M.Tech. / With the recent cholera outbreak in Zimbabwe and the outbreak taking a sub-regional dimension with cholera cases being reported from neighbouring countries like Botswana and South Africa, there was a need to monitor drinking water from environmental water sources as well as household water-storage containers at the point-of-use in rural communities. Although conventional culture-based microbiological methods for the identification of Vibrio species from environmental water samples are reliable, they require several days to complete (Khan and Cerniglia, 1994). Culture dependent and culture independent methods for the detection of Vibrio cholerae and Vibrio parahaemolyticus from water samples were optimised during the current study. With these methods, the occurrence and distribution of V. cholerae and V. parahaemolyticus in source waters as well as in household container stored-waters at the point-of-use in the Nwanedi Catchment, was determined. The culture based approach analyses involved the enrichment of water samples in alkaline peptone water (APW) for 18 hours at 37°C followed by culture on selective thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Typical colonies on TCBS agar were confirmed using the API 20NE as well as the two multiplex polymerase chain reactions (m-PCR). The culture independent PCR approach was done by filtering 100 ml of the water sample onto polycarbonate membranes followed by DNA extraction from the bacteria captured on the membranes using an adaptation of the in-house DNA extraction method used in the laboratory. This DNA was used as template for the m-PCR’s. For the culture based PCR detection, 100 ml water was filtered onto nitrocellulose membranes followed by 18 hours enrichment in APW. DNA was then extracted from the enrichment broth and subsequently used as template for the m-PCR’s. All water samples were analysed with all three methods to compare the results and determine the most effective method for the detection of the two-selected Vibrio species present in water samples. PCR analyses were performed using two m-PCR assays targeting the SodB (V. cholerae species), FlaE (V. parahaemolyticus species) and 16S rRNA (Vibrio and Enterobacteriacea species) genes (Multiplex 1) and the V. cholerae O1 and V. cholerae O139 rfb genes, ctxA (cholera toxin) gene and 16S rRNA gene (Multiplex 2). The 16S rRNA primers were included in the Multiplex PCR’s as an internal control. The m-PCR assays were 100% specific for total and toxigenic V. cholerae and total V. parahaemolyticus when using target bacteria and various other non-target bacteria. The m-PCR assays when coupled with an 18 hours enrichment step could detect as few as 4-10 V. cholerae and V. parahaemolyticus cells in pure cultures as well as in spiked environmental water samples. Fifty water-storage containers and 56 environmental water samples (river, spring and borehole) from rural households in the Vhembe district of the Limpopo Province of South Africa were tested for the presence of selected Vibrio’s, using (1) the standard culture based approach, (2) PCR detection without enrichment and (3) PCR with a brief pre-enrichment. Container water samples were collected before [referred to as free volume (FV) of water] and after dislodging of the biofilm [referred to as dislodged biofilm (BD)] from the inner sidewalls of containers. Of the samples analysed with the standard cultured based technique combined with colony confirmation using m-PCR 1, 34 (12.8%) tested positive for the presence of V. cholerae (SodB gene), 2 (1.3%) for the presence of V. parahaemolyticus (FlaE gene) and all the samples tested positive for the 16S rRNA gene. In contrast, only 1 (0.6%) tested positive for the presence of V. cholerae and 0 (0%) for the presence of V. parahaemolyticus when the isolates were confirmed with API 20NE. With the culture dependant PCR method, 65 (41.7%) of the samples tested positive for the presence of V. cholerae, 3 (1.9%) for the presence of V. parahaemolyticus and all the samples tested positive for the 16S rRNA gene. Seventeen (10.9%) of the samples tested positive for the presence of V. cholerae (SodB) and 16S rRNA genes, 0 (0%) for the presence of V. parahaemolyticus (FlaE gene) with the culture independent direct PCR detection protocol. All the samples that tested positive for V. cholerae with any of the three methods were tested for the presence of toxigenic V. cholerae species with the second multiplex PCR. Six of the source water samples tested positive for V. cholerae O1 as well as the cholera toxin genes. Of the 56 source water samples, 14 (25%) were positive for V. cholerae and 0 (0%) were positive for V. parahaemolyticus with one or all of the methods. Six (10.7%) of the V. cholerae positive samples tested positive for V. cholerae O1 rfb gene, and ctxA gene (cholera toxin). Thirty (60%) of the 50 FV and 28 (56%) of the DB water samples tested positive for V. cholerae, and 3 (6%) of the FV and 0 (0%) of the DB samples tested positive for V. parahaemolyticus with one or all of the methods. None of the positive V. cholerae samples tested positive for the presence of toxigenic V. cholerae. The results presented suggest that the use of culture-based techniques alone is inadequate for detection of selected Vibrio’s in the environmental water samples and that such techniques are not enough to guarantee satisfactory