Submitted in fulfillment of the requirements for the degree of Doctorate of Technology: Biotechnology, Durban University of Technology, Durban, South Africa, 2014. / All over the world concerns have been raised over the possible adverse effects that may occur when exposed to chemicals that have the potential to interfere and affect the endocrine system. The concern is directed at both humans and wildlife. There is still a lack of public awareness regarding Endocrine Disrupting Chemicals (EDCs) and the harmful effects on humans and wildlife. It has only been within the last decade that South Africa began the actual task for proper management and control for water and wastewater quality. There are many ways to detect these EDCs all of which are very laborious and most of the cases these EDCs are either in the pico or nano gram per litre range, too minute for many methods to detect effectively; so therefore the research project aimed to use rapid and sensitive techniques to determine the quickest means to detect the very low concentrations of theses EDCs. Two techniques were researched, i.e., Enzyme Linked immunoassays (ELISAs) and Radio-immunoassays (RIAs). The research study thus assessed the solid phase extraction (SPE) technique for total recovery of hormones; the ELISA and RIA techniques for rapid detection of natural (estrone (E1), estradiol (E2) and estriol (E3) and synthetic ethinylestradiol (EE2) by validating the precision and reproducibility . These techniques were then applied to determine hormone EDC removal first at laboratory scale investigations and then applied to full scale wastewater treatment plants (WWTP) with different configurations in order to deduce removal efficiency of each type of plant. The next phase assessed the toxicity of individual and combined estrogen standards as well as the toxicity in the WWTPs and classify and to determine if there was a correlation between hormone concentration and toxicity in final effluents. The assessment of the SPE and the immunoassay procedures (ELISA AND RIA) using standards and controls found that both these assays can be utilised to quantify hormone estrogens in wastewater. The small sample volume required reduced the labour time and application of the procedure made it cost effective and reliable techniques. The intra-assay and inter-assay validation procedures as well as the standard recoveries confirmed reproducibility and precision of the immunoassays. The % CV were <10% for both the intra-assay and inter-assay validations. The laboratory scale investigations included the operation of a modified Ludzak-Ettinger (MLE) process which enabled control and manipulation over the operational parameters in order to establish how certain parameters influenced the removal of hormone EDCs. One such parameter that was manipulated was the sludge retention time (SRT). The MLE tests showed that the SRTs definitely have an effect on the removal of hormones from the influent as well as the overall performance of sewage treatment. The 10 day SRT proved that longer SRTs will definitely aid in the removal of hormones and possibly other EDCs in raw sewage. During the 10 day SRT the influent hormone concentrations (E1: 59.11 ng/L, E2: 61.40 ng/L) were almost double than the influent hormone concentrations (E1: 26.46 ng/L, E2: 27.60 ng/L) during the 5 day SRT, which impacted on the removal efficiency. The 5 day SRT had an overall average E2 and E1 removal of 78.11% and 81.71% respectively while the 10 day SRT had average E2 and E1 removal of 91.24 % and 80.56% respectively. The 24 hour batch test provided evidence of the reversible metabolism of the E2 hormone. This was seen by the rapid decrease of E2 and the rapid increase of E1 in less than 3 hours, which proved that E2 can be metabolized in to E1. An average reduction of 94.44% of E2 was seen after 5 hours and after 10 hours was no longer detected. After 13 hours E1 could no longer be detected. This finding also provided clarity as to the lower percentage removal of E1 during the 10 day SRT of the MLE process. The Vibrio fischeri biotox method implemented was the most economic and easiest way to conduct the toxicity tests. The validation of the test used a 52.9 mg/L K2Cr2O7 standard which provided a Cr (VI) concentration of 18.7 mg/L in the final test suspension which is the theoretical effective concentration causing 50% inhibition (EC50). This specific concentration of the Cr (VI) exhibited an EC50 at 20.08 mg/L. The toxicity investigations of the individual and mixed hormone standards revealed that at the 10 ng/L concentration the individual E2 standard had the highest percentage inhibition (%INH) of 45.99% after the 30 minute contact time (T30), and when this standard was further diluted to 5 and 1 ng/L also showed higher % INH (26.04 and 23.66 %INH, respectively) than the individual EE2 standard (21.92 %INH) at 10 ng/L. . According to the toxicity classification system and after interpretation of the data, all the hormone standards were classified as Class II as they all exhibited slight acute toxicity. The 10 ng/L E2 standard had Toxicity Units (TU) of 0.8 which was close to the Class III level; however when it was in a mixture with E1 and E3, the TU was much lower (0.6 TU). The synthetic EE2 hormone also showed slight acute toxicity and had the lowest TU of 0.4. The application of the above mentioned techniques to full scale WWTPs with different configurations showed different removal efficiencies. The WWTPs ranged from the most primary consisting of just oxidation ponds to biological trickling filters, to biological nutrient removal (BNR) to conventional activated sludge (AS) plants. Removal rates ranged from 29% to 96% for E2, 0% to 89% for E1 and 0% to 100% for EE2. The overall ranking of the WWTPs from the most efficient to least efficient in terms of hormone removal were as follows: Plant E (91%) = Plant D (before UF) (91%) > Plant B (east side) (88%) > Plant B (west side) (77%) > Plant C (east side) (71%) > Plant D (after UF) (57%) > Plant A (56%) > Plant C (west side) (12%). Using the Vibrio fischeri method to evaluate the reduction of toxicity in WWTPs C, D and E proved effective. It was seen immediately after secondary biological treatment in the clarifier effluent the toxicity was reduced. Plants C, D and E had reduced the toxicities by 100, 80 and 97 % immediately after secondary biological treatment, while after the addition of the Chlorine disinfectant in the final stage of treatment the toxicity increased having %INH of 99.9, 15.7 and 99.9 respectively. In conclusion the SPE can be used as an extraction procedure for hormones in wastewater and the immunoassays can be used as rapid techniques for quantification of hormone EDCs in wastewater. The ELISA technique proved to be the slightly superior to the RIA in terms of facilities required. The laboratory scale procedures proved that some hormones can be oxidised to other hormones and therefore longer sludge retention times may be required to improve the removal. The study of the different WWTPs configuration showed that plant configuration and operational parameters impact the removal of hormone EDCs. The composition of the influent received by the plant also has an effect on the removal, i.e., whether it’s industrial, domestic or a mixture of both. Results concluded that plants which have either mixing and/or aeration with activated sludge and longer SRTs of more than 10 days have a higher rate of hormone removal than those plants with shorter SRTs and that the activated sludge processes were capable of reducing the toxicity of the influent. Overall results indicated that hormone EDCs are indeed being discharged with the effluents from WWTPs in South Africa. However whether the concentrations left in the final effluents will still have an adverse effect on the aquatic life is a question that still remains unanswered. The aquatic ecosystems are inevitably being polluted with these EDCs and their breakdown products. / D

