The overall goal of this work was to investigate microbial activity leading to the anaerobic degradation of polycyclic aromatic hydrocarbons and an organophilic clay sediment cap used at a creosote-contaminated Superfund site. To determine whether or not PAHs were being degraded under anaerobic conditions in situ, groundwater and sediment porewater samples were analyzed for metabolic biomarkers, or metabolites, formed in the anaerobic degradation of naphthalene (a low-molecular weight PAH). In addition, a groundwater push-pull method was developed to evaluate whether the transformation of deuterated naphthalene to a deuterated metabolite could be monitored in situ and if conservative rates of transformation can be defined using this method. Metabolites of anaerobic naphthalene degradation were detected in all samples that also contained significant levels of naphthalene. Anaerobic degradation of naphthalene appears to be widespread in the upland contaminated aquifer, as well as within the adjacent river sediments. A zero-order rate of transformation of naphthalene-D₈ to naphthoic acid-D₇was calculated as 31 nM·d-¹. This study is the first reported use of deuterated naphthalene to provide both conclusive evidence of the in situ production of breakdown metabolites and an in situ rate of transformation. Methane ebullition was observed in areas of the sediment cap footprint associated with organophilic clay that was used a reactive capping material to sequester mobile non-aqueous phase liquid (NAPL) at the site. Anaerobic slurry incubations were constructed using sediment core samples to quantify the contribution of the native sediment and the different layers of capping material (sand and organophilic clay) to the overall methane production. Substrate addition experiments using fresh, unused organophilic clay, as well as measured changes in total carbon in organophilic clay over time supported the hypothesis that microbes can use organophilic clay as a carbon source. Quantitative PCR (qPCR) directed at the mcrA gene enumerated methanogens in field samples and incubations of native sediment and capping materials. Denaturing gradient gel electrophoresis (DGGE) was also performed on DNA extracted from these samples to identify some of the predominant microorganisms within the sediment cap footprint. The organophilic clay incubations produced up to 1500 times more methane than the native sediment and sand cap incubations. The organophilic clay field sample contained the greatest number of methanogens and the native sediment contained the least. However, the native sediment incubations had greater numbers of methanogens compared to their respective field sample and comparable numbers to the organophilic clay incubation. An increase in methane production was observed with the addition of fresh, unused organophilic clay to the already active organophilic clay incubations indicating that organophilic clay stimulates methanogenesis. In addition, organophilic clay retrieved from the field lost about 10% of its total carbon over a 300-day incubation period suggesting that some component of organophilic clay may be converted to methane. DGGE results revealed that some of the predominant groups within the native sediment and sediment cap were Bacteriodetes, Firmicutes, Chloroflexi, and Deltaproteobacteria. An organism 98% similar to Syntrophus sp. was identified in the organophilic clay suggesting this organism may be working in concert with methanogens to convert the organic component of organophilic clay ultimately to methane. The capacity of organophilic clay to sequester organic contaminants will likely change over time as the organic component is removed from the clay. This, in turn, affects the use of this material as a long-term remedial strategy in reduced, contaminated environments.