A HISTOLOGY-BASED HEALTH ASSESSMENT OF SELECTED FISH SPECIES FROM TWO RIVERS IN THE KRUGER NATIONAL PARK BY WARREN CLIFFORD SMITH DISSERTATION SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE MAGISTER SCIENTIAE IN AQUATIC HEALTH IN THE FACULTY OF SCIENCE AT THE UNIVERSITY OF JOHANNESBURG SUPERVISOR: DR. G.M. WAGENAAR CO-SUPERVISOR: PROF. N.J. SMIT MAY 2012 Contents
Acknowledgements ______6
Abstract ______8
List of abbreviations ______11
List of figures ______14
List of Tables ______17
Chapter 1: General Introduction ______19
1.1 Introduction ______19
1.2 Study motivation ______19
1.3 Hypotheses ______21
1.4 Aim of the study ______21
1.5 Objectives ______21
1.6 Dissertation outline ______21
Chapter 2: Literature Review ______23
2.1 Introduction ______23
2.2 Study Sites ______23 2.2.1 Olifants River (OR) ______23 2.2.2 Luvuvhu River (LR) ______25
2.3 Metals in the aquatic environment ______26 2.3.1 Aluminium (Al) ______26 2.3.2 Arsenic (As) ______27 2.3.3 Cadmium (Cd) ______28 2.3.4 Chromium (Cr) ______29 2.3.5 Copper (Cu) ______30 2.3.6 Iron (Fe) ______31 2.3.7 Lead (Pb) ______31 2.3.8 Manganese (Mn) ______32 [1]
2.3.9 Zinc (Zn) ______33
2.4 Study organisms ______34 2.4.1 Hydrocynus vittatus ______34 2.4.2 Labeobarbus marequensis ______35 2.4.3 Labeo cylindricus ______37 2.4.4 Labeo rosae ______38
2.5 Health Assessment Index ______39
2.6 Blood constituents’ analysis ______39
2.7 Biometric indices ______40 2.7.1 Condition factor ______40 2.7.2 Hepatosomatic index ______40 2.7.3 Splenosomatic index ______40 2.7.4 Gonadosomatic index ______41
2.8 Histology and histopathology of selected target organs ______41 2.8.1 Liver as a target organ ______41 2.8.2 Kidney as a target organ ______43 2.8.3 Gill as a target organ ______46 2.8.4 Testis as a target organ ______52 2.8.5 Ovary as a target organ ______53 2.8.6 Semi-quantitative histological assessment ______54
2.9 Ageing ______54
Chapter 3: Materials and Methods ______56
3.1 Introduction ______56
3.2 Study period ______56
3.3 Water and sediment collection ______60
3.4 Physical water quality parameters ______60
3.5 Field surveys ______61
3.6 Histology-based health assessment protocol ______64 3.6.1 Blood constituents analysis ______64 3.6.2 Necropsy ______64 3.6.3 HAI ______64
[2]
3.6.4 Biometric indices ______64
3.7 Tissue processing ______65
3.8 Qualitative histological assessment ______67 3.8.1 Gonadal reproductive stage determination: ______67
3.9 Semi-quantitative histological assessment ______69
3.10 Ageing ______72
3.11 Statistical analysis ______73
Chapter 4: Results ______74
4.1 Introduction ______74
4.2 Physical water quality parameters ______74
4.2.1 Temperature ______74 4.2.2 pH ______74 4.2.3 Oxygen percentage ______74 4.2.4 Conductivity ______74 4.2.5 Total dissolved salts ______75
4.3 Metal concentrations ______75 4.3.1 Water metal concentrations ______75 4.3.2 Sediment metal concentrations ______77
4.4 Biometric indices ______78 4.4.1 Condition factor (Cf) ______78 4.4.2 Hepatosomatic index (HSI) ______78 4.4.3 Splenosomatic index (SSI) ______78 4.4.4 Male Gonadosomatic index (GSI) ______79 4.4.5 Female Gonadosomatic index (GSI) ______79
4.5 Blood parameters ______79 4.5.1 Haematocrit (Hct) ______79 4.5.2 Leukocrit (Lct) ______80 4.5.3 Total plasma protein (TP) ______81
4.6 Health assessment index ______83
4.7 Normal histology ______85 4.7.1 Normal liver histology ______85
[3]
4.7.2 Normal kidney tissue ______88 4.7.3 Normal gill tissue ______91 4.7.4 Normal testis tissue ______93 4.7.5 Normal ovary tissue ______96
4.8 Gonadal staging ______99 4.8.1 Testis staging ______99 4.8.2 Ovary staging ______99
4.9 Histopathology ______99 4.9.1 Liver histopathology ______100 4.9.2 Kidney histopathology ______104 4.9.3 Gill histopathology ______108 4.9.4 Testis histopathology ______109 4.9.5 Ovary histopathology ______111 4.9.6 Fish index ______113
4.10 Age ______115
4.11 Age – Histopathology correlations ______118 4.11.1 Age – Liver Index Correlations ______118 4.11.2 Age – Fish Index Correlations ______118
Chapter 5: Discussion ______121
5.1 Introduction ______121
5.2 Physical water quality parameters ______121 5.2.1 Temperature ______121 5.2.2 pH ______121 5.2.3 Dissolved oxygen ______122 5.2.4 TDS/Conductivity ______122
5.3 Metal concentrations in water and sediment ______122
5.4 Biometric indices ______125 5.4.1 Condition factor (Cf) ______125 5.4.2 Hepatosomatic index (HSI) ______126 5.4.3 Splenosomatic index (SSI) ______126 5.4.4 Gonadosomatic index (GSI) ______126
5.5 Blood parameters ______127
[4]
5.5.1 Haematocrit (Hct) ______127 5.5.2 Leukocrit (Lct) ______127 5.5.3 Total plasma protein (TP) ______127
5.6 Health assessment index (HAI) ______128
5.7 Normal histology ______129 5.7.1 Liver histology ______129 5.7.2 Kidney histology ______129 5.7.3 Gill histology ______129 5.7.4 Testis histology ______130 5.7.5 Ovary histology ______130 5.7.6 Gonadal staging ______130
5.8 Histopathology ______132 5.8.1 Liver histopathology ______132 5.8.2 Kidney histopathology ______133 5.8.3 Gill histopathology ______134 5.8.4 Testis histopathogy ______135 5.8.5 Ovary histopathology ______136
5.8.6 Fish index (Ifish) values ______136
5.9 Age ______137 5.9.1 Age – Liver index correlation ______137 5.9.2 Age – Fish index correlation ______137
Chapter 6: Conclusion and recommendations ______138
Chapter 7: References ______140
Appendix A: HAI score sheet ______158
Appendix B: Histological assessment sheets ______159
Appendix C: HAI score sheet results ______164
Appendix D: Gonadal staging sheets ______172
[5]
Acknowledgements
The LORD, my God for giving me the opportunity to study and the strength to finish
My supervisor, Dr. G.M. Wagenaar for the support, guidance and motivation she provided me with throughout this project
My co-supervisor, Prof. N.J. Smit, for guidance, motivation and organization he provided during this project
Dr. J.C. Van Dyk for sharing his considerable knowledge on histology in particular as well as help in the field with fishing and dissections
Dr. M.J. Cochrane for helping with histology and other lab techniques and always having a kind word to say
Dr. R. Greenfield for help with fishing and fixing various pieces of equipment
Dr. W. Vlok for help with fishing and sharing his considerable local knowledge
Mr. C. Renshaw for help with fishing as well as keeping the rest of the team safe as our field ranger
Ms. E. Fisher for help with dissections and metal analysis
Mr. R. Gerber for help with fishing, dissections and ageing techniques
Ms. T.L. Botha for help with the histological assessments and various other lab techniques
Mr. N. Sikhakhane for help with the histological assessments and various other lab techniques and for being a true friend to me
Mr. K. McHugh for help with the histological assessments and reading of blood samples
Ms. A. Mooney for help with the histological assessments
Ms. E. Lutsch for help with histological techniques and for everything else she does for the UJ Zoology Department
Ms. L. Mokae for her advice on histology and being a source of encouragement
[6]
Mr. W. Malherbe for doing the GIS maps of the sampling sites
Ms. L. Whitlow for help with reading of blood samples
My parents, Roger and Carien for their support, both financial and otherwise, as well as motivation
The Department of Zoology at the University of Johannesburg for the use of laboratories and facilities
The National Research Foundation and the University of Johannesburg for funding.