protection of human health. The combination of filtration, enrichment, DNA extraction and m-PCR method provide a sensitive and specific method for the detection of V. cholerae and V. parahaemolyticus in environmental water samples. This method proved to be the most effective for detection and identification of selected Vibrio’s when compared to the culture based method and PCR without enrichment method. The inclusion of an enrichment period allows for the detection of culturable bacteria which is crucial as PCR detection does not give indications on the viability of the detected material. The enrichment period will also dilute any inhibitors for the m-PCR’s that may be present. Detection of V. cholerae and V. parahaemolyticus in the source water used by the population and in the water-storage containers indicates possible seeding of containers with Vibrio species from the source water. Furthermore, the detection of these organisms in DB samples indicates that these organisms attach to containers’ inner sidewalls, forming biofilms, further sustaining their occurrence and proliferation. The detection of V. cholerae and V. parahaemolyticus in household water-storage containers certainly places the consumers at risk of infection of diseases caused by these organisms.

M.Sc. / South Africa is a developing country in which the rapid increase in population size is accompanied by expanding mining and industrial sectors. All these factors have resulted in an increase in the demand for good quality water, but various anthropogenic activities continually result in pollution of the freshwater reserves in the country, via either diffuse or point sources. Metals resulting from both mining and industry are common pollutants of South African aquatic systems. Although some are essential elements that are necessary for normal growth and metabolism in organisms, all metals can become toxic at elevated levels, thus resulting in either lethal and sublethal effects. Aquatic organisms can also bioaccumulate metals thereby transferring them to their predators, including man. Monitoring of freshwater systems is thus necessary in order to protect the environment from these toxic substances, and ultimately to protect mankind. Most monitoring programmes should use an integrated approach in which both physico-chemical and biological aspects are monitored. The present study employed an integrated approach in order to investigate the state of metal pollution in two water bodies that are subjected to differential levels of metal contamination. Selected physico-chemical characteristics of the water and sediment were analysed, as were the levels of metals bioaccumulated by the Freshwater River crab, Potamonautes warreni. The specific object of this study was to assess the potential of P. warreni as a suitable bioaccumulative indicator of metal pollution in the aquatic environment. Water and sediment samples were collected from Germiston Lake and Potchefstroom Dam every alternate month between February 1995 and February 1996. Water quality data revealed that although the quality of the water at both sites was relatively high, Germiston Lake appeared to be more anthropogenically impacted than Potchefstroom Dam. Analysis of metal levels in the water and sediment indicated that while levels of copper, manganese, nickel, lead and zinc were higher in Germiston Lake, calcium and iron were present in higher concentrations in Potchefstroom Dam. Cadmium levels differed little between the two sites. With the exception of iron in Potchefstroom Dam, nickel in Germiston Lake, and zinc at both sites, all of the physical and chemical characteristics of the water from the two sites were within the boundaries of the South African guideline values laid down for the protection of aquatic life. Potamonautes warreni individuals were also collected every alternate month, and metal concentrations in these organisms were determined using atomic absorption spectrophotometry. The results of this investigation showed that metal levels in these organisms did tend to reflect those in the environment, with lower levels of calcium and iron and significantly higher (p < 0.05) levels of copper, manganese, lead, nickel, and zinc in P. warreni from Germiston Lake. Cadmium concentrations detected in these crabs were essentially the same at the two sites. All metals, except for cadmium, were influenced by the size of the crabs at one or other site, but never at both, thus suggesting that the presence of size-related trends is influenced by ambient metal levels in the system. Iron and lead concentrations in P. warreni from Potchefstroom Dam were the only metals that were influenced by the sex of the organisms, with higher levels found in female crabs. The results of the present study clearly indicate that metal levels in P. warreni are indicative of the environmental levels of metals to which these crabs are exposed since higher metal levels were found in the organisms from the site that had higher environmental metal levels. These organisms have proven to be useful bioaccumulative indicators of metal pollution in the aquatic environments from which they were captured, and they should therefore be incorporated into biomonitoring protocols. It must, however, be stressed that further research is required to increase our database on metal levels in these crabs from different aquatic systems.

