Impacts of Anthropogenic Activities on the Stable Isotopic Dynamics of Particulate Organic Matter in Mbashe River, Eastern Cape, South Africa

IMPACTS OF ANTHROPOGENIC ACTIVITIES ON THE STABLE ISOTOPIC DYNAMICS OF PARTICULATE ORGANIC MATTER IN MBASHE RIVER, EASTERN CAPE, SOUTH AFRICA

MUNETSI ZVAVAHERA

A dissertation submitted in fulfilment of the Requirements for the Degree of

MASTER OF SCIENCE (MSc) (ZOOLOGY)

WALTER SISULU UNIVERSITY

Supervisor: Dr Kuriah F.K.

September 2014

Abstract

Mbashe River passes through three districts of Eastern Cape, South Africa, with catchments that have been modified by anthropogenic activities. To determine the effects of anthropogenic activities on the river particulate organic matter, 13 15 (δ CPOM), (δ NPOM) and C/N ratios of particulate organic matter were investigated. Six sampling sites from three sections of the river were identified and selected according to anthropogenic activity occurring close to the river and sampled over a period of twelve months. The results indicated that different anthropogenic activities had significant effect on the POM stable isotope dynamics, resulting in uniquely distinct stable isotopes signatures varying both 13 15 temporally and spatially. The C/N ratios, (δ CPOM), and (δ NPOM) isotopic values varied significantly (95% confidence interval) and revealed that POM was derived from different sources in the river catchment. Anthropogenic activities affected 15 C/N ratios and δ NPOM temporally, spatially and between river sections. The 15 15 upstream was δ NPOM depleted (4.5‰) while downstream the river δ NPOM was 13 enriched (5.8 ‰). The δ CPOM values ranged from -12‰ to -32‰ temporally during the study period. The study revealed that POM was mainly derived from 15 allochthonous sources (C/N ratios >8). The (δ NPOM) revealed that upstream was more affected by anthropogenic activities than downstream. Our findings suggest that anthropogenic activities had more temporal effect than site to site. Further research is recommended and required to check whether isotopic dynamics observed can be replicated and determine whether the effect of anthropogenic activities is increasing or decreasing.

Key words: stable isotopes, anthropogenic activities, enrichment, depletion

Declaration

I, Munetsi Zvavahera student number 212204513, declare that this research entitled “Impacts of anthropogenic activities on stable isotope dynamics of particulate organic matter in Mbashe River, Eastern Cape, South Africa”, which I submit for the degree of MASTER OF SCIENCE (ZOOLOGY) at Walter Sisulu University is my own work and all relevant references are shown in the reference list. This study has not previously in its entirety or in part been submitted at any university in order to obtain an academic qualification.

MASTERS CANDIDATE : ZVAVAHERA MUNETSI

SIGNATURES : ______

DATE : ______

SUPERVISOR : DR F.K KURIAH

SIGNATURE : ______

DATE : ______

iii

Declaration on Plagiarism

(i) I am aware that plagiarism is defined at Walter Sisulu University as the

inclusion of another’s or others’ ideas, writing, works, discoveries and

inventions from any source in an assignment or research output without due,

correct and appropriate acknowledgement to the author(s) or source(s) in

breach of values, conventions, ethics and norms of the different professional,

academic and research disciplines and includes unacknowledged copying

from intra – and internet and peers /fellow students.

(ii) I have duly and appropriately acknowledged all references and conformed to

avoid plagiarism as defined by WSU.

(iii) I have made use of the citation and referencing style stipulated by my

supervisor/ lecturer.

(iv) The submitted work is my own.

(v) I did not and will not allow anyone to present my work as his/hers own.

(vi) I am aware of the consequences of engaging in plagiarism.

______Signature Date

Acknowledgements

I am greatly indebted to Dr F.K.Kuriah who mentored, supervised and assisted me academically and socially throughout my studies. My due acknowledgements go to the Walter Sisulu University for funding my studies and for that I am very grateful. I also thank the Cwebe Nature Reserve management for allowing us to use estuary as part of the sampling sites. To my wife Chido and my two sons,

Alistair and Douglas Jr, whom I deprived the luxury of having a father during my long absence from home are gratefully appreciated and acknowledged. My parents who financially and morally supported all the way from the start to the end are greatly appreciated. Special thanks also go to Dr E. Okuthe personal and social support and Dr S.K Kuria for statistical analysis. Friends who made it possible to complete my studies and unconditional support; Dr S. Matope, W

Masomera, T. Makonese, J. Majatame, K. Brown and Ms V. Magubane. The colleagues from the department of zoology are greatly acknowledged for their support academically, and after I was involved in a car accident during the course of my study.

TABLE OF CONTENTS

Abstract ...... ii

Declaration ...... iii

Declaration on plagiarism ...... iv

Acknowledgements ...... v

Acronyms ...... xi

CHAPTER 1 ...... 1

1.1 GENERAL INTRODUCTION ...... 1

1.2 Riverine problems ...... 3

1.3 Background to the study ...... 3 1.4 Problem statement ...... 6 1.5 Aim ...... 7 1.6 Hypotheses ...... 7 1.7 Objectives ...... 8 1.8.1 Main objectives ...... 8 1.8.2 Specific objectives ...... 8 1.9 Rationale ...... 8 1.10 Significance of the study ...... 8 1.11 Justification of the study ...... 10 1.12 Assumptions...... 11 1.13 Limitations ...... 12 1.14 Delimitations ...... 12

CHAPTER 2 ...... 13

LITERATURE REVIEW ...... 13

2.1 Introduction ...... 13

2.2 Rivers ...... 14

2.3 Stable isotopes ...... 15

2.4 Carbon/ Nitrogen ratios ...... 17

2.5 Carbon isotopes ...... 19

2.6 Nitrogen isotopes ...... 19

2.7 Particulate organic matter ...... 21

2.8 Anthropogenic activities ...... 22

2.9 Human Settlements ...... 25

2.10 Agriculture ...... 26

2.11 Damming ...... 28

2.12 Forestry and Deforestation ...... 29

2.13 Sand mining ...... 29

2.14 Wild fires ...... 30

2.15 Grazing land/ pasture land ...... 30

Chapter 3 ...... 32

MATERIALS AND METHODS ...... 32

3.1 Experimental Design ...... 32

3.2 Study area ...... 32

3.2.1 Water use ...... 36

3.2.2 Land use/ anthropogenic activities ...... 36

3.2.3 Vegetation ...... 36

Methodology ...... 37

3.3 Sample collection ...... 37

3.4 Particulate Organic matter ...... 37

vii

3.5 Isotopic analysis ...... 38

3.6 Statistical analysis ...... 39

CHAPTER 4 ...... 40

RESULTS ...... 40

4.1 C/N ratios ...... 40

4.2 The δ13C isotopic signatures of POM...... 40

15 4.3 The δ NPOM Variations ...... 41

4.4 Correlation results ...... 41

CHAPTER 5 ...... 54

5.0 DISCUSSION ...... 54

6.0 CONCLUSION ...... 59

Recommendations...... 61

Outcomes ...... 61

References ...... 62

Table 1: Sampling sites and anthropogenic activities in Mbashe River...... 35

13 15 Table 2: p values from Kruskal Wallis ANOVA of C/N ratios, δ CPOM and δ NPOM isotopic fingerprints of particulate organic matter in Mbashe River for seasons and river sections...... 43

Table 3: Mann Whitney U- test values to compare different river sections along Mbashe River...... 43

13 Table 4: The W and p values from Mann-Whitney test for δ CPOM variations for different for seasons...... 43

Table 5: The W and p values from Mann Whitney test for comparisons between 15 seasonal changes in δ NPOM...... 44

viii

Table 6: The W and p values from Mann Whitney test for comparisons between seasons using C/N ratios of particulate organic matter from for Mbashe River...... 44

Figure 1: The sampling sites and land use of the study area along Mbashe River...... 34

Figure 2: C/N ratios for different Mbashe River sections...... 45

Figure 3: Seasonal C/N ratio variations of POM for Mbashe River...... 45

Figure 4: Monthly C/N ratio variations of POM for Mbashe River...... 45

13 Figure 5: Monthly δ CPOM (‰) variations for Mbashe River for the sampling period ...... 45

13 Figure 6: The δ CPOM isotopic variations for the three Mbashe River sections (upstream, midstream and downstream)...... 47

13 Figure 7: Seasonal variation of δ CPOM...... 47

15 Figure 8: Monthly δ NPOM (‰) during the twelve months sampling along Mbashe River...... 48

15 Figure 9: Seasonal δ NPOM variations along Mbashe River...... 48

15 Figure 10: Spatial variation of δ NPOM among sites along Mbashe River (1 & 2 – upstream, 3 & 4 –midstream and 5 & 6 –downstream)...... 49

Figure 11: The δ15 values of POM for river sections along Mbashe River...... 49

15 Figure 12: Correlation analyses between C/N and δ NPOM along Mbashe River...... 50

Figure 13: The correlation between δ15C and C/N ratios of river POM for Mbashe River...... 50

13 15 Figure 14: Correlation analyses between δ CPOM and δ NPOM of Mbashe River...... 51

Figure 15: MDS ordination map for Mbashe River – testing the effect of sites ...... 52

Figure 16: MDS ordination maps for Mbashe River ...... 52

Appendices ...... 90

Appendix A: Riparian grazing at Isixhotyweni near Clarkeburry with clear water flowing in the river...... 90

Appendix B: Burning of the grazing area at Lota...... 91

Appendix C: Subsistence crop cultivation at Msana...... 91

Appendix D: Dam at Msana location with turbid water...... 92

Appendix E: Temporary construction company at N2 Bridge ...... 92

Appendix F: Road construction between N2 and Ludondolo...... 93

Appendix G: Bridge construction at Mvezo...... 93

Appendix H: Quarry mining near Mthendu...... 94

Appendix I: Sand mining at Tsholora/Noshange...... 94

Appendix J: Irrigation near Tsholora Bridge...... 95

Appendix K: Nature reserve and protected area Cwebe...... 95

Appendix L : Application letter to conduct a study along Mbashe River and Cwebe marine Estuary ...... 97

Appendix M: Walter Sisulu University: Directorate of Postgraduate studies on plagiarism ...... 98

ACRONYMS

DWAF Department of Water Affairs and Forestry

POM Particulate Organic Matter

SIA Stable isotope analysis

C/N δ13C to δ15N ratio

13 (δ CPOM) change in carbon 13 isotope

15 (δ NPOM) change in nitrogen 15 isotope

GFF Glass fibre filters

SSA Statistics South Africa

CF-IRMS Continuous flow isotope ratio mass spectrometry

CHAPTER 1

1.1 GENERAL INTRODUCTION The main purpose of the research was to determine whether anthropogenic activities in the river catchment have any effect on the aquatic ecosystem through the use of stable isotopic analysis of POM. The Mbashe River catchment area is characterised by different anthropogenic activities which vary (Table 1). The main objective of the research was to determine how and whether different anthropogenic activities in the catchment area produced different isotopic fingerprints spatially and temporally.

Generally there are very few remaining pristine aquatic ecosystems in most river catchments worldwide (Michener and Lajtha, 2007). Some of the Eastern Cape Rivers have impaired water quality due to anthropogenic activities (Kuriah, 2008). Most river systems have been modified by anthropogenic activities in the watersheds worldwide. Mbashe River passes through catchments that have been modified by different anthropogenic activities. Though the activities are predominantly agro based there are many other non-agricultural activities in Mbashe drainage basin. The main anthropogenic activities include agriculture mainly subsistence, road construction, irrigation, riparian grazing, human settlements, deforestation, afforestation and nature reserves.

