STABLE ISOTOPES DYNAMICS OF MACROPHYTES ALONG

UMTATA RIVER IN THE EASTERN CAPE OF SOUTH AFRICA

by

MZAMO SANELE CALEB

A dissertation submitted in fulfillment of the requirements for the degree of

MASTER OF SCIENCE (MSc) (Zoology)

at

WALTER SISULU UNIVERSITY

SUPERVISOR: DR KURIAH, F.K.

SEPTEMBER 2013

i

Abstract

The decline of freshwater ecosystems, generally result from land use activities in

the river catchment and is of great concern worldwide. This study was conducted

along Umtata River in the Eastern Cape province of South Africa between May

2010 and April 2011. The study was aimed at identifying macrophytes families

(to species level) and determining the stable isotope signatures (C:N ratios, δ13C

and δ15N) and to relate their isotopic signatures to the land use activities along

the river catchment. Analysis of variance was performed to test the effect of sites

and sampling period on the C:N ratios, δ13C and δ15N signatures. There were 16

macrophyte families represented by 26 species recorded along the river. There

was only a significant difference in sites and sampling period in δ15N (p< 0.05).

The highest C:N ratios value (30.75±9.65‰) was recorded in the upper reaches

while the lowest value (6.10±2.35‰) occurred in the lower reaches. The δ13C

values varied throughout the length of the river with highest values (-

19.63±5.44‰) obtained in the middle reaches. Spatial variation was evident in

δ15N throughout the length of the river and showed increase from the upper

reaches to middle reaches and decreased towards lower reaches. The δ15N

ranged from 3.92±2.43‰ in the upper reaches to 10.02±4.56‰ in the middle

reaches. Temporal variation in δ15N was also evident throughout the sampling

period with highest peak in May (9.77± 4.49‰) and lowest in February

(0.50±2.49‰) respectively. The highest values of isotope signatures at spatial

ii

level demonstrated the true reflection of urban development, sewage discharge

and agricultural activities taking place along the river system.

Continued monitoring is recommended that may ultimately come up with a

better management options for the communities living within the study area, and

also to better enhanced land utilization.

Key words: Macrophytes, anthropogenic activities, carbon:nitrogen ratios,

stable isotopes of carbon, nitrogen and Umtata River.

iii

Declaration

I ‘Mzamo Sanele Caleb’ Student Number 205623204, hereby declare that the

information contained in this thesis is my original work.

Signature………………………………………………………………

Date………………………………………………………………………

Supervisor: Dr F.K. Kuriah

Signature:

Date………………………………………………………………………

iv

Declaration on Plagiarism

I “Mzamo Sanele Caleb” is aware that plagiarism is regarded as a theft in

a whole or piece of information recklessly taken from the source without

acknowledging the original author of the information or the whole paper is

reproduced and presented as someone’s original work.

All the authors cited in this thesis are acknowledged and confirmed to

avoid plagiarism as defined by WSU.

I have followed the citation and referencing style stipulated by Walter

Sisulu University.

This submitted thesis is my original work.

Any unauthorized use of this document is prohibited, no duplication of this

document for academic or any other practice is allowed.

This document was submitted for academic purposes (Master of Science

in Zoology).

__________________ __________________

Signature Date

v

Acknowledgements

I would particularly thank Dr Kuriah, F.K for his commitment and

dedication in assisting and guiding me during the course of my study.

I acknowledge the help of Mr. Nasila offered in statistical data analysis.

I would also like to thank the department of Zoology for giving me an

opportunity to do my masters degree.

I cannot forget my colleagues (Zoology masters students) and my friends

for their support during my study.

Finally, I would like to thank my family for their unwavering support they

have given me throughout my time at the university.

This project was funded by National Research Foundation (NRF) to whom

I am grateful.

vi

Dedication

I’d like to dedicate the work that I have put into this thesis to my parents (Mrs.

Mzamo F.N.N and Mr. Mzamo G.M) and my sister (Ms. Mzamo Vuyelwa Mavuyi),

for their dedication and encouragement during my study period at Walter Sisulu

University.

Mzamo Sanele Caleb

Lusikisiki

Republic of South Africa

JUNE 2013.

vii

TABLE OF CONTENTS

Chapter 1

Introduction 1

1.1 Study background 1

1.2 Problem statement 2

1.3 Agricultural practices and urban development as sources of nutrients to

aquatic ecosystems 3

1.4 Stable isotopes 3

1.5 Rationale of the study 5

Chapter 2

Literature review 8

2 Macrophytes 8

2.1 The C:N ratios 10

2.2 The δ13C 12

2.3 The δ15N 14

2.4 The use of δ15N in studying aquatic ecosystem functioning 15

2.5 Justification of the study 15

2.6 Aims and objectives 16

2.7 The research hypothesis 17

viii

Chapter 3

Materials and methods 18

3.1 Description of the study area 18

3.2 Selection of sampling sites 19

3.3 Macrophytes 20

3.3 Isotopic analysis 23

3.5 Statistical data analysis 24

Chapter 4

Results 25

4.1 The macrophyte species composition of Umtata River 25

4.2 The C:N ratios at family level 26

4.3 The δ13C values at family level 28

4.4 The δ15N at family level 28

4.5 The effect of sampling sites on C:N ratios of macrophytes 31

4.6 The effect of sampling period on C:N ratios of macrophytes collected

along Umtata River 32

4.7 The effect of sampling sites on δ13C signatures of macrophytes 34

4.8 The effect of sampling period on δ13C signatures 36

4.9 The effect of sites on δ15N signatures of macrophytes 38

4.10 The effect of sampling season on δ15N signatures of macrophytes 40

ix

Chapter 5

Discussion 43

Conclusion 52

References 53

Appendix 64

x

LIST OF TABLES

Table 1: Showing sampling locations, coordinates and activities along the

sampling sites 20

Table 2: Showing the number of macrophytes families and species from

the sites along Umtata River 22

Table 3: Showing the stable isotope composition of macrophytes families

from the sites along Umtata River 28

Table 4: Showing the effect of sampling sites and seasons on the C:N

ratios, δ13C and δ15N signatures of macrophytes along Umtata

River 29

Table 5: Showing the mean spatial values and standard deviations of

C:N ratios of macrophytes recorded along Umtata River 30

Table 6: Showing the mean seasonal values and standard deviations of C:N

ratios of macrophytes recorded along Umtata River 32

Table 7: Showing the mean spatial values and standard deviations of δ13C

of macrophytes recorded along Umtata River 34

Table 8: Showing the mean seasonal values and standard deviations of

δ13C of macrophytes recorded along Umtata River 36

Table 9: Showing the mean spatial values and standard deviations of

δ15N signatures of macrophytes recorded along Umtata River 38

xi

Table 10: Showing the seasonal mean values and standard deviations of δ15N

signatures of macrophytes recorded along Umtata River 40

Table 11: The list of all macrophyte families and species collected

along Umtata River 63

xii

LIST OF FIGURES

Figure1: Map showing study area and sampling sites (Source: Department

of Water Affairs, King Sabata Dalindyebo Municipality) 19

Figure 2: Graph showing mean spatial C:N values of macrophytes recorded

along Umtata River 31

Figure 3: Graph showing mean seasonal C:N values of macrophytes recorded

along Umtata River 33

Figure 4: Graph showing mean spatial δ13C values of macrophytes recorded

along Umtata River 35

Figure 5: Graph showing mean seasonal δ13C values of macrophytes recorded

along Umtata River 37

Figure 6: Graph showing mean spatial δ15N values of macrophytes recorded

along Umtata River 39

Figure 7: Graph showing mean spatial δ15N values of macrophytes recorded

along Umtata River 42

xiii

List of abbreviations

Carbon: Nitrogen ratio (C:N ratio).

Carbon stable isotope (δ13C).

Nitrogen stable isotope (δ15N).

Stable isotope signatures (SIS).

C3 plants: plants that use ribulose-1.5biphosphate carboxylase/ oxgenase

to fix carbon.

C4 plants: plants that use phosphoenol pyruvate carboxylase to fix CO2..

CAM plants: plants that use Crassulacean Acid Metabolism to fix carbon.

