Table Of ContentCOPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION
Attribution — You must give appropriate credit, provide a link to the license, and indicate if
o
changes were made. You may do so in any reasonable manner, but not in any way that
suggests the licensor endorses you or your use.
NonCommercial — You may not use the material for commercial purposes.
o
ShareAlike — If you remix, transform, or build upon the material, you must distribute your
o
contributions under the same license as the original.
How to cite this thesis
Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/
M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved
from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed:
Date).
Biomarker responses in three aquatic crustacean species
from the lower Phongolo Floodplain, KwaZulu-Natal, South
Africa.
By
Gregg Jansen van Rensburg
DISSERTATION
Submitted in Fulfilment of the Requirements for the Degree
MAGISTER SCIENTIAE
In
Aquatic Health
In the
FACULTY OF SCIENCE
At the
UNIVERSITY OF JOHANNESBURG
Supervisor: Prof. JHJ van Vuren
Co-supervisor: Prof L Bervoets
February 2016
The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged.
Opinions expressed and conclusions arrived at are those of the author and are not necessarily to be attributed to the NRF
Contents
List of Figures .................................................................................................................. IV
List of Tables ................................................................................................................... IX
List of Abbreviations ........................................................................................................ XI
Acknowledgements .......................................................................................................... 1
Summary .......................................................................................................................... 2
Chapter 1 – General introduction ...................................................................................... 4
1.1 Motivation .............................................................................................................. 4
1.2 Hypothesis ............................................................................................................. 7
1.3 Aims ....................................................................................................................... 7
1.4 Objectives .............................................................................................................. 7
1.5 Layout of dissertation ............................................................................................. 7
1.5.1 Chapter 1 – General Introduction .................................................................... 7
1.5.2 Chapter 2 – Background literature and site selection ...................................... 8
1.5.3 Chapter 3 – Materials and methods. ............................................................... 8
1.5.4 Chapter 4 – Results. ....................................................................................... 8
1.5.5 Chapter 5 – Discussion................................................................................... 8
1.5.6 Chapter 6 – Conclusion and recommendations. ............................................. 8
1.5.7 Chapter 7 – Reference list. ............................................................................. 8
Chapter 2 - Background literature ..................................................................................... 9
2.1 Background ............................................................................................................ 9
2.2 Pesticides ............................................................................................................ 10
2.3 Biomonitoring/bio-indicator ................................................................................... 12
2.4 Selected Study Organisms ................................................................................... 13
2.4.1 Cherax quadricarinatus (von Martens, 1868) ................................................ 13
2.4.2 Macrobrachium petersii (Hilgendorf, 1878) ................................................... 16
2.4.3 Caridina nilotica (Roux, 1833)....................................................................... 17
2.5 Biomarkers ........................................................................................................... 18
2.5.1 Biomarkers of Exposure ............................................................................... 20
2.5.2 Biomarkers of Effect ..................................................................................... 21
2.6 Study Area ........................................................................................................... 23
2.6.1 Phongolo River ............................................................................................. 23
2.6.2 Usuthu River ................................................................................................. 24
2.6.3 Ngwavuma River .......................................................................................... 25
2.6.4 Nyamithi Pan ................................................................................................ 25
I | Pag e
Chapter 3 Materials and Methods ................................................................................... 26
3.1 Site Selection ....................................................................................................... 26
3.2 Collection and processing of organisms ............................................................... 31
3.3 Organochlorine Pesticide Analysis ....................................................................... 33
3.3.1 Sample Preparation ...................................................................................... 33
3.3.2 OCP analysis ................................................................................................ 33
3.3.3 Quality assurance ......................................................................................... 34
3.4 Biomarker protocols ............................................................................................. 35