Aquatic toxicology has been identified as a valuable tool in the identification and management of chemical pollution in aquatic ecosystems. Standardised methodologies for acute aquatic bioassays have been adopted from international agencies. As a result of these standard methods, the use of laboratory cultured organisms for toxicity testing has been more popular than that of indigenous field-caught organisms. Included in these adopted methods are those for the cultured crustacean Daphnia pUlex. D.pulex is adapted to living in standing water and the suitability of this species to determine toxic effects for South African riverine environments, which are largely flowing, has been questioned. Thus this thesis is a case-study ofthe use of D.pulex and indigenous site-specific macroinvertebrates as toxicity test organisms for setting acute water quality guidelines to protect aquatic ecosystems. The study highlights site-specific problems such as reference sites and organism identification. The acute tolerance of selected indigenous invertebrates was compared to that of D. pulex, using both a single-substance reference toxicant (zinc) and selected whole efiluents. The significance of source population and culture age as a potential source of biological variability between D.pulex cultures was also investigated. D.pulex cultures have been initiated in South Africa from females collected from a number of different local populations; also it is assumed that no genetic change (due to mutation) occurs within a D.pulex culture over time. In order to establish if source population and culture age are a source of biological variability between D.pulex experiments, the acute tolerJuce to zinc of two different D.pulex populations and three different generations within a population were compared. Due to experimental variability results were inconclusive, and differences in tolerance as a result of population difference or culture age could not be determined with confidence. The acute tolerance of D.pulex to a single reference chemical (zinc) and selected whole efiluents was compared to that of selected indigenous invertebrates. Acute 48 h D.pulex zinc tolerance (LC50 range: 0.22 - 0.60 mg/l Zn) was found to be more sensitive than acute 96 h tolerances shown by mayfly species A.fconurus peringueyi (Heptageniidae) (LC50: 17.42 mg/l Zn), Euthrauluselegans (Leptophlebiidae) (LC50: 0.98 mg/IZn), Ba~tidae (LC50: 0.94mg/IZn) and shrimp, Caradina nilotica (Atyidae) (LC50: 3.17 mg/l Zn). This result suggests that guidelines for zinc set using D.pulex will protect the selected indigenous invertebrates. Selected whole eftluents were not acutely toxic to either D.pulex or selected indigenous invertebrates. These experiments were used as a case study for method development regarding the comparative use of D.pulex and indigenous invertebrates in acute whole eftluent toxicity testing. Finally, it is recommended that a suite of indigenous organisms (e.g. macroinvertebrates, fish and algae), as well as laboratory cultured D.pulex, be used in the initial setting of guidelines and that D.pulex be used for routine compliance monitoring. It is futher recommended that a suite of available monitoring methods, such as chemical and biomonitoring methodologies, be used in conjuction with toxicity testing in water quality management.

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