The author would like to thank the Water Research Commission (WRC) for providing funding for this research on the Olifants and Luvuvhu Rivers in the Kruger National Park (WRC PROJECT no. K5/1922).
[7]
Abstract
Title:
A histology-based health assessment of selected fish species from two rivers in the Kruger National Park
Author:
WC Smith
Supervisors:
GM Wagenaar and NJ Smit
Contact details:
Department of Zoology
University of Johannesburg
P.O. Box 524, Auckland Park, 2006
Johannesburg, South Africa [email protected]
+27 11 559 2440
The Olifants- and Luvuvhu rivers both flow through the Kruger National Park (KNP). The Olifants River (OR) is a major tributary of the Limpopo River with water quality being less than desirable due to high concentrations of pollutants as a result of the activities in the upper catchment. The crocodile population of the river declined from 1000 in 2008 to 347 in 2009 due to pansteatitis. Labeobarbus marequensis in the upper catchment also showed symptoms of the disease (Templehoff, 2010). This raised concern about the health of the fish in the OR, particularly in the section flowing through the KNP. The Luvuvhu River (LR) is also a tributary of the Limpopo River with main land uses in the catchment being agriculture, mining and communal lands. Of concern is the finding of DDT residues in water, sediment, and domestic and indigenous biota upstream of the sites sampled in this study (Barnhoorn et al., 2009) which raised concerns about the health of fish in the lower reaches of the river flowing through the KNP. Therefore, the aim of this study was to assess the health status of
[8]
selected fish species from the OR and LR in the KNP using a histology-based health assessment protocol (HBHA).
The species selected for this study included Hydrocynus vittatus, Labeobarbus marequensis, Labeo cylindricus and Labeo rosae. The OR was sampled in September 2009 and May 2010 while the LR was sampled in November 2009 and April 2010. A necropsy was done to determine if there were any internal or external macroscopic alterations with any alterations noted on a score sheet. Blood was taken for haematocrit (Hct), leukocrit (Lct) and total plasma protein (TP) determination. As part of the HBHA, a necropsy-based health assessment index (HAI) was applied using a modified protocol of Adams et al. (1993) where the necropsy and blood parameters were used to calculate the mean and sum HAI for the different species sampled on each sampling trip. Biometric indices including the condition factor, hepatosomatic index, splenosomatic index and the gonadosomatic index were calculated. Age was determined using otoliths for tigerfish and scales for the other species. Samples of selected organs (liver, kidney, gill, testes or ovaries) were taken for histological analysis. Microscope slides were assessed qualitatively to identify any histological alterations present. These results were semi-quantitatively assessed according to the protocol of Van Dyk et al. (2009a) from which an organ index and fish index were calculated. The organ index is an indication of the number and severity of histological alterations in a particular organ of a selected species. The organ indices were classed according to the classes of Van Dyk et al. (2009a) with Class 1 (<10) being tissue with slight histological alterations; Class 2 (10-25) being tissue with moderate histological alterations. The fish index is a sum of all of the organ indices for any given fish sampled.
The fishes from the OR had overall higher HAI values compared to fishes from the LR. Higher HAI values indicate more macroscopic alterations present. Biometric indices were within the normal range, however, the hepatosomatic index values were lower than the normal range stipulated by Munshi and Dutta (1996). Haematocrit values varied with some values being above, below and in the normal range. Leukocrit values were within the normal range apart from L. marequensis from the OR in 2009 which were higher than the normal range. H. vittatus had the highest fish index values of all the species with no significant differences between sites for the species. The mean liver index values of H. vittatus from OR was 13 in 2009 and 10.33 in 2010 and was thus in Class 2, meaning the tissue had moderate histological alterations. All other mean liver index values were in Class 1, meaning the tissue had slight histological alterations, but were all 8 or higher, which is almost Class 2. All mean kidney index, gill index, testis index, and ovary index values were also in Class 1.
[9]
The results of the biometric indices, blood parameters, HAI, and histological alterations would indicate that the fish were healthy in terms of these parameters. This may be because the fish have adapted to chronic levels of pollution.
The water quality of the rivers may also be positively affected by flowing through the KNP, which is a protected area with little to no anthropogenic activities in it, as opposed to being exposed to direct anthropogenic activities outside of the park. Nevertheless, the health of fish from the OR and LR needs to be continually assessed in the future so that any early negative effects on the fish can be detected.
