for cement and it will continue to be produced for decades to come. Unfortunately, this industry is now known to be amongst the major environmental polluters. Less has been done to address the adverse effects that comes with the production of cement, especially in the developing countries where there is huge demand for cement. This study focusses on dust emanating from production processes especially cement manufacturing from rotary kiln stage during production of cement and cement bricks. The production of cement and cement bricks generate dust, which is distributed over large areas of the environment. In South Africa, there are a number of factories in operation without proper planning of pollution prevention and compliance to environmental legislature. Since the production of cement is associated with the release of dust containing heavy metals, the dust is atmospherically deposited on the land, water surfaces and residential areas. The soil, street pavements, wetlands and water surfaces have become the sinks of heavy metals. Heavy metals that are being deposited include arsenic, cadmium, chromium, manganese, cobalt, copper, barium, antimony, selenium, vanadium, nickel and lead. Such metals pose health threat to the animals, plants and human beings living around the cement factories. These metals can easily be leached out from the soil and washed to the water bodies causing water pollution. Old processing techniques have been found to be inefficient to prevent emission of dust to the atmosphere. Hence, the emission of the toxic heavy metals to the environment was uncontrollable. Since cement is used to produce cement bricks, the whole process is subjected to heavy metals being discharged with dust from the factory to the surrounding environment. Four papers (I, II, III and V) were written to assess the level of heavy metals. In paper I, water and plants samples (Bidens Pilosa, Phragimites Australis and Xanthium Strumarium) were collected in the Mvudi River nearby a cement factory. Sampling was done before, within and after the wetland. Samples were digested with nitric acid for analysis. The concentration of zinc, chromium and lead were determined in the samples using a graphite furnace atomic absorption spectromentry. Results showed that the concentrations of zinc, chromium and lead were above the permissible limits in different parts of the plants analysed and water. The pH of water samples were below the threshold recommended by Department of water affairs and forestry (DWAF) and World health organisation (WHO). In paper II, seven soils at different distance, seven soils below soil surface at seven different layers and a bulk were sampled nearest to the cement brick making factory. Bulk sample was separated into five particle sizes (2 - 3 mm, 1 - 2 mm, 0.5 - 1 mm, 0.5 mm). Five sediments samples were also collected before, within and after the wetland along Mvudi river. Modified three step BCR sequential extraction was applied to the 23 samples in order to obtain the metal distribution in the samples. Heavy metal concentrations of nickel and chromium were determined using graphite atomic absorption spectrometry. Results showed that the levels of nickel and chromium exceeded the permissible limits recommended by WHO. Elevated concentrations Ni and Cr in soil and sediments also showed that the cement brick making factory is the main source of pollution in the area. To assess the contribution of cement dust to heavy metal pollution from the cement plants to the surrounding environment, two studies were carried out in the vicinity of two cement plants one in Thabazimbi and the other in Pretoria. Two papers (III and IV) were written from the studies and were summarised as follows: In paper III, dust samples were collected along the road leading to and passing by the cement plant in Thabazimbi, South Africa. The samples were collected using a brush and pan into sampling bags. After sampling dust samples were sieved into three particle size fractions (PM125, PM75, and PM32). A bulk and five samples were collected beneath the soil at different depth for depth analysis nearest to the cement plant. Water samples were collected along the Crocodile River before and after the cement plant site. The samples were digested using aqua ragia and extracted using Modified BCR sequential extraction. The samples were analysed using inductive coupled plasma optical emission spectrometry (ICP-OES) for concentration of platinum group metals and x-ray fluorescence for elementary analysis (XRF). Analysis of samples included characterisation of the dust samples using x-ray diffraction (XRD). The vi concentrations were also compared to that of the control study (blank) area to find out if the metals were discharged from the cement factories of interest. In paper IV, street dust samples were collected randomly on the paved surfaces, on the streets and accessible residential and roadsides on locations close to the cement plant in Pretoria. Some samples were collected along the road leading to the gate of the factory and also on the road near the cement plant. The samples were collected into sampling bags using a brush and pan. After sampling dust samples were sieved into three particle size fractions (PM125, PM75 and PM32). A bulk and five samples were collected beneath the soil at different depth for depth analysis nearest to the cement plant. Water samples were collected along the Apies River before and after the