3.4.1 Biomarkers of Exposure ............................................................................... 35
3.4.1.1 Acetylcholinesterase activity.................................................................... 35
3.4.1.2 Metallothioneins ...................................................................................... 35
3.4.1.3 Cytochrome P450 ................................................................................... 36
3.4.2 Biomarkers of Effect ..................................................................................... 36
3.4.2.1 Reduced glutathione content ................................................................... 36
3.4.2.2 Superoxide dismutase ............................................................................. 37
3.4.2.3 Catalase activity ...................................................................................... 37
3.4.2.4 Malondialdehyde content ........................................................................ 37
3.4.2.5 Protein carbonyl induction ....................................................................... 38
3.4.2.6 Cellular energy allocation ........................................................................ 38
3.4.2.6.1 Energy consumption ......................................................................... 39
3.4.2.6.2 Energy availability ............................................................................. 39
3.5 Statistical Analysis ............................................................................................... 40
Chapter 4 – Results ........................................................................................................ 41
4.1 Organochlorine pesticide bioaccumulation ........................................................... 41
4.2 Biomarker responses ........................................................................................... 42
4.2.1 Cherax quadricarinatus................................................................................. 42
4.2.1.1 Biomarkers of exposure and effect .......................................................... 42
4.2.1.2 Discriminant function analysis .................................................................... 49
4.2.1.3 Redundancy analysis................................................................................. 54
4.2.2 Macrobrachium petersii ................................................................................ 56
4.2.2.1 Biomarkers of exposure and effect .......................................................... 56
4.2.2.2 Discriminant function analysis ................................................................. 59
4.2.3 Caridina nilotica ............................................................................................ 63
4.2.3.1 Biomarkers of exposure and effect .......................................................... 63
4.2.3.2 Discriminant function analysis ................................................................. 71
Chapter 5 – Discussion .................................................................................................. 77
5.1 Organochlorine pesticide accumulation ................................................................ 77
II | Pag e
5.2 Biomarker responses ........................................................................................... 80
5.2.1 Relationship between biomarker responses and OCP accumulated levels ... 85
Chapter 6 - Conclusion and Recommendations .............................................................. 86
6.1 Conclusion ........................................................................................................... 86
6.2 Recommendations ............................................................................................... 88
Chapter 7 - Reference List .............................................................................................. 90
Appendix A – Raw bioaccumulation data ...................................................................... 104
Appendix B – Raw biomarker data ............................................................................... 105
III | Pag e
List of Figures
Figure 2.1: Adult male (A) and female (B) Cherax quadricarinatus…………………………….15
Figure 2.2: Adult Macrobrachium petersii………………………………………………………...16
Figure 2.3: Adult Caridina nilotica…………………………………………………………………17
Figure 3.1: Map of the lower Phongolo River region indicating the location of sampling sites
and corresponding site number. Insets illustrating location of South Africa (A) and the
KwaZulu-Natal province (B)………………………………………………………………………..28
Figure 3.2: Photographs of sampling sites within the Ndumo Game Reserve. A - Nyamithi
Pan (Site 1), B - Usuthu River (Site 2) and C - Phongolo River (Site 5)…………………………29
Figure 3.3: Photographs of sampling sites outside the Ndumo Game Reserve. A - Ngwavuma
River (Site 3), B - Phongolo River (Site 4) and C - Phongolo River below the dam wall (Site
6)………………………………………………………………………………………………………30
Figure 4.1: Box-and-whisker plots representing 5 – 95 percentile range of biomarkers of
exposure: acetylcholinesterase (AChE, A), metallothionein (MT, B) and cytochrome P450
(CYP, C) in Cherax quadricarinatus. High flow survey (Apr 2014) indicated by plots grey fill,
low flow survey (Oct 2014) indicated by plots with no fill. NP – Nyamithi Pan, UR – Usuthu
River, PR up – Phongolo River upstream of Ndumo Game Reserve and PR down – Phongolo
River inside the Ndumo Game Reserve. Common superscripts indicate no statistically
significant differences (p < 0.05)…………………………………………………………………43
Figure 4.2: Box-and-whisker plots representing 5 – 95 percentile range of biomarkers of
effect: reduced glutathione (GSH, A), superoxide dismutase (SOD, B) and catalase (CAT, C),
in Cherax quadricarinatus. High flow survey (Apr 2014) indicated by plots with grey fill, low
flow survey (Oct 2014) indicated by plots with no fill. NP – Nyamithi Pan, UR – Usuthu River,
PR up – Phongolo River upstream of Ndumo Game Reserve and PR down – Phongolo River
inside the Ndumo Game Reserve. Common superscripts indicate no statistically significant
differences (p < 0.05)………………………………………………………………………………..44
Figure 4.3: Box-and-whisker plots representing 5 – 95 percentile range of biomarkers of
effect: malondialdehyde (MDA, A) and protein carbonyls (PC, B), in Cherax quadricarinatus.
