by Wong Suk Ying. / Thesis submitted in: November 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 144-157). / Text in English; abstracts in English and Chinese. / by Wong Suk Ying. / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.v / List of Figures --- p.x / List of Tables --- p.xvi / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Electroplating Industry in Hong Kong --- p.1 / Chapter 1.1.1 --- Typical stages in electroplating process --- p.1 / Chapter 1.1.1.1 --- Pre-treatment --- p.1 / Chapter 1.1.1.2 --- Electroplating --- p.3 / Chapter 1.1.1.3 --- Post-treatment --- p.3 / Chapter 1.1.2 --- Typical characteristics of wastestreams from electroplating industry --- p.3 / Chapter 1.2 --- Chemical Specific Approach against Toxicity Based Approach --- p.6 / Chapter 1.3 --- Ecotoxicological Study on Electroplating Effluent --- p.7 / Chapter 1.4 --- Toxicity Identification Evaluation --- p.8 / Chapter 1.4.1 --- Phase I: Toxicity Characterization --- p.9 / Chapter 1.4.2 --- Phase II: Toxicity Identification --- p.10 / Chapter 1.4.3 --- Phase III: Toxicity Confirmation --- p.12 / Chapter 1.5 --- Toxicity Identification Evaluation on Electroplating Effluent --- p.14 / Chapter 1.6 --- Selection of Organisms for Bioassays --- p.15 / Chapter 1.6.1 --- Organism used for toxicity identification evaluation --- p.17 / Chapter 2. --- OBJECTIVES --- p.20 / Chapter 3. --- MATERIALS AND METHODS --- p.21 / Chapter 3.1 --- Source of Samples --- p.21 / Chapter 3.2 --- Toxicity Identification Evaluation: Phase I Baseline Toxicity Test --- p.21 / Chapter 3.2.1 --- Microtox® test --- p.23 / Chapter 3.2.2 --- Growth inhibition test of a marine unicellular microalga Chlorella pyrenoidosa CU-2 --- p.25 / Chapter 3.2.3 --- Survival test of a marine amphipod Hylae crassicornis --- p.28 / Chapter 3.2.4 --- Survival test of a marine shrimp juvenile Metapenaeus ensis --- p.31 / Chapter 3.3 --- Toxicity Identification Evaluation: Phase I Toxicity Characterization --- p.34 / Chapter 3.3.1 --- pH adjustment filtration test --- p.35 / Chapter 3.3.2 --- Aeration test --- p.36 / Chapter 3.3.3 --- C18 solid phase extraction test --- p.37 / Chapter 3.3.4 --- EDTA chelation test --- p.38 / Chapter 3.3.5 --- Graduated pH test --- p.40 / Chapter 3.4 --- Toxicity Identification Evaluation: Phase II Toxicity Identification --- p.41 / Chapter 3.4.1 --- Filter extraction test --- p.41 / Chapter 3.4.2 --- Total metal content analysis --- p.42 / Chapter 3.5 --- Toxicity Identification Evaluation: Phase III Toxicity Confirmation --- p.43 / Chapter 3.5.1 --- Chemicals --- p.44 / Chapter 3.5.2 --- Mass balance test --- p.44 / Chapter 3.5.3 --- Spiking test --- p.44 / Chapter 4. --- RESULTS --- p.46 / Chapter 4.1 --- Chemical Characteristics of the Electroplating Effluent Samples --- p.46 / Chapter 4.2 --- Toxicity Identification Evaluation: Phase I Baseline Toxicity --- p.46 / Chapter 4.2.1 --- Toxicity of electroplating effluent samples on Microtox® test --- p.46 / Chapter 4.2.2 --- Toxicity of electroplating effluent samples on growth inhibition test of microalga Chlorella pyrenoidosa CU-2 --- p.46 / Chapter 4.2.3 --- Toxicity of electroplating effluent samples on survival test of amphipod Hyale crassicornis --- p.52 / Chapter 4.2.4 --- Toxicity of electroplating effluent samples on survival test of shrimp juvenile Metapenaeus ensis --- p.52 / Chapter 4.3 --- Toxicity Identification Evaluation: Phase I Toxicity Characterization --- p.52 / Chapter 4.3.1 --- Toxicity Characterization of electroplating effluent samples using Microtox® test --- p.56 / Chapter 4.3.2 --- Toxicity Characterization of electroplating effluent samples using microalgal growth inhibition test of Chlorella pyrenoidosa CU-2 --- p.59 / Chapter 4.3.3 --- Toxicity Characterization of electroplating effluent samples using survival test of amphipod Hyale crassicornis --- p.65 / Chapter 4.3.4 --- Toxicity Characterization of electroplating effluent samples using survival test of shrimp juvenile Metapenaeus ensis --- p.68 / Chapter 4.4 --- Toxicity Identification