1

CHAPTER 1

Introduction

1.1 Study background

This study was aimed at investigating the effect of different land uses occurring

from the catchment of the Umtata River system. The Umtata River is under

anthropogenic pressure due to different land uses which include agricultural and

urban development (Table 1).

Rivers and their runoff, including groundwater, both of which are impacted by

anthropogenic activities provide freshwater to estuarine systems. Most rivers

receive nitrogen-enriched runoff loaded with agricultural fertilizers, animal and

human waste from many diverse sources (Tappin, 2002). Various forms of land

uses influence runoff quality and quantity during and following rainfall. The

middle reaches of the Umtata River are severely affected by urban development.

This part of the river receives waste water from waste treatment plants and

leaking drainage pipes. This waste water is rich in nitrogen and phosphorus both

of which are required in large quantities by living organisms. This has led to the

growth of water hyacinth and deterioration of water quality in this area and the

river downstream.

The Umtata River system is mainly used for agricultural, domestic and

recreational purposes which include drinking, swimming and laundry in the lower

reaches. The poor water quality from middle to lower reaches becomes a limiting

2

factor to the importance of this river to human population along this section. This

raises a concern to carry out extensive research to provide suggestions for water

quality improvement of the river.

1.2 Problem statement

Land use within river catchments are widely known to be major donors of

nutrients to aquatic environments where they cause eutrophication (Kohzu, et

al., 2008; Nyenje, et al., 2010). Eutrophication is a complex process in which

fresh and marine waters become enriched by nitrogen, phosphorus etc from both

internal and external sources (Xu, et al., 2007; Nyenje, et al., 2010). It is one of

the most prevalent global problems (Nyenje, et al., 2010; Wozniak, et al., 2012),

and is the most pressing environmental problem in both developing and

developed countries (Xu, et al., 2007; Kohzu, et al 2008). It alters ecological

integrity, cause fish extinction and toxic algal blooms (Xu, et al., 2007). The

major external sources of nutrients to rivers are agricultural practices (Forsberg,

1993; Lake, et al., 2001; Martinelli, et al., 2002; Birgand, et al., 2007; Kohzu, et

al., 2008) and urban development (Kohzu, et al., 2008; Miller, et al., 2010;

Kendall, et al., 2010; Nyenje, et al., 2010). The Umtata River experiences both

industrial and agricultural nutrient inputs. As a result the river has been found to

accumulate alarming levels of heavier metals (Fatoki, et al., 2001).

3

1.3 Agricultural practices and urban development as sources of

nutrients to aquatic ecosystems

In areas with extensive agricultural practices, aerosols from biomass burning are

a cause of change in chemical composition of the atmosphere, altering rain water

leading to changes in aquatic systems (Martinelli, et al., 2002). Agriculture has

been found to be the only single largest nutrient source to aquatic systems

(Birgand, et al., 2007). The nutrients from agricultural activities include nitrogen

(N), phosphorus (P) and carbon (C) (Nyenje, et al., 2010). Sewage and industrial

waste in both developed and developing countries add nutrients in elevated

quantities to river systems (Kendall, et al., 2010; Miller, et al., 2010; Nyenje, et

al., 2010). Human waste contains both N and P in the form of ammonium,

ammonia, nitrates, urea and phosphates respectively (Nyenje, et al., 2010).

These inputs make nutrients available to macrophytes (Miller, et al., 2010), and

become limiting factors for primary producers (Forsberg, 1993; Flair and

Heikoop, 2006; Birgand, et al., 2007).

1.4 Stable isotopes

Isotopes are elements that have same numbers of protons but different numbers

of neutrons (Ehleringer, et al., 1993; Madder, 1996). They are classified into two

categories based on the physical chemistry properties or reactive characteristics

namely stable isotopes and radio-active isotopes (Ehleringer, et al., 1993;

Madder, 1996; Southwood, et al., 2000). Stable isotopes are those that do not

4

decay over time, while radio-active isotopes are subjected to decay and have

shorter life span (Southwood, et al., 2000). Stable isotope composition is

expressed in terms of the delta (δ) notation which are parts per thousands (‰)

or “mils” (δ=relative, ∆= absolute) (Ehleringer, et al., 1993). Stable isotopes of

biological interest include carbon, hydrogen, nitrogen, oxygen, and sulfur and

exist in two or more forms e.g. carbon isotopes include δ12C, δ13C and δ14C &

nitrogen isotopes include δ14N and δ15N (Ehleringer, et al., 1993). The use of

stable isotopes in studying aquatic systems is complex and involves complex

processes (Otero, et al., 2000), and as a result the measurement of stable

isotopes in ecology has emerged as an approach that integrates ecophysiological

processes over time (Smedley, et al., 1991). For this study the δ13C, δ15N and

their C:N ratios have been chosen for a number of reasons which include:

Their ability to trace sources and sinks of nutrients to aquatic systems and

pinpointing areas that need immediate attention (Kendall, et al., 2010).

Their ability to differentiate between autochthonous (within a system) and

allochthonous (from outside system) nutrient inputs (Otero, et al., 2000).

Their ability to mark plants of different photosynthetic pathways

(Ehleringer, et al., 1997).

Their ability to indicate the effect of environmental change and

contamination in aquatic ecosystem functioning (Ehleringer, et al., 1991;

Wozniak, et al., 2012).

5

Their ability to act as useful tracers of changes in nutrients loads to

aquatic ecosystems, and to detect spatial and seasonal variations in C:N,

δ13C and δ15N signatures of macrophytes (Smedley, et al., 1991; Baeta,

et al., 2009).

They serve as powerful tools to study biogeochemical cycles (Xu, et al.,

2007).

1.5 Rationale of the study

Human activities such as urban sewage, industrial waste disposal and runoff

from agricultural fields negatively affect water bodies (Kohzu, et al., 2008;

Chang, et al., 2009; Kendall, et al., 2010; Miller, et al., 2010; Nyenje, et al.,

2010). These activities are associated with high nitrogen values while agricultural

activities are associated with both high carbon and low nitrogen loads to water

bodies (Flair & Heikoop, 2006; Chang, et al., 2009; Kohzu et al., 2008; Miller, et

al., 2010). Enriched carbon and nitrogen nutrient loads cause algal blooms,

faunal extinction and loss of socio-economic importance of aquatic systems (Flair

and Heikoop, 2006; Chang, et al., 2009; Miller, et al., 2010; Nyenje, et al.,

2010).

The effect of human activities in aquatic environments is mainly caused by

transformation of lateral flows of water, air, soil, or organisms from the

catchment, and they exact pressure in offsite areas (Van Noordwirjk, et al.,

6

2004). Pollution problems which are caused by sewage disposal and agricultural

activities have caused a detrimental change in sub-tropical wetlands (Chang, et

al., 2009; Nyenje, et al., 2010). Studies conducted in the 21st century on the

effect of land use have shown interdependency on the physical and biological

concepts of aquatic systems and terrestrial landscapes (Cole, et al., 2006; Kohzu,

et al., 2008; Van Noordwijk, et al., 2004). Furthermore, it has been noted that

species composition and richness change in response to different land use

activities in the river and the drainage system (Maltais-Landry, et al., 2009).

The studying of spatial and temporal patterns of nutrient loads is important as it

can identify areas or locations that are severely affected by anthropogenic

activities (Kendall, et al., 2010). Samples taken in one sampling station are

representative of what is happening around and above the catchment area,

because nutrients are mainly transported by ground water, wind, and runoff and

by the river itself (Cole, et al., 2006; Kendall, et al., 2010).

The human population growth, urban development and human settlements

closer to river systems cause disturbances and water quality deterioration (Xu, et

al., 2007; Kohzu, et al 2008; Kendall, et al., 2010). The occupation of river flood

plains is associated with rapid economic growth and organic pollution (Miller, et

al., 2010). Elevated nutrient loads to aquatic ecosystems stimulate production of

phytopkton and macrophytes, leading to loss of other organisms and aesthetical

7

value of the surrounding environment (Flair and Heikoop, 2006; Miller, et al.,

2010; Nyenje, et al., 2010). To estimate the amount of nutrient loads, it is vital

to examine the linkage between land uses and land derived nutrients loads so as

to identify point sources and their contributions (Kendall, et al., 2010).