cement plant. All samples were kept in a cooler box with ice bags to keep them in good condition. The samples were digested using aqua ragia and extracted using Modified BCR sequential extraction. Results were used to establish spatial distribution of the heavy metals around the urban streets. The samples were analysed using ICP-OES for concentration of heavy metals and XRF. Analysis of samples included characterisation of the dust samples using XRD. The concentrations were also compared to that of the control study (blank) area to find out if the metals were discharged from the cement factories of interest. In paper V, seven different vegetables (spinach/Spinacia oleracea, Chinese cabbage/Brassica rapa, onion/Allium cepa, beetroot/Beta vulgaris, sweet potatoes/Ipomoea batatas, tomatoes/ Lycopersicon esculentum and cabbage/Brassica pekinensis), fruits (bananas/Musa acuminate) and their soils taken after uprooting them were sampled in farming area close to Thohoyandou town and the cement factory. The concentrations of cadmium, nickel and manganese were measured using the graphite atomic absorption spectrometry (GFAAS). Cadmium, nickel and manganese levels were found above permissible limits proposed by Food agricultural organisation (FAO) and WHO in edible parts of vegetables, fruits and soils and hence, may pose a health risk to consumers. Similarly the results from XRF also showed high concentration of the heavy metals in soil analysed. The aim of this project is to determine the levels of toxic heavy metals carried with dust emanating from cement factories. This assessment is meant to identify and highlight the levels of heavy metals in areas that are close to cement factories. The study will develop a database of heavy metals in affected areas and the pollution impact to the affected environments. / NRF

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Environmental Health in the Faculty of Applied Sciences at the Cape Peninsula University of Technology, 2013 / Water is an important daily requirement and in a clean, pure form, it promotes health and well-being. In addition to South Africa being one of the driest countries in the world, water availability is also being compromised by massive pollution of remaining water sources. The Berg- and Plankenburg Rivers are two of the surface water sources in the Western Cape, South Africa, which are highly polluted by sewage, industrial and agricultural run-off. The current investigation was aimed at comparing diagnostic tools, which are employed by municipalities and food industries, and molecular based techniques to routinely monitor water for indicator organisms in time- and cost-effective manner. These rivers were sampled twice a month (July 2010 to January 2011) at the sites closest to the informal settlements of Kayamandi in Stellenbosch (Plankenburg River) and Mbekweni in Paarl (Berg River). The contamination levels of the two river systems were evaluated by the enumeration of Escherichia coli and coliforms using the Colilert 18® system, Membrane Filtration (MF) and Multiple Tube Fermentation (MTF) techniques. The highest faecal coliform count of 9.2 × 106 microorganisms/100 ml was obtained in weeks 21 and 28 from the Plankenburg River system by the MTF technique, while the lowest count of 1.1 × 103 microorganisms/100 ml was obtained in week one for both river systems by the MTF technique. The highest E. coli count of 1.7 × 106 microorganisms/100 ml was obtained from the Berg River system (week 9) using the MTF technique, while the lowest count of 3.6 × 102 microorganisms/100 ml was obtained by the MF technique from the Plankenburg River system. The coliform and E. coli counts obtained by the enumeration techniques thus significantly (p > 0.05) exceeded the guidelines of 2000 microorganisms/100 ml stipulated by the Department of Water Affairs and Forestry (DWAF, 1996) for water used in recreational purposes. Overall the results obtained in this study showed that the water in the Berg- and Plankenburg River systems is highly polluted, especially where these water sources are used for irrigational and recreational purposes. For the coliform and E. coli counts obtained using the three enumeration techniques, it was noted that the MTF technique was more sensitive and obtained higher counts for most of the sampling weeks. However, the media (Membrane lactose glucuronide agar) used in the MF technique also effectively recovered environmentally stressed microbial cells and it was also better for the routine selection and growth of coliforms and E. coli. While E. coli and total coliforms were detected utilising the Colilert 18® system, accurate enumeration values for these two indicator groups was not obtained for the entire sampling period for both river systems. It has previously been shown that dilutions (up to 10-3) of highly polluted waters increase the accuracy of the Colilert 18® system to enumerate colifoms and E. coli in marine waters. As the results obtained utilising the Colilert 18® system were also not comparable to the MF and MTF techniques it is recommended that highly polluted water samples be diluted to increase the accuracy of this system as a routine enumeration technique. Water samples were directly inoculated onto MacConkey, Vile Red Bile (VRB) agar and the Chromocult Coliform agar (CCA) and single colonies were inoculated onto nutrient agar. Chromocult coliform agar proved to be more sensitive than MacConkey and VRB agar