Evaluation: Phase II Toxicity Identification --- p.73 / Chapter 4.4.1 --- Metal analysis on the electroplating effluents --- p.75 / Chapter 4.4.2 --- Effect of filter extraction test on toxicity recovery of the electroplating effluent samples --- p.75 / Chapter 4.4.2.1 --- Microtox® test --- p.75 / Chapter 4.4.2.2 --- Growth inhibition test of microalga Chlorella pyrenoidosa CU-2 --- p.75 / Chapter 4.4.2.3 --- Survival test of amphipod Hyale crassicornis --- p.81 / Chapter 4.4.2.4 --- Survival test of shrimp juvenile Metapenaeus ensis --- p.90 / Chapter 4.4.3 --- Effect of filter extraction test on metal ions recovery of the electroplating effluent samples --- p.90 / Chapter 4.5 --- Toxicity Identification Evaluation: Phase III Toxicity Confirmation --- p.96 / Chapter 4.5.1 --- Mass balance test results on Microtox® test --- p.96 / Chapter 4.5.2 --- Mass balance test results on survival test of amphipod Hyale crassicornis --- p.104 / Chapter 4.5.3 --- Spiking test results on Microtox® test --- p.106 / Chapter 4.5.4 --- Spiking test results on survival test of amphipod Hyale crassicornis --- p.113 / Chapter 5. --- DISCUSSION --- p.118 / Chapter 5.1 --- Toxicity Identification Evaluation: Phase I Baseline Toxicity --- p.118 / Chapter 5.2 --- Toxicity Identification Evaluation: Phase I Toxicity Characterization --- p.119 / Chapter 5.2.1 --- pH adjustment filtration test --- p.119 / Chapter 5.2.2 --- Aeration test --- p.120 / Chapter 5.2.3 --- C18 solid phase extraction test --- p.120 / Chapter 5.2.4 --- EDTA chelation test --- p.120 / Chapter 5.2.5 --- Graduated pH test --- p.121 / Chapter 5.3 --- Toxicity Identification Evaluation: Phase II Toxicity Identification --- p.122 / Chapter 5.3.1 --- Metal analysis on the electroplating effluents --- p.122 / Chapter 5.3.2 --- Effect of filter extraction test on toxicity and metal ions recovery of the electroplating effluent samples --- p.123 / Chapter 5.3.3 --- Comparison between the concentrations of the metal ions in the electroplating effluent samples with the Technical Memorandum on standards for effluent discharged --- p.124 / Chapter 5.3.4 --- Comparison between the concentrations of the metal ions in the electroplating effluent samples with the toxicity of the metal ions reported in the literature --- p.124 / Chapter 5.3.4.1 --- Microtox® test --- p.126 / Chapter 5.3.4.2 --- Microalga --- p.126 / Chapter 5.3.4.3 --- Amphipod --- p.126 / Chapter 5.3.4.4 --- Shrimp --- p.126 / Chapter 5.4 --- Toxicity Identification Evaluation: Phase III Toxicity Confirmation --- p.131 / Chapter 5.4.1 --- Mass balance test on Microtox® test --- p.132 / Chapter 5.4.2 --- Mass balance test on survival test of amphipod Hyale crassicornis --- p.133 / Chapter 5.4.3 --- Spiking test on Microtox® test --- p.133 / Chapter 5.4.4 --- Spiking test on survival test of amphipod Hyale crassicornis --- p.134 / Chapter 5.5 --- Toxicity of the Metal Ions Identified as the Toxicants in the Electroplating Effluent --- p.135 / Chapter 5.5.1 --- Copper --- p.135 / Chapter 5.5.2 --- Nickel --- p.137 / Chapter 5.5.3 --- Zinc --- p.138 / Chapter 5.6 --- Summary --- p.140 / Chapter 6. --- CONCLUSIONS --- p.142 / Chapter 7. --- REFERENCES --- p.144 / Chapter 7.1 --- APPENDIXES --- p.158

The production of final effluents that meet discharged requirements and guidelines remain a major challenge particularly in the developing world with the resultant problem of surface water pollution. This study assessed the physicochemical and microbiological qualities of two wastewater treatment works in the Eastern Cape Province of South Africa in terms of the prevalence of faecal coliforms and Escherichia coli O157:H7 over a five month period. All physicochemical and microbiological analyses were carried out using standard methods. Data were collected in triplicates and analysed statistically using IBM SPSS version 20.0. The ranges of some of the physicochemical parameters that complied with set guidelines include pH (6.7 – 7.6), TDS (107 – 171 mg/L), EC (168 – 266 μS/cm), Temperature (15 – 