The effect of conventional herbicides, used for the control of alien vegetation on microbial communities within freshwater biofilms

Thesis (MSc)--Stellenbosch University, 2017.ENGLISH ABSTRACT: Freshwater streams in South Africa are heavily permeated with invasive plant species. These invasive plant species pose a major threat to native vegetations by competing for available ecosystem resources. A common method used for the removal of these species, is herbicide application. The overuse of these chemicals and the surface runoff into the nearby rivers, has become problematic. The effect of these chemicals on the freshwater biofilms, which consists of various microbial communities, is, however, still unknown. Microbial communities play an important role in the functioning of riparian ecosystems especially in biogeochemical cycling. Biofilms are mostly beneficial to their surrounding environment and the microbial communities change depending on the exposure to external factors. These factors are mostly abiotic factors and also include exposure to chemicals. These chemicals can either be harmful or beneficial to the microbial community. Some microbial species are able to break down these chemicals and utilise it as a nutrient source. In this study, the effect of three conventional herbicides, Springbok, Garlon and Plenum, on freshwater biofilms was investigated. The Berg River in the Western Cape was chosen based on the fact that it flows through herbicide applied zones. An optimized herbicide detection method using liquid chromatography coupled to mass spectrometry (LC-MS) was developed and used to determine the current levels of herbicide active ingredients present in the river system. A preliminary study was done to determine the effect of glyphosate on freshwater biofilms. This study showed that a significant microbial community shift occurs after the application of the herbicide. A physical disruption of the biofilm was also observed at the concentrations that were applied. In a follow-up study, river water collected from the Berg River which was used to set up a laboratory-scale artificial river system. Flow cells were used to cultivate the biofilms, using the river water as the natural inoculum. The biofilms were allowed to establish for 10 days after which the herbicide and the active ingredient were applied. The flow cells were then used for fluorescent microscopy and DNA extractions. Garlon, Plenum and Springbok were found to decrease the biofilm biomass significantly (p<0.05) for all treatments analysed. Finally, the DNA of the biofilm was extracted from the flow-cells using the standard DNA extraction protocol. The DNA was then used for PCR amplification with bacterial and fungal specific primers. Automated ribosomal intergenic spacer analysis (ARISA) primer sets were used. Significant shifts (p<0.05) were found for the herbicides, Springbok (glyphosate) and Plenum (fluroxypyr and picloram). The Shannon diversity index showed a decrease in diversity for both these treatments. This suggest that some species tend to dominate the biofilm because they can tolerate the environmental change. The hypothesis for this study which states that conventional herbicides influence the growth of microbial communities within freshwater biofilms, is not rejected. This is supported by the significant (p<0.05) decrease in biofilm biomass that was seen after herbicide application and also a microbial community shift occurred after herbicide application.AFRIKAANSE OPSOMMING: Varswaterstrome in Suid-Afrika word swaar binnegeval deur indringerplant spesies. Hierdie indringerspesies vorm 'n groot bedreiging vir inheemse plantegroei en die beskikbaarheid van water. 'n Algemene metode wat gebruik word vir die verwydering van hierdie spesies is, onkruiddodertoediening. Die oorbenutting van hierdie chemikalieë en die afloop van die oppervlak in nabygeleë riviere, het probleme veroorsaak. Die effek van hierdie chemikalieë op die varswater biofilms, wat uit verskeie mikrobiese gemeenskappe bestaan, is egter nog onbekend. Mikrobiese gemeenskappe speel 'n belangrike rol in die funksionering van oewer-ekostelsels in die biogeochemiese sirkulering. Biofilms is meestal voordelig vir hul omliggende omgewing, maar die mikrobiese gemeenskappe verander afhangend van die blootstelling aan eksterne faktore. Hierdie faktore is meestal abiotiese faktore, en sluit die blootstelling aan chemikalieë in. Hierdie chemikalieë kan beide skadelik of voordelig vir die mikrobiese gemeenskap wees. Sommige mikrobiese spesies breuk hierdie chemikalieë af en gebruik dit as ‘n voedingsbron. Hierdie studie ondersoek die effek van drie konvensionele onkruiddoders, Springbok, Garlon en Plenum op varswater biofilms. Die Bergrivier in die Wes-Kaap is gekies omrede, dit deur gebiede vloei waar onkruiddoders toegedien word. 'n Geoptimaliseerde onkruiddoder opsporings metode met behulp van vloeistof chromatografie gekoppel aan massaspektrometrie (LC-MS) is ontwikkel en gebruik om die huidige vlakke van aktiewe bestanddele in die rivier te bepaal. 'n Voorlopige studie is uitgevoer om die effek van glifosaat op varswater biofilms te bepaal. Hierdie studie het getoon dat 'n beduidende mikrobiese gemeenskapsverskuiwing na die toediening van die onkruiddoder plaasvind, 'n fisiese ontwrigting van die biofilm is ook waargeneem teen dieselfde toegediende konsentrasie. Na afloop van die ondersoek studie is rivierwater in die Bergrivier versamel wat gebruik is om 'n kunsmatige rivierstelsel in die laboratorium op te rig. Vloei selle is gebruik om die biofilms te kweek, met die rivierwater as die inokulum. Die biofilms is toegelaat om te groei vir tien dae voor die onkruiddoder en die aktiewe bestanddeel toegedien is. Die vloei selle was gebruik vir fluoresserende mikroskopie en DNA ekstraksies. Garlon, Plenum en Springbok het bevind dat die biomassa aansienlik verminder het (p <0.05) vir alle behandelings. Laastens is die DNA van die biofilm uit die vloeiselle verkry deur gebruik van die standaard DNA-ekstraksie protokol. Die DNA was gebruik vir polimerase kettingreaksie (PKR) amplifikasie met bakteriese en swamspesifieke as inleiers. Geoutomatiseerde ribosomale intergeniese afstand ontleding (ARISA) inleier stelle is gebruik. Beduidende verskuiwings (p