Developing countries face numerous challenges in balancing economic development with environmental conservation (Banadda, et al., 2009), especially aquatic ecosystems. Anthropogenic pressure has resulted in the severe reduction of plant and animal species both in terrestrial and in aquatic ecosystems (Papa, et al., 2011). Water resource protection has been given high priority in natural resource management and is very important in sustaining human and ecological communities (Randhir and Hawes, 2009). Our research study on the

1 anthropogenic effects will be of great importance in the identification of major sources of organic pollution and can be used to determine how human activities are impacting on aquatic ecosystem. This will also inform ecologists and environmentalists on how to have effective land-use management systems.

1.2 Riverine problem Human life is greatly dependent on rivers for survival but has severely impacted the riverine resources (Dawson and Siegwolf, 2007). Activities on the terrestrial ecosystems have direct or indirect influence on the aquatic ecosystems (Ashkenas, et al., 2004). Effects of different anthropogenic activities are variable on the aquatic ecosystems having direct/ indirect effect (Baby, et al., 2014).

Proper management of pollution is critical in the maintenance of good water quality in rivers (Wu and Chen, 2013). Environmental and ecological changes due to anthropogenic activities are rapidly increasing (Dawson and Siegwolf, 2007), hence there is need to have very accurate methods to determine the causes of changes. Variation of isotopic composition of organic matter in rivers is poorly understood (Michener and Lajtha, 2007). Management of pollution requires accurate identification of the sources of pollution (Drolc and Koncan, 2008).

Irrigation is higher during the dry season and usually causes water stress in rivers (Dessu, et al., 2014). Irrigation results in the transportation of denitrified organic material to rivers which results from leaching (Delconte, et al., 2014). Nitrates are naturally found in aquatic ecosystems but anthropogenic inputs from terrestrial ecosystem increase nitrate loads in freshwater ecosystems (Fenech et al., 2012).

1.3 BACKGROUND OF THE STUDY Terrestrial ecosystems that are close to rivers require effective management in order to reduce the effects of anthropogenic activities. Anthropogenic modification of the physical structure of the landscape in river catchments is mainly for commercial, agriculture and recreational purposes (Poff, et al., 2002). Rapid human population growth worldwide in general has led to the increase of anthropogenic activities in river catchments (Kennish, 2002; Thompson, et al., 2005; Wu, et al., 2007; Leu, et al., 2008). The increase in anthropogenic activities are due to the continuous increase need of resources for human survival worldwide (Gupta, et al., 2012). Anthropogenic activities are very dynamic, changing with the changes in human needs (Ayivor and Gordon, 2012).

Massive economic developments in watersheds have resulted in the production of large amounts of waste which find their way to stream and rivers (Wu, et al., 2009). Top contributors to South African economy include agriculture, fisheries and forestry and such activities have exerted great risk to South African Rivers, of which many rivers are being over exploited and polluted (Statistics South Africa, 2010). Eastern Cape river water mainly in rural areas is highly polluted and does not meet the WHO standards (Igbinosa and Okoh, 2009). Anthropogenic activities in river catchments greatly influence POM in rivers through land use (Miserendino, et al., 2011; Zhang, et al., 2011), causing serious contamination of river water to unacceptable levels (Michener and Lajtha, 2007). Severe river pollution has threatened immensely public and environmental health (Dsikowitzky, et al., 2004; Kennish, 2002), and also has impaired and altered the balance in aquatic ecosystems (Randhir and Hawes, 2009). Anthropogenic pollution in rivers comes from terrestrial sources such as mining, industries, and urbanization along the rivers (Azrina, 2006).

The demand for agricultural products is likely to double thereby directly impacting on both aquatic and terrestrial biodiversity (Baudron and Giller, 2014). Running water is 3 greatly affected by anthropogenic activities because of direct contact with their catchments that are continuously modified by humans (Malmqvist and Rundle, 2002; Pokhrel, 2012). Rivers along their course pick up a variety of organic pollutants from point (urban and industrial effluents) and non-point (agricultural land runoff) pollution sources (Angelids, et al., 1995).

Rivers and streams are very sensitive and complex aquatic ecosystems (Fatoki, et al., 2002; Dsikowitzky, et al., 2004, Al-Shami, et al., 2011), and are major contributors of global biodiversity and production that are constantly disturbed and destroyed by human activities in river catchments (Poff, et al., 2002). Such activities severely affect areas with high biodiversity (Leu, et al., 2008). There is strong correlation between anthropogenic activities and species richness with intense anthropogenic activities posing risk to biodiversity (Findlay and Houlahan, 1997; Al-Shami, et al., 2011; Allan, 2013). Plant and animal biodiversity in aquatic ecosystems is lost when water is heavily polluted by human activities (Malmqvist and Rundle, 2002; Leu, et al., 2008).

Organic matter from anthropogenic activities or sources influences biomass production and thereby modifying the nature of natural organic matter in aquatic ecosystems (Micic, et al., 2011). Anthropogenic activities in river catchments contribute significantly to loss of biodiversity through the habitat destruction (Kennish, 2002). River water transports allochthonous organic matter (derived from terrestrial sources) and autochthonous organic matter from production within the river itself (Richard, et al., 1997; Neatrour, et al., 2004; Parkyn, et al., 2005; Gorecki, et al., 2006; Michener and Lajtha, 2007). Rivers are major and important routes of terrestrial organic matter from river catchments to the oceans (Raymond, et al., 2004). Anthropogenic activities in river catchments generate the greatest proportion of organic pollution that is carried to rivers by rain water as runoff (Leu, et al., 2008; Micic, et al., 2011). Allochthonous particulate organic matter (POM) severely impacts aquatic ecosystems causing new changes that may be detrimental or lethal to aquatic organisms (Michener and Lajtha, 2007; Igbinosa

4 and Okoh, 2009). This may subsequently affect the natural ecological balance in aquatic ecosystems.

Organic matter in rivers shows the attributes of the catchment area from which it is derived (Kaplan, et al., 2006). The sources of such organic matter can be traced through the determination of carbon (δ13C), nitrogen (δ15N) isotopes and C/N ratios of POM (Liu, et al., 2007). The stable isotope analysis (SIA) can be used to detect environmental changes especially those that are products of anthropogenic activities in river basins (Michener and Lajtha, 2007).

Isotopes are atoms of the same element that have same proton and electron number but differ in neutron numbers (Michener and Lajtha, 2007). Most elements have more than one different isotopes which can be distinguished from each other by mass (Yakir, 2002; Dawson and Siegwolf, 2007). Usually one of the isotopes is found in high abundance (mostly the lighter/common isotope) and the other found in low abundances (heavier /rare isotope) in biological compounds (Dawson and Siegwolf, 2007).

Isotopic differences does not affect how elements react physically or chemically, but provide a way of their identification by isotope ratio mass spectrometry (IRMS), for all substances carry a unique isotopic fingerprint (proportion) of given variable forms (Jardine, et al., 2003). Stable isotopes are those isotopes that do not spontaneously decay but remains energetically stable for a long period (Michener and Lajtha, 2007). The use of stable isotope analyses has become an important application to determine the potential sources of particulate organic matter in rivers (Chang, et al., 2009). The SIA has been also applicable in estuaries to determine the dynamics (Boonphakdee, et al., 2008).

Lighter isotopes are commonly used for ecological investigations because their natural abundance (Michener and Lajtha, 2007). Carbon (δ13C) and Nitrogen (δ15N) isotopes are used because elements are central to the normal functioning of aquatic ecosystems,

5 however their alterations in the river catchments results in severe water quality problems. It is of ecological importance to study δ13C and δ15N isotopes as they determine the functioning and processes of aquatic ecosystems (Dodds and Whiles, 2010).

Carbon and nitrogen elements are mainly used in stable isotope analysis (SIA) for ecological research on environmental processes (Jardine, et al., 2003; Harmelin-Vivien, et al., 2010). The element carbon exists primarily as the carbon-12 (δ12C) isotope but a small fraction presents as heavy isotope carbon-13 (δ13C), while nitrogen’s most abundant form is the nitrogen-14 (δ14N) isotope, with nitrogen-15 (δ15N) making up a small heavier percentage (Jardine, et al., 2003). Ratios of the rare 13C to the common 12C and 15N to 14N in POM samples are reported using “δ” (delta) notation in units of parts per thousand (‰) or ‘parts per mil’ (Boonphakdee, et al., 2008). This research thesis investigated the impact of different anthropogenic activities on stable isotope dynamics of particulate organic matter using stable isotope analysis in order to determine the causes of pollution to the river.

1.4 Problem statement Anthropogenic activities in the Mbashe River catchment have great potential of altering water quality in the river (DWAF, 2008). A follow up study revealed that the number of taxa in the studied area has drastically decreased downstream along Mbashe River (DWAF, 2010) hence the research may be used to give explanation for the observed trends. Other Eastern Cape rivers have been altered by anthropogenic activities which have affected the isotopic composition of particulate organic matter (Kuriah, 2008). Isotopic response of river water quality to anthropogenic activities in the catchments is not fully understood (Wilson and Weng, 2011). The isotopic fingerprints of anthropogenic activities haven’t been documented for Mbashe River. This has created a large knowledge gap for aquatic ecologists to explain the dynamics of the river’s catchment and connect them to terrestrial activities. Spatial and temporal variation in organic matter is poorly understood compared to other processes of aquatic ecosystem, 6 and this results from the fact that rivers vary greatly in physical and chemical conditions (Michener and Lajtha, 2007). It is against this background the research had to be undertaken.

Sound management of river catchments requires clear-cut identification of anthropogenic pressures, and how they impact aquatic ecosystems (Drolc and Koncan, 2008). There has been an increase in anthropogenic activities along the Mbashe River catchment area. These include bridge, dam constructions, small scale irrigation schemes, sand-gravel mining, deforestation and introduction of alien species of plants (DWAF, 2010), quarry mining and several gravel road which may be altering the quality of Mbashe River.

1.5 Aim  To assess whether the current anthropogenic activities in the catchment area influence the stable isotopic dynamics of POM.  To determine whether there are spatial and temporal impacts of the anthropogenic activities on allochthonous POM stable isotopes dynamics of the Mbashe River. 1.6 Hypotheses

 H0 Anthropogenic activities in river catchments have no effect on the stable isotope dynamics of river POM.

 H1 Anthropogenic activities in the river catchment influence stable isotope dynamics of POM.

1.7 Main objective  To investigate how different anthropogenic activities in the river catchment are influencing on the stable isotopic composition of POM in Mbashe River.

1.8.1 Specific objectives  Determine whether there are spatial/temporal variations in the isotopic composition of POM of Mbashe River.  To investigate the isotopic of anthropogenic activities on the δ13C and δ15N of POM dynamics in Mbashe River.

1.9 Rationale For ecological managers and ecologist to improve the health status of river systems, there is need to accurately assess the ecological state and this is important in tracking /rehabilitation efforts (Young, et al., 2008). The stable isotopic analyses of POM provides relevant information on the extent to which anthropogenic activities have impacted the river ecology (Martinelle, et al., 1999), hence this research can be used in tracing the sources of environmental changes and rehabilitating the ecosystem. Sound knowledge of isotopic composition of POM will help in bio-monitoring of Mbashe River as isotopic composition can be used to trace source of many pollutants in the river system (Michener and Lajtha, 2007). Policies on the conservation of biodiversity in wetlands must also include the activities that are taking places on the adjacent land as they have a significant effect on the processes taking place in rivers (Findlay and Houlahan, 1997). This has prompted the research to be undertaken, in order to provide detailed information on isotopic dynamics of POM.

1.10 Significance of the study The research will be used in the determination of how best to strike a balance between human activities in river catchments and aquatic ecological health state can be achieved and maintained. Mbashe estuary is one of the largest in South Africa supporting very valuable biodiversity in the area (Van Niekerk and Tupie, 2012), and this makes it important to study how anthropogenic activities occurring upstream the river catchment, impact on the aquatic ecosystem. Identifying how the current activities are

8 altering the isotopic composition will be of great significance in preserving endangered species and biodiversity in general in the study area.