[10]
List of abbreviations
General content
HV – Hydrocynus vittatus
KNP – Kruger National Park
LC – Labeo cylindricus
LMar – Labeobarbus marequensis
LRos – Labeo rosae
LR – Luvuvhu River
OR – Olifants River
USEPA – United States Environmental Protection Agency
Histology based fish health assessment protocol
Cf – condition factor
GSI – gonadosomatic index
H&E – Haemotoxylin and eosin
HAI – health assessment index
Hct – haematocrit
[11]
HSI – hepatosomatic index
ICP – MS – Inductively couples mass spectroscopy
ICP – OES – Inductively coupled plasma atomic emission spectroscopy
Lct – leukocrit
NBF – neutrally buffered formalin
SSI – splenosomatic index
TDS – total dissolved salts
TP – total plasma protein
TWQR – target water quality range
Fish necropsy and histology
a – score value alt – alteration
CD – circulatory disturbances
I – inflammation
Ifish – fish index
Iorg – organ index
Iorgrp – reaction index of an organ
IS – intersex
MMC – melanomacrophage centre org – organ
PC – progressive changes
[12]
RC – regressive changes rp – reaction pattern
T – tumour w – importance factor
Metals
Al – aluminium
As – arsenic
Cd – cadmium
Cr – chromium
Cu – copper
Fe – iron
Mn – manganese
Pb- lead
Zn – zinc
[13]
List of figures
Chapter 2
Figure 2.1: Olifants River within the KNP______24
Figure 2.2: Luvuvhu River within the KNP______25
Figure 2.3: Tigerfish (Hydrocynus vittatus) ______34
Figure 2.4: Lowveld Largescale Yellowfish (Labeobarbus marequensis) ______36
Figure 2.5: Redeye Labeo (Labeo cylindricus) ______37
Figure 2.6: Rednose Labeo (Labeo rosae) ______38
Chapter 3
Figure 3.1: Map of the Kruger National Park (figure adapted from http://www.accomodation- in-kruger-park.com/images/maps/map_of_krugerpark.gif), highlighting the Olifants – and Luvuvhu Rivers in red______58
Figure 3.2: Map of the Olifants River in the Kruger National Park______59
Figure 3.3: Map of the Luvuvhu River in the Kruger National Park______59
Figure 3.4: Fish sampling and measurement______62
Figure 3.5: Necropsy and blood collection______63
Figure 3.6: Histological processing and assessment______66
[14]
Chapter 4
Figure 4.1: Mean Haematocrit values for all species from all sampling trips ______80
Figure 4.2: Mean Leukocrit values for all species from all sampling trips ______81
Figure 4.3: Mean Total Protein values for all species from all sampling trips ______82
Figure 4.4: Mean Health Assessment Index values for all species from all sampling trips______84
Figure 4.5: Normal liver histology showing hepatic parenchyma ______86
Figure 4.6: Normal liver histology showing hepatopancreatic tissue and bile ducts______87
Figure 4.7: Normal kidney histology showing renal corpuscles ______89
Figure 4.8: Normal kidney histology showing tubules and thyroid follicles ______90
Figure 4.9: Normal gill histology ______92
Figure 4.10: Normal testis histology showing interstitial tissue and interstitial tissue ___94
Figure 4.11: Normal testis histology showing spermatocytes, spermatogonia and spermatids______95
Figure 4.12: Normal ovary histology showing oogonia and peri-nucleolar oocytes______97
Figure 4.13: Normal ovary histology showing cortical alveolar, vittelogenic and maturation oocytes ______98
Figure 4.14: Liver histopathology ______101
Figure 4.15: Mean liver index values for all species from all sampling trips ______103
Figure 4.16: Kidney histopathology ______106
Figure 4.17: Mean kidney index values for all species from all sampling trips______107
Figure 4.18: Monogean parasite on the gills of L. marequensis (40X) ______108
Figure 4.19: Melanomacrophage centres in testis tissue of L. rosae (40X) ______109
[15]
Figure 4.20: Mean testis index values for all species from all sampling trips ______110
Figure 4.21: Melanomacrophage centres in ovary tissue of H. vittatus ______111
Figure 4.22: Mean ovary index values for all species from all sampling trips ______113
Figure 4.23: Mean fish index values for all species from all sampling trips ______114
Figure 4.24: Age – liver index correlation - Hydrocynus vittatus ______119
Figure 4.25: Age – liver index correlation - Labeobrabus marequensis ______119
Figure 4.26: Age – fish index correlation - Hydrocynus vittatus ______120
Figure 4.27: Age – fish index correlation - Labeobarbus marequensis ______120
[16]
List of Tables
Chapter 2
Table 2.1: Description of common liver histopathological alterations ______44
Table 2.2: Liver histopathological alterations found in literature ______45
Table 2.3: Description of common kidney histopathological alterations ______47
Table 2.4: Kidney histopathological alterations found in literature______48
Table 2.5: Description of common gill histopathological alterations______50
Table 2.6: Gill histopathological alterations found in literature______51
Table 2.7: Testis histopathological alterations found in literature______52
Table 2.8: Ovary histopathological alterations found in literature______53
Chapter 3
Table 3.1: Olifants River sites and GPS co-ordinates ______57
Table 3.2: Luvuvhu River sites and GPS co-ordinates______57
Table 3.3: Gonadal reproductive stage determination ______68
Chapter 4
Table 4.1: Physical water quality parameters results ______75
Table 4.2: Metal concentrations in Olifants River (OR) water ______76
[17]
Table 4.3: Metal concentrations in Luvuvhu River (LR) water ______76
Table 4.4: Metal concentrations in Olifants River (OR) sediment ______77
Table 4.5: Metal concentrations in Luvuvhu River (LR) sediment ______77
Table 4.6: Summary of the mean values for mass, length and Cf for the different fish species at the corresponding sites ______78
Table 4.7: Summary of biometric indices of all specimens ______79
Table 4.8: Liver frequency of alterations (Olifants River) ______99
Table 4.9: Liver frequency of alterations (Luvuvhu River) ______99
Table 4.10: Kidney frequency of alterations (Olifants River) ______102
Table 4.11: Kidney frequency of alterations (Luvuvhu River) ______102
Table 4.12: Testis frequency of alterations ______104
Table 4.13: Ovary frequency of alterations (Olifants River) ______105
Table 4.14: Ovary frequency of alterations (Luvuvhu River)______109
Table 4.15: Hydrocynus vittatus Olifants 2009 Age ______112
Table 4.16: Hydrocynus vittatus Olifants 2010 Age ______112
Table 4.17: Hydrocynus vittatus Luvuvhu 2009 Age ______115
Table 4.18: Hydrocynus vittatus Luvuvhu 2010 Age ______115
Table 4.19: Labeobarbus marequensis Olifants 2009 Age ______116
Table 4.20: Labeobarbus marequensis Olifants 2010 Age ______116
Table 4.21: Labeobarbus marequensis Olifants 2009 Age ______117
Table 4.22: Labeobarbus marequensis Olifants 2010 Age ______117
[18]
Chapter 1: General Introduction
1.1 Introduction South Africa is an arid country, having only 8.6% of rainfall available as surface water. South Africa‘s groundwaters are also relatively small compared to world averages (Walmsey et al., 1999). Further problems are created by this water not being evenly distributed throughout the country and variability of water resources over seasons. As a result of scarce water resources and variable hydrological conditions, in many catchments the need for water exceeds the supply (Walmsey et al., 1999). The condition of a river system which includes biodiversity is a direct reflection of the conditions in its catchment (Ashton, 2007) and even rivers which are within protected areas are impacted by activities in their catchment since those rivers are generally not fully contained in those protected areas (Nel et al., 2007). Fish can be used as indicators of ecosystem health as they are widespread; important to their ecosystem; have a long generation time; have economic value; are easy to identify; are easy to sample and have good background information relating to pollution tolerance and response to disturbance (Resh, 2008).
