Biological indicators of copper-induced stress in soil

Du Plessis, Keith R. (Keith Roland) (2002-03)

Thesis (MSc)--University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: The concentrations of copper (Cu) in vineyard soils of the Western Cape range from 0.1 to 20 ppm. However, more than 160 tons of the fungicide copper oxychloride are annually being sprayed on these vineyards. This has raised concerns that Cu may accumulate in these soils, resulting in a negative impact on the soil biological processes, especially since the soils in the Western Cape are slightly acidic, making Cu more mobile and available for soil organisms than would have been the case in alkaline soils. The goal of the initial part of this study was therefore to identify those soil microbial communities indigenous to the Western Cape, which are most susceptible to Cu-induced stress as a result of the addition of copper oxychloride. These potential bioindicators of Cu-induced stress were first searched for in uncultivated agricultural soil from Nietvoorbij experimental farm. Consequently, a series of soil microcosms was prepared by adding various concentrations of Cu as a component of copper oxychloride, to each of eight aliquots of soil: 0 (control), 10, 20, 30, 40, 50, 100, 500 and 1000 ppm. The resulting concentrations of exchangeable Cu in these microcosms were found to be 2 (control), 12,23,34,42,59, 126,516 and 1112 ppm. Selected microbial communities in each microcosm were subsequently monitored over a period of 245 days. It was found that the culturable microbial numbers did not provide a reliable indication of the effect of Cu on community integrity. However, analyses of terminal-restriction fragment length polymorphism (T-RFLP) community fingerprints and especially analyses of the whole community metabolic profiles, revealed that shifts in the soil microbial communities took place as the Cu concentration increased. Direct counts of soil protozoa also revealed that the addition of Cu to the soil impacted negatively on the numbers of these eukaryotes. To confirm these findings in other soil ecosystems, the impact of copper oxychloride on whole community metabolic profiles and protozoan numbers were investigated in soils from Koopmanskloof commercial farm and Nietvoorbij experimental farm. These potential bioindicators were subsequently monitored in a series of soil microcosms prepared for each soil type by adding the estimated amounts of 0 (control), 30, 100 and 1000 ppm Cu as a component of copper oxychloride to the soil. The results confirmed the fmdings that elevated levels of copper impact negatively on the metabolic potential and protozoan numbers of soil. Consequently, it was decided to investigate a combination of protozoan counts and metabolic profiling as a potential bioindicator for Cu-induced stress in soil. Data collected from all the microcosms containing exchangeable Cu concentrations ranging from 1 ppm to 1112 ppm was used to construct a dendrogram using carbon source utilization profiles in combination with protozoan counts. It was found that the microcosms grouped into clusters, which correlated with the concentration of exchangeable Cu in the soil. Under the experimental conditions used in this study, the combination of protozoan counts and metabolic profiling seemed to be a reliable indicator of Cu-induced stress. However, this bioindicator must be further investigated in other soil types using other types of stress inducing pollutants. In addition to the above fmdings it was also found that the numbers of soil protozoa was particularly susceptible to Cu-induced stress in soils with a low soil pH. This is in agreement with the fmdings of others on the bio-availability of heavy metals in low pH soils. In these soils, nutrient cycling as a result of protozoan activity, may therefore be particularly susceptible to the negative impact of copper to the soil.