The spatial and temporal variation of isotopic fingerprints of organic matter in rivers is not well understood (Michener and Lajtha, 2007). The SIA of POM used in this research and other studies may be used by environmental managers as early warning detection tool for environmental and ecological change. SIA of riverine POM will be used to identify major sources of organic pollution that are critically causing environmental degradation. The identification of the sources of POM pollution is vital for managers as it enables them to react swiftly to ecological problems (Dawson and Siegwolf, 2007), that affect biodiversity directly or indirectly. Given the potential increase of human activities in the ecosystems due to demand for social, economic and recreational services, there is great need to understand how ecosystems will evolve under the stresses of increased anthropogenic activities (Leu, et al., 2008).

Most of South Africa’s largest river catchments have been greatly modified by anthropogenic activities from their pristine condition (Van Niekerk and Turpie, 2012). Large river catchments experience significant changes due to rapid developments in them. The anthropogenic activities in catchments have a cumulative effect on aquatic ecosystem and require sound management interventions. Such activities greatly modify the ecological processes and functions, such as productivity and food webs (Jardine, et al., 2003). The research will be of great importance for ecological management in determining how developments in the catchment are impacting on the aquatic ecosystems.

Human activities in the river basin often change the absolute and relative amounts of POM in aquatic ecosystems (Parkyn, et al., 2005). Stable isotope analyses of POM are useable in detecting the impacts of such activities (Jardine, et al., 2003; Bianchi, et al., 2007). A sound knowledge of SIA of POM can be the basis for good and sustainable use and management of river systems and the identification of the sources of POM are of

9 great importance in bio-monitoring (Schubert, et al., 2012). The research will help in accurate assessment and proper monitoring of the impact of human activities on the river ecosystem and habitat protection for aquatic organisms. The data may give a strong and sound understanding of how human activities are altering the water quality as a habitat for aquatic organisms.

Land anthropogenic activities transform landscapes and disrupt the river ecosystems thus posing a need for restoration in order to attain ecological sustainability (Zhou, et al., 2012). The evaluation of source contribution to the POM of aquatic ecosystems is very valuable in the assessment of the anthropogenic impact (Barros, et al., 2010). The research will be important as it will be used to assess current status of rivers and also to predict future changes to the ecosystem (Dawson and Siegwolf, 2007).

The goal of this research thesis was to determine or identify the major sources of anthropogenic POM from the catchment in order to determine how human activities may be impacting aquatic ecosystem. This will fill the knowledge gap on the impact of anthropogenic activities. The study of such anthropogenic activities helps ecologist to understand how aquatic ecosystems respond to these changes (Randhir and Hawes, 2009; Costea and Haidu, 2010). Analysis of anthropogenic activities is used to predict how the ecological communities will be affected by such activities (Wilson and Weng, 2011).

1.11 Justification of the study POM is important in the understanding of dynamics of river ecosystems, yet little is known about changes and the origins of POM in relation to physical and seasonal change along rivers (Akamatsu, et al., 2011). Very few ecological studies have looked at the spatial and temporal variation of POM in rivers (Michner and Lajtha, 2007). Sources and fate of organic matter in aquatic ecosystem is poorly understood (Finlay, et al., 2010). The same applies to Mbashe River where the POM dynamics is undocumented, despite the fact that it is a good indicator of the effect of anthropogenic activities on the 10 aquatic ecosystem. Lack of knowledge on the impacts of anthropogenic activities on the river system affects the interpretation of ecological processes (Osenberg and Schmitt, 1996). It is against this background that the research had to be undertaken, mainly to bridge the knowledge gap on POM dynamics and the related effects on aquatic ecosystem as this will have a bearing on the land use programmes.

The Mbashe River catchment has multiple anthropogenic activities from its source to the river mouth, hence it is important to isolate the activities that are responsible for polluting the aquatic ecosystems. The activities vary both spatially and temporally in the studied watershed. The extent to which human activities are affecting aquatic habitats need to be correctly assessed so as to achieve sound information and meaningful ecological management (Young, et al., 2008; Sakamaki and Richardson, 2011). Despite the fact that there are no heavy industries in the catchment area that cause serious pollution, there is need for constant monitoring of river water (Perillo, et al., 2005).

The Dwesa- Cwebe Estuary is a reserve for many fish species hence there is need to protect the habitats in its pristine state since poor agricultural practices along the catchment have affected the river (Eastern Cape Parks Board - ECPB, n.d). Mbashe Estuary/Cwebe Estuary is partly protected yet it has been identified as requiring full protection. It is the only spawning estuary in South Africa (Van Niekerk and Tupie, 2012), hence it is of great importance to know how the current anthropogenic activities upstream are impacting aquatic ecosystem. Such activities have the potential to extremely modify the aquatic ecosystem.

1.12 Assumptions Allochthonous organic matter contributes the greatest proportion of river particulate organic matter.

1.13 Limitations Time and financial constraints were limiting as the research may be improved by doing over longer period. The study only focused on the main river and did not include the tributaries.

1.14 Delimitations of the study The research was restricted to the effect of major anthropogenic activities on the isotopic composition of particulate organic matter in Mbashe River. The main focus of the research was the major sources of allochthonous POM and did not look at autochthonous POM as it is not directly as a result from human activities. The study was conducted in Mbashe River catchment area only.

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction Generally, rivers carry large amounts of organic matter from land surface areas in catchments through runoff (Dinakaran and Krishnayya, 2011). Riverine POM is related to the landscape and geochemical characteristics of the watersheds (Gordeev, et al., 2004). Changes in land use and anthropogenic activities may severely upset the natural isotopic dynamics of organic matter in the rivers (Neatrour, et al., 2004). Organic pollutants resulting from anthropogenic activities accumulate into river systems, disturbing their natural state (Schubert, et al., 2012). The particulate phase of organic matter is sensitive to anthropogenic impacts (Durchane, et al., 2007).

River catchments exert an economic heave and support relatively high human population densities, because of their encouraging living conditions such as the availability of fertile lands, water for irrigation, industrial, domestic purposes, and efficiency of transportation (Mouri, et al., 2011). This has led to a rapid swell in the number of anthropogenic activities in river catchments and in turn translated to ever increasing environmental problems (Osenberg and Schmitt, 1996). Many large rivers have been extensively modified during the past few decades by anthropogenic activities (Ayivor and Gordon, 2012). Change in the water quality, mainly the organic content has become a major ecological distress worldwide as it impacts ecological state of rivers (Kennish, 2002; Miserendino, et al., 2011). Most of these anthropogenic influences are part of the larger process of catchment land use or land cover change that can affect water quality in rivers as well as downstream estuarine and coastal water (Peters and Meybeck, 2000; Mouri, et al., 2011; Zhou, et al., 2012).

Aquatic ecosystems are constantly changing in response to anthropogenic processes and changes may have significant effects on the environment (Kendall, et al., 2010; Jennerjahn, et al., 2004). Some anthropogenic activities severely and extensively 13 transform the landscape of river catchments (DWAF, 2005) and may have dire effects on the river ecology (Peters and Meybeck, 2000; Isik, et al., 2008). Activities such as deforestation, agriculture, fertilizer and pesticide use, waste discharge, and destabilization of river beds lead to changes in composition of substances transported by the rivers (Jennerjahn, et al., 2004). Other land use activities adjacent to rivers create significant ecological risks to river/wetland biodiversity reducing species richness (Findlay and Houlahan, 1997; Kennish, 2002).

Anthropogenic activities have conspicuous effects on water quality, biodiversity and distribution of species, with high species diversity being in unpolluted areas (Azrina, et al., 2006). These activities have presented an array of problems in the preservation and protection of biodiversity (Papa, et al., 2011). Human activities in the river catchments upset the usual development of susceptible and fragile ecosystems especially downstream of the river (Azrina, et al., 2006). Anthropogenic activities alter biological communities in ecosystems and more severely in aquatic systems (Anderson and Cabana, 2009). Large numbers of plant and animal species are fast disappearing because of ever increasing anthropogenic pressure due to the demand people place upon the natural recourses (Papa, et al., 2011).

2.2 Rivers River systems are always evolving, responding to parameter changes of the river catchment system itself (Chapman, 1996), however they may fail to cope with the rate at which humans are modifying these catchments. Land use changes alter the composition of organic matter in rivers (Bernardes, et al., 2004). Rivers carry a wide range of organic matter from different sources (Michener and Lajtha, 2007). Estuaries receive water directly from rivers and are regarded amongst the most impacted ecosystems and are exposed to multiple stresses which affect ecological communities and biodiversity (Kennish, 2002).

2.3 Stable isotopes Traditionally river health monitoring most commonly employed the use of structural measurement such as taxonomic appearance/absence of organisms in aquatic ecosystems (Young, et al., 2008). In most cases such biological communities are not often frequent because of factors other than anthropogenic activities (Miserendino, et al., 2011). On the other hand the use of stable isotopes in ecological studies has gained edge over the traditional methods for tracing anthropogenic induced changes in freshwater ecosystems (Thompson, et al., 2005; Grey, 2006; Bannon and Roman, 2008). Stable isotope analysis of POM has become handy in the ecological research and provides crucial and unique information about high precedence issues and ecological sciences (Dawson and Siegwolf, 2007). Stable isotopes can be used in the conservation of ecosystems that are being affected by agriculture and assessing soil erosion (Bhat, et al., 2010).

Anthropogenic activities and natural disturbances to the ecosystems leave isotopic fingerprints that are uniquely different (Dawson and Siegwolf, 2007). Organic substances derived from anthropogenic activities record changes in ratio of isotopes contained in the material of which natural variation in isotopic fingerprints (Dawson and Siegwolf, 2007), hence making it possible and easy to trace the origins of organic matter in rivers. The origins of organic matter informs on the scale of anthropogenic alteration in the drainage basin (Michener and Lajtha, 2007).

Elements such as carbon, nitrogen, sulphur and hydrogen have more than one isotope of which the isotopic composition can be determined with great precision with a mass spectrometer (Peterson and Fry, 1987). The stable isotopes can be expressed as percentages however for convenience reason are expressed in ‰ (parts per thousand/ per mill) (Ehleringer and Matheson, 2010). Different isotopes of elements have the matching chemical properties but vary in characteristics that result of their atomic properties (Jardine, et al., 2003). The ecological applicability of SIA comes from the predictable processes such as fractionation or discrimination of isotopes in biological

15 and non-biological substances (Grey, 2006), that will favour either the lighter or heavier isotopes, leading to depletion or enrichment, respectively (Jardine, et al., 2003).

The ratio of rare to common (heavy to lighter) in any particular material provides valuable information on the source information of organic substances (Dawson and Siegwolf, 2007). Variation in the stable isotope ratios provides the basis for many ecological applications (Boonphakdee, et al., 2008). Isotopic differences are as result of various processes occurring in the watershed (Michner and Lajtha, 2007).

Carbon (δ13C) and Nitrogen (δ15N) isotopic fingerprints are widely used to trace the sources of POM in river catchments (Lobbes, et al., 2000; Countway, 2007; Boonphakdee, et al., 2008; Botter, et al., 2010;Harmelin-Vivien, et al., 2010; Akamatsu, et al., 2011; Hatten, et al., 2012). Materials such as POM acts as archives of environmental change (Dawson and Siegwolf, 2007). Changes in stable isotope signatures indicates changes in rivers/wetlands show that they have been exposed to multiple or complex anthropogenic activities (Wu, et al., 2007; Chang, et al., 2009). Different isotopic values and ratios indicate that organic matter is derived from different sources (McCallister, et al., 2006; Liu, et al., 2007; Boonphakdee, et al., 2008). Stable isotopes of POM can be used to show anthropogenic manipulation of terrestrial ecosystem and their effects on aquatic ecosystems (Chang, et al., 2009).

Isotopic standards have been developed for individual isotopes to accurately compare isotopic values across experimental studies. These standards were chosen because they demonstrated consistent values after repeated and multiple measurements (Jardine, et al., 2003).