1.2 Study motivation The Kruger National Park (KNP) is an important tourist destination in South Africa and the value of river conservation within the park also has economic benefits (Turpie and Joubert, 2001). Two rivers which flow through the KNP were chosen for this study, namely the Olifants and Luvuvhu Rivers. Both of these rivers have upstream activities which are likely to affect the water quality and consequently the health of organisms living in those rivers. The study sites for this project are situated within the KNP. Although the sites are within a protected environment, the activities upstream of the selected sites could have an effect on the water quality and ecosystem health of the river sections in the KNP. The activities in the upper reaches of the Olifants River include extensive mining (Ashton, 2010). In 2008 crocodiles in the Olifants River gorge in the KNP started dying of pansteatitis and this raised concern about the water quality as well as the health of fish in the river not far upstream (Van Vuuren, 2009). The Luvuvhu River was selected as the more natural of the two rivers; however, this river is also affected by pollution with DDT residues being found in the water and fish not far from where the river flows into the KNP (Barnhoorn et al., 2009).
[19]
A necropsy of fish by observing and noting internal and external macroscopic changes would give an indication of the environmental stress of a fish population (Adams et al., 1993). Histopathological alterations are biomarkers of effect exposure to environmental stressors, revealing prior alterations in physiological and or biochemical function (Hinton et al., 1992). This biomarker also incorporates biotic factors as well as water quality, therefore it is able to give a more holistic view of fish health (Handy et al., 2002). The histological assessment would show changes before they manifest macroscopically and can therefore be used as an early warning system for pathological changes in the fish tested (Marchand et al., 2008). Histopathology-based bio-assessment studies showed liver alterations (Marchand et al., 2008) and gill alterations (Van Dyk et al., 2009a) in Clarias gariepinus from a polluted urban nature reserve in South Africa. By using the protocol of Bernet et al. (1999) adapted by Van Dyk et al. (2009a) histological alterations noted can be quantified to allow for meaningful comparison between species, sites, as well as between organs to a certain extent. Together with a necropsy, organosomatic indices, the quantified histological assessment (Bernet et al., 1999) adapted by Van Dyk et al. (2009a) would give an indication of the health of the fish sampled. The identification of tissue lesions requires a baseline appreciation of normal tissue conditions (Yonkos et al., 2000) and for this reason a brief description of the normal histological features of selected organs (liver, kidney, gills, testis, ovary) of H. vittatus L. marequensis, L. cylindricus and L. rosae specimens was also done.
The fishes selected for this study include the tigerfish (Hydrocynus vittatus) which is an important predator which feeds on fish (Skelton, 2001). Numbers of tigerfish have declined due to water abstraction, pollution, obstructions such as dams and weirs and fishing pressure (Steyn et al., 1996; Skelton, 2001) and has been included on the protected and threatened (TOPS) species list of South Africa (DEAT, 2007). The Lowveld largescale yellowfish (Labeobarbus marequensis) above 110 mm feeds on insects (Fouche et al., 2003) and although this species is regarded as being non-sensitive (Kleynhans, 1991) the species is vulnerable to reduction in flow rates and increased levels of siltation, especially at breeding sites (Fouché and Gaigher, 2001). The Redeye Labeo (Labeo cylindricus) feeds on algae and ―aufwachs‖ from the surface of rocks (Skelton, 2001). The Rednose Labeo (Labeo rosae) feeds on detritus, algae and small invertebrates (Skelton, 2001). The four species sampled would give an indication of the health status of fish from different trophic levels in the ecosystems of the Olifants and Luvuvhu Rivers so that the effects of possible bioaccumulation of pollution in the rivers studied could be determined.
[20]
1.3 Hypotheses There will be a difference in the histology-based health status of the fish of the Olifants – and Luvuvhu River, with the Olifants River being the most impacted river and thus the more affected fish, with regards to fish health.
There will be a difference in the histology-based health status of the fish species sampled, with H. vittatus as top predators being the most affected species in terms of the HAI and histopathological biomarkers.
1.4 Aim of the study In order to test the above hypotheses, the aim of the study is to:
Determine the health status of four species of fish from the Olifants – and Luvuvhu Rivers using a histology-based health assessment protocol.
1.5 Objectives In order to achieve the aim of this study, the following objectives were set:
Assess the physical and chemical parameters of the water in the Olifants and Luvuvhu Rivers.
Compare the health status of fish from the Olifants – and Luvuvhu rivers to each other using a histology-based health assessment protocol
Compare health status of fish from species in different trophic levels to each other using a histology-based health assessment protocol
Describe the normal histological structures of H. vittatus, L. marequensis, L. cylindricus and L. rosae specimens
1.6 Dissertation outline Chapter 2 is a review of literature relevant to the study. Literature regarding both rivers sampled as well as the fish species sampled is included. Background information about water quality variables and metals is also included. An explanation of biometric indices, blood constituents and health assessment index values is also given. The role of histopathology as a biomarker is discussed with more detail given to specific organs studied in this project (liver, kidney, gills, testis, ovary).
[21]
Chapter 3 is a description of the materials and methods used in this study. It includes a description of work done in the field and the laboratory. Field work done includes the taking of physical water quality parameters and the collection of water and sediment for chemical analysis. The collection of fish, calculation of biometric indices (condition factor, hepatosomatic index, splenosomatic index and gonadosomatic index), blood collection and analysis of constituents (haematocrit, leukocrit and total plasma protein) and necropsy as well as collection of histological samples are described. Histological processing and the protocol for the quantitative (normal and alterations) and semi-quantitative assessments are described. Ageing techniques and statistical analysis is also described.
Chapter 4 is the results obtained in this study. The results include physical and chemical water quality, biometric indices, blood constituents analysis, health assessment index, qualitative and semi-quantitative histological assessments as well as ageing and age – histopathology correlation.
Chapter 5 is the discussion of the results obtained. All of the results are discussed and related to similar work to give a clear picture of the health of the fish studied according to the parameters studied.
Chapter 6 is the conclusion and recommendations. The results are concluded and the main outcomes are summarised. Recommendations for future studies in a similar field are also set out.
Chapter 7 is the references. The references used in the preceding six chapters are listed here.