Surface water and sediments from the St. Lawrence River system (Quebec region) were analysed for genotoxicity using nonlinear SOS Chromotest parameters, as well as for their chemical concentrations of polycyclic aromatic hydrocarbons and heavy metals. Additionally, sediments chlorobenzenes, polychlorinated biphenyls, organochlorinated pesticides, ammonia and nitrites concentrations were determined. Organic contaminants were extracted dichloromethane. For surface water, fifteen extracts of filtered water and seven of particulates, and for sediments, one extract of pore water and three of particulates proved to be weakly genotoxic. All but one of the genotoxic responses observed in the surface water were obtained from samples taken from the highly industrial portion of the St. Lawrence River system, with the strongest responses observed in Lake St-Louis. Surface water genotoxicants partitioning favors the particulate fraction. Bottom particulates genotoxicity was one thousand fold weaker than suspended particulates. Additionally, whole sediments were extracted with a 10% dimethylsulfoxide-saline solution. Genotoxicity of hydrophilic contaminants was detected in all extracts. The observed distributions of genotoxicity values did not correlate with observed concentrations of demonstrated SOS inducers, mutagens and/or carcinogens, nor with the presence of other toxic chemicals.

No description available.

Zebra mussels (Dreissena polymorpha) have invaded a great proportion of the waters of the eastern part of North America. This mollusk may be a useful sentinel species for bioaccumulation of heavy metals and organic contaminants in aquatic ecosystems. The zebra mussel's capacity to bioaccumulate cadmium at environmentally relevant exposure and to sequester metals in metal-binding proteins, metallothioneins, was investigated. Elevated (relative to control) concentrations of Cd$ sp{2+}$ were detected in Dreissena exposed to $ ge$2 $ mu$g Cd/liter suggesting that zebra mussels cannot regulate Cd$ sp{2+}$ of trace exposure concentration. More than 85% of the measured Cd$ sp{2+}$ was bound to metallothioneins. / Lesser scaup (Aythya affinis) was assessed as a model species for potential impacts of zebra mussels on higher trophic levels. The feasibility of incubating and rearing scaup in semi-natural conditions was evaluated. A series of biomarkers was assessed in 3 groups of scaup fed a contaminant-free diet or diets containing zebra mussels from the St-Lawrence River or Lake Erie. / Lesser scaup proved to be a suitable species to raise in captivity providing daily water misting through out incubation and strict hygiene conditions during brooding. A hatching success of 89% was achieved. Appropriate housing, feeding and bathing conditions yielded low (3%) post-hatching mortality. / Phagocytosis and respiratory burst activities of heterophils of scaup were suppressed after 6 weeks of feeding on zebra mussels, compared to the control group. These two heterophilic functions were negatively correlated with the incidence of pododermatitis (bacterial feet infection), suggesting a suppression of the non specific immunity. Increased liver/body mass ratio and decreased hepatic vitamin A (retinol and retinyl palmitate) concentrations were observed in scaups fed zebra mussels. Lipid accumulation and glycogen overload were found in the livers of scaups fed mussels from the St-Lawrence River and the Lake Erie groups, respectively. These immunological, biochemical and histopathological biomarkers show promise for monitoring "early" injury and may help in the understanding of health impairment of different species of waterfowl exposed to xenobiotics via contaminated food sources.

