Thesis (M.S.)--University of Wisconsin, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 126-129).

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For the protection of the ecological Reserve in South Africa, the proposed introduction of compulsory toxicity testing in the licensing of effluent discharges necessitates the development of whole effluent toxicity testing. The elucidation of the effects of effluent on the local indigenous populations of organisms is essential before hazard and risk assessment can be undertaken. The limpet Burnupia stenochorias, prevalent in the Eastern Cape of South Africa, was chosen to represent the freshwater molluscs as a potential toxicity indicator. Using potassium dichromate (as a reference toxicant) and a textile whole effluent, the suitability of B. stenochorias was assessed under both acute and chronic toxicity conditions in the laboratory. In support of the toxicity studies, aspects of the biology of B. stenochorias were investigated under both natural and laboratory conditions. Using Principal Component and Discriminant Function Analyses, the relative shell morphometrics of three feral populations of B. stenochorias were found to vary. Length was shown to adequately represent growth of the shell, although the inclusion of width measurements is more statistically preferable. Two of the feral populations, one in impacted water, were studied weekly for 52 weeks to assess natural population dynamics. Based on the Von Bertalanffy Growth Equation, estimates of growth and longevity were made for this species, with growth highly seasonal. Age is not easily discerned from shell size. Egg laying occurred all year round, with early summer (peak egg lay), mid summer (a second, smaller peak in egg lay), and winter (limited presence of eggs) phases. In toxicity testing, consideration is given to the choice of the test organism based on age and sexual development. Consequently, the sexual development of B. stenochorias relative to shell length was determined with the aid of histological examinations of transverse sections of limpets, of all sizes, collected over one year. Limpets less than 3mm shell length were found to be immature in the development of the oocytes and spermatozoa, and were later chosen for acute toxicity tests. A laboratory diet was developed, for both culturing and maintaining of the limpets during toxicity tests; however, the diet requires optimisation. Under laboratory conditions, growth was linear, and individual fecundity highly variable. Successful methods for the collection of limpets from naturally occurring populations, and their acclimation to the laboratory were developed. Three B. stenochorias populations, representing different hydrological and water quality conditions, were compared to a laboratory population (maintained for three years) in their responses to the textile whole effluent and potassium dichromate. Under acute conditions, variability of mortality between limpet populations and between seasons was consistent with acceptable international standards. However, seasonal differences between feral limpets were apparent, with early summer limpets significantly more susceptible to both potassium dichromate and textile effluent than winter limpets. Although mortality occurred within the effluent at all concentrations, no 96 hour LC₅₀ values were obtained. The chronic toxicity effects of the textile whole effluent were assessed over the entire life cycle of B. stenochorias, based on survival, growth and reproductive effects. Lower concentrations of effluent (# 10%) gave greater variability of responses and toxicity than higher concentrations, with a 43 day LC₅₀ of 3.9% effluent. The No Observed Effect Concentrations for survival (over 43 days) were calculated in consecutive years as 0.1% and 1% effluent. Survival is considered a useful tool for determining toxicity endpoints using B. stenochorias. Limpet growth remained linear in effluent, with an apparent stimulation of growth at the 3-10% effluent concentration, confusing the toxicity and variability assessments. The possible addition of nutrients from the effluent points to either a potential inadequacy of the food quality provided in the chronic assessment, or the presence in the effluent of growth stimulants. Growth was also found to be too variable to allow adequate statistical conclusions about the toxicity of the effluent, although it is suggested that growth may be useful in the assessment of single compounds. Despite large individual variability in fecundity, statistical differences were discernible between effluent concentrations. The application of fecundity of B. stenochorias in hazard assessment therefore warrants further investigation. It was concluded that an assessment of textile whole effluent toxicity to B. stenochorias over an entire life cycle, and an F1 generation, is unnecessary. The development of the bucket/plastic bag method for both acute and chronic toxicity assessment of B. stenochorias was useful. In the final assessment of the usefulness of B. stenochorias as a toxicity indicator, toxicity endpoints were compared with those of the standard laboratory organism Daphnia pulex. Both in acute and chronic toxicity, B. stenochorias was found to be more sensitive. B. stenochorias is therefore considered valuable as a South African freshwater molluscan ecotoxicological indicator, with a place in hazard assessment, although further development and research is necessary before the limpet can be effectively used.