8

Chapter 2

Literature review

2.0 Macrophytes

In studying aquatic systems, it is important to look at those factors that influence

energy sources at the base of the food web (Estevez & Suzuki, 2010). These

factors include the importance of macrophytes, seasonality, anthropogenic

nutrient loading (Kendall, et al., 2010). Macrophytes have wide but restricted

distribution in river networks (Michener & Lajtha, 2007), and this spatial

distribution is the indication of species dispersal and sensitivity function.

Reduction and deterioration of macrophytes result from enhanced anthropogenic

nitrogen (Chang, et al., 2009; Kohzu, et al., 2008) and phosphorus nutrient

loading (Estevez and Suzuki, 2010; Cohen and Bradman, 2010).

The difference in nutrient loads among sites, forces macrophytes to be isotopically

distinct, variable and less predictable than terrestrial plants (Michener & Lajtha,

2007). This is due to the large variation in the amount and stable isotope ratios of

dissolved inorganic nutrients in the water column (Kendall, et al., 2010). The plant

isotopic composition is influenced largely by environmental processes (temperature

and humidity), making their isotopic composition fluctuate with environmental

changes (Smedley, et al., 1991). For an example, the seasonal increase in

temperature and evaporative demands leads to a decline in soil moisture which in

turn lead to nutrient concentration and availability to plants (Smedley, et al., 1991;

9

Nordt, et al., 2002). The other factors that influence the isotopic composition of

macrophytes include stream geomorphology, water current and plant form and part

being analyzed (Kendall, et al., 2010). The isotopic signatures of plants reflect the

available elemental sources in the atmosphere, soil, aquatic and the whole host of

environmental conditions (Kendall, et al., 2010). Consequently, submerged

macrophytes differ in the sensitivity of their isotopic signatures to environmental

change (Chang, et al., 2009). Research in isotopic composition and variation on

aquatic ecosystems over spatial and temporal scales provides information on

natural ecosystem function and the effects of land use (Kohzu, et al., 2008; Chang,

et al., 2009; Kendall, et al., 2010). As a result, macrophytes growing in areas with

elevated levels of nitrogen and phosphorus are expected to be distinctively higher

in N & P levels compared to those in lower levels (Kohzu, et al., 2008; Cohen and

Bradman, 2010).

Macrophytes provide habitat and food for different species of invertebrates and

fish (Esteves & Suzuki, 2010). The type of macrophytes and their nutritive quality

is important in fishery industry, as the contribution of different macrophytes

groups to fauna production variation depends on their relative importance as

primary producers (Forsberg, et al., 1993).

Plants exhibit different life forms which include herbaceous macrophytes, trees,

periphyton and phytoplankton (Ehleringer, et al., 1991; Forsberg, et al., 1993).

10

They also exhibit different modes of photosynthesis and they are grouped into

C3, C4 and CAM plants (Ehleringer, et al., 1991; Smedley, et al., 1991; Forsberg,

et al., 1993). The C4 plants represent 15% of all species found on the planet and

are only found in Angiosperms (Smedley, et al., 1991). They are common in hot,

low CO2 areas and in open environments (Ehleringer, et al., 1991). C4 plants use

phosphoenol pyruvate carboxylase to fix CO2 (Ehleringer, et al., 1991; Smedley,

et al., 1991). The second group (C3 plants), accounts for 75% of the planet's

plant species. They are found in cold to warm areas with high CO2 levels

(Ehleringer, et al., 1991; Smedley, et al., 1991; Forsberg, et al., 1993). The C3

Plants use Ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco)

(Ehleringer, et al., 1991; Smedley, et al., 1991). The last group (CAM plants) is

mostly found in hot to dry areas and account for only 10% of the planet's flora

(Ehleringer, et al 1991).

2.1 The C:N ratios

The C:N ratios are the measure of the dry weight of total organic carbon to total

nitrogen of the sample analyzed (Durga, et al., 2011). The C:N ratios of

macrophytes are influenced by both natural factors and anthropogenic nutrient

loads (Durga, et al., 2003; Baeta, et al., 2009). The isotopic ratios of carbon and

nitrogen in freshwater plants act as indicators of ecosystem functioning and the

effect of land use change (Durga, et al., 2003; Cohen and Bradman, 2010). The

use of C:N ratios in ecological studies is based on the assumption that they

11

remain relatively constant as they are passed from producer to consumer

(Forsberg, et al., 1993), and that their suitability as bio-indicators depends on

the nature of nutrient supply (e.g. Inorganic C & N supply) (Baeta, et al., 2009).

It has been noted that seasonal nitrogen variations reflect the variation in C:N

ratios of submerged macrophytes (Herzschuh, et al., 2010). The change in C:N

ratios are also believed to be influenced by the selectivity in biochemical

processes of the organisms (Lake, et al., 2001). The C:N ratios in detritus based

systems are higher compared to macrophytes because of the decrease in carbon

as a result of the function of microbial organisms (Greenwood & Rosemond,

2007). it has been noted that in estuarine systems, C:N ratio signatures of

aquatic plants decline in relation to the higher proportion of phytoplankton rich

detritus in the sediment’s organic pool (Otero, et al., 2000). This is because soil

carbonate layers take on the δ13C composition of the material that was buried on

the site during carbon formation (Ehleringer, et al., 1991).

The isotopic composition of plants differ based on their bio-physiological

adaptations and environmental conditions (Zhao, et al., 2010), their biochemical

processes (Ehleringer, et al., 1991; Forsberg, et al., 1993), plant life forms and

life cycles (LaZerte & Szalados, 1982; Smedley, et al., 1991; Forsberg, et al.,

1993). The total organic carbon to total nitrogen (C:N ratio) of C3, C4 and CAM

plants differ considerably due to plant’s habitat requirements and bio-

12

physiological adaptations (Ehleringer, et al 1991; Durga, et al., 2003). The C:N

ratios of phytoplankton range from (5‰ to 6‰), planktonic detritus (6‰ to

9‰), and phytogenic material (4‰ to 10‰) (Durga, et al., 2003). Further, C3

plants (trees, shrubs and some grasses) and (macrophytes and grasses) range

from 15‰ to 20‰ and 20‰ to 39.4‰ respectively (Durga, et al., 2003;

Fellerhoff, et al., 2003; Wu, et al., 2007).

2.2 The δ13C

The δ13C signatures have been used extensively in tracing different carbon

sources and in studying food relations (Peterson, and Whitfield, 1997; Baeta, et

al., 2009) and quantifying the off-site effects of biomass burning (van Noordwijk,

et al., 2004). The plant biochemical processes and anthropogenic activities are

the major factors affecting the δ13C signatures of macrophytes (Chang, et al.,

2009; Cohen, et al., 2010). Therefore, the δ13C of macrophytes depend on the

tissue being analyzed and nature of land - use changes (Chang, et al., 2009).

The δ13C signatures are sensitive to land-use changes in the river catchment

because of the different amounts of nutrients added in the agricultural field close

to the river systems (Lake, et al., 2001). The δ13C signatures in urban areas are

lower compared to agricultural areas (Kendall, et al., 2010). These nutrient loads

cause spatial and temporal variation in δ13C signatures of macrophyte (Troxler

and Richards, 2009). The aquatic environments that receive large inflows of

13

allochthonous (nutrients derived from outside the river system) inputs, are

expected to have depleted δ13C signatures due to the abundance of trees in their

upper reaches (Otero, et al., 2000).

