M.Sc. (Aquatic Health) / Sediments can contain a variety of organic and inorganic contaminants. These contaminants accumulate, resulting in extremely high concentrations even once the overlying water concentrations are at or below acceptable water quality guidelines. Any changes in the physical parameters'of the overlying water can cause these pollutants to be released back into solution. Accumulated contaminants can be released at even higher concentrations than previously detected. In recent years, sediment contamination has highlighted the need to monitor these previously overlooked pollutant sources that have accumulated in aquatic ecosystems. South Africa does not currently have standardised methods to assess sediment toxicity. Although international methods exist, they are largely untested in South Africa and the organisms needed to conduct these tests are not readily available. Over the years numerous culture methods have been develop globally for culturing organism to be used for water and sediment toxicity tests. In South Africa, the focus has mainly been on culturing organisms for water. toxicity testing. Sediment toxicity testing with indigenous organism however, was not developed. Established international culture methods from the United States Environmental Protection Agency, the Organisation for Economic Cooperation and Development, and Environment Canada were taken into consideration when developing the laboratory culture method for two (2)of the selected organisms (Chironomus spp. & Hydra sp.) from this study. A preliminary culture method was also developed for the third selected organism, Melanoides tuberculata (gastropod). The organisms cultured in this study were selected based on their extent of contact with the substrate, ease of handling, availability, culture maintenance as well as their reproductive cycle. The Hydra, Chironomids and M. tuberculata cultures were successfully breeding under laboratory conditions and remained stable. The Chironomus sp. and M. tuberculata maintain contact with the sediment making them suitable as ecologically relevant organisms for use in whole sediment toxicity testing in South Africa.

Aquatic toxicity tests are increasingly being used in water resource management worldwide, and currently in South Africa, policy and legislation are being drafted to reflect this international trend. While standard toxicity test methods and test organisms are being considered to develop and set water quality guidelines and effluent discharge limits, it is not clear whether guidelines and discharge limits set using these standard test organisms will be sufficient to protect South Africa’s scarce water resources. As part of ongoing research to investigate the use of indigenous riverine organisms as toxicity test organisms a number of potential species have been identified, including the freshwater shrimp Caridina nilotica. For much of the history of aquatic toxicological data the bulk of the data has been generated by acute toxicity testing, based on short exposures and using mortality as the response end point. There are relatively few chronic, longterm tests with sub-lethal endpoints. However, it was recognized that information about longer exposure durations and non lethal response endpoints was needed, instead of mortality. Chronic tests can provide a more environmentally realistic measure of chemical toxicity than acute toxicity tests. Caridina nilotica has been identified as a potential standard toxicity test organism, as it is widely distributed, easy to find and it occurs in flowing waters. It is an indigenous species which can be easily cultured and maintained in the laboratory and is also ecologically important. Both adults and juveniles have been used successfully in acute toxicity tests at the Institute for Water Research (Rhodes University) and the ability to rear the organisms under laboratory conditions has allowed the development of chronic toxicity tests using C. nilotica. Chronic early life stage tests include continuous exposure of the early life stages, which are presumed to be the most sensitive for aquatic organisms. This study reports on the embryonic development of C. nilotica at the culture temperature of 24⁰C. Morphological developmental stages were monitored and measured and 7 developmental stages were identified. Based on the measurements of the features that were identified, toxicity tests using the reference chemicals sodium chloride (NaCl), sodium sulphate (Na₂SO₄) and cadmium chloride (CdCl₂) were undertaken to test the suitability of C. nilotica embryonic development for chronic toxicity tests for use in water resource management. The length, width, length:width ratios and area of the features decreased in size when exposed to the chemicals. The Lowest Observed Effect Concentration (LOEC) values were 2000mg/L for Na₂SO₄, 3000mg/L for NaCl and 0.31mg/L for CdCl₂. The No Observed Effect Concentration (NOEC) values were 1000mg/L for Na₂SO₄, 2000mg/L for NaCl and <0.31mg/L for CdCl₂. Further research on the teratogenic effects of single chemicals and industrial effluent on developing C. nilotica embryos needs to be undertaken in order to evaluate the described test protocol for use in water resource management.