SIA of POM can be used in conjunction with other traditional methods aimed at quantifying and mitigating the impacts of alterations to ecosystems through anthropogenic activities (Boonphakdee, et al., 2007). This has been made possible because different physical and biogeochemical processes in landscapes often create

16 distinctive and unique isotopic signatures (Kendall, et al., 2010), with impacted areas showing isotopic signatures that are uniquely different from pristine areas (Vizzini and Mazolla, 2006). Spatial and temporal stable isotopic variation of POM in a river indicates effects of different land use and land cover in river catchments (Jennerjahn, et al., 2008). Isotopic techniques have proved to be useful for tracing sources of various pollutants in large river basins (Kendall, et al., 2010).

The standards used in SIA have values of 0‰, substance with positive values having ratio higher than the standard whilst negative values have values less than the standard (Dawson and Siegwolf, 2007). Consistent isotopic depletion and enrichment is an indicator of the effect of anthropogenic activities on the freshwater ecosystems (Hou, et al., 2013).

2.4 Carbon/ Nitrogen ratios The C/N ratios, δ13C, and δ15N values can be used together in ecological studies and are a powerful tool for showing the dominant source of POM found in aquatic ecosystem (Flite, et al., 2008; Dolenec, et al., 2011). The notation δ13C shows the ratio of the isotopes (13C/12C) relative to the ratio of a standard that has a predefined value of 0‰ using the international carbon standard Vienna Pee Dee Belemnite (V-PDB) (Boonphakdee, et al., 2008). Isotopic variation in C/N ratios of river organic matter is important in providing information of what is happening in the river catchments (Michener and Lajtha, 2007).

C/N ratios can also be used to estimate the relative proportions of autochthonous and allochthonous organic matter in rivers (Perdue and Koprivnjak, 2007), also the variations of such sources in different times (Hellings, et al., 1999). River particulate carbon is mainly of allochthonous origin (Ran, et al., 2013). C/N ratios are very important in ecological studies because they are predictable and indicate the major source of organic matter in rivers (Michner and Lajtha, 2007). Isotopic differences are due to unevenness of lighter and heavier isotope abundances (fractionation) which 17 make it possible for ecologist to predict their origin (Dawson and Siegwolf, 2007). The rate of fractionation mainly depends on the attributes of landscape processes or ecological processes in the river catchment (Bedard - Haughn, et al., 2003). Ecological processes have an impact on the C/N ratios, making it possible to give insights and sources of materials carried by rivers (Boonphakdee, et al., 2008). Spatial and temporal variations in C/N ratios of POM are mainly due to the combination of natural and human factors (Ran, et al., 2013).

2.5 Carbon isotopes The carbon derived from allochthonous and autochthonous organic matter varies considerably in rivers (Michener and Lajtha, 2007). Generally pristine rivers have depleted isotopic δ13C values for POM (Lafon, et al., 2014). Anthropogenic activities in drainage basin influence carbon isotopes in rivers (Hilton, et al., 2010). The element carbon is essential for many forms of life hence can be found in a number of organic compounds. It has three isotopes (14C, 13C, and 12C) which differ in their masses (Balakrishna and Probst, 2005). Dissimilar isotopic carbon values spatially and temporally make it easy to map out the exact sources of organic matter in the aquatic ecosystems (Michener and Lajtha, 2007). The 14Cis highly unstable and is of limited use in environmental application however13C and 12C isotopes are very stable and can be used for ecological investigations. The applicability of carbon isotopes in environmental analysis lies in the fact that they are spatially precise and integrate a range of information into one isotopic signal (Boonphakdee, et al., 2008). The δ13C of river POM indicates the isotopic characteristics of the soil from which it is derived (Cravotta, 1997).

2.6 Nitrogen isotopes The element nitrogen is very important in the nutrient system of aquatic ecosystems 15 (Gang, et al., 2005). Analyses of δ NPOM in pristine rivers are very important as they provide an ecological foundation for assessment of nitrogen dynamics in rivers (Tank, et

18 al., 2000). Many anthropogenic activities in river catchments (e.g. deforestation removes leaf detritus), alter organic nitrogen dynamics in the aquatic ecosystems (Deegan, et al., 2011). Anthropogenic alteration of nitrogen cycle in watersheds changes δ15N of organic matter in the river (Michener and Lajtha, 2007). Many rivers around the world have experienced rapid increases in nitrogen with non-point source pollution being the chief contributor to fresh water nitrogen which varies spatially (Wang, et al., 2012).

Transport of terrestrial nitrogen through runoff and seepage to rivers exerts a strong influence on the development, productivity and biodiversity of ecosystems (Miyajima, et al., 2009). Aquatic nitrogen is directly related to anthropogenic activities such as fertilizer application, livestock waste, agriculture, land use and other activities in the drainage basin (Wang, et al., 2012; Michner and Lajtha, 2007). A positive δ (delta) indicates the sample contains more of the heavy isotopes than the standard, while a negative δ shows the sample contains less of the heavy isotope than the standard (Michner and Lajtha, 2007). The differences in the nitrogen isotopic values result from fractionation (Bedard-Haughn, et al., 2003).

15 δ NPOM isotope analyses provides a complete depiction of the flow and fate of N from associated river catchments and can be used to determine the primary sources of Nitrogen in the aquatic ecosystems (Bedard-Haughn, et al., 2003). Characteristics of landscape are major determinants of how nitrogen is lost from terrestrial to aquatic ecosystem (Wang, et al., 2009). The δ15N isotopic signatures of organic material in aquatic ecosystems vary spatially (Barnes and Raymond, 2010). There is need to identify the sources of nitrogen because of the ever increasing amounts of nitrogen being discharged into rivers (Bedard-Haughn, et al., 2003). Nitrogen pollution has rapidly increased due to massive economic, agricultural and social developments in river catchments (Yue, et al., 2013). Anthropogenic nitrogen contamination has increased in ecosystems around the world and stable isotope analysis can be used to trace the sources anthropogenic nitrogen pollution (Karthic, et al., 2013). Nitrogen applied to

19 soils to increase crop production is the major pollutant of aquatic ecosystems with heavy discharge of nitrogen leading to environmental problems such as eutrophication 15 (Drolc and Koncan, 2008; Bednarek, et al., 2014). Seasonal variation in δ NPOM in aquatic ecosystem is mainly caused by human activities in river catchments and increases with nitrogen usage and varies from region to region (Wang, et al., 2012).

Knowing the sources of nitrogen in the rivers will be of great importance in the prevention of pollution of freshwater ecosystems (Bao, et al., 2006). Riverine particulate nitrogen is high during times when there is high river discharge (Duan, et al., 2008), and also isotopic fingerprints vary accordingly to show isotopic variation (Yue, et al., 2013).

Nitrogen is one of the most common nutrients transported by rivers as runoff (Hubbard, et al., 2004), and it varies spatially and temporally in river catchments (Schulte, et al., 2006), depending on soil fertility. Solids mainly in the organic form such as pesticides and nutrients from grazing areas impair water quality (Ohlenbusch, et al., 2002).

2.7 Particulate organic matter Rivers, as freshwater ecosystems, have distinct spatial flows of organic matter worldwide (Lafon, et al., 2014). Organic matter in rivers is mainly a mixture of terrestrial and autochthonous produced organic matter (Perdue and Koprivnjak, 2007). A small proportion of river POM is autochthonous whilst the larger portion is allochthonous POM (Hein, et al., 2003). The amount of allochthonous POM decreases with the distances down the river course indicating that the spatial influence of human activities decreases towards the sea (Vinagre, et al., 2011). Allochthonous POM is important in natural ecosystems and in agricultural catchments with spatial and temporal differences indicating that POM is derived from different sources (Cravotta, 1997; Martinelli, et al., 1999). Allochthonous POM in rivers is mostly derived from soil

20 resulting from erosion (Onstad, et al., 2000; Wu, et al., 2007; Lafon, et al., 2014). Isotopically heavy and lighter nitrogen results from anthropogenic waste and autochthonous production such as phytoplankton, respectively (Liu, et al., 2007).

SIA of river water POM has been proven to be a powerful tool in the ecological investigation of aquatic ecosystems (Cravotta, 1997; Kaplan, et al., 2006; Countway, et al., 2007), however very little information is known about stable isotopes (Akamatsu, et al., 2011). Riverine POM plays an important role in aquatic ecosystems (Hakanson, et al., 2005). Organic substances in rivers record important information on processes within the drainage basins, and contributions of terrestrial inorganic and organic material to aquatic ecosystems (Lobbes, et al., 2000).

Distressed aquatic ecosystems transport extensively large amounts of particulate organic matter more than pristine ecosystems (Webster, et al., 1990). The particulate phase of organic matter in rivers is sensitive to anthropogenic activities in the catchments and can be used to appreciate how aquatic ecosystems respond to such activities (Thomas, et al., 2004). Rivers plays an important role as pathways of POM from terrestrial sources to the sea (Gordeev, et al., 2004). POM isotopic composition is influenced by watershed attributes such as land use (Kaplan, et al., 2006).

Stable isotope analysis of POM is not only important to the area with particular anthropogenic activities but also downstream where it may impact on ecosystem (Peters and Meybeck, 2000). During wet or rainy seasons the POM is largely allochthonous that is flushed from the catchments (Gupta, et al., 1997). Normally, lighter C isotopes would be likely in the wetter periods when either terrestrial or freshwater C sources are important (Matson and Brinson, 1990).

2.8 Anthropogenic activities Anthropogenic activities in the river catchments influence the isotopic attributes of POM thereby affecting aquatic ecosystems (Young, et al., 2008). Anthropogenic activities 21 affect the all the river sections (Fan, et al., 2006). Anthropogenic activities may have drastic effects on the river ecology, with alterations in the upstream part of the catchment having important implications for the downstream (Isik, et al., 2008).

Terrestrial ecosystems and aquatic ecosystems are closely related (Ashkenas, et al., 2004), with activities on the terrestrial directly affecting rivers. Anthropogenic activities can be divided into activities that affect direct and indirect activities that affect aquatic ecosystem directly or indirectly (Chapman, 1996; Allan, 2004). Activities which directly affect the ecosystem include; dam construction, diversions, recreation, channelization and riparian grazing (Wohl, 2006). Indirect include activities such deforestation, agriculture, urbanization, mining, grazing, chemical fertilizers and wildfires (Ayivor and Gordon, 2012). Such anthropogenic effects on river ecosystems need to be correctly assessed and measured to achieve sound ecosystem management (Sakamaki and Richardson, 2011).

Human influences on ecosystem will continue in the twentieth century and is likely to accelerate due to poor ecological management systems and rapid industrialisation of developing countries (Dawson and Siegwolf, 2007). Pollution has been projected to rapidly increase in developing countries more than developed countries (Liu C, et al., 2012). Anthropogenic activities contribute significantly to amounts of organic matter in water thereby greatly changing the water composition and quality to varying scales (Masu, 2010). Knowledge of such activities is of paramount importance in the prevention of pollution (Shen, et al., 2011).

Variability in anthropogenic activities and land use in river catchments alters the natural functions of rivers and significantly impact the water quality as a habitat and ecosystem dynamics (Randhir and Hawes, 2009, Jing, et al., 2012). Intensive exploitation of resources results in severe degradation of river catchments and result in ecological problems (Huang, et al., 2006; Randhir and Hawes, 2009). Anthropogenic activities affect the abiotic factors which in turn affect the aquatic biota (Kuriah, 2008).

Anthropogenic activities are by far more responsible for altering aquatic habitats than natural processes (Leu, et al., 2008; Randhir and Hawes, 2009; Filoso, et al., 2013) and may modify aquatic ecosystems at different levels directly or indirectly (Allan, 2004). Surface water bodies are exposed to a number of anthropogenic stresses and rivers in the watershed receive waste from industries and agriculture (Malmqvist and Rundle, 2002; Wang, et al., 2007; Zhou and Peng, 2012). Negative impacts of anthropogenic activities range from slight to very chronic levels (Baby, et al., 2014). Human activities in the river system itself may affect migration patterns and routes of aquatic organisms (Gong and Xu, 1987).

