A COMPARATIVE ECOTOXICOLOGICAL STUDY OF TWO MAJOR SOUTH AFRICAN RIVER SYSTEMS: A CATCHMENT SCALE APPROACH FOR IMPROVED RISK ASSESSMENT AND ENVIRONMENTAL MANAGEMENT THROUGH THE INTEGRATION OF ABIOTIC, BIOTIC, BIOCHEMICAL AND MOLECULAR ENDPOINTS By Arno Reed de Klerk Dissertation presented for the degree of Doctor of Philosophy Faculty of Natural Sciences Stellenbosch University Supervisor: Prof. Anna-Maria Botha-Oberholster (Genetics Department) Co-supervisor: Prof. Johannes H van Wyk (Department of Botany and Zoology) Co-supervisor: Prof. Paul J Oberholster (Department of Botany and Zoology; CSIR: NRE) December 2016 Stellenbosch University https://scholar.sun.ac.za DECLARATION 1 By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), and that I have not previously in its entirety or in part submitted it for obtaining any qualification. This dissertation includes two original papers published in peer-reviewed journals and three unpublished publications. The development and writing of the papers (published and unpublished) were the principal responsibility of myself and, for each of the cases where this is not the case, a declaration is included in the dissertation, indicating the nature and extent of the contributions of the co-authors. Signature: Declaration with signature in possession of candidate and supervisor Date: Copyright © 2016 Stellenbosch University All rights reserved ii Stellenbosch University https://scholar.sun.ac.za ABSTRACT Along with the prosperity of humankind, the inevitable increase in developments to support this often results in water resources becoming repositories for the waste generated by such progress. To ensure sustainable development, anthropogenic impacts need to be properly assessed and anticipated in order to respond accordingly to mitigate such events. With the Mokolo River being at the center of plans to exploit the largest remaining coalfield in South Africa, this study was initiated to improve our understanding of the range of expected water resource impacts that may occur, by taking lessons learned from the upper Olifants River system, which has been severely impacted by coal mining for more than a century. The aim of this study was to conduct a comparative ecotoxicological study at a catchment scale on both the Mokolo River and upper Olifants River systems. Study sites in the upper Olifants and Mokolo rivers, their major tributaries, as well as major impoundments were selected and assessed seasonally for a number of years. Water and sediment samples were analyzed for various constituents, whilst biological samples (invertebrates and fish) were also sampled to gain perspectives on the impact of pollution at molecular, cellular and community levels. Lastly, a case study to examine the potential use of rehabilitation measures on ecological infrastructure to mitigate the potential impact of acid mine drainage (associated with coal mining activities) was conducted. Important abiotic ecosystem drivers were identified, whilst the influence of the tributaries and impoundments on the main stream rivers were determined. The major exposure routes of bioaccumulation were found to be through the dietary uptake of contaminated sediment and algae. The use of the fresh water crab, Potamonautes warreni, provided an excellent model organism to study the fate, transport and impact of pollution and also to monitor various degrees of molecular and cellular impacts. In contrast to the general use of fish as bioindicators, the use of aquatic invertebrate community structures and freshwater crabs proved more useful to characterize the ecosystem integrity of these systems, detect turnover and determine the links with possible contaminants. From the various biochemical and molecular markers used, several endpoints proved useful in monitoring chronic impacts on biota, which may be used as early warning indicators, whilst others are linked to acute anthropogenic impacts. A significant improvement in water quality occurred after the wetland rehabilitation case study, including increased productivity, reduced toxicity (embryotoxicity and teratogenicity) and changes in the biotic community structures. Therefore, the benefit of the rehabilitation of ecological infrastructure to mitigate the impact of coal mining associated pollution was iii Stellenbosch University https://scholar.sun.ac.za proven. Overall, the use of a comparative catchment approach, employing both an impacted and less impacted river system, proved extremely useful and valuable to support the future management of the Mokolo River in the face of ever increasing land use activities. This catchment scale comparative study is a rare and unique opportunity which few