The physiological processes of C3 plants increase the ratio of carbon fixation

during growing season leading to the decline of δ13C of these plants (Smedley, et

al., 1991). The δ13C of plants also differ due to stomatal opening and because of

variation in the chlorophyll-a carbon demand (Otero, et al., 2000). The δ13C

values in C3 plants have known ranges of -32 to -20‰ in freshwater systems, -

36‰ to -21.5‰ in marine systems (Boutton, 1991), and -37‰ to -22‰ in

terrestrial environments (Zhao et al 2010). The global δ13C mean value of C3

plants is -27‰ (Otero, et al., 2000; Zhao, et al., 2010). The δ13C in C4 plants

have been found to range from -17 to -9‰ with a mean of -13‰ in aquatic

systems (Boutton, 1991) and -15 to -9‰ in terrestrial environments (Zhao, et

al., 2010). The known δ13C signatures of macro-algae are higher than those of

C3 and C4 as macro-algae assimilate detritus from both plant forms (Boutton,

1991). The δ13C in CAM plants have also been found to range from -28 to -10‰

but their values are variable as these plants can change from CAM to C3 plants

under stressful conditions (Boutton, 1991).

14

2.3 The δ15N

Nitrogen (N) has two stable isotopes, lighter 14N, which is the most abundant

element of all naturally occurring nitrogen, and heavier 15N (Ehleringer, et al.,

1993). The least abundant δ15N is often used in ecological studies to investigate

the effect of fertilizers, urbanization etc on aquatic and terrestrial ecosystems

(Lake, et al., 2001; Lepointa, et al., 2004; Kohzu, et al., 2008; Chang, et al.,

2009). Its behaviour, form, abundance and rate processes have been studied in

deeper details in aquatic systems (Wada and Hattori, 1991).

Nitrogen stable isotopes provide information on sources and sinks of nutrients

(Kohzu, et al., 2008; Kendall, et al., 2010). The atmosphere has a value of ~

0‰ of δ15N. Consequently any value above that has a different origin (Forsberg,

et al., 1993). The δ15N is known to increase with increase in land use activities

and the values of δ15N range from –3‰ to +3 ‰ in agricultural areas and

+10‰ to + 20‰ in human dominated areas with human waste (Forsberg, et

al., 1993). The δ15N in all plants is variable and depends on the nutrient supply

and environmental conditions (i.e. soil type) and is reported to range from 0‰

to 8‰ in undisturbed areas (Chang, et al., 2009). The δ15N of allochthonous

inputs to river is known to range from -5‰ to + 18‰ with an average value of

~ 3‰ (Wada and Hattori, 1991). This is in support of Cohen and Bradman

(2009) suggestions that, δ15N has high values in anthropogenic impacted areas

than those thought to be pristine. This difference is the result of the δ15N inputs

15

of nutrient sources, the rate of isotopic fractionation and the type of nitrogen

transformation (Wozniak, et al., 2012). As a result the analysis in macro-algae

and macrophytes has helped identify sources of anthropogenic nitrogen loads at

sites directly impaired by land use changes (Wozniak, et al., 2012).

2.4 The use of δ15N in studying aquatic ecosystem functioning

Nitrogen is a limiting factor in highly nitrogen enriched ecosystems (Wozniak, et

al., 2012). Nutrients from human induced activities can severely change aquatic

food webs and ecosystem functioning (Greenwood, et al., 2007; Kohzu et al.,

2008; Miller, et al., 2010; Nyenje, et al., 2010). The δ15N is the most preferred

nitrogen stable isotope by plants and has been found to be high in macrophytes

raised or naturally growing in waste water receiving sites of river systems

(Kohzu, et al., 2008; Cohen and Bradman, 2010). The δ15N from anthropogenic

activities is important in understanding nitrogen dynamics and aquatic

contamination (Wozniak, et al., 2012). It is widely used to trace nitrogen sources

which include precipitation, fertilizers, animal and human wastes (Cohen and

Bradhman, 2009) and to study lake paleo-productivity (Herzschuh et al., 2010).

16

2.5 Justification of the study

Rivers and coastal oceans are active sites for carbon cycling because of larger

autochthonous production relative to the open oceans and closed lakes (Kuzyk,

et al., 2010). Human activities such as agricultural and sewage discharge put

pressure on aquatic systems and cause algal blooms (Xu, et al., 2007; Kohzu et

al., 2008; Chang, et al., 2009; Nyenje, et al., 2010). The effects of land use in

the study area are important in view of the fact that Umtata River runs through

forested plantation in its upper reaches, is densely populated along Umtata town

and subsistence farming in its middle/ lower reaches (Fatoki, et al., 2001). These

activities are the major causes of eutrophication to this river.

2.6 Aims

To identify macrophytes species composition along Umtata River system.

To determine SIS of C:N ratios, δ13C and δ15N composition of

macrophytes along Umtata River.

To determine effect of selected sites and sampling period on the SIS of

C:N ratios, δ13C and δ15N of macrophytes along Umtata River.

To relate the C:N ratios, δ13C and δ15N composition of macrophytes to

land use activities occurring along Umtata River system.

17

2.7 The research hypothesis

Ho: The δ13C, δ15N and C:N ratio signatures of submerged macrophytes

are the same in all sites in all seasons.

Ha: The δ13C, δ15N and C:N ratio signatures of submerged macrophytes

are not the same in all sites in all seasons.

The overall hypothesis was to test whether the δ13C, δ15N and C:N ratio

signatures of submerged macrophytes reflect the land-uses occurring in

the Umtata River catchment.

18

CHAPTER 3

Materials and methods

3.1 Study area

The study was conducted along the Umtata River which originates in the plateau

region of the former Transkei region of the Eastern Cape Province, South Africa,

approximately midway between the Drakensberg escarpment and the Indian

Ocean. The catchment of the river is about 100km long and up to 50km wide

(Fatoki, et al., 2001). The terrain is generally undulating and in Mthatha town

vicinity it flows through a wider flat plain with a shallow gradient (Mafereka, et

al., 2009). Soils in the catchment are moderate to deep, and vary between

sandy-loam in the upper half to clay-loam in the middle reaches to sandy soils in

the lower reaches and the mouth (Fatoki, et al., 2001). The river flows through a

steep slope in its upper reaches which are extensively covered with both natural

and artificial forests, and has clean water in this area owing to the vegetation

cover (Fatoki, et al., 2001). In the middle to lower reaches water hyacinth

(Eichhornia crassipes) covers much of the surface water.

For simplicity of data interpretation, the sampling period was divided into winter

(May, June, July), spring (August, September, October) summer (November,

December, January) autumn (February, March, April). The study area was

divided into seven sampling points (S1 to S7, Table 1). Sites (S1 to S3) were

at (upper reaches), sites (S4 to S6) (middle reaches) and site (S7) (lower

19

reaches). All the land use activities taking place in the study area were recorded

(Table 1).

3.2 Selection of sampling sites

Seven sampling sites were recorded using Global Positioning System (GPS)

device along Umtata River. The sampling sites were chosen to reflect different

land use activities in the catchment - upper stream, midstream and downstream,

Figure1 & Table 1.

3.3 Macrophytes

Submerged macrophytes were handpicked in the river banks along the seven

selected sites, placed in plastic Ziploc bags, transferred to a portable refrigerator

and taken to the laboratory. In the laboratory all macrophyte samples were

identified into species level according to guides to aquatic plants (Gerber, et al.,

2004). All samples were rinsed severally with distilled water to remove sediment

particles and thereafter oven dried at 60oC for 48 hours. The dried samples were

later crushed in a mortar using a pestle. Samples were then sealed in plastic

eppendorf tubes for later stable isotope analysis.

20

Figure1: Map showing study area and sampling sites (Source: Department of

Water Affairs, King Sabata Dalindyebo Municipality).

21

Table 1: Showing sampling locations, coordinates and activities along the sites

Sampling sites Coordinates Anthropogenic activities

Langeni forest1

(S1)

31º 29, 58’’S:

28º 28, 61’’E

Indigenous, artificial forests,

deforestation, grazing land and road

construction.

Langeni forest 2

(S2)

31º 29, 92’’S:

0 28º 29, 61’’E

Artificial forest, subsistence farming,

deforestation and grazing land.

Kambi

(S3)

31º 29, 92’’S:

0 28º 36, 1’’E

Human settlement, mixed forest,

deforestation and grazing land.

Mthatha dam

(S4)

31º 33, 02’’S:

0 28º 44, 5’’E

Human settlement, domestic water use,

wood processing industry, subsistence

farming and grazing land.