[22]
Chapter 2: Literature Review
2.1 Introduction As mentioned in Chapter 1, this chapter is a review of literature relevant to the study. Literature regarding both rivers sampled as well as the fish species sampled is included. Background information about water quality variables and metals is also included. An explanation of biometric indices, blood constituents and health assessment index values is given. The normal histology of selected organs (liver, kidney, gills, testis, ovary) in teleosts is discussed. The role of histopathology as a biomarker is discussed with a motivation for the selection of the target organs studied (liver, kidney, gills, testes, ovaries).
2.2 Study Sites As mentioned previously, the Kruger National Park (KNP) is a popular international tourism destination and thus generates much revenue from tourism. The recreational use value of the KNP will decrease if the river quality decreases (Turpie and Joubert 2001) which will result in a loss of revenue. This gives more importance to the determination of the health of rivers in the park and the subsequent management of those rivers. Two rivers were chosen for this study, namely the Olifants - and Luvuvhu River. Both rivers have upstream activities which are likely to affect the water quality and consequently the health of organisms living in those rivers.
2.2.1 Olifants River (OR) The Olifants River (OR) (Fig. 2.1) is a major tributary of the Limpopo River. The river rises in Trichardt, East of Johannesburg, in Gauteng and flows Northeast, through the provinces of Mpumalanga and Limpopo, into Mozambique (McCartney and Arranz, 2007). The region is a lowland area (200-600 m amsl) with rolling plains, moderate rainfall (400-800 mm per year) and high temperatures (20-22 °C and above). The water quality in the area studied in the KNP was said to be lower than desirable, with high concentrations of dissolved salts having accumulated due to the activities in the upper reaches of the catchment (WRC, 2001a).
[23]
Figure 2.1: Olifants River within the Kruger National Park, view from the Olifants rest camp
The Massingire dam in Mozambique causes the river‘s flow to decrease during floods and results in sediment being deposited in the Olifants River gorge (WRC, 2001a). The upper reaches of the catchment is influenced by agriculture, large-scale coal mining, small dams, power-generation plants, alien vegetation and fauna as well as several towns and smaller urban centres (WRC, 2001a; Driescher, 2008; Ashton et al., 2010). The middle reaches of the catchment contain extensive areas of irrigated agriculture as well as several platinum, chrome and vanadium mines, two ferro-chrome refineries and numerous smaller urban centres (Ashton, 2001; 2010). The lower reaches of the catchment contain several small mines and the Cu and phosphate mining around the Phalaborwa area (Mussagy, 2008; Ashton, 2010). High sediment loads have been attributed to industrial and mining activities which accumulate in the Phalaborwa barrage and are flushed out from time to time. This causes large quantities of sediment to be released causing severe damage to in-stream habitats and biota in the downstream part of the OR. Fish have also been known to die from oxygen depletion or being smothered by silt clogging their gills (WRC, 2001a). Despite a new Cu mine being opened close to Phalaborwa, water quality monitoring has not been
[24]
systematically (De Villiers and Mkwelo, 2009). Many studies have been done showing the effects of the activities on the water quality in the OR in the KNP and its subsequent effect on fish. Metals found in fish in the OR include: Mn (Seymore et al.,1995; Robinson and Avenant-Oldewage, 1997), Pb (Seymore et al.,1995), strontium (Seymore et al.,1995), Cr (Robinson and Avenant-Oldewage, 1997; Marx, 2000), Cu (Robinson and Avenant- Oldewage, 1997; Kotze et al.,1999; Marx, 2000; Coetzee et al., 2002), Fe (Robinson and Avenant-Oldewage, 1997; Avenant-Oldewage and Marx, 2000) and Zn (Kotze et al.,1999; Coetzee et al., 2002). Of great concern was the decline of the crocodile population in the river in the KNP from 1000 in 2008 to 347 in 2009 due to pansteatitis with Labeobarbus marequensis also showing effects of the disease (Templehoff, 2010).
2.2.2 Luvuvhu River (LR)
Figure 2.2: Luvuvhu River within the Kruger National Park, view of site 2.
The Luvuvhu River (LR) (Fig. 2.2) rises as a steep mountain stream in the southeasterly slopes of the Soutpansberg Mountains (WRC, 2001b) and drains an area of 5940 km² (Jewitt et al., 2004). The section of the LR surrounded by the KNP is desired to be in a natural state and is largely unspoilt (WRC, 2001b) with the biggest threats to the area being from influences outside the KNP e.g. flow regulation and reduction, increased silt loads and spreading of alien plants (WRC, 2001b). Land-uses in the catchment include state and [25]
private forestry plantations (pine, eucalyptus and wattle) as well as irrigated dryland agriculture, woodland, conservation, mining and communal lands (Jewitt et al., 2004). According to Griscom et al. (2010), considerable land cover changes have occurred in the LR catchment in the last two decades with these changes being associated with human population growth as well as changes in winter river baseflows and increased episodes of river drying within the KNP. As water demands increase in the future, river drying will be the greatest threat to riparian and riverine communities along the LR within the KNP (Kleynhans, 1996) as cited by (Griscom et al., 2010). Of concern is the finding of DDT residues in water, sediment, domestic and indigineous biota upstream of the sites sampled (Barnhoorn et al., 2009).
2.3 Metals in the aquatic environment Metals are found naturally in aquatic ecosystems or as a result of human activities. The concentrations vary from trace metals e.g. Cd and Hg to major metals e.g. Ca, Mg and Na. The effects of these metals range from beneficial (Ca, Zn) through troublesome to being dangerously toxic in some case (Pb, Hg). The toxicity of metals depends on their concentrations and physiological behaviour (Galvin, 1996).
2.3.1 Aluminium (Al)
Aluminium is one of the most abundant metals and the third most abundant element, after oxygen and silicon, in the earth's crust. It is widely distributed and constitutes 8.8% of the earth‘s crust (ATDSR, 2006). It occurs primarily as aluminosilicate minerals which are too insoluble to participate readily in bio-geochemical reaction. Aluminium is described as a non- critical element, though there is growing concern over the effects of elevated concentrations of Al in the environment, primarily that mobilized as a result of acid mine drainage and acid precipitation. Aluminium can be mobilised from soils and sediments by both natural weathering and accelerated acidification processes, resulting in detectable concentrations in surface waters. Aluminium is found in soluble forms mainly in acid mine drainage waters and is also of concern in natural waters affected by acid rain. Aluminium is one of the principal particulates emitted from the combustion of coal, and aluminium fluoride is emitted from Al smelters. Industries using Al in their processes or in their products include the following: the paper industry; the metal construction industry; the leather industry; and the textile industry. In addition to liquid effluents that may be generated from the above industries, alum or aluminium sulphate is used in most water treatment processes as a flocculating agent for
[26]
suspended solids, including colloidal materials, micro-organisms and "humic rich" dissolved organics (DWAF, 1996).