Anthropogenic activities in the catchments alter river water quality through the addition of foreign substances and wastes to the landscape (Peters and Meybeck, 2000; Neill, et al., 2001; Malqvist and Rundle, 2002; Durchane, et al., 2007; Randhir and Hawes, 2009). Changes in water quality result from point and non-point sources of pollution (Angelids, et al., 1995; Miserendino, et al., 2011; Shen, et al., 2011). Physical alterations of the landscape include urbanization, deforestation and afforestation, land drainage, damming, and mining (Peters and Meybeck, 2000; Fan, et al., 2006). Such activities have increased the amount of organic matter and nutrients in rivers considerably worldwide (Hopkinson, et al., 1998; Bernardes, et al., 2004; Neatrour, et al., 2004; Leu, et al., 2008).

Anthropogenic land degradation induced by deforestation, wetland transformation, overgrazing, poor housing, poor farming methods, horticulture practices and poor traffic networks have all had a marked impact on the pollution of water (Banadda, et al., 2009). Land use changes affect the composition of the river POM (Martinelli, et al., 1999). Anthropogenic activities significantly affect the transport of organic matter in rivers (Marttila, et al., 2013). Anthropogenic pressure alters resilience of ecosystems to other forces acting on them (Ayivor and Gordon, 2012).

The effects of anthropogenic activities differ spatially and may have extreme effects on aquatic ecosystems. These activities include diversion of water for irrigation which often turns perennial rivers into seasonal streams (Xu, 2004 Wu, and Chen, 2013). Manipulation of aquatic ecosystem of this kind also affects biodiversity by reducing the availability of habitat for aquatic organisms (Gong and Xu, 1987).

Rapid economic development and urbanisation cause an increase in the organic compounds. It has created large amounts of pollutants (Bao et al., 2012). Environmental problems caused by anthropogenic activities can be diverse whilst human activities pose an ecological risk affecting biodiversity among other things (Cahoon and Mallin, 2013). Landscaping has strong effects to aquatic ecosystems producing eco-toxicological risks that seem to be less discriminative (Michner and Lajtha, 2007). Changing landscapes and anthropogenic activities poses a great challenge to management of aquatic ecosystems thereby making it difficult to understand their impact on aquatic ecosystems (Allan, 2004).

2.9 Human Settlements Population growth has led to unsustainable expansion of human settlements into areas with high ecological value (Aguilar and Santos, 2011). Rapid population growth leads to development of settlements without proper services (sewage, sanitation, waste disposal) which result in pollution of the environment and eventually affect aquatic ecosystems (Aguilar and Santos, 2011).

When landscape is used for housing and commercial purposes, vegetation is removed and has direct effect on water quality (Mallin, 2009). Rural settlements in river catchments also contribute significantly to non-point source pollution in rivers (Yang, et al., 2012). Residential areas and intensive agriculture contribute a lot to aquatic pollution through the transport of nutrients to rivers (Lang, et al., 2013). There should be reconciliation between human settlements and sustainability of environments (Aguilar and Santos, 2011). 24

High human population densities in the river catchments are responsible for heavy loading of anthropogenic wastes to rivers (Liu, et al., 2007). Most river basins support high population densities because of favourable conditions for many activities such as agriculture, industries and irrigation (Masu, 2010; Mouri, et al., 2011). Infrastructural development, such as the construction of houses is a characteristic of many developing and developed countries. Such development has been adversely impacts the environment thus resulting in severe loss of biodiversity and deterioration of aquatic ecosystem (Kennish, 2002; Fu, et al., 2009; Costea and Haidu, 2010; Baudron and Giller, 2014).

2.10 Agriculture Agriculture is the main source of income for poor communities with livestock and crop cultivation being the most dominant activities in most rural communities (Gadzirayi, et al., 2007). Increased crop production in drainage basins results in higher fertiliser usage which will eventually be carried to rivers (Sun, et al., 2012; Baudron and Giller, 2014). Demand for food resources worldwide has led to the fast expansion of irrigation and agro-industry which have posed great threat to sustainability of the aquatic ecological systems (Dessu, et al., 2014). The increased demand is due to rapidly increasing human population leading to intensification of agriculture in watersheds (Hochman, et al., 2013). Economic developments cause changes in agriculture too which in turn changes pollution levels in rivers (Pastuszak, et al., 2012).

Agricultural activities have also resulted in increased addition of foreign and organic compounds to aquatic ecosystem through fertilizer and pesticides applications. Organic pollution has become a major risk to aquatic ecosystems and agriculture has become the major source of river pollution (Chapman, 1996). Pesticides applied to crops find their way to rivers and the effects vary spatially along river course (Liu, et al., 2013; Lafontaine, et al., 2014).

Agricultural land use affects the quality, quantity and sources of organic matter in rivers and intensive cultivation contributes large proportion of POM in aquatic ecosystems (Munson and Curry, 2004). Allochthonous sources of organic matter plays an important role in the composition of the river POM in natural ecosystems and agricultural system (Martinelli, et al., 1999). Agriculture and densely populated areas leave distinguishable isotopic fingerprints on particulate organic matter (Jennerjahn, et al., 2008).

Despite the efforts to manage water pollution, non-point sources like agriculture and other activities pose continual environmental problems (Diebel, et al., 2008). Environmental problems in aquatic ecosystems are very complex usually arising from a variety stresses (Kennish, 2002; Shen et al 2013). Agriculture in most cases is not sustainable and contributes a lot to non-point source pollution (Guo, et al., 2014). Poor farming methods have further worsened pollution through large amounts organic fertilizers and inorganic fertilizers being transported to rivers (McClelland and Valiela, 1998; Gang, et al., 2005), resulting in elevated nitrogen in the rivers (Bao, et al., 2006).

Fertilizer application in the catchments has been identified as contributing to heavy nitrogen loading in rivers (Cravotta, 1997, Wang, et al., 2012). Activities such as land tillage and other cropping practices make the soil/ landscape vulnerable to erosion (Vorosmarty and Sahagian, 2000). Land use types in river catchments results the soil erosion and sediment deposition in rivers, this include the type of crop to be planted (Zhang, et al., 2014).

Irrigation in the river catchments a large extent, contributes to aquatic pollution through nitrates (Skoulikidis, 2009; Monteagudo, et al., 2012). Due to increased demand for food, much of the landscapes have been converted into irrigated land affecting both terrestrial and this affects terrestrial and aquatic ecosystems (Arroita, et al., 2013).

Nitrogen pollution is as a result of the use of livestock manure in agriculture. The problem has been exacerbated by the increase in demand for improved crop production. Worldwide non-point pollution contributes the largest pollution to water sources (Howarth, 2004). The main causes of water quality change in most rural rivers, comes from extensive fertiliser applications and use of pesticides (Sun, et al., 2012). Pesticides and herbicides reach the rivers as runoff (Mallin, 2009). Anthropogenic activities that are agro based have significant effects to aquatic ecosystems by affecting nitrogen dynamics (Delconte, et al., 2014).

Seasonal variation in anthropogenic activities is important as it determines fertilizer usage in the watersheds where seasonal crop cultivation is practised (Jennerjahn, et al., 2008). Most farming activities are correlated to fertiliser usage in croplands (Kuriah, 2008). In areas with low population density, fertiliser applications in cropland are the major cause of water quality deterioration through runoff (Fisher, et al., 2006). The intensity of agriculture in close proximity to rivers is important as it produces adverse effects on rivers (Monteagudo, et al., 2012). Irrigation has been used to improve crop production worldwide (Bourrie, et al 2013). Due to climate change which has caused rainfall patterns to change, irrigation is likely to increase or even double in South Africa (Calzadilla, et al., 2014). This causes significant soil erosion, with the eroded material affecting the organic content of rivers (Fisher, et al., 2006). Lack of pollution awareness by subsistence farmers has increased pollution of river water through excessive application of fertiliser and pesticides (Sun, et al., 2012). Insufficient information of pollution is detrimental to the management of such ecosystems (Bao, et al., 2012). Agriculture is the major source of source pollution to freshwater ecosystems in agricultural watersheds (Baudron and Giller, 2014).

2.11 Damming Dams constructed along the river course are responsible for holding a lot of fresh water runoff from the catchments (Vorosmarty and Sahagian, 2000). Damming affects the natural flow of river water thereby affecting the quality of water (Kuriah, 2008; Eng, et 27 al., 2013). Dam construction contributes ecological consequences that seriously impair organic matter exchange among ecosystems (Isik, et al., 2008; Lafon, et al., 2014). Regardless of the main use, dams trap water and alter the natural functioning of the river through interfering and modifying the flow peaks and distribution of flow (Kundolf, et al., 2002). The construction of dams affects river processes by reducing fluctuations in most of the aquatic parameters (Kuriah, 2008), this increases eutrophication because of higher nitrogen and organic matter storage being retained (Chapman, 1996; Ahearn and Dahlgren, 2005).

2.12 Forestry and Deforestation Presence or absence of forest in a drainage basin affects important river processes with forests in river catchments storing water and the removal of forest will eventually affect both terrestrial and aquatic ecosystems (Vorosmarty and Sahagian, 2000). Terrestrial and riparian vegetation plays a role in regulating river POM concentration, with higher values observed in forested streams (Webster and Meyer, 1997). Rivers in forested areas receive higher amounts of POM than those passing through non-forested areas (Golladay, 1997).

Terrestrial vegetation controls organic matter concentration in aquatic ecosystems (Golladay, 1997). This means that anthropogenic activities, such as deforestation, in the river catchments will affect POM dynamics in aquatic ecosystems. Afforestation projects affect runoff considerably in river catchments (Iroume and Palacios, 2013). This practice has positive effects on freshwater ecosystems through reduction of pollutants carried to river (Liu, et al., 2013). Riparian vegetation in river catchments acts as buffer of non-point source pollution thereby reducing effects of anthropogenic activities on aquatic ecosystems (Zhao, et al., 2009; Zaimes and Schultz, 2011). Deforestation in river catchment areas increases with increase in human population sizes in the river basins (Gorman, et al., 2009), and causes significant ecological changes to aquatic ecosystems (Deegan, et al., 2011).

2.13 Sand mining Sand mining in rivers is ever increasing (Cao, et al., 2004). Sand mining in river channels may cause severe effects to river systems. Excavations done along river channels and catchments interfere with river flow and sediments in the river. The worst effects of sediment mining are experienced when accompanied by other anthropogenic activities (Rinaldi, et al., 2005; Luo, et al., 2007).

2.14 Wild fires Wildfires in river catchments will continue to occur in the near future (Brown, et al., 2008). Fires affect the river ecosystems to varying degrees (Smith et al., 2011) and may have adverse effect on the ecosystems (Brown et al 2008). Wildfires are responsible for directly destroying terrestrial ecosystems and indirectly affecting the aquatic one (Smith, et al., 2011; Stein, et al., 2012). Fires in the river catchments have pronounced effects on the water quality of rivers through the removal of organic matter (Certini, 2005; Adeniyi and Imevbore, A.M., 2008, Britton, et al., 2008; Kuriah, 2008,). The occurrence of wildfires in drainage basins is important as it encourages surface flow of water and erosion which results in nitrogen loss through affecting water infiltration (Certini, 2005; Aranibar, et al., 2010). Fires produce ashes that are δ13C and δ15N that are isotopically deplete which may be transported to rivers as runoff (Gomez-Rey, et al., 2013), and decrease C/N ratios of organic matter (Pereira, et al., 2012). Slug derived from fires in the river catchment severely affects biodiversity in aquatic ecosystem which may result in the extinction of certain species (Lyon, et al., 2008).