for the culturing of E. coli and coliforms. Preliminary identification of these colonies was done using the RapID ONE and API 20 E systems. The most isolated Enterobacteriaceae species by both systems, included Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli and Enterobacter cloacae in both river systems. The API 20 E system was more sensitive in the preliminary identification of the various isolates, as greater species diversity was obtained in comparison to the RapID ONE system. The Polymerase Chain Reaction (PCR) was firstly optimised using positive Enterobacteriaceae species. The optimised method was then applied to the analysis of river water samples, which were centrifuged to harvest the bacterial cells, with DNA extracted using the boiling method. The extracted DNA was amplified using conventional PCR with the aid of species specific primers. The Enterobacteriaceae species that were detected throughout the study period in both river systems include Serratia marcescens, Escherichia coli, Klebsiella pneumoniae and Bacillus cereus. Conventional PCR was the most reliable and sensitive technique to detect Enterobacteriaceae to species level in a short period of time when compared to RapID ONE and the API 20 E systems. Multiplex PCR was optimised using the positive pathogenic E. coli strains namely, Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAEC). It was then employed in river water sample analysis and enabled the detection of EAEC, EHEC, and EIEC strains in Berg River system, with only the EAEC detected in the Plankenburg River system. Real-time PCR was used to optimise the multiplex PCR in the amplification of E. coli strains and successfully reduced the time to obtain final results when using control organisms. Real-time PCR was found to be more sensitive and time-effective in the identification of E. coli strains, and also more pronounced DNA bands were observed in real-time PCR products compared to conventional-multiplex PCR amplicons. To sustain the services provided by the Berg- and Plankenburg Rivers in the Western Cape (South Africa), these water sources should frequently be monitored, results assessed and reported according to the practices acknowledged by responsible bodies. It is therefore recommended that the enumeration techniques be used in conjunction with the very sensitive PCR technique for the accurate detection of coliforms and E. coli in river water samples.

MENVSC / Department of Hydrology and Water Resources / Sewage sludge (biosolids) from wastewater treatment plants (WWTPs) has been widely used as a soil improver in Europe, United States of America and some developing countries including South Africa. It has its benefits for farmers as a good source of organic matter and minerals, however, sludge after treatment still contains pathogenic organisms, heavy metals and persistent organic pollutants (POPs). The POP and heavy metal contaminants that accumulate in sludge may transfer through the food chain and cause adverse effects on human beings. In this study, a field experiment was carried out on farmland fertilized with sewage sludge from a wastewater treatment plant (WWTP) that vasically receives domestic wastewater and storm water. Vegetable spinach (Spinacia oleracea) was used for this study and was planted on a farmland under controlled conditions. Ten ridges each of dimensions 20 m × 0.3 m was made and dry sludge weights of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 kg were applied as manure on each of the ridges, respectively. Representative samples of sludge and soil were taken for analysis of heavy metals and POPs. At maturity, in twelve weeks, the root and leave samples of the vegetable were taken from all the ridges including the control. The soil, sludge, and vegetable samples were analyzed for total heavy metal content (Cd, Cr, Cu, Ni, Pb, Co, Zn, Al, Fe, Mn), speciated heavy metal content and POP (PAH, PCB). Soil and sludge samples were also analyzed for total organic content, pH, cation exchange capacity (CEC), conductivity and alkalinity. The analysis for total heavy metals and speciated heavy metal content was carried out using inductively coupled plasma optical emission spectrophotometer (ICP-OES), and CEC analysis was carried out using atomic absorption spectrophotometer (AAS). A two-dimensional gas chromatograph with time of flight mass spectrometry detector (GC X GC TOFMS) was used for POP measurements. pH measurement was made using a pH meter and conductivity measurement using a conductivity meter. Alkalinity and total organic content analysis was performed using titrimetric apparatus. The highest total heavy metal concentration of 378.9 mg / kg was recorded in Fe metal in soil and Leaf sample while the lowest total metal concentration of 0.0003 mg / kg was in Cu metal in root sample. The highest heavy metal concentration of 1002 mg / kg in speciated forms was in Mn metal in F1 fraction and the lowest of 0.0004 mg / kg was in Cd metal in F5 fraction. PAHs were only found in soil samples and their concentrations ranged from 2.53 mg / kg to 146.5 mg / kg. There were no PCB detected in all the samples analysed. The results indicated that the trace metals concentrations found in the exchangeable fraction were higher than those observed in any of the preceding extractions except in the case of Cd, Cr, Fe and Pb where Fe-Mn oxide and organic matter fractions predominated and were closely followed by exchangeable fraction.