High flow survey (Apr 2014) indicated by plots with grey fill, low flow survey (Oct 2014)
IV | Pag e
indicated by plots with no fill. NP – Nyamithi Pan, UR – Usuthu River, PR up – Phongolo River
upstream of Ndumo Game Reserve and PR down – Phongolo River inside NGR. Common
superscripts indicate no statistically significant differences (p < 0.05)………………………….45
Figure 4.4: Box-and-whisker plots representing 5 – 95 percentile range of cellular energy
allocation (CEA, A) and its components, energy consumption (Ec, B) and energy allocation
(Ea, C), in Cherax quadricarinatus. High flow survey (Apr 2014) indicated by plots with grey
fill, low flow survey (Oct 2014) indicated by plots with no fill. NP – Nyamithi Pan, UR – Usuthu
River, PR up – Phongolo River upstream of Ndumo Game Reserve and PR down – Phongolo
River inside NGR. Common superscripts indicate no statistically significant differences (p <
0.05)…………………………………………………………………………………………………..47
Figure 4.5: Box-and-whisker plots representing 5 – 95 percentile range of energy allocation
components: lipids (A), glucose (B) and protein (C), in Cherax quadricarinatus. High flow
survey (Apr 2014) indicated by plots with grey fill, low flow survey (Oct 2014) indicated by plots
with no fill. NP – Nyamithi Pan, UR – Usuthu River, PR up – Phongolo River upstream of
Ndumo Game Reserve and PR down – Phongolo River inside NGR. Common superscripts
indicate no statistically significant differences (p < 0.05)………………………………………...48
Figure 4.6: Canonical variates derived from a discriminant functions analysis (DFA) using
biomarkers of exposure in Cherax quadricarinatus (CH). All sampled sites are included as NP
– Nyamithi Pan, UR – Usuthu River, PR up – Phongolo River upstream of Ndumo Game
Reserve and PR down – Phongolo River inside Ndumo Game Reserve. High flow (HF, Apr
2014) and low flow (LF, Oct 2014) surveys indicated. Function 1 and 2 indicate the first two
canonical functions of the analysis…………………………………………………………………50
Figure 4.7: Canonical variates derived from a discriminant functions analysis (DFA) using
biomarkers of effect (A) and cellular energy allocation (B) in Cherax quadricarinatus (CH). All
sampled sites are included as NP – Nyamithi Pan, UR – Usuthu River, PR up – Phongolo
River upstream of Ndumo Game Reserve and PR down – Phongolo River inside the Ndumo
Game Reserve. High flow (HF, Apr 2014) and low flow (LF, Oct 2014) surveys indicated.
Function 1 and 2 indicate the first two canonical functions of the analysis……………………..51
Figure 4.8: RDA triplot of biomarker responses and OCP accumulated levels in C.
quadricarinatus. Sites included are the Usuthu River (UR), Phongolo River (PR) and the
Nyamithi Pan (NP). High flow (HF, Apr 2014) and low flow (LF, Oct 2014) surveys indicated.
The ordination explains 81.9% of the variation, with 54.5% explained on the first axis and
27.4% explained on the second axis………………………………………………………………55
V | Pag e
Figure 4.9: Box-and-whisker plots representing 5 – 95 percentile range of biomarkers of
exposure: acetylcholinesterase (AChE, A), metallothionein (MT, B) and cytochrome P450
(CYP, C) and biomarkers of effect: reduced glutathione (GSH, D), superoxide dismutase
(SOD, E) and catalase (CAT, F) malondialdehyde (MDA, G) and protein carbonyls (PC, H), in
Macrobrachium petersii. High flow survey (Apr 2014) indicated in axis label, low flow survey
(Oct 2014) indicated by plots with no fill. UR – Usuthu River, PR down – Phongolo River inside
the Ndumo Game Reserve. Common superscripts indicate no statistically significant
differences (p < 0.05)…………………………………………………………………………….57
Figure 4.10: Box-and-whisker plots representing 5 – 95 percentile range of cellular energy
allocation (A) and its components: energy consumption (Ec, B) and energy allocation (Ea, C)
in Macrobrachium petersii. The components of energy allocation are also included: glucose
(D), lipids (E) and protein (F). High flow survey (Apr 2014) indicated by plots with grey fill, low
flow survey (Oct 2014) indicated by plots with no fill. UR – Usuthu River, PR down – Phongolo
River inside the Ndumo Game Reserve. Common superscripts indicate no statistically
significant differences (p < 0.05)…………………………………………………………………58
Figure 4.11: Canonical variates derived from a discriminant functions analysis (DFA) using
biomarkers of exposure (A) and effect (B) in Macrobrachium petersii (M). All sampled sites are
included as UR – Usuthu River and PR down – Phongolo River inside the Ndumo Game
Reserve. High flow (HF, Apr 2014) and low flow (LF, Oct 2014) surveys indicated. Function 1
and 2 indicate the first two canonical functions of the analysis………………………………….60
Figure 4.12: Canonical variates derived from a discriminant functions analysis (DFA) using
cellular energy allocation and its components in Macrobrachium petersii (M). All sampled sites
are included as UR – Usuthu River and PR down – Phongolo River inside the Ndumo Game