2.15 Grazing land/ pasture land Livestock rearing is a common and at times is a dominant activity in most rural communities (Gadzirayi, et al., 2007). Intensive livestock production release large amounts of organic waste into the watershed and can have both positively and negatively effect on water quality (Ohlenbusch, et al., 2002; Hubbard, et al., 2004). Grazing land in river catchments contribute significantly to non-point pollution of river

29 water (Agouridis, et al., 2005). Problem of pollution increases if there is overstocking of animals in the river catchment (Hubbard, et al., 2004). Activities such as intensive grazing have direct effect on the isotopic composition of the soil in the river catchments, leading to depletion of carbon and nitrogen (Golluscio, et al., 2009).Rivers that flow through grazing areas or pasture land accumulate organic matter more than forested areas (Deegan et al., 2011).

In order to achieve sound freshwater quality protection there is need for proper management of grazing land (Ohlenbusch, et al., 2002). Grazing of animals remove riparian vegetation along the rivers thus influences the structure and function of aquatic ecosystems (Kiffney and Richardson, 2010). Riparian vegetation helps to assimilate sediment, nutrients, and organic matter from grazing animals (Hubbard, et al., 2004), however it is usually destroyed by grazing and deforestation.

Chapter 3

MATERIALS AND METHODS

3.1 Experimental Design Six sampling sites were selected using purposive sampling methods to represent different land use patterns and anthropogenic activities in the river catchment area. A Global Positioning System was used to map out the location of the sampling points. Samples were collected monthly from August 2012 to September 2013 from the upstream, midstream and downstream. Samples collected from the field were immediately cooled (Jardine, et al., 2003), at 4OC using a portable fridge to stop all the biochemical activities and metabolism of the microorganisms in the water (Azrina, et al., 2006). The samples processed at WSU Zoology laboratory later and sent to University of Cape Town for stable isotope analysis.

3.2 Study area Mbashe River is perennial and found in rural Eastern Cape Province of South Africa, located in Mzimvubu - Keiskamma Water Management area with a catchment area of 8679 km2. The river has three major tributaries in the upper course namely Xuka, Ngancule and Mgwali. Xuka and Ngancule tributaries have their sources in the Drakensburg Mountains in Elliot and Mgwali drains from the Ncobo area in Eastern Cape (DWAF, 2008).

Mbashe River Catchment is predominantly rural where subsistence farming is the main land use. Other important activities found in the study area include cattle grazing of which there is overgrazing due to overstocking (DWAF, 2010), irrigation, settlements, quarry, sand mining and construction.

The study area was marked from Clarkeburry at Isixhotyweni Bridge located upstream to river mouth at Cwebe marine Sanctuary elevation (Figure 1). Two sites were selected from each of the three sections of the river and they predominantly drain water from rural areas.

The climate of Mbashe River catchment is characterized by seasonal rainfall, with mean annual rainfall of approximately 810mm and total annual rainfall of 1129 million m3 /a (DWAF, 2008). The river is perennial, flowing through three districts (OR Tambo, Amatole and Chris Hani).

Close to Cwebe Nature Reserve land use is mainly for human settlement, gardens fields and grazing land. The fields in that part of the catchment are actively cultivated. Cwebe village area which is found near sampling (Cwebe), considerable land is under forest. Gardens and the area use large amounts of organic matter. About 83.1% of Cwebe Nature Reserve area is under forest cover and the Dwesa side of the area is forested. The nature reserve has animals such as zebra, blesbok, Blue Wildebeest and buffalos. Mbashe / Cwebe Estuary is mainly pristine however there is no monitoring (ECPB, n.d), which may cause it to deteriorate.

Figure 1: Map of Mbashe River showing the six sampling sites from the upstream to downstream.

Table 1: The sampling sites showing land use of the study area along Mbashe River.

RIVER SITE NAME CLOSEST CO- MAJOR LAND SECTION TOWN ORDINATES USES Upstream 1 Isixhotyweni Engcobo 31048’11’’S Subsistence farming @ 28020’11’’ E (stream bank Clarkeburry cultivation), grazing land 2 Msana Idutywa 31051’04’’ S Pasture land, 28023’32’’ E damming Midstream 3 N2 Bridge Idutywa 31055’09’’ S Deforestation, 28026’52’’ E subsistence crop cultivation, human settlements 4 Mvezo Idutywa 31055’29’’ S Crop cultivation, 28028’20’’ E road construction, bridge construction human settlements, and hydro electric power generation. Downstream 5 Noshange Willowvale 32008’51’’ S Forested area, /Tsholora 28048’00’’ E extensive sand bridge mining, schools and irrigation. 6 Cwebe Willowvale 32014’38’’ S Wild life and human Estuary 28053’52’’ E settlements

3.2.1 Water use Mbashe water is mainly used for house hold use, livestock, irrigation schemes, community garden projects and hydro-electrical power generation (Collywobbles) which has changed the flow regime of the midstream (DWAF, 2010). There are very few activities that consume large amounts of water (DWAF, 2005).

3.2.2 Land use/ anthropogenic activities Anthropogenic pollution from non-point sources is higher than that from point sources (Banadda, et al., 2009). The research area is exposed mainly to non-point source pollution since approximately 94% of the Mbashe population lives in rural areas (DWAF, 2008). Subsistence farming and cattle rearing dominates in the catchment area. Overstocking and poor agricultural practices have resulted in serious environmental degradation. Land use in the Mbashe River catchment includes also to a lesser scale commercial agriculture and forestry (DWAF, 2008; DWAF, 2010). Subsistence farming in Mbashe River Catchment area is mainly for maize and vegetables (DWAF, 2008; DWAF 2010). Upstream there are large scale irrigation schemes at Ncora and sediment mining (DWAF, 2010).

3.2.3 Vegetation There is a variety of vegetation types in the Mbashe catchment area. Grasslands are very dominant in the upstream/ upper course but there are patches of coastal forests downstream (DWAF, 2010). Massive afforestation projects results in the reduction of runoff and this is projected to impact on the ability to satisfy the ecological component of the Mbashe River (DWAF, 2005). Alien vegetation has invaded the river catchment and has led to the disappearance of indigenous vegetation. Deforestation is evident in the study area (DWAF, 2010).

3.3 Sample collection Six sampling points were identified and selected along the main course of Mbashe River. Water samples were collected from below the water surface (0.5m depth) and bottled in pre-cleaned plastic bottles (Boonphakdee, et al., 2008; Harmelin-Vivien, et al., 2010).Collection of samples for twelve months started in August 2012 to September 2013 to cover all the four seasons experienced in the province. Two samples were collected per site for each sampling month. The glass fibre filters were preheated in a furnace at 5000Cfor 24 hours to remove contaminants to enable accurate measurement of isotopic values of POM. The oven was preheated at 480C for 24 hours prior to drying of the samples.

3.4 Particulate Organic matter Initial processing of the samples was done in the zoology laboratory at Walter Sisulu University. POM was obtained by filtering river water through 25mm Glass Fibre Filters (GFF) (Hobson and Welch, 1992). The GFF filters were used in the collection of POM from water samples to obtain higher particle loadings approximately 10 mg for the analysis (Ernestberger, et al., 2004). The filters with POM samples were heated in at 60°C for 48 hours in a furnace and then treated with 2N: HCL to remove carbonates and inorganic carbon from POM, followed by rinsing with distilled water to remove excess acid. After acid treatment, the filters were oven dried at 60°C for 48 hours (Sato, et al., 2006; Harmelin-Vivien, et al., 2010; Lorenzini, et al., 2010). Dried samples were then packed into clean eppendorf tubes to avoid contamination of samples (Jardine, et al., 2003), for spectrometry isotopic analyses at University of Cape Town.

3.5 ISOTOPIC ANALYSIS Continuous flow isotope ratio mass spectrometry (CF-IRMS) analysis was used for the identification of isotopic signatures of POM. Filters with POM samples were cut and weighed to approximately 1mg. Isotopic composition in samples was reported using the conventional delta notation (δ) as parts per thousand (‰) deviations in isotope ratio

36 from international standards, namely Vienna-Pee Dee Belemnite (V-PDB) for carbon and atmospheric N2 (AIR) for nitrogen (Michener et al 2007). Stable isotopes are expressed as natural differences in the isotopes relative to a known international standard. Isotope ratios for carbon and nitrogen is presented as δ values, The SIS was expressed using the standard notation:

δX (‰) = ((Rsample/ Rstandard) 1) 1000 ...... (1)

13 15 13 12 15 14 Where X is C or N and Rsample is the C/ C or N/ N respectively. Pee Dee 13 15 Belemnite (PDB) is used as Rstandard for C; for N it is atmospheric N2 (air). The standards expressed the ratios of 13C/12C and 15N/14N, relative to PDB and air and the values are in ‘mils’ (‰).

Where δ(X) = 1,000 x [(Rsample / Rstandard) -1],

And X is 13C or 15N, R = 15N/14N and 13C/12C, respectively (Chamberlain, et al., 2005; Sugimoto, et al., 2006; Lorenzini, 2010).

The isotopic abundance (δ) is often very small and hence the final result is multiplied by 1000 to allow for convenient expression with the isotopic standards having values of 0‰ (Dawson and Siegwolf, 2007). A positive (δ) delta indicates that the sample has more of the heavy isotope than the standard and the sample is said to be enriched whereas a negative indicates the sample has less of the heavy isotope than the standard or depleted (Michener and Lajtha, 2007; Dawson and Siegwolf, 2007).

3.6 STATISTICAL ANALYSIS Mann-Whitney U test was used to test for the significance of the isotopic differences between sites, seasons and river section. Stastica version 9 was used to analyse the isotopic data obtained analysis of POM. A Kruskal- Wallis ANOVA was used to establish whether there were temporal or spatial variations in POM isotopic dynamics of Mbashe 37

River. Mann Whitney U test was used to assess the statistical significance of differences among means from various stations and sampling times. Statistical differences were analyzed at 95% confidence interval (p-value of 0.05).

Correlation analysis was used to determine relationships between different investigated variables. Multidimensional scaling (MDS) was used to visualize the level of similarity of different seasons, river sections and sampling sites to establish whether different activities have similar or different effects on the isotopic composition of POM.

CHAPTER 4

RESULTS

4.1 C/N ratios The Kruskal – Wallis ANOVA was performed followed by Mann Whitney U test, revealed that the C/N ratios of POM varied significantly temporally (p<0.05) but insignificantly spatially (p>0.05) during the study period (Table 2). All the river sections recorded generally similar values however downstream had wider range of values (5-20) than other sections (Figure 2). The C/N ratios of POM showed that there were high significant seasonal variations (p<0.05). Spring season had the highest median C/N ratios approximately 15 with a range of 10-18, followed by the summer and autumn season whilst winter had the lowest median ratios of 6 but with a very wide variation of values 6-17 (Figure 3). Monthly C/N ratio variations were statistically different with August September and October showing very high C/N ratios (>12) (Figure 4). The month of August depicted the widest C/N range of isotopic values of 7-27. The period from May- July recorded the lowest C/N ratios with a range of 6-7 (Figure 4).

13 4.2 The δ CPOM isotopic signatures A Kruskal-Wallis ANOVA results revealed that there were no significant variations 13 spatially (p˃0.05) for δ CPOMTable 2, however downstream recorded the widest variations and upstream recorded narrowest variations (Figure 6).

13 There were significant monthly δ CPOM variations with median values ranging from - 17‰ to -28 ‰. The months of August, September and October recorded most 13 depleted values δ CPOM (negative values) and widest variations from -26‰ to -29‰ 13 while February and March had enriched δ CPOM values with values around -16‰ 13 (Figure 5). A very narrow range of median δ CPOMvalues was recorded (-19‰ to - 13 22‰) during the months of June and July of 2013 (Figure 5). Spatially, δ CPOM did 13 not show significant variations (Figure 7). The seasonal median δ CPOM values varied

13 from -17‰ to -24‰ for the whole year. Spring season δ CPOM values indicated much isotopic depletion with a median of -24‰ while winter season had the widest variation (-12‰ to -31‰) and summer had the narrowest variation ranging from -19‰ to - 21‰ (Figure 7).

