Thesis (M.S.)--Northern Michigan University, 2006. / Includes bibliographical references.

Thesis (M.C.E.)--University of Delaware, 2006. / Principal faculty advisor: William Ritter, Dept. of Civil & Environmental Engineering. Includes bibliographical references.

M. Tech. / Background: Waterborne disease contributes significantly to the total global disease burden. Populations in rural areas of South Africa depend on untreated waters for consumption and sanitation. Contamination of public water supplies by harmful bacteria such as pathogenic E. coli poses a major risk for public health. Ingestion of these pathogenic microorganisms present in the contaminated and untreated waters could cause infection, leading to systemic inflammatory responses manifested by the production of various proinflammatory cytokines. To date, there is no human system test available to detect whether water, following ingestion, would cause disease (i.e. whether the water is infectious). The current water testing methods only test for the presence of indicator organisms, such as faecal coliforms, total coliforms, and Escherichia coli. A reliable in-vitro bioassay that could assess whether the water would cause an inflammatory response was investigated in this study. Objectives: Pro-inflammatory cytokines and whole-blood have been used in similar studies to detect the inflammatory responses following exposure to specific stimulants such as dust, lipopolysaccharide (LPS), E. coli and various others. It has been reported that larger numbers of these contaminants induced higher levels of pro-inflammatory cytokine expression. This implies that the pro-inflammatory cytokine expression could be used as a marker of infection since, inflammation occurs in response to infection. Successful infection is thus necessary for inflammation to occur, and high levels of pro-inflammatory cytokine expression confirm that infection has occurred. Thus if pro-inflammatory cytokines could serve as indicators for infection, these cytokines could be used as indicators for bacterial pathogenicity of water.

The problem of this thesis consists of a bacterial analysis of twenty-five representative wells within a radius of thirty miles of Canyon, Texas. An attempt has been made to determine the possible presence of the typhoid organism in these wells.

A bacterial analysis of the water from thirty-six sources of consumption by the white population of Trinidad, Henderson County, Texas, was made to determine the potability of each of these in regard to infection from typhoid or related organisms.

The purpose of this study was to assess the effects of water-soluble fractions (WSF) of naphthalene, phenanthrene, No. 2 fuel oil, and coal-tar creosote on the survival, growth, reproduction, feeding, and metabolism of Daphnia pulex. The 48 hr LC50 values after acute exposure (as percent WSF) for creosote, No. 2 fuel oil, phenanthrene and naphthalene were 2.91, 34.10, >>100, and 57.52 percent, respectively. Gas chromatography analysis indicated naphthalene and phenanthrene 48 hr LC50 values (as mg/1) were 2.92-3.89 and 0.96-1.28, respectively. Up to 40 peaks were noted in each stock WSF of creosote and No. 2 fuel oil. For chronic studies, young (24 hr) Daphnia were exposed to calculated LC20 and LC30 concentrations of WSF's for their entire life. The LC30 concentrations of creosote and phenanthrene showed a significant reduction in growth rate and number of live young, as well as reduced number of broods, impairment of molting, and significant delay in reproductive maturation; instances of possible neoplasms were also noted in one organism from each of these test groups. No. 2 fuel oil produced similar effects on growth and reproduction, but results were not as significant. Increased longevity and slight reduction in growth rate were noted for both naphthalene test groups. The effects upon oxygen consumption after exposure to test WSF's were variable. The LC30 concentration of creosote and both naphthalene concentrations were significantly different from each other; both naphthalene concentrations elicited the lowest oxygen consumption rates recorded, while the creosote LC30 group exhibited the highest rate of oxygen consumption. However, no experimental means were significantly different from controls. Highly significant differences existed between filtering rates of organisms after exposure to the WSF's. The LC20 concentrations of creosote and phenanthrene produced the highest and lowest filtering rates, respectively. Both oil test groups demonstrated significantly higher filtering rates. Monitoring zooplankton filtering rates appears to be a promising parameter to evaluate physiological stress on these organisms. This chronic study and data from other comparable chronic studies indicate that the length of a pre-adult Daphnia after 7 days of exposure to stress can be used to predict chronic reproductive effects with the same degree of accuracy as the 21-day test. Adoption of this test would eliminate difficulties with starvation, nutrition, and competition for food which contribute to the variability in reproductive impairment tests. A possible mechanism of action of polynuclear aromatic hydrocarbons upon endocrine systems is strongly suggested by the dramatic and diverse effects upon growth and reproduction in Daphnia pulex. / Ph. D.