Reserve. High flow (HF, Apr 2014) and low flow (LF, Oct 2014) surveys indicated. Function 1
and 2 indicate the first two canonical functions of the analysis…………………………………61
Figure 4.13: Box-and-whisker plots representing 5 – 95 percentile range of biomarkers of
exposure: acetylcholinesterase (AChE, A), metallothionein (MT, B) and cytochrome P450
(CYP, C), in Caridina nilotica. High flow survey (Apr 2014) indicated by plots grey fill, low flow
survey (Oct 2014) indicated by plots with no fill. NP – Nyamithi Pan, UR – Usuthu River, NR
– Ngwavuma River, PR dam – Phongolo River downstream of the Pongolapoort Dam wall,
PR up – Phongolo River upstream of Ndumo Game Reserve and PR down – Phongolo River
inside the Ndumo Game Reserve. Common superscripts indicate no statistically significant
differences (p < 0.05)………………………………………………………………………………..66
VI | Pag e
Figure 4.14: Box-and-whisker plots representing 5 – 95 percentile range of biomarkers of
effect: reduced glutathione (GSH, A), superoxide dismutase (SOD, B) and catalase (CAT, C),
in Caridina nilotica. High flow survey (Apr 2014) indicated by plots with grey fill, low flow survey
(Oct 2014) indicated by plots with no fill. NP – Nyamithi Pan, UR – Usuthu River, NR –
Ngwavuma River, PR dam – Phongolo River downstream of the Pongolapoort Dam wall, PR
up – Phongolo River upstream of Ndumo Game Reserve and PR down – Phongolo River
inside the Ndumo Game Reserve. Common superscripts indicate no statistically significant
differences (p < 0.05)……………………………………………………………………………….67
Figure 4.15: Box-and-whisker plots representing 5 – 95 percentile range of biomarkers of
effect: malondialdehyde (MDA, A) and protein carbonyls (PC, B), in Caridina nilotica. High flow
survey (Apr 2014) indicated by plots with grey fill, low flow survey (Oct 2014) indicated by plots
with no fill. NP – Nyamithi Pan, UR – Usuthu River, NR – Ngwavuma River, PR dam –
Phongolo River downstream of the Pongolapoort Dam wall, PR up – Phongolo River upstream
of Ndumo Game Reserve and PR down – Phongolo River inside the Ndumo Game Reserve.
Common superscripts indicate no statistically significant differences (p < 0.05)………………68
Figure 4.16: Box-and-whisker plots representing 5 – 95 percentile range of cellular energy
allocation (CEA, A) and its components, energy consumption (Ec, B) and energy allocation
(Ea, C), in Caridina nilotica. High flow survey (Apr 2014) indicated by plots with grey fill, low
flow survey (Oct 2014) indicated by plots with no fill. NP – Nyamithi Pan, UR – Usuthu River,
NR – Ngwavuma River, PR dam – Phongolo River downstream of the Pongolapoort Dam wall,
PR up – Phongolo River upstream of Ndumo Game Reserve and PR down – Phongolo River
inside the Ndumo Game Reserve. Common superscripts indicate no statistically significant
differences (p < 0.05)………………………………………………………………………………..69
Figure 4.17: Box-and-whisker plots representing 5 – 95 percentile range of energy allocation
components, glucose (A), lipids (B) and protein (C), in Caridina nilotica. High flow survey (Apr
2014) indicated by plots with grey fill, low flow survey (Oct 2014) indicated by plots with no fill.
NP – Nyamithi Pan, UR – Usuthu River, NR – Ngwavuma River, PR dam – Phongolo River
downstream of the Pongolapoort Dam wall, PR up – Phongolo River upstream of Ndumo
Game Reserve and PR down – Phongolo River inside the Ndumo Game Reserve. Common
superscripts indicate no statistically significant differences (p < 0.05)…………………………70
Figure 4.18: Canonical variates derived from a discriminant functions analysis (DFA) using
biomarkers of exposure in Caridina nilotica (Car). All sampled sites are included as NP –
Nyamithi Pan, UR – Usuthu River, NR – Ngwavuma River, PR dam – Phongolo River
downstream of the Pongolapoort Dam wall, PR up – Phongolo River upstream of Ndumo
VII | Pag e
Game Reserve and PR down – Phongolo River inside the Ndumo Game Reserve. High flow
(HF, Apr 2014) and low flow (LF, Oct 2014) surveys indicated. Function 1 and 2 indicate the
first two canonical functions of the analysis……………………………………………………….72
Figure 4.19: Canonical variates derived from a discriminant functions analysis (DFA) using
biomarkers of effect (A) and cellular energy allocation (B) in Caridina nilotica (Car). All sampled
sites are included as NP – Nyamithi Pan, UR – Usuthu River, NR – Ngwavuma River, PR dam
– Phongolo River downstream of the Pongolapoort Dam wall, PR up – Phongolo River
upstream of Ndumo Game Reserve and PR down – Phongolo River inside the Ndumo Game
Reserve. High flow (HF, Apr 2014) and low flow (LF, Oct 2014) surveys indicated. Function 1
and 2 indicate the first two canonical functions of the analysis………………………………….73
VIII | Pag e
Description:The trends observed between species were not completely identical, though there were some responses that were similar . Biomonitoring can be done with the use of bioindicators. A bioindicator is a .. but also in the cycling of organic matter (Hart, 2001; Mensah et al., 2012). The species selected w