Tipin

(S5)

31º 31, 57’’S:

0 28º 48,10’’E

Informal human settlement, poor

sanitation, subsistence farming,

wastewater discharge point, brick making

and dumping site.

Ntsaka

(S6)

31º 4, 13’’S:

028º 49,25’’E

Human settlement, domestic water use,

subsistence farming and grazing land.

Mdumbi

(S7)

31º55,52’’S:

0 29º 08, 19’’E

Human settlement, domestic water use,

road construction, subsistence farming

and grazing land

22

3.4 Isotopic analysis

The stable isotope signatures (SIS) analysis were done at the Archaeometry

laboratory, University of Cape Town using Finnegan-MAT 252 stable light mass

spectrometer. Stable isotope signatures (SIS) were expressed in the following

standard notion:

δX (%)= (Rsample/ Rstandard)-1* 100

where X is 13C or 15N and Rsample is the 13C/12C or 15N/14N respectively. R standard

for 13C is Pee Dee Belemnite (PDB). All resultant values were expressed in mils

(o/oo) (Lepointa, et al., 2004).

3.5 Statistical data analysis

The data was analyzed using two-way Analysis of Variance (ANOVA). The two-

way ANOVA(s) were followed by post hoc test. The Sites and seasons were

independent variables while stable isotopes of C:N ratios, δ13C and δ15N of

macrophytes were dependent variables. The statistical data analysis was

performed using Statistical 7.

23

Chapter 4

Results

4.1 The macrophyte species composition of Umtata River

A total number of 126 macrophyte samples consisting of 16 families and 26

species were recorded. The highest number of macrophytes family was 7 at site

(S5), 6 families at sites S6 and 4 families at S7 compared to sites (S1, S2, S3

& S4 which had 3 families at each) (Table 2). The upper reaches (S1, S2 & S3)

showed a least number of families (5 in total) compared to middle reaches (S4,

S5, S6) (10 families in total) and lower reaches (S7) (4) (Table 2).

Most macrophyte families showed site specific occurrences while others occurred

in more than two sites (Table 2). The family Cyperaceae was the most

abundant with 8 species and occurred throughout the length of the river (Table

2). The only site specific families which did not occur at any other site were;

freshwater algae at site (S4), Brassicaceae, Onagraceae (S5), Typhaceae (S6)

and Commelinaceae at site (S7). At the species level, the species composition

decreased from upper to lower reaches. A large number of exotic species (Alisma

plantago-aquatica, Eichhornia crassipes, Phragmites lapathifolia, Nasturtium

officinale, Myriophyllum aquaticum) was recorded in the middle reaches (Table

2) The Juncus effusus occurred only at the upper (S1) and Isolepis fluitans at

lower reaches (S7) respectively (Table 2). The following two species: Berula

erecta, Typha capensis and were recorded only in the middle reaches (S5 & S6)

24

(Table 2). The following species were recorded only at S7: Cyperus

sexangularis, Floscopa glomerata and Phragmites australis (Table 2). The

Eichhornia crassipes species covered the river in the middle reaches (S5 & S6)

and was also collected at the lower reaches (S7) after heavy rain falls (Table 2).

4.2 The C:N ratios at family level

For ease of data interpretation, the macrophytes species were grouped into

families and the average values of C:N ratios were taken for each family at each

site (Table 3).

The isotopic composition of macrophytes and C:N ratios showed variation in all

families collected from the upper to lower reaches of the river. There were site

specific differences in C:N ratios values of macrophytes observed along Umtata

river (Table 3). The most depleted C:N ratios signatures (6.01‰) were

recorded in family Poaceae at site 7 while the most enriched were recorded in

family Cyperaceae (30.75‰) at site (S2, Table 3).

25

Table 2: Showing the number of macrophytes families and species from the

sites along Umtata River. *Alien species.

Sites Family Species No.

Families

No. Species

S1 Cyperaceae Cladium mariscus 3 6

Cyperaceae Schoenoplectus brachyceras

Polygonaceae Persicaria senegalensis Junacaceae Juncus effusus Junacaceae Juncus lomatophyllus

S2 Cyperaceae C. mariscus 3 6

Potamogetonaceae Potamogeton pectinatus

Cyperaceae S. brachyceras Potamogetonaceae P. corex-austra-africana

Poaceae Phragmites mauritianus * Persicaria lapathifolia S3 Cyperaceae S. brachyceras 3 4

Cyperaceae C. margmatus Polygonaceae P. lomatophyllus Potamogetonaceae P. schweinfurthii S4 Potamogetonaceae P. pectinatus 3 3

Freshwater algae Spirogyra Alismataceae *Alisma plantago-

aquatica S5 Apiaceae Berula erecta 7 6

Brassicaeae *Nasturtium officinale

Cyperaceae Cyperaceae sexangularis

Pontederiaceae *Eichhornia crassipes Lemnaceaeae Lemna gibba Polygonaceae * Persicaria lapathifolia Polygonaceae Persicaria decipiens Onagraceae Ludwigia adsendens S6 Apiaceae Berula erecta 6 6

Haloragidaceae *Myriophyllum aquaticum

Poaceae P. mauritiatus Polygonaceae P. decipiens Pontederiaceae *E. crassipes Typhaceae Typha capensis S7 Cyperaceae Isolepis fluitans 4 8

Cyperaceae C. marginatus Cyperaceae C. sexangularis Commelinaceae Floscopa glomerata Haloragaceae P. australis Polygonaceae P. senegalensis

26

4.3 The δ13C values at family level

The δ13C values in macrophytes showed site-specific variations and longitudinal

variation in all sites sampled. The Juncaceae at site (S7) had the most depleted

δ13C value of less than -31‰, while the most enriched family, the

Potamogetonaceae at site (S6) was -15.14‰ (Table 3). The family Cyperaceae

occurred in all river stages sampled except sites (S4 and S6) and its δ13C values

fell between -19.50‰ to -29.25‰. The family Polygonaceae showed

longitudinal variation in δ13C signatures and followed family Cyperaceae with its

values ranging between -18.0‰ to -29.02‰.

4.4. The δ15N at family level

The families that showed longitudinal distribution along the river system also had

varying values of δ15N signatures (Table 3). The most depleted δ15N values

were recorded in family Cyperaceae at site (S1) (3.42‰), while the most

enriched δ15N value of about 24.32‰ was recorded in Pontederiaceae at site

(S6). All the families in the middle reaches (Apiaceae, Brassicaceae, Cyparaceae,

Holaragidaceae, Onagraceae, Poaceae, Polygonaceae, Pontederiaceae,

Potamogetonaceae and Typhaceae had highest values compared to other

families in the upper and lower reaches (Table 3).

27

Table 3: Showing the stable isotope composition of macrophytes families from

the sites along Umtata River. Each family represents average SIS of species

collected.

Sites Family δ15N (‰) δ13C (‰) C:N Ratio (‰)

S1 Cyperaceae 3.42 -20.75 19.75

Juncaceae 3.35 -21.51 24.51

Polygonaceae 7.4 -21.50 17.50

S2 Cyperaceae 6.5 -29.25 30.75

Poaceae 5.0 -28.38 23.25

Polygonaceae 6.13 -26.75 13.26

S3 Cyperaceae 8.26 -20.23 23.25

Polygonaceae 4.75 -24.75 13.76

Potamogetonaceae 5.27 -28.0 17.69

S4 Potamogetonaceae 7.83 -21.67 17.83

Poaceae 6.01 -17.5 19.33

Potamogetonaceae 6.81 -15.14 16.05

S5 Apiaceae 7.64 -25.24 18.08

Brassicaceae 8.99 -25.01 11.98

Cyperaceae 8.47 -19.50 6.47

Onagraceae 21.05 -21.64 10.01

Poaceae 7.46 -22.23 20.15

Polygonaceae 16.27 -29.02 9.50

Pontederiaceae 8.91 -29.14 11.45

S6 Apiaceae 14.98 -15.40 15.7

Haloragidaceae 20.43 -20.35 10.45

Poaceae 12.04 -20.34 6.52

Polygonaceae 18.61 -18.04 21.0

Pontederiaceae 24.32 -24.03 8.78

Typhaceae 17.11 -22.15 19.86

S7 Cyperaceae 9.88 -22.61 14.79

Commelinaceae 7.0 -22.43 27.35

Cyperaceae 6.33 -19.99 17.33

Cyperaceae 7.50 -23.81 22.0

Haloragidaceae 8.52 -24.52 13.41

Juncaceae 8.99 -31.21 17.22

Poaceae 8.67 -22.50 6.01

Polygonaceae 9.45 -23.54 7.65

28

The ANOVA(s) showed no statistical significances (p>0.05) between sampling

sites and seasons on C:N ratios and δ13C signatures of macrophytes collected

along Umtata River (Table 4). The effect of both sampling sites and seasons

was only observed in δ15N signatures (p<0.05) (Table 4).