Mine abandonment and the discharge of contaminated mine water is recognised globally as a major source of surface water and groundwater pollution. Contamination generally arises from the oxidation of sulphide minerals, principally pyrite, by the mining process, and the subsequent chemical reactions can lead to the discharge of mineralised, often acidic, iron, and sulphate rich waters. In many historically mined river catchments, mine water discharge is the main cause of poor water quality. Within the UK, managing the legacy of abandoned mines is one of the principal challenges presented by modern environmental legislation, particularly the EU Water Framework Directive, a challenge that is exacerbated by the diverse and widespread nature of historical mining. The impact and hazard associated with abandoned mining in one of the UK’s most intensively mined regions, the Almond River Catchment, Scotland, was examined via: 1) a detailed GIS mapping and investigation of historical mining processes in the catchment, 2) mine site discharge sampling, 3) detailed site investigations, 4) geochemical modelling of four mine waste sites and 5) analysis of temporal and spatial river water quality in the catchment. The results are then brought together to produce a catchment scale mine water hazard map. Mapping has identified over 300 mine sites in the catchment including coal, oil shale and ironstone mine wastes and flooded coal and oil shale mines. The historical development of oil shale retort methods has been shown to have an impact on potential hazard. Sampling of discharge waters from the different mining activities, in conjunction with detailed mineralogical analysis and geochemical modelling at the four mine waste sites has characterised the main hazards. Ironstone and pyrite bearing coal mine wastes discharge waters with highly elevated Fe and sulphate concentrations, up to 160mgl-1 and 1900mgl-1 respectively, due to extensive pyrite oxidation and acid generating salt dissolution (principally jarosite). Coal mine wastes show variable mineralogy, due to the diverse nature of coal bearing strata, and discharge waters with variable chemistry. Oil Shale mine wastes are generally depleted in pyrite due to historic processing and discharge low sulphate waters with moderately elevated Fe concentrations, up to 5mgl-1. Flooded coal mines discharge sulphate dominant alkaline waters, due to the availability of carbonate minerals in the mine complex, with elevated Fe concentrations, up to 50mgl-1, while flooded oil shale mines discharge waters with moderately elevated Fe concentrations, up to 4mgl-1, due to lower pyrite content in mine strata and reduced availability of oxygen related to mine abandonment age. Once in the surface water environment iron and sulphate display significant concentration-flow dependence: iron increases at high flows due to the re-suspension of river bed iron precipitates (Fe(OH)3); sulphate concentrations decrease with increased flow as a result of dilution. Further examination of iron and sulphate loading at low flows indicates a close correlation of iron and sulphate with mined areas; cumulative low flow load calculations indicate that coal and oil shale mining regions contribute 0.21 and 0.31 g/s of iron, respectively, to the main Almond tributary. Decreases in iron loading on river sections demonstrate the deposition and diffuse storage of iron within the river channel. This river bed iron is re-suspended with increased flow resulting in significant transport of diffuse iron downstream with load values of up to 50 g/s iron. Based on this hazard classification, a catchment scale mine water hazard map has been developed. The map allows the prioritisation of actions for future mine water management.