M.Sc. / This study was initiated after a clear need to test and establish a more userfriendly fish species for use in routine laboratory bioassays was identified. This led to a literature review of current toxicity testing species of fish being used, internationally as well as nationally, and identifying which species could possibly be the most suited for use in South African laboratories. From this literature review, it was evident that much emphasis is placed on the practicability of the chosen fish species, and the fact that it can easily be bred within the laboratory, as well as the particular fish specie’s general sensitivity to various toxicants over a wide range of concentrations. The objective of this study is therefore to profile various species of fish to determine which species would be most suited to routine toxicity testing under South African laboratory conditions. This will be done through assimilation of available literature as well as personal communications with people with various expertise and experience in working with the particular fish species. This choice will then be based on the ability of the particular species of fish to ‘balance’ amenability to laboratory conditions with general sensitivity to toxicants. Various indigenous as well as exotic species were therefore selected and tested for suitability for routine testing. Exotic species included Poecilia reticulata and Danio rerio, while the indigenous species tested were Barbus trimaculatus, Barbus argenteus, Tilapia sparrmanii, Oreochromis mossambicus and Pseudocrenilabrus philander philander. Breeding experiments were conducted with all of the abovementioned species and, based on these results; recommendations are made as to which species of fish showed the highest degree of amenability to maintenance within the laboratory.

Waterborne diarrhoeal infections continue to be a major health setback in developing countries, especially in rural areas which lack adequate supply of portable water and sanitation facilities. Globally, waterborne diarrhoeal infections occur with an estimated mortality rate of 10–25 million deaths per year, 95% of which are children under the age of 5 years. The Vibrio species is one of the major groups of enteric pathogens that are responsible for diarrhoeal infections. Many strains of these bacterial species continue to cause epidemics of diarrhoea throughout the world. In this study, the prevalence of Vibrio pathogens in wastewater final effluents was assessed. Wastewater final effluent and discharge point samples were collected monthly between September 2012 and August 2013. All samples were collected aseptically using sterile 1 L Nalgene bottles containing 0.5 ml of sterile sodium thiosulphate solution and transported on ice to the laboratory for analyses within 6 h of collection. The membrane filtration method was used for enumeration of presumptive Vibrio densities on thiosulfate citrate bile salt (TCBS) agar plates. Polymerase chain reaction (PCR) was then used to confirm the identities of the presumptive Vibrio species using the species-specific primers. The confirmed isolates were further subjected to molecular characterization to confirm their respective pathotypes. Presumptive Vibrio densities varied from 0 to 2.11 × 102 cfu/100 ml. Out of 300 confirmed Vibrio isolates; 13.3% (40/300) were Vibrio fluvialis, 22% (66/300) were confirmed to be Vibrio parahaemolyticus, and 24.7% (74/300) proved to be Vibrio vulnificus, and 40% (120/300) were other Vibrio species which were not assessed for in this study. The strains of Vibrio fluvialis were found to exhibit 100% resistance to Polymixin and Tetracycline. However, Gentamicin was active against all the three Vibrio species selected for the purpose of this research. The recovery of Vibrio species in the discharged effluents throughout the sampling period even in adequately disinfected effluents is not acceptable considering the fact that Vibrio is a pathogenic bacterium. The findings of this study underline the need for constant monitoring of the microbiological qualities of discharged effluents and might also be suggestive for a review of the disinfection methods used at the treatment works.