Elevated concentrations of bio-available Al in water are toxic to a wide variety of organisms. The mechanism of toxicity in fish seems to be related to interference with ionic and osmotic balance and with respiratory problems resulting from coagulation of mucous on the gills of fish (DWAF, 1996; Abdel-Latif, 2008) and has been found to cause severe fusion of lamellae and filaments in the gills (Abdel-Latif, 2008). Al is considered to be an endocrine disrupting chemical in mature Oreochromis niloticus (Nile tilapia) females (Correia et al., 2010). Fish exposed to Al showed significantly higer total erythrocyte counts; haematocrit (Hct); mean cell haemoglobin concentration (MCHC) and mean cell haemoglobin (MCH) while mean corpuscle volume (MCV) was significantly lower (Alwan et al., 2009). Salmo salmar (Atlantic salmon) smolts exposed to acid and moderate to high Al concentrations showed impaired seawater tolerance which resulted from extensive gill Al accumulation; damage to the epithelium; reduced mitochondria-rich cells and protein abundance and a synergistic stimulation of apoptosis in the gill upon seawater exposure (Monette et al., 2010). Blue mussels (Mytilus edulis) offshore of an Al smelter showed tubular dilation or atrophy; melanomacrophage centres; large lipid vacuoles in basophilic cells and eosinophilic bodies in digestive cells (Aarab et al., 2008).
2.3.2 Arsenic (As) Arsenic is a metalloid element and is one of the most important and concerning global toxicants (Bears et al., 2006; Gonzalez et al., 2006). It is common in a great variety of minerals and has a mean concentration of 2 mg/kg in the earth‘s crust (Galvin, 1996). It is toxic to marine and freshwater aquatic life and is a known carcinogen. Elemental As is insoluble in water, although many of its compounds are highly soluble. The United States Environmental Protection Agency (USEPA) has classified As as "very toxic and relatively accessible" to aquatic organisms (DWAF, 1996). Elemental As is found to a limited extent in nature, mostly as a result of weathering of As-containing rocks and of volcanic activity. Arsenic may occur at high concentrations in water bodies subject to industrial pollution, or in the vicinity of industrial activities utilising or discharging As or arsenic compounds (DWAF, 1996; Galvin, 1996). Manufacturers that use As in their processes, or in their products, include: the mining industry; the metal processing industry; producers of pesticides and fertilizers; producers of glass and ceramics; tanneries; dye manufacturers; producers of wood preservation products; the chemical industry and producers of detergents (DWAF, 1996). Fish appear to be particularly susceptible to aquatic As toxicity as they are continually
[27]
exposed to it through their gills and intake of As-contaminated food (Ahmed et al., 2008). Arsenic has been found to cause the head kidney cells to be swollen with intercellular oedema in Clarias batrachus (Walking catfish) (Ghosh et al., 2007) while vacuolation has also been found in Clarias batrachus (Walking catfish) exposed to As (Datta et al., 2009). Arsenic was also found to cause a depletion of lymphocytes and melano-macrophage centres in Clarias batrachus (Walking catfish) (Ghosh et al., 2007; Datta et al., 2009). Medaka (Oryzias latipes) exposed to As had a reduction in hatching success as well as reduction in time to hatching (Ishaque et al., 2004).
2.3.3 Cadmium (Cd) Cadmium is a trace element in the earth‘s crust which is generally associated with Zn, Pb and copper sulphide ore bodies (Galvin, 1996; DWAF, 1996). Cadmium is a metal with no known beneficial properties that support life and there is no evidence that it is either biologically necessary or beneficial (Eisler, 1985; Eisler, 2000; Nordberg et al., 2007; ATDSR, 2008). Cadmium is defined by the USEPA as potentially hazardous to most forms of life, and is considered to be toxic and relatively accessible to aquatic organisms (DWAF, 1996). In one study that used comparative acute toxicity testing of 63 heavy metals, Cd was the most toxic metal (Borgmann et al., 2005). Cadmium is present in the earth's crust at an average concentration of 0.2 mg/kg. Due to its abundance, large quantities of Cd enter the global environment annually as a result of natural weathering processes. Cadmium is found at trace concentrations in fresh waters and mostly a result of industrial activity. The main sources of Cd in the environment are due to: emissions to air and water from mining, metal (Zn, Pb and Cu) smelters, and industries involved in manufacturing alloys, paints, batteries and plastics; agricultural use of sludges, fertilizers and pesticides containing Cd; burning of fossil fuels (very limited effect); and the deterioration of galvanized materials and Cd-plated containers (DWAF, 1996).
Because it is a nondegradable, cumulative pollutant, continued releases are of global concern (ATDSR, 2008). Cadmium accumulates in the kidney, liver and gills of freshwater fish (Dallinger et al., 1997; Chowdry et al., 2004). Cadmium accumulation in these organs appears to be related to the presence of Cd-binding molecules called metallothionens (Dallinger et al., 1997). Cadmium is an endocrine disrupter (Vettillard and Bailhache, 2004) and has been shown to interfere with the formation of steroids, eggs and sperm in rainbow trout (Oncorynchus mykiss) and it alters hormone synthesis in testes. In carp (Cyprinus carpio) it inhibits steroid formation and ovarian formation (Vettillard and Bailhache, 2004). Exposures to low levels of Cd can cause DNA damage and stress in common carp
[28]
(Cyprinus carpio) (Jia et al., 2010). Cadmium toxicity has been shown to cause gill alterations such as hypertrophy and vacuolation in fish exposed to a solution of Cd as well as Cu, nickel and iron (Pandey et al., 2007). Oreochromis mossambicus exposed to a mixture of Cd and Zn showed liver alterations in the form of hyalinisation, hepatocyte vacuolation, cellular swelling and congestion of blood vessels (Van Dyk et al., 2007). Radhakrishan and Hemalatha (2010) found cytoplasmic vacuolation of hepatocytes, blood vessel congestion, inflammatory leucocytic infiltration and necrosis in fish exposed to Cadmium chloride.
Histological alterations were observed in O. mossambicus liver (structural alterations; congestion of blood vessels and sinusoids; granular degeneration of hepatocytes; fat accumulation; intercellular deposits; nuclear alterations; necrosis; hypertrophy of hepatocytes; mild inflammation), gills (general loss of gill lamellar structure; vacuolation of primary lamellar epithelium; intercellular deposits; hyperplasia of primary and secondary epithelium; enlargement of mucous cells; increase of mucous secretion), ovaries (deposits in interstitial tissue) and testes (vacuolation of spermatogonia; spermatocyte plasma alterations; vacuolated spermatocytes; hypertrophy of interstitial tissue; tumour) after exposure to sublethal Cd (Ackermann, 2008).