Several sediment characteristics were analyzed to determine their suitability for use as potential normalization factors for the bioavailability of neutral organic compounds sorbed to sediments. Percent organic carbon, cation exchange capacity and particle surface area were measured sediment characteristics that varied sufficiently to encompass the range in observed sediment toxicity. Laboratory sediment toxicity test data using fluoranthene suggest that there is no biologically significant correlation between sediment toxicity and sediment characteristics (organic carbon, cation exchange capacity, particle size distribution, particle surface area). Fluoranthene amended sediments with similar organic carbon contents do not yield similar toxicities due to sorbed fluoranthene and thus do not support the organic carbon normalization approach for evaluating sediment quality or for sediment criteria development.

PhD. (Chemistry) / This thesis focuses on functionalising the silicone membrane by filling it with synthesised γ-Fe2O3 nanoparticles for the purpose of using it as a passive sampling device for monitoring microcystins and cylindrospermopsin in aquatic environments. The identity and purity of the synthesised nanoparticles was verified by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscope (SEM), surface zeta potential analyser and Brunauer-Emmet-Teller (BET). There was no chemical uptake of microcystin compounds into the silicone membrane without a suspension of maghemite nanoparticles. But, a linear uptake of microcystin compounds was observed into the functionalised silicone membrane passive sampler throughout the exposure period. These findings suggest that the silicone membrane filled with a suspension of synthesised γ-Fe2O3 nanoparticles can be an efficient, green and less costly instrument useful in estimating biotoxin concentrations in South African waters and the world at large.

Three artificial stream designs, termed Large Artificial Stream Units (LASUs), Raceways, and Channels, at two major scales (1700 L, 12.5 L and 20 L recirculated volume) were developed at the Institute for Water Research, Rhodes University, Grahamstown, in order to explore the possibilities of using indigenous rheophilic macroinvertebrates in routine toxicity tests. This study compared these systems, using 96h-EC50 values from sodium sulphate toxicity tests as the experimental response. Two local Ephemeroptera (Leptophlebiidae: Adenophlebia auriculata Eaton, and Baetidae: Afroptilum sudafricanum Lestage) were evaluated for their suitability in routine toxicity tests; and the possible effects of elevated salinity levels in South African rivers on the test species were assessed. Two sets of experiments with each mayfly species were conducted, following an unreplicated regression design. Dechlorinated tap water was used as the water source. Experiments in the Channels were repeated to determine experimental variability. Results were compared statistically by testing for overlap of 95% confidence limits (95%Cls) of EC50 values. The differences between A. auriculata EC50 values in the different systems were statistically significant (no overlap of 95%CLs), but they were not more variable than has been considered normal for biological systems (Coefficient of variation 20.1 %; ratio of greatest EC50 / smallest EC50 1.63). The differences were not related to the scale or the average current velocity characteristic of each stream design (average current velocity LASUs - Raceways - Channels 0.090 - 0.083 - 0.038 m/s). The Channels proved to be most efficient with regard to practical performance as they are portable and easily transportable, user-friendly, reliable, splash-free, cost effective to construct, and can easily be adapted to specific requirements. These systems are therefore recommended for regular use. The suitability of the two mayfly species for routine toxicity testing was evaluated. A. auriculata EC50 values showed a significant negative correlation with the corresponding average body-size (range 1476 - 1610 μm, mean 1555 μm). The different average body-sizes probably reflected the abundance of a certain size range present in the Palmiet River at the time of collection. Both species reacted similarly to Na₂S0₄ (similar slopes of the toxicity curves), identifying this salt as a slow acting toxicant. A. sudafricanum populations were more sensitive to Na₂S0₄ (EC50 3.404 g/L) than A. auriculata (EC50 8.090 g/L), probably because of its smaller body-size (mean 709 μm) and a lack of extremely tolerant individuals. In comparison to other freshwater macro invertebrates, including the standard toxicity test organism Daphnia spp., both mayfly species seemed to be moderately tolerant of Na₂S0₄; therefore there was no particular advantage to using these indigenous taxa rather than Daphnia spp. An assessment of the effects of elevated salinity/TDS levels on the test taxa yielded preliminary insights. A NaCI-EC50 for A. sudafricanum could be extrapolated and suggested a higher sensitivity to Na₂S0₄ than to NaCl. When Na₂S0₄ EC50 values of both species were compared to selected TDS levels of South African rivers, 4. auriculata would mostly not be affected, but A. sudafricanum might occasionally suffer from sub-lethal effects, depending on the sulphate proportion of the TDS. The South African guideline for TDS seemed to protect both species sufficiently.