Worldwide, water reuse is promoted as an alternative for water scarcity, however, wastewater effluents have been reported as possible contaminants to surface water. The failure of some wastewater treatment processes to completely remove organic matter and some pathogenic microorganisms allows them to initiate infections. This manifests more in communities where surface water is used directly for drinking. To assess water quality, bacteria alone cannot be used as it may be absent in virus-contaminated water. This study was carried out to assess the quality of two wastewater treatment plant effluents from the Eastern Cape Province of South Africa. Physicochemical parameters and microbiological parameters like faecal coliforms, adenovirus, rotavirus, hepatitis A virus, norovirus and enterovirus were evaluated over a projected period of one year. Physicochemical parameters were measured on site using multiparameters, faecal coliforms enumerated using culture-based methods and viruses are detected using both conventional and real-time PCR. Physicochemical parameters like electrical conductivity, turbidity, free chlorine and phosphates were incompliant with the standards set by the Department of Water affairs for effluents to be discharged. Faecal coliform counts were nil for one plant (WWTP-R) where they correlated inversely (P < 0.01) with the high free chlorine. For WWTP-K, faecal coliforms were detected in 27% of samples in the range of 9.9 × 101 to 6.4× 104 CFU/100ml. From the five viruses assessed, three viruses were detected with Rotavirus being the most abundant (0-2034176 genome copies/L) followed by Adenovirus (0–275 genome copies/L) then Hepatitis A virus (0–71 genome copies/L) in the WWTP-K while none of the viruses was detected in WWTP-R. Species B, species C and Adv41 serotypes were detected from the May 2013 and June 2013 samples where almost all parameters were incompliant in the plant. The detection of these viruses in supposedly treated effluents is suggestive of these being the sources of contamination to surface water and therefore renders surface waters unsafe for direct use and to aquatic life. Although real-time PCR is more sensitive and reliable in detection of viruses, use of cell-culture techniques in this study would have been more efficient in confirming the infectivity of the viruses detected, hence the recommendation of these techniques in future projects of this nature.

Water is an essential need that stimulates health and well being. Increase in population size and urbanization negatively affect water resources due to high demands of effluent outputs. Wastewater is an important reservoir for Escherichia coli and can present significant acute toxicity if released into receiving water sources without being adequately treated. E. coli is used as indicator organism for the detection of faecal contamination. These strains have been considered to be one of the primary causes of diarrhoeal infections worldwide. The present study was conducted between September 2012 and June 2013 to assess the prevalence of pathogenic E. coli strains in the final effluents of four wastewater treatment plants in Chris Hani and Buffalo City Municipalities in the Eastern Cape Province of South Africa. Standard membrane filtration technique was used for bacteriological analysis and molecular based technique was used for identification of E. coli pathotypes. The results were recorded in colony forming units/100 ml. Faecal coliforms ranged between 0-9.6×10³ CFU/100 ml for the wwtp-Q and E. coli densities ranged between 0-8.4×10³ CFU/100ml. Faecal coliforms ranged between 4×10²-9.7×10³ CFU/100 ml for wwtp-M and E. coli densities ranged between 1.2×10¹-8.4×10³ CFU/100 ml. The wwtp-E showed to have bacterial counts of faecal coliforms ranging between 4.0×10³-8.2×10³ CFU/100 ml and E. coli densities ranging between 3.5×10¹-7.1×10³ CFU/100 ml. The WWTP-K in this study was only assessed for the presence of E. coli. Faecal coliforms were assessed by the other members of the group. This plant showed to have E. coli densities ranging between 0-7.5×10²CFU/100 ml. A total of 200 presumptive E. coli isolates were subjected to screening by conventional PCR in which (29%) of the wwtp-M isolates were positively identified as E. coli, (16%) of the wwtp-K, (22%) of the wwtp-Q and (34%) of the wwtp-E isolates were positively confirmed as E. coli. A total of 100 randomly selected E. coli isolates were characterised into different pathotypes. (16%) of positive isolates were detected as EPEC and 11% were detected as UPEC strains. There was no detection for the ETEC strains. Antibiotic susceptibility patterns of E. coli strains showed high levels of resistance to Penicillin G, Erythromycin, Tetracycline and Sulfamethoxazole. High levels of Susceptibility were observed in antibiotics such as Chloramphenicol, Amoxicillin and Tetracycline. The results of this study reveal that the plants were above the recommended Standard limit of zero CFU/100 ml for effluents meant to be discharge into receiving water sources. This study reveals inadequacy of the plants studied to produce effluents of acceptable quality.