Thesis (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Several genera of cyanobacteria produce a range of toxins. The increased rate of eutrophication of surface fresh waters due to anthropogenic inputs has resulted in more frequent and severe cyanobacterial bloom events. Such bloom events make impoundments unsuitable for recreational use and increase the cost of production of potable water due to the necessity for removal of toxins released from cells during the purification process. Microcystis aeruginosa is the major freshwater bloom-forming toxic cyanobacterium. Concentrations of the hepatotoxin, microcystin, are highly variable in blooms. Published literature on environmental conditions leading to increased microcystin production was often contradictory and in many cases did not consider all relevant parameters. However, environmental nitrogen and phosphorus, temperature and light, and growth rate were implicated in regulation of toxin content. The purpose of this work was therefore to investigate environmental factors (specifically nitrogen and phosphorus) and cellular activities (specifically carbon fixation and nitrogen uptake rates and growth rate) involved in the modulation of microcystin production in M. aeruginosa in order to clarify the role of these parameters, and in an attempt to identify regulatory mechanisms for microcystin production. Environmental nitrogen, phosphorus and growth rate were shown to co-modulate microcystin production in M. aeruginosa. Adequate phosphorus is required for photosynthetic carbon fixation. Phosphorus uptake by M. aeruginosa is strongly correlated with carbon fixation rate. Although microcystin content increased with increasing nitrogen:phosphorus ratios in culture medium, under phosphorus limitation microcystin content was lower irrespective of nitrogen concentrations. This observation and the requirements for fixed carbon for nitrogen assimilation therefore prompted investigation of the effects of cellular carbon fixation and nitrogen uptake in the modulation of microcystin production. Microcystin production was found to be enhanced when nitrogen uptake rate relative to carbon fixation rate was higher than that required for balanced growth. The cellular nitrogen:carbon ratio above which microcystin concentrations increased substantially, corresponded to the Redfield ratio for balanced growth. Investigation of potential regulatory mechanisms involving the cyanobacterial nitrogen regulator, NtcA, yielded putative NtcA binding sites indicative of repression in the microcystin synthetase gene cluster. In culture, the polypeptide synthetase module gene, mcyA, and ntcA were inversely expressed as a function of carbon-fixation:nitrogen-uptake potential. However, no increase or decrease in microcystin production could be linked to either glutamine, glutamate or a-ketoglutarate, metabolites that are involved in regulation of ntcA. The role of NtcA in regulation of microcystin production could therefore not be confirmed. In conclusion, these data suggest that microcystin production is metabolically regulated by cellular C:N balance and specific growth rate. The primary importance of nitrogen and carbon was demonstrated by a simple model where only nitrogen uptake, carbon fixation and growth rate were used to predict microcystin levels. The model also explains results previously described in literature. Similarly, an artificial neural network model was used to show that the carbon fixation dependence on phosphorus allows accurate prediction of microcystin levels based on growth rate and environmental nitrogen and phosphorus. / AFRIKAANSE OPSOMMING: Verskeie genera van sianobakterieë produseer 'n verskeidenheid van toksiene. Die toename in die tempo van eutrofikasie van varswater oppervlaktes as gevolg van antropogeniese insette veroorsaak al hoe meer en al hoe erger sianobakteriële infestasies. Dit veroorsaak probleme vir ontspanninggebruik van hierdie waters en verhoog die koste van produksie van drinkbare water as gevolg van die noodsaak om die toksiene wat deur die selle gedurende die suiweringsproses vrygelaat word te verwyder. Microcystis aeruginosa is die belangrikste varswater bloeisel-vormende toksiese sianobakterium. Die konsentrasie van die hepatotoksien mikrosistien is hoogs varieerbaar in sulke bloeisels. Gepubliseerde literatuur oor die omgewingskondisies wat lei na verhoogde mikrosistienproduksie is dikwels weersprekend en neem in vele gevalle nie al die relevante parameters in ag nie. Desnieteenstaande word omgewingstikstof, fosfor, temperatuur en lig, asook groeisnelheid, geïmpliseer in die regulering van toksieninhoud. Die doel van hierdie navorsing was dus om omgewingsfaktore (spesifiek stikstof en fosfor) en sellulêre aktiwiteite (spesifiek koolstoffiskering en die snelheid van stikstofopname en van groei) betrokke by die modulering van mikrosistienproduksie in M. aeruginosa te ondersoek in 'n poging om die rol van hierdie parameters te verstaan en om regulatoriese meganismes vir mikrosistienproduksie te identifiseer. In hierdie studie is aangetoon dat omgewingstikstof en fosfor sowel as groeisnelheid mikrosistienproduksie in M. aeruginosa ko-moduleer. Genoegsame fosfor word benodig vir fotosintetiese koolstoffiksering. Fosforopname deur M. aeruginosa korreleer sterk met die snelheid van koolstoffiksering. Alhoewel mikrosistieninhoud toegeneem het met 'n toename in die stikstof:fosfor verhouding in die kultuurmedium, was die mikrosistieninhoud onder kondisies van fosforlimitering laer ongeag die stikstofkonsentrasie. Hierdie waarneming, tesame met die noodsaak van gefikseerde koolstof vir stikstofassimilering, het gelei na 'n studie van die effekte van sellulêre koolstoffiksering and stikstofopname op die modulering van mikrosistienproduksie. Dit is gevind dat mikrosistienproduksie verhoog was wanneer die snelheid van stikstofopname relatief tot die snelheid van koolstoffiksering hoër was as die waarde wat benodig word vir gebalanseerde groei. Die sellulêre stikstof:koolstof verhouding waarbo mikrosistienkonsentrasies beduidend verhoog is stem ooreen met die Redfield verhouding vir gebalanseerde groei. 'n Ondersoek na potensiële reguleringsmeganismes waarby die sianobakteriële stikstofreguleerder NtcA betrokke is het gelei na die ontdekking van moontlike NtcA bindingseteis; dit kan dui op die repressie van die mikrosistiensintetase geengroepering. Onder kultuurkondisies is gevind dat die geen vir die polipeptiedsintetase module, mcyA, en ntcA omgekeerd uitgedruk word as 'n funksie van koolstofopname:stikstofopname potensiale. Geen toename of afname in mikrosistienproduksie kon egter gekoppel word aan óf glutamien, óf glutamaat, óf a-ketoglutaraat nie, metaboliete wat betrokke is by die regulering van ntcA. Die rol van NtcA in die regulering van mikrosistienproduksie kon dus nie bevestig word nie. Die gevolgtrekking is dus gemaak dat mikrosistienproduksie metabolies gereguleer word deur die C:N balans en die spesifieke groeisnelheid. Die primêre belang van stikstof en koolstof is gedemonstreer deur 'n eenvoudige model waarin slegs stikstofopname, koolstoffiksering en groeisnelheid gebruik word om mikrosistienvlakke te voorspel. Die model verklaar ook resultate wat tevore in die literatuur beskryf is. Soortgelyk is 'n artifisiële neurale netwerkmodel gebruik om te toon dat die afhanklikheid van koolstoffiksering van fosfor akkurate voorspelling van mikrosistienvlakke gebaseer of groeisnelheid en omgewingstikstof en fosfor moontlik maak.

Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water samples. Nonradioactive probes offer a safe, inexpensive alternative to radiolabeled probes and tissue culture for the detection of viruses in the environment when ultrasensitivity is not required.

Rotaviruses are one of the most significant causes of diarrheal disease in the world. Their presence in groundwater and drinking water supplies constitutes a health risk to the population. The study of rotaviruses in the environment has been hampered by the lack of accessible and consistent detection methodologies. Gene probes and other molecular techniques are a novel approach for the detection of these viruses in water. The feasibility of these new techniques for the detection and study of rotaviruses in the environment has been assessed using the simian SA-11 and the culturable human Wa rotavirus strains as models. Two general approaches have been undertaken consisting of hybridization of probes with genomic RNA and hybridization with mRNA produced by the virion-incorporated transcriptase. Hybridization of gene probes with genomic dsRNA of rotaviruses in environmental concentrates resulted in the detection of 10 4 immunofoci of Wa rotavirus. In vitro transcription serves as an amplification method with sensitivity 100- to 1000-fold greater than when probing for genomic RNA. The sensitivity obtained in Wa-seeded distilled water and environmental concentrates after in vitro transcription is 2 and 20 immunofoci, respectively. Proteins in environmental concentrates decrease the efficiency of probe hybridization by 10-100 fold. Also, transcriptase-inhibiting factors found in environmental samples decrease the production of mRNA. Both proteins and transcriptase-inhibiting factors can be reduced significantly with Sephadex G-200 columns. Passage of environmental concentrate through Sephadex G-200 spun columns, followed by in vitro transcription, was used to detect rotaviruses in environmental samples. Rotaviruses were detected by this combination of techniques in eight of 20 sewage samples, one of 16 tap water samples, five of 32 ground water samples, and two of nine surface water samples. Only one of 17 samples which tested positive with Wa cDNA 4 was positive for non-specific probe binding. The probing of rotavirus mRNA, amplified by the virion-incorporated transcriptase, is a practical and feasible method for monitoring these viruses in the environment.