The distribution and potential sources of 15 polycyclic aromatic hydrocarbons (PAHs) in soils and Digitaria eriantha in the vicinity of three South African coal-fired power plants, Matla, Lethabo and Rooiwal were determined by gas chromatography–mass spectrometry. An ultrasonic assisted dispersive liquid-liquid microextraction (UA-DLLME) method was developed for the extraction of polycyclic aromatic hydrocarbon in soil, followed by determination using gas chromatography mass spectrometry. The study showed that an extraction protocol based on acetonitrile as dispersive solvent and C2H2Cl2 as extracting solvent, gave extraction efficiencies comparable to conventional soxhlet extraction for soil samples. The extraction time using ultrasonication and the volume of the extraction solvent was also investigated. Using a certified reference material soil (CRM), the extraction efficiency of UA-DLLME ranged from 64 to 86% in comparison with the Soxhlet result of 73 to 95%. In comparison with the real sample, the CRM result did not show a significant difference at 95% C.I. The UA-DLLME proved to be a convenient, rapid, cost-effective and greener sample preparation approach for the determination of PAHs in soil samples. PAH compound ratios such as phenanthrene/phenanthrene + anthracene (Phen/ Phen + Anth) were used to provide a reliable estimation of emission sources. The total PAH concentration in the soils around three power plants ranged from 9.73 to 61.24 μg g−1, a range above the Agency for Toxic Substances and Disease Registry levels of 1.0 μg g−1 for a significantly contaminated site. Calculated values of the Phen/Phen + Anth ratio were 0.48±0.08, 0.44±0.05, and 0.38+0.04 for Matla, Lethabo and Rooiwal, respectively. The flouranthene/fluoranthene + pyrene (Flan/ Flan + Pyr) levels were found to be 0.49±0.03 for Matla, 0.44±0.05 for Lethabo, and 0.53±0.08 for Rooiwal. Such values indicate a xx pyrolytic source of PAHs. Higher molecular weight PAHs (five to six rings) were predominant, suggesting coal combustion sources. The carcinogenic potency B[a]P equivalent concentration (B[a] Peq) at the three power plants ranged from 3.61 to 25.25, indicating a high carcinogenic burden. The highest (B[a] Peq) was found in samples collected around Matla power station. It can, therefore, be concluded that the soils were contaminated with PAHs originating from coal-fired power stations. Nine metals (Fe, Cu, Mn, Ni, Cd, Pb, Hg, Cr and Zn) were analysed in soil and the Digitaria eriantha plant around three coal power plants (Matla, Lethabo and Rooiwal), using ICP-OES and GFAAS. The total metal concentration in soil ranged from 0.05 ± 0.02 to 1835.70 ± 70 μg g-1, 0.08 ± 0.05 to 1743.90 ± 29 μg g-1 and 0.07 ± 0.04 to 1735.20 ± 91 μg g-1 at Matla, Lethabo and Rooiwal respectively. The total metal concentration in the plant (Digitaria eriantha) ranged from 0.005 ± 0.003 to 534.87 ± 43 μg g-1 at Matla, 0.002 ± 0.001 to 400.49 ± 269 μg g-1 at Lethabo and 0.002 ± 0.001 to 426.91 ± 201 μg g-1 at Rooiwal. The accumulation factor (A) of less than 1 (i.e. 0.003 to 0.37) at power plants indicates a low transfer of metal from soil to plant (excluder). The enrichment factor values obtained (2.4 – 5) indicate that the soils are moderately enriched, with the exception of Pb that had significant enrichment of 20. The Geo-accumulation Index values of metals indicate that the soils are moderately polluted (0.005 – 0.65), except for Pb that showed moderate to strong pollution (1.74 – 2.53). / Chemistry / D. Phil. (Chemistry)