Table 4: Showing the effect of sampling sites and seasons on the C:N ratios,

δ13C and δ15N signatures of macrophytes along Umtata River.

Sources of

variation

SS DF MS F P

C:N ratios Months 163.33 11 52.43 3.19 0.07

Sites 117.18 6 52.53 2.24 0.16

Interaction 37.56 11 30.58 2.37 0.79

δ13C Months 37.56 11 30.58 1.22 0.40

Site 72.34 6 30.58 2.36 0.14

Interaction 26.77 66 30.58 0.87 0.65

δ5N Months 39.07 11 6.35 6.15 0.01

Sites 45.22 6 20.42 7.12 0.01

Interaction 6.84 66 57.58 1.08 0.51

29

4.5 The effect of sampling sites on C:N ratios of macrophytes

The C:N ratio signatures varied throughout the length of the river (Table 5).

Although there was no statistical significance (P>0.05), the trends showed a

decrease in C:N ratios from upper to lower reaches (Figure 2). The highest C:N

ratios value (21.95±9.65‰) was recorded at site (S2) while the lowest value

(12.14±5.0‰) occurred at site S6 (Table 5, Figure 2).

Table 5: Showing the mean spatial values and standard deviation of C:N ratios

of macrophytes recorded along Umtata River, N= 84 .

SAMPLING SITES MEAN VALUES (‰) STANDARD DEVIATION

S1 19.68 9.05

S2 21.29 9.65

S3 17.02 5.92

S4 18.90 8.03

S5 14.47 7.21

S6 12.14 5.0

S7 13.73 3.92

30

0

5

10

15

20

25

S1 S2 S3 S4 S5 S6 S7

Sampling sites

C:N

ratios (

‰)

Figure 2: Graph showing mean spatial C:N values of macrophytes recorded

along Umtata River.

4.6 The effect of sampling period on C:N ratios of macrophytes

collected along Umtata river

The mean C:N ratio values varied throughout the sampling period with February

recording the lowest value of 5.8‰ (Table 6, Figure 3). The monthly C:N ratio

mean values that exceeded 20‰ were recorded in May, November and January

(Table 6). The trends of seasonal C:N mean values decreased from May to

October and then increased variably between November to April with highest and

lowest peaks recorded in January and February respectively (Table 6, Figure

3).

31

Table 6: Showing the mean seasonal values and standard deviation of C:N

ratios of macrophytes recorded along Umtata River, N= 84.

SAMPLING PERIOD MEAN VALUES (‰) STANDARD DEVIATION

May 21.06 10.45

June 17.02 9.01

July 15.50 7.95

August 17.72 7.45

September 15.10 4.52

October 11.67 1.97

November 20.31 8.34

December 17.30 3.51

January 24.0 7.44

February 5.80 1.53

March 19.01 6.98

April 16.31 2.54

32

0

5

10

15

20

25

30

May

June

July

August

Septe

mber

Octo

ber

Novem

ber

Decem

ber

January

Febru

ary

Marc

h

April

Sampling period (Months: 2010 - 2011)

C:N

ratios (

‰)

Figure 3: Graph mean showing seasonal C:N values of macrophytes recorded

along Umtata River.

4.7 The effect of sampling sites on δ13C signatures of macrophytes

The mean δ13C signature values varied across the length of the river, with the

highest enriched value recorded at site S4 and lowest depleted values at sites

S5, S6, and S7 ( Table 7, Figure 4). The upper sites (S1, S2, S3 and S4)

were marginally enriched compared to sites (S5, S6 and S7) (Table 7, Figure

4).

33

Table 7: Showing the mean spatial values and standard deviation of δ13C of

macrophytes recorded along Umtata River, N= 84.

SAMPLING SITES MEAN VALUES (‰) STANDARD DEVIATION

S1 -22.64 4.08

S2 -24.23 5.19

S3 -24.65 5.31

S4 -19.63 6.12

S5 -26.33 5.77

S6 -25.65 4.70

S7 -26.37 4.14

34

-30

-25

-20

-15

-10

-5

0

S1 S2 S3 S4 S5 S6 S7

Sampling sites

Carb

on s

table

isoto

pe (

‰)

Figure 4: Graph showing mean spatial δ13C values of macrophytes recorded

along Umtata River.

4.8 The effect of sampling period on δ13C signatures

The mean monthly δ13C signatures varied throughout the sampling period

(Table 8, Figure 5). The most depleted δ13C signatures were recorded in the

months of July (-27.05±3.27‰), September (-26.52±3.05‰) and April (-

26.10±6.84‰) (Table 8). The mean δ13C signatures values appeared to

variably increase and decrease between the sampling period of May to April

(Table 8, Figure 5).

35

Table 8: Showing the mean seasonal values and standard deviation of δ13C of

macrophytes recorded along Umtata River, N=84.

SAMPLING PERIOD MEAN VALUES (‰) STANDARD DEVIATION

May -21.45 6.83

June -24.47 5.33

July -27.05 3.27

August -24.85 3.96

September -26.52 3.05

October -24.89 3.35

November -25.13 4.41

December -20.92 5.65

January -24.61 7.72

February -21.6 1.14

March -25.20 6.55

April -26.10 6.84

36

-30

-25

-20

-15

-10

-5

0

May

June

July

August

Septe

mber

Octo

ber

Novem

ber

Decem

ber

January

Febru

ary

Marc

h

April

Sampling period (Months: 2010 - 2011)

Carb

on s

table

isoto

pe (

‰)

Figure 5: Graph showing mean seasonal δ13C values of macrophytes recorded

along Umtata River.

4.9 The effect of sites on δ15N signatures of macrophytes

The mean δ15N signatures of macrophytes collected along Umtata river revealed

statistical significance (p<0.05) on sampling sites. The trends of δ15N signatures

and δ15N site mean values increased variably from upper to middle sites (S1 to

S6) and decreased towards lower reaches site (S7) respectively (Figure 6,

Table 9). The middle sites (S5, S6) and lower site (S7) had highest mean δ15N

values compared to lower values at the other sites respectively (Figure 6 &

Table 9).

37

Table 9: Showing the mean spatial values and standard deviation of δ15N

signatures of macrophytes recorded along Umtata River, N=84.

SAMPLING SITES MEAN VALUES (‰) STANDARD DEVIATION

S1 3.92 2.43

S2 6.61 2.12

S3 6.22 3.35

S4 6.98 2.11

S5 9.12 4.81

S6 10.02 4.56

S7 8.12 3.10

38

0

2

4

6

8

10

12

S1 S2 S3 S4 S5 S6 S7

Sampling sites

Nitro

gen s

table

isoto

pe (

‰)

Figure 6: Showing mean spatial δ15N values of macrophytes recorded along

Umtata River.

4.10 The effect of sampling season on δ15N signatures of macrophytes

The mean δ15N signatures of macrophytes had statistical significance (p<0.05,

N=84) for sampling period. The δ15N signatures mean values between the

sampling period ranged from approximately 7‰ to 10‰ but dropped sharply to

less than 1‰ in February (Figure 7 & Table 10).

39

Table 10: Showing the mean seasonal values and standard deviation of δ15N

signatures of macrophytes recorded along Umtata River, N=84.