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The quality of wine is largely dependent on environmental conditions and recent studies have therefore focused on agricultural practices in terms of water, soil and biodiversity conservation. The industry aims to create sustainable practice and to protect the natural resources available. Sources of potential grape contamination include: vehicle pollution, pesticides, bushfires and wood preservatives used for trellising systems. The latter have come to the attention of the South African wine industry (e.g. creosote and Copper Chromium Arsenate (CCA) products) as they may have consequences for the environment and wine quality. Creosote is a known pollutant of soil and ground water and the volatile fraction has been monitored in air. Plants may also accumulate polycyclic aromatic hydrocarbons (PAHs), which constitute up to 85% of the mass of creosote, and of which some have been proven carcinogenic. Because of the health risks associated with it, creosote has therefore been restricted for use in most applications in Europe, and also in the United States, Canada and Australia. This study focussed on the sensorial and chemical analyses of environmental and wine samples taken from the area around the creosote stockyard to determine accumulation of creosote-derived compounds. Environmental samples were collected and analysed at different distances from the affected area, over two vintages. Wines were made from grapes grown in vineyard blocks adjacent to the stockyard, to determine the effect of distance and skin contact during alcoholic fermentation treatments on wine taint. A sensory panel was trained for descriptive analysis to determine the intensity of the taint. Analytical methods were developed for the analysis of volatile organic compounds (VOCs) by gas chromatography mass spectrometry (GC-MS) and polycyclic aromatic hydrocarbons by high performance liquid chromatography with diode array detection (HPLC-DAD). The sensory results obtained showed white and rosé wines were perceived as clean, whilst the red wines were associated with burnt rubber/tar taint. The perceived taint decreased as distance from the stockyard increased. Wines made from the Cabernet Sauvignon blocks adjacent to the stockyard also showed an increase of taint with the increase of skin contact. Chemical data obtained showed that the taint consisted of a complex mixture of compounds, each with its own pattern of retention within the vineyard and wine. Only m-cresol was found above odour threshold, and only in red wines. The synergistic effect of some compounds may lead to an increase in the perceived taint. Berries and leaves had higher concentrations of volatile compounds than wines. Leaf contamination varied and followed the general trend in literature where the plants with high lipid content and exposed leaf area were the most contaminated. There may be other compounds present in creosote emissions, aside from those investigated here, with sensory attributes related to the taint found in wines. These compounds are styrene, indene, benzene, toluene, isoquinoline and quinoline and should be included in further investigations. From the PAH analysis of environmental samples and wines, it is evident that the samples closest to the stockyard were affected the most. The contamination varied with the depth of the soil: some of the heavier compounds were found in the samples taken from the deeper levels, whilst nearly all other target compounds were present in the top layer of soil. The concentrations found in the environmental samples were lower than reported in literature. Wines had few PAHs present, but at much higher concentrations than is allowed by EU legislation. From this study it is evident that the stockyard had negative effects on the surrounding environment in terms of sensory and chemical contamination. Recommendations include area rehabilitation by means of bioremediation to protect resources and ensure sustainable and safe production of crops. Industrial emissions should also be regulated and restricted in agricultural areas. Furthermore the use of creosote should be revised, and prohibited for agricultural use. / AFRIKAANSE OPSOMMING: Die kwaliteit van wyn is grootliks afhanklik van die omgewingstoestande. Daarom fokus verskeie onlangse studies op landbou aktiwiteite en die invloed daarvan op die omgewing in terme van water, grond en biodiversiteit bewaring. The wyn industrie se doelwitte sluit volhoubare praktyke in, wat die natuurlike hulpbronne bewaar en beskerm. Druiwe kan deur middel van die volgende bronne besoedel word: brandstof uitlaatgasse, insekdoders, veldbrande, gifstowwe wat gebruik word vir houtperservering wat dan verder aangewend word vir opleistelsels. Houtperservering (Koper chroom arsenaat (CCA) en kreosoot) het veral in die laaste paar jaar onder aandag gekom in die wynbedryf van Suid-Afrika as gevolg van die invloed daarvan op die omgewing en die uiteindelike wynkwaliteit. Kreosoot