The distribution and potential sources of 15 polycyclic aromatic hydrocarbons (PAHs) in soils and Digitaria eriantha in the vicinity of three South African coal-fired power plants, Matla, Lethabo and Rooiwal were determined by gas chromatography–mass spectrometry. An ultrasonic assisted dispersive liquid-liquid microextraction (UA-DLLME) method was developed for the extraction of polycyclic aromatic hydrocarbon in soil, followed by determination using gas chromatography mass spectrometry. The study showed that an extraction protocol based on acetonitrile as dispersive solvent and C2H2Cl2 as extracting solvent, gave extraction efficiencies comparable to conventional soxhlet extraction for soil samples. The extraction time using ultrasonication and the volume of the extraction solvent was also investigated. Using a certified reference material soil (CRM), the extraction efficiency of UA-DLLME ranged from 64 to 86% in comparison with the Soxhlet result of 73 to 95%. In comparison with the real sample, the CRM result did not show a significant difference at 95% C.I. The UA-DLLME proved to be a convenient, rapid, cost-effective and greener sample preparation approach for the determination of PAHs in soil samples. PAH compound ratios such as phenanthrene/phenanthrene + anthracene (Phen/ Phen + Anth) were used to provide a reliable estimation of emission sources. The total PAH concentration in the soils around three power plants ranged from 9.73 to 61.24 μg g−1, a range above the Agency for Toxic Substances and Disease Registry levels of 1.0 μg g−1 for a significantly contaminated site. Calculated values of the Phen/Phen + Anth ratio were 0.48±0.08, 0.44±0.05, and 0.38+0.04 for Matla, Lethabo and Rooiwal, respectively. The flouranthene/fluoranthene + pyrene (Flan/ Flan + Pyr) levels were found to be 0.49±0.03 for Matla, 0.44±0.05 for Lethabo, and 0.53±0.08 for Rooiwal. Such values indicate a xx pyrolytic source of PAHs. Higher molecular weight PAHs (five to six rings) were predominant, suggesting coal combustion sources. The carcinogenic potency B[a]P equivalent concentration (B[a] Peq) at the three power plants ranged from 3.61 to 25.25, indicating a high carcinogenic burden. The highest (B[a] Peq) was found in samples collected around Matla power station. It can, therefore, be concluded that the soils were contaminated with PAHs originating from coal-fired power stations. Nine metals (Fe, Cu, Mn, Ni, Cd, Pb, Hg, Cr and Zn) were analysed in soil and the Digitaria eriantha plant around three coal power plants (Matla, Lethabo and Rooiwal), using ICP-OES and GFAAS. The total metal concentration in soil ranged from 0.05 ± 0.02 to 1835.70 ± 70 μg g-1, 0.08 ± 0.05 to 1743.90 ± 29 μg g-1 and 0.07 ± 0.04 to 1735.20 ± 91 μg g-1 at Matla, Lethabo and Rooiwal respectively. The total metal concentration in the plant (Digitaria eriantha) ranged from 0.005 ± 0.003 to 534.87 ± 43 μg g-1 at Matla, 0.002 ± 0.001 to 400.49 ± 269 μg g-1 at Lethabo and 0.002 ± 0.001 to 426.91 ± 201 μg g-1 at Rooiwal. The accumulation factor (A) of less than 1 (i.e. 0.003 to 0.37) at power plants indicates a low transfer of metal from soil to plant (excluder). The enrichment factor values obtained (2.4 – 5) indicate that the soils are moderately enriched, with the exception of Pb that had significant enrichment of 20. The Geo-accumulation Index values of metals indicate that the soils are moderately polluted (0.005 – 0.65), except for Pb that showed moderate to strong pollution (1.74 – 2.53). / Chemistry / D. Phil. (Chemistry)