SAMPLING PERIOD MEAN VALUES (‰) STANDARD

DEVIATION

May 9.77 4.35

June 9.25 5.81

July 9.67 2.97

August 7.19 2.55

September 8.33 2.94

October 7.01 3.35

November 7.01 1.45

December 6.70 2.42

January 7.50 3.88

February 0.50 2.49

March 7.21 1.95

April 7.50 2.10

40

02

46

810

May

June

July

August

Septe

mber

Octo

ber

Novem

ber

Decem

ber

January

Febru

ary

Marc

h

April

Sampling period (Months: 2010 - 2011)

Nitro

gen s

table

isoto

pe(‰

)

Figure 7: Graph showing mean spatial δ15N values of macrophytes recorded

along Umtata River.

41

CHAPTER 5

Discussion

The occurrence of macrophytes in rivers is mainly determined by their

biogeographic distribution, nutrient availability, and anthropogenic activities

(Cole, at al., 2006; Benoit, et al., 2009; Esteves & Suzuki, 2010). The dominant

agricultural activities along Umtata River catchment may account for the

macrophytes family composition along this river system. The addition of sewage

and industrial waste discharged in the middle reaches sites (S5) to (S6),

favoured the presence of the highest macrophyte groups collected with alien

plants dominating over the local plants (Table 2, Eastern Cape River Health

Programme, 2004-2006).

The lowest number of species was recorded at the impounded site, Umtata dam

(S4), and it’s vegetation along its banks is mostly grass and algae (Table 2).

The development of macrophytes in lentic and lotic systems is complex and

involves the interaction between physiological, physic-chemical and ecological

factors (Esteves & Suzuki, 2010). The longitudinal distribution of macrophytes in

Umtata River may be attributed to habitat transformations due to different land

uses along its catchment (Benoit, et al., 2009; Esteves and Suzuki, 2010) and

the difference in the number of families in sampling sites which results from

availability of different nutrient sources (Michener & Lajtha, 2007).

42

The isotopic composition recorded along Umtata River indicated high values in

middle reaches of the river and this may have been the case, for macrophytes as

bio-indicators of eutrophication depends on the nature of nutrient supply to river

systems (Troxler and Richards, 2009), and they differ from one macrophyte to

another (Cohen & Bradham, 2010). These results (Table 3) indicated nutrient

accumulation and availability at sites sampled and the response of submerged

macrophytes to environmental changes (Cole, et al., 2006; Chang, et. al., 2009).

The C:N ratios and δ15N signatures of the river were lower in the upper reaches

compared to middle reaches (Table 3). This may be due to contribution of

depleted δ15N forest derived values compared to urban inputs (Cohen &

Bradham, 2010, Kendall, et. al., 2010; Ogrinc, et al., 2008). The anthropogenic

activities taking place along Umtata River system may have altered sediment and

water chemistry leading to modified habitat for macrophytes growing in this river

and also great variation in isotopic values measured in the study site (Michener

and Lajtha, 2007; Ogrinc, et. al., 2008; Baeta, et al., 2009; Chang, et al., 2009;

Troxler and Richards, 2009) (Table 3).

The signatures of the three variables (C:N ratios, δ13C, δ15N) investigated in this

study had higher values both in dry and wet periods (Tables 6, 8 & 10,

Figures 3, 5 & 7). These high values maybe attributed to biomass burning

(Martinelli et. al., 2002), less effluents received by water systems (Kendall, et al.,

43

2010), nature of nutrient supply (Ehleringer, et al., 1991; Baeta, et al., 2009;

Otero, et al., 2000), plant biochemical processes (Forsberg, et al., 1993; Lake et

al., 2001) and plant life forms and life cycles (LaZerte & Szalados, 1982;

Smedley, et al., 1991; Forsberg, et al., 1993). Other factors reported in the

literature include temporal variation in the nitrogen source (Cohen & Bradham,

2010), the transport and accumulation of nutrients from one site to another

(Cole, et al., 2006) and the response of aquatic organisms to seasonal changes

(Esteves and Suzuki, 2010).

The C:N ratios trends indicated areas with elevated nutrient inputs with the

maximum values of 37.05‰ (Figure 2, Table 5). This value represents the

total dry weight of organic carbon and total nitrogen present in water column

signaling high nutrient content in the river (Durga, et al., 2011). However, the

findings of Troxler and Richards, (2009) suggest that the nutrient retention and

seasonal nutrient limitations by submerged macrophytes and productivity

change, have minor effect on their C:N ratios. These may also account for the

findings of the varying mean C:N ratio trends in this study (Tables 2 & 3,

Figures 3 & 4) Moreover, the C:N ratios recorded along Umtata river fell

between 3.57‰ to 37.05‰ with an average value of 16.03‰. This range was

lower compared to those reported in the literature on macrophytes (20‰ to

39.4‰ and C4, 15‰ to 20‰ respectively) (Fellerhoff, et al., 2003; Durga, et

al., 2011), and even lower than that reported in planktonic detritus (6‰ to

44

9‰) (Fellerhoff, et al 2003; Kuriah, 2008, Durga, et al., 2011). This C:N ratio

range recorded in this study can be attributed to different anthropogenic

activities in the catchment. The forested upper reaches of Umtata River seemed

to derive its nutrient from leaf litter and detritus from the mixed forest around it

as they had higher values. In this area decomposing plant material was

abundant and blocked water current in some parts forming temporal

impediments (Personal observations). Similar observations have been reported in

USA freshwater systems (Kendall, et. al., 2010) and in freshwater dominated

estuaries of southeastern USA (Ogrinc, et. al., 2008).

The variation of seasonal mean values of C:N ratios throughout the length of the

river and sampling period (Figures 3, Table 6), is thought to be a result of

stream geology and the river physics (Maltias-Landry et al., 2009), agricultural

and sewage nutrient inputs (Boullion, et. al., 2002; Ogrinc, et. al., 2008). This

variation is mainly related to contribution of different nutrient inputs which are

driven by both seasonality and spatiality to these systems (Kennedy, et al.,

2005). For this study the values of C:N ratios may be the result of dominant

agricultural practices in upper reaches (Doucett, et. al., 1996), waste water

discharge in the urban dominated middle reaches (Baeta, et al., 2009, Durga, et

al., 2011), the mixture of both land-use activities and within river nutrient inputs

towards the lower reaches (Doucett, et. al,. 1996).

45

The high winter C:N ratios mean values found in this study are not different from

those reported in the literature in terms of seasonality. This is probably due to

the fact that the dry winter months of May and June promoted detritus and

reduced water level in rivers leading to higher carbonates and nitrates

concentrations in the water column (Salas and Dudgeon, 2001; Chang, et al.,

2009; Esteves and Suzuki, 2010; Kendal, et al., 2010), and terrestrial

environment (Zhao, et al., 2010) (Figure 3, Table 6). The rainfall may account

for the difference in C:N ratios between spring and autumn. The transport and

transformation of nutrients along the river in undeveloped and developed sites

account for isotopic differences (Cole, et al., 2006).

The δ13C signatures fell between -26.37‰ to-19.63‰ with an average of -

24.41‰. The δ13C signatures recorded in this study were within the known

range for freshwater macrophytes (C3 plants, -32 to -20‰ and C4, -17 to -9‰)

respectively (Boullion, 2002), but their mean value was lower than that reported

in the literature (-27‰ for C3 and -13‰ for C4 plants) (Paterson and Whitfield,

1997; Otero, et al., 2000; Zhao, et al., 2010). These C:N ratios and δ13C

signatures in macrophytes closely compared to the range observed for C3 plants

(-24‰ to -34‰) for two rivers in the Eastern Cape Province of South Africa

(Kuriah and Pakhomov, 2008). This may probably be due to the nature of land

uses occurring along the sites selected for this study and to δ13C sensitivity to

land use activities (Lake, et al., 2001). Slow river current flow is closely

46

associated with high δ13C values in impoundments (Kendall, et. al., 2010),

leading to high nutrient retention by macrophytes (Maltias-landry, et al., 2009).