Olive mill wastewater is produced in large quantities during the production of olive oil and olive production effluents are produced during the processing of olives. This project was planned to find a use for constituents found in olive production wastewater. The task was carried out by first characterizing the olive effluents, then screening microorganisms for growth in the effluents and reduction of the pollutant properties of the effluents. An investigation into the biotransformation of aromatic compounds present in the effluents into useful chemicals, was carried out. The olive production effluents were collected from different stages in the process for treating olive wastewater, viz, a fermentation tank (FB), the surface of a digester (LV) and an evaporation pond (SO). The three effluents were characterized by investigating their phenolic composition. Protocatechuic acid, vanillic acid, syringic acid, hydroxyphenyl acetic acid, coumaric acid and ferulic acid were identified in an olive effluent, FB, using thin layer chromatography (TLC) and High perfomance liquid chromatography (HPLC). Hydroxyphenyl acetic acid constitutes almost 60% of the organics in olive effluent FB. Five bacteria, namely RU-LV1; RU-FBI and RU-FB2; RU-SOI and RU-S02, were isolated from the olive effluents LV, FB and SO respectively. These isolates were found to be halotolerant and were able to grow over a broad temperature and pH range, with the maximum temperature and pH for growth being 28°C and pH 7 respectively. A range of microorganisms were evaluated for their ability to grow and reduce the total phenolic content of the olive effluents. Among these Neurospora crassa showed the highest potential for the biological reduction of total phenolics in olive effluents. Approximately 70% of the total phenolic content was removed by N. crassa. Trametes verscilor, Pseudomonas putida strains, RU-KMI and RU-KM3s, and the bacteria isolated from olive effluents could also degrade the total phenolic content of olive effluents, but to a lesser extent. The ability of the five bacterial isolates to grow and degrade aromatic compounds was assessed by growing them in medium with standard aromatic compounds. RU-L V1 degraded 96%, 100%, 73% and 100% of caffeic acid, protocatechuic acid, p-coumaric acid and vanillic acid respectively. The other isolates degraded caffeic acid and protocatechuic acid, but their ability to degraded p-coumaric acid and vanillic acid was found to be lesser than the ability of RU-LV1 to degrade the same aromatic compounds. Whole cells of RU-LV1 degraded vanillic acid but no metabolic products were observed on HPLC analysis. Resting cells, French pressed extract, cell free extracts and cell debris from RU-LV1 cells induced with vanillic acid degraded vanillic acid, ferulic acid and vanillin at rates higher than those obtained from non-induced cultures. No products were observed during the degradation of vanillic acid. Ferulic acid was converted into vanillic acid by French pressed extract, cell free extract and cell debris of RU-LV1. The maximum yield of vanillic acid as a product (0 .23 mM, 50 %yield) was obtained when cell free extracts of RU-LVI, grown in glucose and induced by vanillic acid, were used for the degradation of 0.4 mM ferulic acid. Vanillin was rapidly converted into vanillic acid by resting cells, cell free extracts and French pressed extract of RU-LVI. Using molecular techniques, the similarity ranking of the RU-LVI 16S rRNA gene and its clone showed a high similarity to Corynebacterium glutamicum and Corynebacterium acedopltilum. The rapid degradation of vanillin to vanillic acid suggests that extracts from RU-LV1 degrade ferulic acid into vanillin which is immediately oxidized to vanillic acid. Vanillic acid is also considered as a high value chemical. This project has a potential of producing useful chemicals from cheap substrates that can be found in olive effluents. / KMBT_363