15 4.3 The δ NPOM Variations 15 The δ NPOM isotopic signatures varied significantly seasonally (p<0.05), the values ranged from 1.5‰ to 10‰ but the median values ranged from (4.30‰ – 7.03‰). Winter season had the widest isotopic variation (1.5‰ – 10‰) and summer had the narrowest variations ranging from (4‰ – 6‰) (Figure 10). Autumn months had the most enriched values (6‰) while winter had the most depleted values (4‰) of 15 15 15 δ NPOM (Figure 9). There was δ NPOM depletion upstream while δ NPOM enrichment downstream near the river mouth (Figure 10). The upstream section of the river had 15 the lowest δ NPOM isotopic values (4.4‰) values followed by midstream section (5‰) and downstream section (where river enters the sea) had the highest of (5.5‰).At 15 13 different river sections δ NPOM isotopic signatures varied more in comparison to δ CPOM p<0.05 (Figure 11).

A Kruskal Wallis ANOVA test followed by Mann-Whitney U tests revealed noteworthy 15 monthly median variations in δ NPOM (2‰– 7.5‰) during the study period (Figure 15 8). Months of August - October and March recorded much enriched δ NPOM values with September having the widest variations as indicated by the error bars (2.5‰ – 11.5‰), whilst June and July had the smallest variations (Figure 8). There were 15 significant δ NPOMvariations between sites (Isixhotyweni, Msana, N2, Mvezo, Tsholora and Cwebe) as revealed by Kruskal Wallis ANOVA p<0.05 (Table 2).

4.4 Correlation results 15 15 Correlation analysis was done to determine whether δ CPOM, δ NPOM and C/N ratios co- 15 varied with each other. The δ NPOM and C/N ratios were positively correlated (Figure 15 15 12). The C/N ratios and δ CPOM were negatively correlated (Figure 13). The δ CPOM 40

15 and δ NPOMwere negatively correlated (Figure 14). C/N ratios and were positively 15 correlated to δ NPOM (Figure 12).

MDS ordination map was used to show (whether sampling site, months, seasons and river sections) of various anthropogenic activities had effect on the isotopic composition of POM (Figures 15-16). The stress values for all the MDS maps were very low 0 indicating that the variation was significant (Figures 15-16). MDS ordination map 13 showed fewer similarities between site effects on δ CPOM variations as indicated by longer Eucledian distances between variables but different for other sites (Figure 15a). The MDS ordination map showed that different anthropogenic activities had similar 15 influence on δ NPOM, with sites (5 and 3) but different from, (2 and 4) and (1 and 6) being clustered closer to each other as indicated by shorter Eucledian distances (Figure 15b). C/N ratios also showed some similarities between different sites. Site 1 and 2 were similar, sites 3 and 6 were similar while 4 and 5 had similar effect on isotopic of POM (Figure 15c).

13 The effect of anthropogenic activities on the δ CPOM was evident but only spring was 13 very different from the seasons (Figure 16a). Summer, autumn and winter δ CPOM values were clustered together showing that the effect of these seasons was not 15 different. The seasonal effect of anthropogenic on δ NPOM was also noticeable with only summer and autumn having similar effect (Figure 16b). Winter season showed that the effect of anthropogenic activities were different from other seasons on the MDS ordination map. The ordination map showed that variations between grouping of sites and seasons together for C/N ratios (Figure 16c). The values for each season regardless of the river were closer to each other and distinctly separate from other season.

15 15 Table 2: p values from Kruskal Wallis ANOVA of C/N ratios, δ NPOM and δ NPOM isotopic fingerprints of particulate organic matter in Mbashe River for seasons and river sections.

Variable Season River section Carbon 0.001* 0.95** Nitrogen 0.001* 0.001* C/N ratios 0.001* 0.001* *The values are significantly different and those with ** are not statistically different.

Table 3: Mann Whitney U- test values to compare different river sections along Mbashe River. Group W P-value Upstream: midstream 617.000 0.73** Upstream : downstream 401.000 0.001* Midstream : downstream 429.000 0.001* *Values are statistically different and those with ** are not statistically different.

13 Table 4: The W and p values from Mann-Whitney test for δ CPOM variations for different for seasons. Group W P- values Spring: summer 499.00 0.095** Spring: autumn 309.00 0.064** Spring: winter 152.500 0.131** Summer: autumn 373.500 0.38** Summer : winter 119.000 0.02* Autumn: winter 75.000 0.001* *Values are statistically significant and ** are not statistically different.

Table 5: The W and p values from Mann Whitney test for comparisons between 15 seasonal changes in δ NPOM. Group W P- values Spring: summer 396.000 0.001* Spring: autumn 318.000 0.087** Spring: winter 147.000 0.1** Summer: autumn 413.000 0.78** Summer : winter 110.000 0.001* Autumn: winter 60.000 0.003* *Values are statistically different and ** are not statistically different.

Table 6: The W and p values from Mann Whitney test for comparisons between seasons using C/N ratios of particulate organic matter from August 2012 to July 2013 for Mbashe River. Group W p- values Spring: summer 253.000 0.001* Spring: autumn 156.000 0.001* Spring: winter 192.000 0.58** Summer: autumn 264.000 0.001* Summer : winter 53.000 0.001* Autumn: winter 35.000 0.001* *Values are statistically different and ** are not statistically different

C/Nratio 10

Non-Outlier Max Non-Outlier Min 0 Upstream Midstream Downstream Median River section

Figure 2: C/N ratios for different Mbashe River sections.

C/N ratio C/N 10

Non-Outlier Max Non-Outlier Min 0 Spring Summer Autumn Winter Median Seasons

Figure 3: Seasonal C/N ratios variations for Mbashe River.

15 C/N ratio C/N

5 Non-Outlier Max Non-Outlier Min 0 Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Median Month

Figure 4: Monthly C/N ratio variations of POM for Mbashe River.

-5

-10  ‰  -15 POM C CARBON 13

 -20

-25

-30 Non-Outlier Max Non-Outlier Min -35 Aug Sep Oct Nov Dec Jan Feb Mar Apr May June July Median Month

13 Figure 5: Monthly δ CPOM (‰) variations for Mbashe River.

-5

 -10 ‰  -15 POM CARBON C -20 13  -25

-30 Non-Outlier Max Non-Outlier Min -35 Upstream Midstream Downstream Median River section

13 Figure 6: The δ CPOM isotopic variations for the three Mbashe River sections.

-5

 -10 ‰  -15 POM CARBON C -20 13  -25

-30 Non-Outlier Max Non-Outlier Min -35 Spring Summer Autumn Winter Median Seasons

13 Figure 7: Seasonal variation of δ CPOM along Mbashe River.

 8 ‰  POM

N 6 NITROGEN   4

2 Non-Outlier Max Non-Outlier Min 0 Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Median Month

15 Figure 8: Monthly δ NPOM (‰) during the twelve months sampling for Mbashe River.

8  ‰

 6 POM N NITROGEN

 4 

2 Non-Outlier Max Non-Outlier Min 0 Spring Summer Autumn Winter Median Seasons

15 Figure 9: Seasonal δ NPOM variations along Mbashe River.

8  ‰  6 POM N NITROGEN   4

2 Non-Outlier Max Non-Outlier Min 0 1 2 3 4 5 6 Median Sites

15 Figure 10: Spatial variation of δ NPOM among sites along Mbashe River (1 &2 – upstream, 3 & 4 –midstream and 5 & 6 –downstream).

 6 ‰  5 POM

N 4 NITROGEN 15  3

1 Non-Outlier Max Non-Outlier Min 0 Upstream Midstream Downstream Median River section

Figure 11: The δ15N values of POM for river sections along Mbashe River.

14 2 r 0.16 12

 8 ‰  POM

N 6 NITROGEN   4

Regression 0 0 6 12 18 24 30 36 95% confid. C/N ratio

15 Figure 12: Correlation analyses between C/N and δ NPOMfor Mbashe River.

-6 2 r 0.06 -10

-14  -18 ‰  POM

C -22 CARBON 13  -26

-30

Regression -34 4 10 16 22 28 34 95% confid. C/N ratio

Figure 13: The regression between δ15Cand C/N ratios of river POM for Mbashe River.

-8 2 r  -12

-16  ‰  -20 POM C CARBON 13  -24

-28

-32 Regression 0 2 4 6 8 10 12 95% confid. NITROGEN  NPOM‰

Figure 14: Correlation analyses between δ13C and δ15N of Mbashe River POM.

Figure 15: MDS ordination map for Mbashe River – testing the effect of sites (UP – upstream, MD – midstream, DN – downstream, ST - experimental site on carbon (a), nitrogen (b) and C/N ratio (C).

Figure 16: MDS ordination maps for Mbashe River generated using (a) carbon (b) nitrogen (c) C/N ratio data collected in different seasons (U – upstream, M- midstream,

D- downstream, S – experimental site, SE – season, digits represents either sites or the different seasons).

CHAPTER 5

5.1 DISCUSSION Our research showed that isotopic composition of POM varied both spatially and temporally. The spatio- temporal differences in δ13C, δ15N and C/N ratio isotopic values of POM could be due to the effects of different anthropogenic activities (Wu and Chen, 2013). The results showed that different anthropogenic activities had different effects on the isotopic values of riverine POM. Spatial differences (river sections) in land uses and anthropogenic activities have significant effect on pollution in different river sections of the river (Shen, et al., 2011). Anthropogenic activities in the Mbashe River catchment affected isotopic values of the rivers from the source to the river mouth as indicated by distinct isotopic values for different activities and this was comparable to earlier findings by other scientists (Fan, et al., 2006).

The research findings showed noticeable differences in C/N ratios throughout the year along river. Isotopic variation could be attributed to temporal variations and distribution in activities (Balakrishna and Probst, 2005). Monthly C/N variations observed could be attributed to different anthropogenic activities in the river catchment at different time of the year. This resulted into distinct C/N seasonal variations. Temporal C/N ratios variation with high values during dry spring and lower values in winter (Figure 3) and this could be caused by seasonal nature of anthropogenic activities such crop. The C/N ratios >15 in spring season may be related to fresh terrestrial plant detritus (Ogrinc, et al., 2008) derived from litter fall of deciduous plants along the river. Relatively high δ13C values and low C/N ratios (7.7±1.6) also indicate contribution from anthropogenic sources such as agriculture, road construction and human settlements (Kuriah, 2008). High C/N ratios in spring season could also be attributed to veld fires which usually results from farmers burning the fields. Usually the veld fires burn large tracts of land in the river catchment removing organic material.

C/N ratios of POM varied widely in the researched area and this could signify the spatial distribution of anthropogenic activities in the catchment area (Michner and Lajtha 2007). Spatial variations in isotopic fingerprints could be indicative of changes in human activities throughout the year (Jennerjahn, et al., 2008). Observed C/N ratio trends at Tsholora could be attributed to irrigation, concentrated human settlements and extensive sand mining. The settlements have no proper sanitation in terms of toilets.

Spring season was the least affected by anthropogenic activities as C/N ratio were around 15 adding fresh plant material. The C/N ratios for the summer, autumn and winter ranged 8.1-14 and this implied possible effect of anthropogenic activities on POM in the river catchment that it was mainly derived from soil (Balakrishna and Probst, 2005). C/N ratios between 8- 9 for the summer season could be attributed to anthropogenic activities in the catchment. Summer crop cultivation and application of organic and inorganic fertiliser could have contributed to the variability. Other than crop cultivation, road cultivation could be responsible for the observed isotopic trends include road construction. Winter season had fewer activities such irrigation and riparian grazing. C/N ratios >7 were recorded throughout the year except for May to July with C/N ratios less than 7, thus reflecting anthropogenic activities (Wu, et al., 2007).