This may well explain the higher δ13C values trends at the dammed sites (S1 &

S4, Figure 4). The varying δ13C values recorded in agricultural dominated upper

and lower sites (Figure 4, Table 7) may also be attributed to vegetation cover

and soil type (Martinelli, et al., 1999). The sites S4 & S6 were in open areas

and showed unequal δ13C values signaling the contribution of nutrient sources

with distinctive isotopic composition (Kohzu, et al., 2008). The effect of different

land uses was evident with distinct isotopic signatures in all sites sampled. The

study revealed high values in agricultural areas in upper and lower reaches

compared to urban and below urban sites in middle reaches (Tables 7, Figure

4), probably due to the fact that forested areas add carbon rich detritus material

to river system than carbon depleted urban sewage (Meskumpumn, et al., 2005;

Salas and Dugeon, 2008). Furthermore, carbon is reported to be higher in

agricultural areas than in human dominated areas (Kohzu, et al., 2008).

However, these results contradict the findings of (Otero, et al., 2000) who

reported lower δ13C values in C3 dominated upper reaches of freshwater

dominated estuaries of South Eastern USA.

The δ13C values varied greatly throughout the sampling period. The high δ13C

values in months December and January (Figure 5, Table 8) may be attributed

to runoff which introduces nutrients to the river (Salas and Dudgeon, 2001; Van

47

Noordwirjk, et al., 2004; Ogrinc., et. al., 2008), whereas high δ13C values in dry

season are associated with low rainfall and windblown terrestrial material to river

systems (Martinelli, et al., 2002; Kennedy, et al., 2005). Other reported driving

forces behind season differences in δ13C values include plant taxonomic

differences in photosynthetic pathways (Ehleringer, et al., 1997; Fellerhoff, et al

2003; Durga, et al., 2011), stomatal aperture and the chlorophyll-a carbon

demand (Otero, et al., 2000) and anthropogenic impacts (Kennedy, et al., 2005).

The seasonal and spatial differences in δ15N signatures of macrophytes depend

on the tissue being analyzed and the rate of exposure to contaminated water

systems (Cohen & Bradham, 2010). The significant results (p<0.05) in δ15N in

both sampling sites and period (Table 4), showed low values in the forested

upper reaches compared to human dominated urban middle reaches and in sites

below urban areas (Figure 6, Table 9). The δ15N is known to increase sharply

in urban dominated areas due to industrial and human waste (Cabana and

Rasmussen, 1996; Kennedy, et al., 2005; Kohzu et al., 2008; Kendall, et al.,

2010). The low δ15N signatures are also reported to result from primary

production in the catchment (Kohzu, et al., 2008; Cohen and Bradman, 2010).

The δ15N values of macrophytes of Umtata River increased from upper to middle

and decreased towards lower reaches indicating the contribution of different

nitrogen inputs with different δ15N composition (Figure 6, Table 9). These

finding are explained by the fact that the unidirectional flow of river water

48

favours mixing and accumulation of nutrients of different origin, leading to

longitudinal differences in δ15N of the river systems (Kendall, et al., 2010).

Agricultural derived nutrient inputs have lower δ15N values than industrial and

human waste δ15N values (Cabana and Rasmussen, 1996, Meksumpun, et al.,

2005, Kendall, et. al., 2010) and this coincided with δ15N values from this study.

The δ15N trends showed enriched values in wastewater receiving middle reaches

of Umtata River (Figure 7, Table 10). Studies in freshwater systems have

found that δ15N values are sensitive to differences in land use (Kennedy, et al.,

2005; Kohzu, et al., 2008; Kendall, et al., 2010). The δ15N is mostly driven by

external factors for example difference in land use (Kennedy, et al., 2005), thus

δ15N values vary along sites which were chosen based on different activities

happening around the selected sites. These external factors are the major

influence of differences in the stable isotopes of δ15N (Kennedy, et al., 2005).

The δ15N mean seasonal values recorded in macrophytes along Umtata river fell

between 6‰ to 10‰ from May to April (Table 10), possibly due to the fact

that runoff is affected by seasonality in Umtata area (Eastern Cape river health

programme, 2004 – 2006). These mean seasonal δ15N values decreased variably

from winter to autumn with a highest peak in May and lowest in February

(9.77‰ and 0.5‰) respectively (Table 10, Figure 7). This variation in δ15N is

known to result from variation of the nutrient flow from pollution points (Cabana

49

& Rasmussen, 1996) which is the function of runoff. The observed high δ15N

macrophytes signatures during the dry period (May to July 2010) coincided with

the low river flow and the fragmentation of the habitat. This likely resulted in

concentrated nutrients within the impoundments to be severely depleted. The

flushing of the terrestrial organic matter runoffs and subsequent decline after the

rainfall event corresponded to the high and low SIS variations. Further, it is

reported that runoff from agricultural sites tends to contain high concentration of

δ15N (Forsberg, et al., 1993; Kohzu, et al., 2008) as is the case with site 2

(Figure 6). Application of nitrogenous fertilizers to agricultural farmlands and

denitrification from human residential areas to drainage system enhance nitrate

and increase of δ15N (Chang, et al., 2008; Kohzu, et al., 2008). The δ15N of

macrophytes vary in watershed including both pristine and agricultural

catchment, with agricultural and urban sites having high values of δ15N

(Forsberg, et al., 1993; Chang, et al., 2009; Cohen and Bradman, 2009;

Wozniak, et al., 2012).

50

Conclusion/recommendations

The macrophyte families and species composition showed both longitudinal and

site specific occurrences along Umtata River. The high abundance of exotic

plants along Umtata River system is an indication of high pollution along the river

system. Although only the stable isotope of nitrogen was statistically significant,

the mean values and trends of all SIS recorded in the river system indicated high

nutrient loading from the land uses from the river catchment. The longitudinal

increase of isotopic composition of macrophytes along the river indicates

accumulation of nutrients from one site to another. The upper reaches of the

river seemed to derive its nutrients from agricultural fields. The middle reaches

received waste water from Mthatha waste treatment facility hence the high δ15N

signatures around this area.

The seasonal variability of the variables tested in this study pointed out rainfall

as major factor controlling the nutrient concentrations along Umtata River. The

dry season seemed to be the most threatening season in terms of nutrient

concentration along the river as δ15N had high values during this season. Based

on the results obtained from this study, there is a need for continued monitoring

of land use of the catchment especially around its middle reaches as it happened

to be an affected area. Further studies on isotopic composition of Umtata River

need to be done as there is no published work on stable isotope ecology of this

system.

51

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61

Table 11: The list of all macrophyte families and species collected along Umtata River.

Macrophyte families

Alismataceae

Apiaceae

Brassicaeae

Commelinaceae

Cyperaceae

Freshwater algae (Unidentified)

Haloragidaceae

Haloragraceae

Junacaceae

Lemnaceaeae

Onagraceae

Poaceae

Polygonaceae

Pontederiaceae

Potamogetonaceae

Typhaceae

Macrophyte species

Alisma plantago-aquatica

Berula erecta

Cladium marginatus

Cladium mariscus

Cladium sexangularis

Eichhornia crassipes

Floscopa glomerata

Isolepis fluitans

Juncus effusus

Juncus lomatophyllus

Lemna gibba

Ludwigia adsendens

Myriophyllum aquaticum

Nasturtium officinale

Persicaria senegalensis

Persicaria decipiens

Persicaria lomatophyllus

Phragmites lapathifolia

Phragmites mauritianus

Potamogeton pectinatus

Potamogeton australis

Potamogeton corex-austra-africana

Potamogeton schweinfurthii

Schoenoplectus brachyceras

Spirogyra Spp

Typha capensis

62

Appendix Study area photographs showing different anthropogenic land use activities along Umtata River Catchment.

S1. The water from this site is used for irrigation by Langeni forest Farm.

S2. The site is reported to be used for fishing and irrigation.

63

S3. Kambi forest- high turbidity levels of the water after heavy rainfall.

S4. The Umtata Dam is mainly used for domestic and recreational purposes.

64

A. Sewage discharged from Mthatha town waste treatment plant. B. leaking sewage

stream from a pipe at Fort Gale area in middle reaches (above Site 5).

A

B

65

Site 5. C. Informal settlements. D. Water hyacinth (Eichhornia crassipes) on the banks.

S6. The site is mainly used by locals for swimming, fishing, domestic and laundry.

C D

66

S7. River system in this area is used by locals for domestic and recreation.

67