studies are able to utilize to assess the likely impacts of future land use change. iv Stellenbosch University https://scholar.sun.ac.za OPSOMMING Tesame met die welvaart van die mensdom, veroorsaak die onvermydelike styging in ontwikkelings om dit te ondersteun dikwels dat waterbronne bewaarplekke word vir afval wat gegenereer word tydens die vordering. Ten einde volhoubare ontwikkeling te verseker, is dit nodig om antropogeniese invloede behoorlik te evalueer en te verwag om sodoende dienooreenkomstig te reageer op sulke gebeurtenisse, sodat die impak verminder kan word. Met die Mokolorivier wat te midde is van planne om die grootste oorblywende steenkoolveld in Suid-Afrika te ontgin, is hierdie studie geïnisieer om ons begrip van die omvang van die verwagte waterbron impakte wat mag voorkom, te verbeter deur te leer uit vorige ervarings in die boonste Olifantsrivier, wat erg geraak is deur steenkoolmynbou vir meer as 'n eeu. Die doel van hierdie studie was om 'n vergelykende ekotoksikologiese studie te doen op 'n opvangsgebied skaal op beide die Mokolo- en boonste Olifantsriviere. Studie areas in die boonste Olifants- en Mokoloriviere, hulle hoof sytakke en damme is gekies en seisoenaal geassesseer vir 'n aantal jare. Water en sediment monsters is ontleed vir verskillende parameters, terwyl biologiese monsters (invertebrata en visse) ook versamel is om die uitwerking van besoedeling te bepaal op 'n molekulêre, sellulêre en gemeenskapsvlak. Laastens was 'n gevallestudie gedoen om die potensiële gebruik van rehabilitasie van ekologiese infrastruktuur om die impak van suur mynwater (geassosieer met steenkoolmyn aktiwiteite) te verminder, ondersoek. Belangrike abiotiese ekosisteem drywers is geïdentifiseer, terwyl die invloed van die sytakke en damme op die hoofstroom riviere bepaal was. Die grootste blootstellingsroetes van bioakkumulering is gevind om deur die opname van gekontamineerde sediment en alge te wees. Die gebruik van die varswater krap, Potamonautes warreni, is 'n uitstekende modelorganisme om die lot, vervoer en impak van besoedeling te bestudeer, asook om verskillende grade van molekulêre en sellulêre impakte te monitor. In kontras met die algemene gebruik van visse as bio-indikators het die gebruik van akwatiese invertebraat gemeenskapstrukture en varswater krappe bewys dat dit meer bruikbaar is om ekosisteem integriteit te beskryf, omsette te bepaal, asook die verbintenisse met moontlike besoedeling te bepaal. Vanuit die verskillende biochemiese en molekulêre biomerkers wat gebruik is, het verskeie eindpunte bewys om nuttig te wees in die monitering van kroniese impakte op biota, wat gebruik kan word as vroeë waarskuwingsindikators, terwyl ander gekoppel is aan akute antropogeniese impakte. Daar was 'n beduidende verbetering in die waterkwaliteit na die rehabilitasieproses van die vleiland, insluitend 'n toename in produktiwiteit, verminderde toksisiteit (embriotoksisiteit en v Stellenbosch University https://scholar.sun.ac.za teratogenisiteit) en verandering in die biotiese gemeenskapstrukture. Hierdeur was die nuttigheid van die rehabilitasie van ekologiese infrastruktuur om die impak van steenkoolmyn geassosieerde besoedeling te verminder, bewys. Dus, die gebruik van 'n vergelykende opvangsgebied benadering, deur die gebruik van beide 'n besoedelde en onbesoedelde rivierstelsel, is baie nuttig om die toekomstige bestuur van die Mokolorivier te ondersteun in die aangesig van toenemende antropogeniese aktiwiteite. Hierdie vergelykende studie op opvangsgebied skaal is 'n seldsame en unieke geleentheid wat min studies in staat is om aan te wend om die waarskynlike impak van veranderings in toekomstige grondgebruik te evalueer. vi Stellenbosch University https://scholar.sun.ac.za ACKNOWLEDGEMENTS I would like to thank the following persons and institutions for their assistance during the undertaking of this study: • My supervisors, Professors A-M Botha, JH van Wyk and PJ Oberholster, for their mentorship, support, patience and guidance during this study. • The Department of Genetics, as well as the Department of Botany and Zoology from the Stellenbosch University for the use of their facilities. • The Coaltech Research Association for their financial support and for all their assistance during the study. • The Water Research Commission of South Africa (WRC) (project K5/2203) for their financial support. • The National Research Foundation (NRF), Technology and Human Resources for Industry Program (THRIP) (Grant No. TP2011072900027) for their financial support. • The Olifants River Forum (ORF) for their financial support. • The South African Department of Environmental Affairs (Working for Wetlands) for their contributions