AFRIKAANSE OPSOMMING: Die konsentrasies van koper (Cu) in wingerdgronde van die Wes-Kaap wissel tussen 0.1 en 20 dpm. Meer as 160 ton van die fungisied koper-oksichloried word egter jaarliks op dié wingerde gespuit, wat kommer laat ontstaan het oor die moontlike akkumulasie van Cu in dié grond en die gevaar van 'n negatiewe impak op die biologiese prosesse in die grond. Die gevaar word vererger deur die feit dat die Wes-Kaapse grond effens suur is, wat Cu meer mobiel en beskikbaar maak vir grondorganismes as wat die geval sou wees in alkaliese grond. Die eerste doelstelling van hierdie studie was dus om die mikrobiese gemeenskappe in die grond, wat inheems is aan die Wes-Kaap, te identifiseer wat die meeste vatbaar is vir Cu-geïnduseerde stres as gevolg van die toevoeging van koper-oksichloried. Hierdie potensiële bioindikatore van Cu-geïnduseerde stres is eerstens gesoek in onbewerkte landbougrond van die Nietvoorbij-proefplaas. 'n Reeks grondmikrokosmosse is gevolglik berei deur verskillende konsentrasies Cu, as 'n komponent van koperoksichloried, by elk van agt hoeveelhede grond te voeg naamlik 0 (kontrole), 10,20, 30, 40, 50, 100, 500 en 1000 dpm. Die gevolglike konsentrasies van uitruilbare Cu in hierdie mikrokosmosse was 2 (kontrole), 12, 23, 34, 42, 59, 126, 516 en 1112 dpm. Geselekteerde mikrobiese gemeenskappe in elke mikrokosmos is vervolgens oor 'n tydperk van 245 dae bestudeer. Daar is gevind dat die kweekbare mikrobiese tellings nie 'n betroubare aanduiding kon gee van die uitwerking van Cu op gemeenskapsintegriteit nie. Die ontledings van terminale-restriksie fragment lengte polymorfisme (T-RFLP) gemeenskapsvingerafdrukke en veral van die metaboliese profiele van die totale gemeenskap, het getoon dat verskuiwings in die grondmikrobiese gemeenskappe plaasgevind het met 'n toename in Cu-konsentrasies. Direkte tellings van grondprotosoë het ook aangedui dat die toevoeging van Cu tot die grond 'n negatiewe uitwerking op die getalle van hierdie eukariote gehad het. Om dié resultate te bevestig, is die impak van koper-oksichloried op die metaboliese profiele van totale gemeenskappe en protosoë-getalle in ander grond-ekosisteme vervolgens bestudeer deur grond van die kommersiële plaas Koopmanskloof en die Nietvoorbij-proefplaas te gebruik. Dié potensiële bioindikatore is vervolgens bestudeer in 'n reeks grondmikrokosmosse, wat vir elke grondtipe voorberei is deur die toevoeging van beraamde hoeveelhede van 0 (kontrole), 30, 100 en 1000 dpm Cu as 'n komponent van koper-oksichloried. Die resultate het die bevindings bevestig dat verhoogde vlakke van Cu 'n negatiewe uitwerking het op die metaboliese potensiaal en op die protosoëgetalle in die grond. Daar is gevolglik besluit om 'n kombinasie van protosoë-tellings en metaboliese profiele te ondersoek as 'n potensiële bioindikator van Cu-geïnduseerde stres in grond. Data van al die mikrokosmosse wat uitruilbare Cu bevat, wisselend van 1 dpm tot 1112 dpm, is gebruik om 'n dendrogram te konstrueer wat koolstofbronbenuttingsprofiele in kombinasie met protosoë tellings gebruik. Daar is gevind dat die mikrokosmosse groepe vorm wat korrelleer met die konsentrasie uitruilbare Cu in die grond. Onder die eksperimentele kondisies wat in dié studie gebruik is, wil dit voorkom of die kombinasie van protosoë-tellings en metaboliese profiele 'n betroubare indikator van Cugeïnduseerde stres is. Hierdie bioindikator moet egter verder in ander grondtipes en met ander tipes stres-induserende besoedeling ondersoek word. By bogenoemde bevindings is daar ook gevind dat die getalle grondprotosoë besonder gevoelig is vir Cu-geïnduseerde stres in grond met In lae pH. Dit is in ooreenstemming met die bevindings van andere met betrekking tot die bio-beskikbaarheid van swaar metale in grond met 'n lae pH. In dié tipe grond mag nutriëntsiklering as gevolg van protosoë aktiwiteit besonder gevoelig wees vir die negatiewe uitwerking van koper in die grond.

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