There is growing concern that commonly used Pharmaceuticals and Personal Care Products (PPCPs) and pesticides are entering and contaminating drinking water supplies. The use of targeted quantitation of PPCP has been well established but there is an emerging trend to also screen for and identify unexpected environmental pollutants. Chemicals like pesticides hormones and antibiotics are especially of interest because of proven endocrine disrupting effects and a possible development of bacterial resistance. Powerful screening methods are required to detect and quantify the presence of these compounds in our environment. PPCP encompass a wide range of pollutants, including Endocrine Disrupting Compounds (EDC), pesticides, hormones, antibiotics, drugs of abuse, x-ray contrast agents and drinking water disinfection by-products to name a few. In order to properly assess the effects of these compounds on our environment, it is necessary to accurately monitor their presence. The diversity of chemical properties of these compounds makes method development challenging. LC/MS/MS is able to analyse polar, semi-volatile, and thermally labile compounds covering a wide molecular weight range. The new AB SCIEX TripleTOF™5600 LC/MS/MS was used to profile environmental samples for unexpected pollutants, to identify and characterise the chemical composition and structure of the pollutants, and to quantify (based on intensity) the concentration in collected water samples. Liquid Chromatography coupled to tandem Mass Spectrometry (LCMS/ MS) is able to analyse polar, semi-volatile, and thermally labile compounds covering a wide molecular weight range, such as pesticides, antibiotics, drugs of abuse, x-ray contrast agents, drinking water disinfection by-products etc. More recently there is a growing interest from environmental researchers to also screen for and identify non-targeted compounds in environmental samples, including metabolites and degradates, but also completely unexpected pollutants. The new AB SCIEX TripleTOF™5600 LC/MS/MS system is capable of performing highly sensitive and fast MS scanning experiments to search for unknown molecular ions while also performing selective and characteristic MS/MS scanning for further compound identification and, therefore, is the instrument of choice for this challenging task. General unknown screening workflows do not use a target analyte list and compound detection is not based on any prior knowledge, including retention times and information on possible molecular and fragment ions. Therefore, acquired chromatograms are very rich in information and can easily contain thousands of ions from both any compounds present in the sample as well as from the sample matrix itself. Thus, powerful software tools are needed to explore such data to identify the unexpected compound. Water samples were collected both upstream and downstream of two WWTPs (Seymour and Fort Beaufort) and were directly injected on the AB SCIEX TripleTOF™5600 LC/MS/MS after being filtered. 15 sample points along the Kat River, ranging from a point as close to the source as possible to a point just before it joins the Great Fish River were used. The samples collected from the source were used as the control in each of the experiments, the assumption being the closer you get to the source, the less contaminated the water would be for the analysis of pesticides. Points were selected where the Kat River crosses the R67 or on farms where the river was accessible using farm roads. Samples were collected from October 2013 to November 2014.The Peak view software and Analyst software were used in the analysis of PPCPs. The XIC Manager allows you to manage large lists of compounds and perform automatic extracted ion chromatogram (XIC) calculations and review results operations. The results were displayed in the chromatogram pane and the XIC table (see results). The results reported here in this thesis indicate that there is contamination in the Kat River water due to both pesticides and PPCPs. The results also indicate that the food products are also contaminated and hence both the Kat River agricultural produce and its water need to be closely monitored for both pesticide and PPCPs contaminants. Further studies to investigate the quantitative levels of pesticides and PPCPs in the Kat river water to determine if the concentration levels of the detected pesticides are below the reported Maximum Residues Limits will be explored in the future.

Concern over sediment toxicity has increased the need for toxicity test information with organisms that inhabit sediments. Oligochaetes are exposed to toxicants through feeding and direct body contact with aquatic sediments. Chronic testing with oligochaetes has historically focused on tubificids with test lengths of one year or more to encompass several generations. Most naidid oligochaetes have generation times of three to seven days and could provide chronic information in a matter of weeks. The cosmopolitan distributed naidid, Pristina leidyi, was evaluated for use as a toxicity test organism. Results of research conducted includes culture methods, effects of temperature on reproduction, growth rates in a reference sediment, acute toxicity tests, and chronic toxicity tests.

No description available.