2.3.4 Chromium (Cr) Chromium is a relatively scarce metal, and the occurrence and amounts thereof in aquatic ecosystems are usually very low (DWAF, 1996). Hexavalent chromium (Cr6+) is a non- essential element and considered to be toxic because of its powerful oxidative potential and ability to cross cell membranes (Eisler, 2000). Hexavalent Cr salts are used extensively: in metal pickling and plating; in the leather industry as tanning agents; and in the manufacture of paints, dyes, explosives, ceramics and paper (DWAF, 1996). Oncorynchus tshawytscha (Chinook salmon) exposed to Cr6+ showed lipid droplets in the liver; increase in gill epithelium; apoptosis of chloride cells; hypereosinophillic chloride cell cytoplasm; pyknosis; karyorrhexis; necrosis of kidney tubules and gross alterations to kidney and spleen (Farag et al., 2006). In another study on the Indian major carp (Labeo rohita) exposed to 96 h LC50 (Lethal concentration, 50%) concentration of hexavalent Cr (39.40 mg/L), the fish showed degeneration of secondary lamellae; hyperplasia of lamellar cells and atrophy of the central axis at the end of exposure (Sesha and Rao, 1998).
Trivalent Cr salts are used much less frequently, but are important as: fixatives in textile dye manufacture; in the ceramic and glass industry; and in photography. Chromium compounds
[29]
may also be discharged in Cr-treated cooling waters where Cr has been used as a corrosion inhibitor (DWAF, 1996). Industrial activity results in elevated levels of Cr in aquatic ecosystems and Cr is associated with the combustion of fossil fuels (Schmitt et al., 2004). Chromium is an essential trace metal involved in glucose metabolism as an insulin co-factor and has a function of increasing protein and amino acid uptake by cells (Galvin, 1996). Sublethal exposure to hexavalent Cr has caused histopathological alterations in the gill, liver and kidney in Channa punctatus (Spotted snakehead) (Mishra and Mohanty, 2008) and liver and kidney alterations in Carassius auratus (Goldfish) (Velma and Tchounwou, 2010). Oreochromis mossambicus showed histological alterations in the liver (congestion of blood vessels; granular degradation of hepatocytes; fat accumulation; intercellular deposits; increase in melano-macrophage centres; nuclear alterations and necrosis), gills (vacuolation of primary lamellar epithelium; intercellular deposits; hyperplasia of primary lamellar epithelium), ovaries (deposits in interstitial tissue) and testes (vacuolation of spermatogonia; hypertrophy of spermatocytes; vacuolation of spermatocytes) after exposure to sublethal Cr (Ackermann, 2008).
2.3.5 Copper (Cu) Copper is one of the world's most widely used metals (DWAF, 1996). Although Cu occurs naturally in most waters, it is regarded as potentially hazardous by the USEPA. Crustal (igneous) rocks contain more Cu (23 - 55 mg/kg) than sedimentary rocks (4 - 45 mg/kg). The occurrence of natural sources of Cu in the aquatic environment is due to weathering processes or from the dissolution of Cu minerals and native Cu. Anthropogenic sources account for 33 - 60 % of the total annual global input of Cu to the aquatic environment. The main anthropogenic sources of Cu in the aquatic environment are: corrosion of brass and Cu pipes by acidic waters; sewage treatment plant effluents; Cu compounds used as aquatic algicides; runoff and ground water contamination from the use of Cu as fungicides and pesticides in the treatment of soils; and liquid effluents and atmospheric fallout from industrial sources such as mining, smelting and refining industries, coal-burning, and iron- and steel-producing industries (DWAF, 1996). Elevated Cu levels in the aquatic environment are also related to the combustion of fossil fuels (Schmitt et al., 2004).
Exposure to sublethal levels of Cu has been shown to cause histopathological alterations in gills (oedema; vasodilation of the lamellar vascular axis) and livers (vacuolation and necrosis) of nile tilapia (Oreochromis niloticus) (Figueiredo-Fernandes et al., 2007). Rainbow trout (Oncorynchus mykiss) exposed to Cu sulphate for 28 days showed histological alterations in the liver (non-homogenous regions; congestion of the central vein; dark-stained [30]
hepatocytes; increasing number of Kupffer cells; vascular degeneration and sinusoidal degenerations) (Atamanalp et al., 2008). Oreochromis mossambicus exposed to Cu showed histopathological alterations in the testes (detachment of the basement membrane; testicular haemorrhage necrosis; disorganisation of lobules; pyknosis; disintegration of primary spermatogonia and disintegration of interstitial tissue) (Pieterse, 2004).
2.3.6 Iron (Fe) Iron is the fourth most abundant element in the earth's crust (Galvin, 1996) and may be present in natural waters in varying quantities depending on the geology of the area and other chemical properties of the water body. Although Fe has toxic properties at high concentrations, inhibiting various enzymes, it is not easily absorbed through the gastro- intestinal tract of vertebrates (DWAF, 1996). On the basis of Fe's limited toxicity and bio- availability, it is classified as a non-critical element. Iron is an essential micronutrient for all organisms, and is required in the enzymatic pathways of chlorophyll and protein synthesis, and in the respiratory enzymes of all organisms (Luschak, 2011). It also forms a basic component of haeme-containing respiratory pigments (for example, haemoglobin), catalyses, cytochromes and peroxidises. Fish obtain Fe from water by uptake across the gill epithelium (Bury et al., 2003). Under certain conditions of restricted availability of Fe, photosynthetic productivity may be limited. Iron is naturally released into the environment from weathering of sulphide ores (pyrite, FeS) and igneous, sedimentary and metamorphic rocks (DWAF, 1996). Iron is also released into the environment by human activities, mainly from the burning of coke and coal, acid mine drainage, mineral processing, sewage, landfill leachates and the corrosion of iron and steel. Various industries that also use Fe in their processes, or in their products, include: the chlor-alkali industry; the household chemical industry; the fungicide industry; the petro-chemical industry. South Africa has extensive gold, uranium and coal mines, drainage from which potentially affects many of our water bodies. Streams may be negatively impacted by high levels of Fe in acid mine drainage. Pyrite, Fe sulphide, is often found in close association with coal deposits (DWAF, 1996). Clarias gariepinus showed restricted growth when fed an Fe-rich diet (Baker et al., 1997).
2.3.7 Lead (Pb) Lead is a common and toxic trace metal which readily accumulates in living tissue. Lead exists in several oxidation states, all of which are of environmental importance. Lead is defined by the USEPA as potentially hazardous to most forms of life, and is considered toxic and relatively accessible to aquatic organisms (DWAF, 1996; USEPA, 2006). Its
[31]
accumulation in tissues may cause several health hazards including neurotoxicity, haemotoxicity and reproductive disturbances (Rodmilans et al., 1996).