Macroalgae such as Ulva are living hosts that are known to perform essential roles in marine ecosystems, and are extensively utilised for several aquaculture operations, including in the integrated production of high value goods such as abalone. Intensive aquaculture operations along the coastline release effluent water into the surrounding coastal waters, which has adverse impact on the environment. As a means to bioremediate abalone effluent, several commercial abalone farms in South Africa use Ulva as a biofilter, after which the Ulva may serve as a feed source for the abalone. Advancements in gene sequencing technology has enabled the assessment of large scale 16S rRNA gene libraries of near full-length sequences. However, studies concerning the epiphytic bacterial communities present on macroalgae grown in effluent systems are scarce, and as a result several commercial farmers have become sceptical about utilising effluent grown Ulva as feed. This study addresses the biosecurity implications associated with the use of Ulva as a biofilter and feed within an integrated multitrophic aquaculture (IMTA) system along with the abalone Haliotis midae by assessing the bacterial communities associated with Ulva and its environment. Water and Ulva samples were collected from an integrated abalone farm along the Western Cape of South Africa and assessed via a culture and a non-culture-based approach. Samples were collected from both fertilised seawater tanks and abalone effluent wastewater raceways. The water samples were collected at the inlets and outlets of each tank/raceway and the Ulva was collected from within each system. The culture-based approach utilised three selective media for the isolation and quantification of culturable bacteria, namely Tryptic Soy Agar (TSA, a general growth media), thiosulfate-citrate-bile-sucrose (TCBS) agar (vibrio selective growth media), and Ulvan agar plates, where the primary carbohydrate of Ulva was utilized as the main carbohydrate source. Post isolation, selected bacteria underwent 16S rDNA gene analysis for identification. The non-culture-based approach utilised the next generation Illumina 16S Metagenomic Sequencing platform (MiSeq). Moreover, the Ulva was sequenced using the rbcL gene to identify the species grown in the aquaculture system. Phylogenetic analysis of Ulva suggests that it falls within the U. rigida clade. The sequenced Ulva cultivated at I&J abalone farm shared close similarity with Ulva rigida (KP233772) and Ulva scandinavica (EU484416) on the GenBank database, and hence was referred to with the name of its corresponding molecular synonym, i.e. U. rigida. The culture-based results indicate that bacterial numbers were significantly higher in the raceways receiving abalone effluent water when compared with the Ulva tanks receiving seawater that were fertilized. Bacterial abundance on all three selective media types was higher on the Ulva cultured in the abalone effluent raceways than on the Ulva cultured in the fertilized seawater tanks. Moreover, it was observed that the Ulva has the potential to significantly reduce the bacterial load of abalone effluent water raceways. Rarefaction results from the non-culture-based approach indicate that the Ulva in both the fertilised seawater and abalone effluent raceways had significantly lower bacterial alpha diversity than the water columns themselves. Principal co-ordinate analysis (PCoA) at phylum level showed that bacterial communities on the Ulva and in the water, columns shared similar phyla diversity. Alternatively, PCoA at genus level demonstrated that microbial communities residing on the Ulva (both effluent and fertilised seawater grown Ulva) had significant differences compared with the water samples obtained from both the inlets and outlets of the effluent and fertilised seawater systems. When assessing the differential abundant bacteria on the Ulva, general marine bacteria appear in high abundance and potentially pathogenic bacteria such as Vibrio appear in low abundance. Moreover, the presence of the Ulva within the wastewater seemed to decrease the bacterial abundance of Vibrio within the fertilised seawater tanks as well as the abalone effluent raceways. Despite the presence of potentially pathogenic bacteria within the abalone effluent raceways, the Ulva does not seem to act as a sink for potentially pathogenic bacteria indicating that feeding effluent grown Ulva to abalone is not of significant biosecurity concern. Even though several commercial abalone farmers consider recirculation within aquaculture feed systems high-risk technology, no papers have reported disease outbreaks due to the use of effluent grown Ulva as abalone feed. These results provide a general basis for the dynamic changes in the bacterial community profiles in a commercial abalone farm associated with utilising effluent grown Ulva as a feed additive for abalone. This effort to profile the bacteria associated with Ulva and its environment under fertilised and effluent conditions provides deeper insight on understanding the biosecurity implications of incorporating effluent grown Ulva into abalone feed.

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It was the purpose of this investigation to determine (1) what organisms are associated with the formation of this slime layer, (2) if certain chemical factors in the environment either enhance or retard formation of the slime layer, (3) whether or not there are certain chemical factors which are detrimental to slime formation, yet not detrimental to purification, or that perhaps enhance purification, (4) whether or not there are chemical factors which enhance purification of the effluent without preventing slime formation, and (5) what effects the chemical treatments have on the microbial flora of the soil system.

This study was undertaken in order to provide further insight into the effects of artificial heating on the fisheries of a small reservoir in the Southwest. The following specific objectives were established: (1) to map the reservoir for the distribution of heated water, (2) to determine the distribution of selected species of sports, rough, and forage fish in areas affected by the effluent and in areas not affected, (3) to compare size and condition of selected species of fish from areas affected by the effluent to size and condition of fish from areas not affected, and (4) to compare food habits of channel catfish in areas affected by the effluent to the food habits of channel catfish in areas not affected.