24oC), NO3- (0 – 8.2 mg/L), NO2- (0.14 – 0.71 mg/L) and PO4 (1.05 – 4.50 mg/L). Others including Turbidity (2.64 – 58.00 NTU), Free Cl (0.13 – 0.65 mg/L), DO (2.20 – 8.48 mg/L), BOD (0.13 – 6.85 mg/L) and COD (40 – 482 mg/L) did not comply with set guidelines. The microbiological parameters ranged 0 – 2.7 × 104 CFU/100 ml for FC and 0 – 9.3 × 103 for EHEC CFU/100 ml, an indication of non-compliance with set guidelines. Preliminary identification of 40 randomly selected presumptive enterohemorrhagic E. coli isolates by Gram’s staining and oxidase test shows 100% (all 40 selected isolates) to be Gram positive while 90% (36 randomly selected isolates) were oxidase negative. Statistical correlation between the physicochemical and the microbiological parameters were generally weak except in the case of free chlorine and DO where they showed inverse correlation with the microbiological parameters. The recovery of EHEC showed the inefficiency of the treatment processes to effectively inactivate the bacteria, and possibly other pathogenic bacteria that may be present in the treated wastewater. The assessment suggested the need for proper monitoring and a review of the treatment procedures used at these treatment works.

Escherichia coli (E. coli) is a common inhabitant of surface waters in the developed and developing worlds. The majority of E. coli cells present in water are not particularly pathogenic to humans; however, there are some present in small proportion that possess virulence genes that allow them to colonize the digestive tract. Pathogenic E. coli causes acute and chronic diarrheal diseases, especially among children in developing countries and in travelers in these locales. The present study, conducted between August 2007 and July 2008, investigated the prevalence and distribution of virulent E. coli strains as either free or attached cells in the final effluents of three wastewater treatment plants located in the Eastern Cape Province of South Africa and its impact on the physico-chemical quality of the receiving water body. The wastewater treatment plants are located in urban (East Bank Reclamation Works, East London), peri-urban (Dimbaza Sewage Treatment Works) and in rural area (Alice Sewage Treatment Works). The effluent quality of the treatment plants were acceptable with respect to pH (6.9-7.8), temperature (13.8-22.0 °C), dissolved oxygen (DO) (4.9-7.8 mg/L), salinity (0.12-0.17 psu), total dissolved solids (TDS) (119-162 mg/ L) and nitrite concentration (0.1-0.4 mg/l). The other xii physicochemical parameters that did not comply with regulated standards include the following: phosphate (0.1-4.0 mg/L); chemical oxygen demand (COD) (5-211 mg/L); electrical conductivity (EC) (237-325 μS/cm) and Turbidity (7.7-62.7 NTU). Results suggest that eutrophication is intensified in the vicinity of the effluent discharge points, where phosphate and nitrate were found in high concentrations. Presumptive E. coli was isolated from the effluent samples by culture-based methods and confirmed using Polymerase Chain Reaction (PCR) techniques. Antibiogram assay was also carried out using standard in vitro methods on Mueller Hinton agar. The viable counts of presumptive E. coli for the effluent samples associated with 180 μm plankton size ranged between 0 – 4.30 × 101 cfu/ml in Dimbaza, 0 – 3.88 × 101 cfu/ml in Alice and 0 – 8.00 × 101 cfu/ml in East London. In the 60 μm plankton size category E. coli densities ranged between 0 and 4.2 × 101 cfu/ml in Dimbaza, 0 and 2.13 × 101 cfu/ml in Alice and 0 and 8.75 × 101 cfu/ml in East London. Whereas in the 20 μm plankton size category presumptive E. coli density varied from 0 to 5.0 × 101 cfu/ml in Dimbaza, 0 to 3.75 × 101 cfu/ml in Alice and 0 to 9.0 × 101 cfu/ml in East London. The free-living presumptive E. coli density ranged between 0 and 3.13 × 101 cfu/ml in Dimbaza, between 0 and 8.0 × 101 cfu/ml in Alice and between 0 and 9.5 × 101 cfu/ml in East London. Molecular analysis successfully amplified target genes (fliCH7, rfbEO157, ial and aap) which