is ‘n gekende gifstof wat verantwoordelik is vir grond en grondwater besoedeling en wat gemonitor word in die atmosfeer. Plante akkummuleer ook poli-sikliese aromatiese hidro-koolstowwe (PAHs), wat to 85% uitmaak van die massa van kreosoot. PAHs is karsinogenies en dus is daar baie navorsing op die molekules reeds gedoen. Die gesondheidsrisikos gepaardgaande met kreosoot het gelei tot die streng geregulasies tans ingestel in Europa, die Verenigde State, Kanada en Australië. Hierdie studie het gefokus op die sensoriese en chemiese analises van omgewings- en wyn-monsters geneem van die omliggende area van die kreosoot palewerf om die akkumulasie van kreosoot-afgeleide-verbindings. Omgewingsmonsters was versamel en geanaliseer om verskillende afstande vanaf die bron van besoedeling (palewerf) te ondersoek oor ‘n twee jaar periode. Wyne is gemaak van die druiwe wat afkomstig is van die blok aangeplant langs die palewerf. Die wyne is ondersoek in terme van afstand vanaf die kreosoot bron asook oenologiese invloede, dopkontak gedurende alkoholiese fermentasie, op die kontaminasie beskryf in wyn. Die wyne is ook oor ‘n twee jaar periode voorberei en sluit die 2011 en 2012 seisoen in. ‘n Sensoriese paneel is opgelei om die beskrywende analises op die wyn uit te voer met die doel om die intensiteit van kontaminasie te identifiseer. Analitiese metodes is ook ontwikkel vir die analise van vlugtige organiese verbindings(VOCs) met gas chromatografie-massa spektrometrie (GCMS) en poli-sikliese aromaties hidro-koolstowwe (PAHs) met hoë druk vloeistof chromatografie. Die sensoriese resultate bekom het wit en rosé wyne as skoon laat blyk, terwyl rooi wyne meer geassosieer was met die gebrande rubber/ teer afgeur beskrywing. Die waargeneemde afgeur het afgeneem soos wat die afstand vanaf die palewerf toegeneem het. Wyne gemaak van die Cabernet Sauvignon blokke langsaan die palewerf het ook ‘n toename in die afgeur gehad met ‘n toename in dopkontak. Chemiese data bekom beeld uit dat die afgeur uit ‘n komplekse mengsel van verbindings bestaan, elk met sy eie patroon van verspreiding en verbinding in die wingerd as ook in die wyn. Net m-kresol was gevind bo die reuk drumpel, dit het ook net in rooi wyne voorgekom. Die sinergistiese effek van die verbindings mag egter bydra tot die waargeneemde afgeur. Druiwekorrels en blare het hoër konsentrasies van die vlugtige verbindings gehad as wat gemeet is in die wyne. Blaar kontaminasie het ook baie gewissel en het ooreengestem met die algemene tendens wat in literatuur beskryf is, naamlik dat plante met ‘n hoër lipid inhoud en grootter blaar oppervlak die meeste gekontamineer word. Daar mag egter nog baie ander verbindings bydra tot die waargeneemde afgeur gevind in die wyn. Spesifieke verbindings wat wel ‘n rol kan speel in kontaminasie en wat voorkom in die vlugtige gedeelte van kreosoot is styreen, indeen, benzeen, tolueen, isoquinoleen die vlugtige verbindings van kreosoot. Die verbindings moet ingesluit word vir verdere studies wat gedoen word op die kreosoot geassosieerde afgeur. Die PAHs analise op die omgewingsmonsters en wyne het gelei tot die bevestiging dat die naasliggende omgewing die meeste geaffekteer is. Die kontaminasie wissel in terme van die diepte in die grond wat die gifstowwe voorkom: die swaarder molekulêre verbindings is tot in die dieper vlakke waargeneem terwyl al die gemete verbindings in die boonste lae teenwoordig was. Die vlakke wat waargeneem is in dié studie is egter laer as wat voorheen in literatuur gevind is in ‘n kreosoot geaffekteerde omgewing. Wyn het PAHs teenwoordig gehad, alhoewel slegs twee verbindings gemeet is, het dit in hoër vlakke voorgekom as wat sekere Europese regulasies as toelaatbaar spesifiseer. Vanaf die studie resultate blyk dit, dat die palewerf se negatiewe invloed op die omliggende omgewing beide meetbaar was in sensories en chemiese kontaminasie. Voorstelle sluit onder andere die rehabilitasie van die omliggende omgewing deur middel van bioremediasie in. Om sodoende die natuurlike hulpbronne in die area te bewaar asook om volhoubare en veilige verbouing van gewasse te verseker. Industriële besoedeling en afval moet ook gereguleer word en beperk word in landbou areas. Verder moet die gebruik van kreosoot heroorweeg word en strenger regulasies moet in plek gestel word om aan internastionale standaarde te voldoen. / Financial support from Winetech, Trip and UFS