Autumn season had ratios that were less than summer but higher than winter suggesting that anthropogenic activities effects were more evident in summer and autumn than in winter (Balakrishna and Probst, 2005). Low C/N ratios could be indicating the effect of autochthonous production than allochthonous. This implies that winter season was far less impacted by anthropogenic activities

C/N ratios increased from the river source to mouth which could indicate the differences in the effect of different anthropogenic activities on the isotopic composition of POM. Activities such irrigation of fields downstream and human settlements resulted in higher C/N ratios while damming and riparian grazing resulted in lower ratios (Gupta, et al., 1997; Akamatsu, et al., 2011). Deforestation could have removed the riparian

54 vegetation which acts as a buffer to the effects of anthropogenic activities. The effect of deforestation is worsened by overgrazing and riparian grazing along the river catchments.

Like many other rivers, the main source of POM could be derived from soil as evidenced by ration ranging from 8-12. Relatively low C/N ratio values (8-12) indicated the possible effect of anthropogenic inputs (Wu2, et al., 2007). In summer the ratios were relatively lower than other seasons and this could be attributed to activities such as crop cultivation during this time of the year (Harmelin- Vivien, et al., 2010).

The δ13C did not vary from site to site and these conformed results from previous studies (Jennerjahn, et al., 2008). Even when the sites where grouped into river 13 sections, no significant isotopic variations were observed. The δ CPOMshowed temporal wide variation and this could signify that POM was derived from different sources along Mbashe River drainage basin (Cravotta, 1997). Spring season had the most depleted 13 δ CPOM values whilst summer and autumn had slightly enriched values. Depletion could be to manure application in preparation of the cropping in summer.

15 Consistent nitrogen (δ NPOM) depletion and enrichment indicated that different anthropogenic activities had significant effects on the isotopic dynamics of river POM 15 (Hou, et al., 2013). The upstream had the most depleted δ NPOM values of around 4.5‰ which indicated that POM was mainly from allochthonous sources (Perdue and Koprivnjak, 2007; Hou, et al., 2013). The allochthonous contribution could be mainly from anthropogenic activities in the river basin. Damming upstream and road 15 construction had the least effect on the δ NPOMwhilst agro-based activities increased 15 15 δ NPOM. Differences in δ NPOM along the river course could be due to variable anthropogenic activities in the catchment (Cifuentes, et al., 1988). Burning of the fields and veld could have contributed to the observed trend. Similar to previous studies, 15 irrigation affected the isotopic values significantly, which was evident by higher δ NPOM 15 downstream (Monteagudo, et al., 2012). High δ NPOM downstream could be

55 alternatively reflect the presence of higher population densities and settlements along river and this conformed to previous studies (Ogawa, et al., 2001).

The spring season had enriched nitrogen values as indicated by ratios ranging from 11- 14 and this could be attributed to organic manure applied in the fields in preparation of 15 the planting season (Spencer, et al., 2012). The δ NPOM enrichment in spring season could be an indicator of lesser effect of anthropogenic activities (Hou, et al., 2013).

15 Both upstream and the midstream sections were had depleted δ NPOM isotopic values than the downstream section of the river suggesting that the upstream was affected more by anthropogenic activities than the downstream (Deegan, et al., 2011). Depletion suggests that POM was mainly of allochthonous origin (Perdue and 15 Koprivnjak, 2007). The δ NPOM depletion in the upstream may signify nitrogen loading from agriculture while the δ15N enrichment in the lower courses could be reflective of autochthonous production by phytoplankton (Hou, et al., 2013). This suggests that the upstream section of the river is affected more by anthropogenic activities (crop cultivation, riparian farming) than downstream (Martineau, et al., 2004).

13 The research findings revealed that δ CPOM varied temporally with spring season having 13 depleted isotopic values and summer with enriched δ CPOM. Depletion and enrichment could be due to the effect of organic and inorganic manure application in the fields to enhance crop production (Ran, et al., 2013). Similarly to other earlier studies indicated 13 13 δ CPOM varied significantly temporally (Bouillon, et al., 2012). Theδ CPOM isotopic composition in summer could be due to erosion of topsoil derived from terrestrial sources (Bouillon, et al., 2012). Summer season was characterized by land tillage and road construction which exposed the soil to erosion during study period.Spatial differences (river sections) in land uses and anthropogenic activities have significant effect on pollution in different river sections of the river (Shen, et al., 2011).

High δ13C and δ15N values downstream could be linked to organic manure-use in surrounding areas and intermediate values in the midstream are attributed to fertilizer usage in areas within irrigation schemes (Cravotta, 1997). This suggests that high δ13C values found in the river were derivatives of organic manure as the river flows through agricultural areas (Dolenec, et al., 2011). Significant differences in δ13C (Figure 4) between seasons indicated that POM was derived from different sources (Prasad and Ramanathan, 2009; Townsend-Small, et al., 2009).

The δ13C values upstream and midstream have an isotopic fingerprint that corresponds to those of subsistence agriculture (around -18‰), which could have been affected by 15 the use organic manure in the fields (Cravotta, 1997). Higher δ NPOM values of POM detected in August could be attributed to pollution which unfortunately could not be identified, and this could be derived from point source (Dolenec, et al., 2011). The effect of anthropogenic activities on POM isotopic composition also extended to the months of September and October.

15 Our research revealed that δ NPOM enrichment of POM increased as we moved downstream with much depleted nitrogen values upstream and enriched values near the river mouth. Elevated δ15N downstream near Cwebe could indicate that the dominant source of POM was animal waste in the game reserve and settlements that are in close proximity to sampling area (Kendall, et al., 2001). Stream-bank cultivation and riparian grazing upstream could be attributed to low nitrogen values. Downstream isotopic values could have been influenced by the fact that the Cwebe Reserve is a protected area and anthropogenic activities are monitored and controlled. The wider isotopic ranges and higher δ15N downstream may also be linked to organic fertilizers animal waste which conformed to previous studies (Michner and Lajtha, 2007). Spatial variation of δ15N isotopes of river POM could be attributed to different activities in the watershed occurring at different time of the year (Michner and Lajtha, 2007; Hou, et al., 2013). Site 2 indicated very impacted δ15N isotopic values and this could be as a

57 result of a number of activities such as damming, subsistence crop cultivation and riparian grazing.

13 The δ CPOM isotopic signatures on the MDS maps showed completely different values implying that POM was derived from different anthropogenic activities. The variation was evident by wider Eucledian distances on MDS map of different. The δ15N isotopic 13 dynamics produced a different trend from that of δ CPOM and this could be due to specific activities in the river catchment (Hou et al., 2013). Some anthropogenic activities produced similar δ15N signatures but also different from the others as indicated distinct groups.

The variables (seasonality, sites, month and river section) showed low stress values on the MDS maps of (0 – 0.01) indicating that the different activities had significant effect on the isotopic composition of POM (Jennerjahn, et al., 2008). The research showed positive correlations (Figure 12) which indicated direct relation between C/N and δ15N. The δ15N increased as C/N ratios increased and δ13C and C/N (Figure 13) whilst negative correlation between δ13C and δ15N were revealed (Figure 14). The findings of this study conformed to other recent studies that have shown strong positive correlation between POM variables (N and C/N, C and C/N) with catchment area, population and fertiliser usage (Kuriah, 2008).

Relatively high δ13C values and low C/N ratios (7.7± 1.6) in POM of Mbashe River water could indicate contribution from anthropogenic sources as shown by earlier studies (Wu, et al., 2007). The organic matter in Mbashe River could be derived from soil because the C/N ratios ranged from 8.1–14 while the intermediate C/N ratios (4 ‰ – 8‰) could be an indicator of mixing of phytoplankton and soil organic matter (Balakrishna and Probst, 2005). This indicates that temporally all the anthropogenic activities had different isotopic influence between seasons. MDS maps

5.2 CONCLUSION The research was the first documented study on isotopic composition along Mbashe 13 15 River which revealed that C/N ratios, Carbon (δ CPOM) and Nitrogen (δ NPOM) varied significantly temporally and spatially. The variations showed that isotopic analysis is of great importance in monitoring the effects of anthropogenic activities on river processes. Temporal variations in C/N ratios indicate that the anthropogenic activities had more effect in summer than other seasons. Very unique and distinctive isotopic 13 values of δ CPOM for different seasons showed that particulate organic matter was derived from different sources during different seasons than between sites.

15 Significant variations in δ NPOM indicated that anthropogenic activities, such as irrigation and human settlements had significant effect on isotopic dynamics of river particulate organic matter. Anthropogenic activities produced significant spatial 15 variations of δ NPOM that varied with part of the river in the catchment. The POM isotopic signatures indicated that anthropogenic activities had significant effect to the 15 stable isotope dynamics in river POM. The study revealed significant δ NPOM temporal variations, which suggests that anthropogenic activities had noteworthy effect on aquatic activities.

15 Upstream was depleted in (δ NPOM) and this meant that anthropogenic activities had 15 significant effect on isotopic composition of POM. Enrichment of δ NPOM downstream could signify the prevalence of phytoplankton and irrigation at Tsholora. Nitrogen depletion shows the effects of agriculture upstream in the river while nitrogen enrichment indicates the effects of phytoplankton downstream at the estuary. This study also revealed that some different activities have similar effect on the isotopic composition of POM both spatially and temporally with others having distinctly different from others.

5.3 Recommendations

There is potential for similar studies along Mbashe River to reveal more interesting processes. The study could also look at the aquatic ecosystem as affected by allochthonous POM in the river drainage basin. Environmental managers in Mbashe River catchment and other areas can benefit from the findings in their efforts to mitigate the effects of anthropogenic activities on the terrestrial and aquatic ecosystems. Tracer studies are recommended as in some cases the sources of nitrogen cannot be accounted for. In the same vein, local governments should fund a continuous research over a period of years to effectively assess the impact of these anthropogenic activities and come up with further recommendations to control activities that negatively affect the environment. This in turn has a direct or indirect effect on the quality of life, the climatic conditions and ecological balance. The extent to which human activities affect aquatic habitats need to be correctly studied and assessed, will be a source of sound and meaningful information to be used for ecological management.

5.4 Outcomes 1. Part of the research was presented at the 6th WSU International conference in March 2014 (submitted for publication) - C/N ratios. 2. Another part of the research was presented at Climate change summit in Alfred Nzo 13 District (Matatiele) in April 2014 (δ CPOM). 15 3. The last part of the research (δ NPOM) has been presented at the Southern African Marine Science Symposium (SAMSS) 15-18 July 2014, Stellenbosch.

4 Part of the research work was presented at SAEON Graduate Student Network Indibano 2014 Conference (Port Elizabeth 05 Sept- 08 Sept 2014).

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APPENDICES

Appendix A: Riparian grazing at Isixhotyweni near Clarkeburry with clear water flowing in the river.

Appendix B: Burning of the grazing area at Lota.

Appendix C: Subsistence crop cultivation at Msana.

Appendix D: Dam at Msana location.

Appendix E: Temporary Construction Company at N2 Bridge

Appendix F: Road construction between N2 and Ludondolo.

Appendix G: Bridge construction at Mvezo with very turbid water.

Appendix H: Quarry mining near Mthendu.

Appendix I: Sand mining at Tsholora/Noshange.

Appendix K: Quarry mining and subsistence farming at

Appendix J: Irrigation near Tsholora Bridge.

Appendix K: Nature reserve and protected area at Cwebe.

Appendix L application letter to conduct research

Walter Sisulu University Faculty of Science, Engineering & Technology Department of Zoology Private Bag X1 Mthatha 5117

March 2012 To whom it may concern The Ecologist Cwebe Marine Sanctuary East London

Dear Sir/Madam RE: Permission to undertake research

I do hereby apply for permission to undertake research study on the “Impacts of anthropogenic activities on the stable isotope dynamics of particulate organic matter, in Mbashe River, Eastern Cape’’. The research will involve collection of water samples from the river and the Cwebe Estuary for a period of twelve months which will be later be tested in Walter Sisulu University and University of Cape Town. I hope my application will meet your favorable response.

Yours faithfully

Munetsi Zvavahera

Appendix M: consent form