are characteristic of pathogenic E. coli strains. The PCR assays using uidA-specific primer confirmed that a genetic region homologous in size to the E. coli uidA structural gene, including the regulatory region, was present in 3 of the E. coli isolates from Alice, 10 from Dimbaza and 8 from East London. Of the 3 E. coli isolates from Alice, 1 (33.3%) was positive for the fliCH7 genes and 3 was positive for rfbEO157 genes. Out of the 10 isolates from Dimbaza, 4 were xiii positive for fliCH7 genes, 6 were positive for the rfbEO157 genes and 1 was positive for the aap genes; and of the 8 isolates from East London, 1 was positive for fliCH7 genes, 2 were for the rfbEO157 genes, 6 were positive for the ial genes. Antimicrobial susceptibility profile revealed that all of the E. coli strains isolated from the effluent water samples were resistant (R) to linezolid, polymyxin B, penicillin G and sulfamethoxazole. The E. coli isolates from Dimbaza (9/10) and East London (8/8) respectively were resistant to erythromycin. All the isolates were found to be susceptible (S) to amikacin, ceftazidime, ciprofloxacin, colistin sulphate, ceftriaxone, cefotaxime, cefuroxime, ertapenem, gatifloxacin, gentamycin, imidazole, kanamycin, meropenem, moxifloxacin, neomycin, netilmicin, norfloxacin and tobramycin. The findings of this study revealed that the Alice wastewater treatment plant was the most efficient as it produced the final effluent with the least pathogenic E. coli followed by the Dimbaza wastewater treatment plant. In addition, the findings showed that the wastewater treatment plant effluents are a veritable source of pathogenic E. coli in the Eastern Cape Province watershed. We suggest that to maximize public health protection, treated wastewater effluent quality should be diligently monitored pursuant to ensuring high quality of final effluents.

Water is an indispensible and yet a difficult resource to be renewed, thus water scarcity has become one of the major challenges faced worldwide, with the Southern regions of Africa being the most impacted and affected, especially the Eastern Cape Province of South Africa where rural communities depend on receiving waterbodies that are often negatively impacted by wastewater final effluents. This present study was conducted between August and December 2010 to assess the physicochemical and microbial qualities of the final effluents of peri-urban and rural communities based wastewater treatment plants in the Eastern Cape Province. The physicochemical parameters were determined on site and in the laboratory, while bacteriological qualities were determined using culture based techniques. The virological qualities were determined by molecular methods using reverse transcriptase polymerase chain reaction for the target RNA virus and the conventional polymerase chain reaction for the target DNA virus. For both wastewater treatment plants, the physicochemical parameters ranged as follows: chemical oxygen demand (5.95-45 mg/L); total dissolved solids (114.5-187.0 mg/L); salinity (0.12-0.20 psu); temperature (14.2-25.7oC); pH (6.0-7.6); nitrate and nitrites (1.55-6.7 mg/L and 0.023-1.15 mg/L respectively); biological oxygen demand (3.5-7.8 mg/L); turbidity (1.49-6.98 NTU); and chlorine residual (0-2.97 mg/L). Feacal indicator bacteria counts ranged as follows: feacal coliforms (0-1.25×104 cfu/100 ml); total coliforms (0-3.95×104 cfu/100 ml); and enterococci (0-5.0×103 cfu/100 ml). xviii Seventy five percent of the rural community based plant and 80 percent of the peri-urban community based plant were positive for coxsackie A virus, while hepatitis A virus was detected in all the rural community based plant 80 percent of the peri-urban community based plant. This study suggests the need for intervention by appropriate regulatory agencies to ensure regular monitoring of the qualities of final effluents of wastewater treatment facilities in the Eastern Cape Province and ensure compliance to established guidelines.

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.