Lead is principally released into the aquatic environment through the weathering of sulphide ores, especially galena. Since metallic Pb and common Pb minerals such as sulphides, sulphates, oxides, carbonates and hydroxides are almost insoluble, levels of dissolved Pb (acetate and chloride salts) in aquatic ecosystems are generally low (DWAF, 1996). Most of the Pb entering aquatic ecosystems are associated with suspended sediments, while Pb in the dissolved phase is usually complexed by organic ligands. The major sources of Pb in the aquatic environment are anthropogenic, these include: precipitation, fallout of Pb dust and street runoff (associated with Pb emissions from gasoline-powered motor vehicles); industrial and municipal wastewater discharge; mining, milling and smelting of Pb and metals associated with Pb, e.g. Zn, Cu, Ag, As and Sb; and combustion of fossil fuels (DWAF, 1996).
There is no demonstrated biological need for Pb, thus uptake and toxicity is likely mediated by the mimicry of other cations (Ballatori et al., 2002). Exposure to low levels of Pb has been associated with behavioural abnormalities, learning impairment, decreased hearing and impaired cognitive function in humans and in experimental animals (Adonalyo and Oteiza, 1999). At high levels it causes damage to almost all organs, especially the central nervous system, kidneys and blood, which culminates in death (Tong et al., 2000). Clarias gariepinus exposed to sublethal Pb had gill histopathological alterations such as occlusion of interlamellar spaces; shrinkage of cartilaginous supporting mass; epithelial cell lysis; intracellular vacuolation; oedema; reduced pillar cell size and epithelial attachment (Olojo et al., 2005). Liver alterations found in C. gariepinus exposed to sublethal concentrations of Pb were: hepatic cirrhosis; detached bile connective tissue; parenchyma degeneration; increase of fibro-connective tissue; blood sinusoid congestion and necrosis (Olojo et al., 2005).
2.3.8 Manganese (Mn) Manganese is an essential micronutrient for plants and animals (DWAF, 1996) and is widely diffused in nature (Galvin, 1996) making up about 0.1% of the earth‘s crust (Lin et al., 2006). It is a functional component of nitrate assimilation and an essential catalyst of numerous enzyme systems in animals, plants and bacteria and is needed for the regulation of reproduction and normal brain functions (Lin et al., 2006). Manganese is the eighth most abundant metal in nature, and occurs in a number of ores (DWAF, 1996). Soils, sediments and metamorphic and sedimentary rocks are significant natural sources of Mn. Industrial [32]
discharges also account for elevated concentrations of Mn in receiving waters and various industries use Mn, its alloys and Mn compounds in their processes, or in their products, examples of which are given below: the steel industry, in the manufacture of dry cell batteries; the fertilizer industry (Mn is used as a micro-nutrient fertilizer additive); and the chemical industry in paints, dyes, glass, ceramics, matches and fireworks (DWAF, 1996). Acid mine drainage also releases a large amount of the Mn. Iron and steel foundries release Mn into the atmosphere, where it is then redistributed through atmospheric deposition (DWAF, 1996).
Manganese exposure can cause cell death which may eventually lead to necrosis, even if Mn-treated cells have have activated apoptosis signalling pathways (Roth and Garrick, 2003). Prolonged exposure to 75 µmol/L and 200 µmol/L of Mn for 48h will induce severe effects in lifespan, development and reproduction in nematodes possibly by affecting the oxidative stress response and metal accumulation and/or bioavailability (Jing et al., 2009). It has been shown that Mn, due to its mitochondrial interactions, induces apoptosis in neuronal cells (Hirata, 2002; Kitazawa et al., 2005). Apoptosis of both the haematopoietic precursor cells and circulating haemocytes seems to be an important contributing factor to the Mn induced haemocytopenia in Nephrops norvegicus (Oweson et al., 2006).
2.3.9 Zinc (Zn) Zinc is an element which occurs frequently being associated with Fe, Cu, Cd and Fe Sulphides (Galvin, 1996). Zinc occurs in rocks and ores and is readily refined into a pure stable metal. It can enter aquatic ecosystems through both natural processes such as weathering and erosion, and through industrial activity. The greatest dissolved Zn concentrations will occur in water with low pH, low alkalinity and high ionic strength. Chemical speciation of Zn is affected primarily by pH and alkalinity. Soluble Zn salts occur readily in industrial wastes (DWAF, 1996). The carbonate, hydroxide and oxide forms of Zn are relatively resistant to corrosion and are used extensively in the following industries: metal galvanising; dye manufacture and processing; pigments (paints and cosmetics); pharmaceuticals; and fertilizer and insecticide (DWAF, 1996).
Zn is an essential micronutrient in fish, but elevated concentrations of waterborne Zn interfere with ion transport at the gill surface. Specifically, Zn acts primarily via competitive inhibition of lamellar chloride cells, reducing the ability of the gills to take up essential Ca, eventually leading to potentially lethal hypocalcemia (Spry and Wood, 1985; Hogstrand et al., 1994; Hogstrand and Wood, 1995; Hogstrand et al., 1996). Zinc exposure has been [33]
shown to induce histopathological alterations in kidney tissue of Channa punctatus (Spotted snakehead) (dilated renal tubules; separation of renal tubule epithelial lining; oedema of renal tubule; hypertrophied nuclei of renal tubule cells; glomerulus vacuolation; disorganised blood capillaries of glomerulus; necrosis and pyknotic nuclei in mesenchymal tissue (Gupta and Srivasta, 2006)), liver tissue of Labeo rohita (Rohior rohu) (severe necrosis; haemorrhage; distended sinusoids and minor vacuolations (Loganathan et al., 2006); liver tissue of Oreochromis mossambicus (Mozambique tilapia) (hyalinisation; hepatocyte vacuolation; cellular swelling and congestion of blood vessels (Van Dyk et al., 2007)) and ovarian tissue of Tilapia nilotica (Nile tilapia) (degeneration and hyperaemia (Carino and Cruz, 1990)).
2.4 Study organisms
2.4.1 Hydrocynus vittatus Class: Actinopterygii
Order: Characiformes
Family: Alestidae
Species: Hydrocynus vittatus
Common name: African tigerfish
Figure 2.3: Tigerfish (Hydrocynus vittatus)
The tigerfish is one of the most sought after sport fishes on the African continent (Gaigher, 1970). The body of H. vittatus is fusiform with pointed fins and a deeply forked caudul fin (Skelton, 2001). The head is large with bony cheeks and strong jaws, each with 8 large, protruding, sharply pointed teeth. The eyes have vertical adipose sleeves. The juveniles are silvery with distinctive parallel stripes that begin to show above 50 mm standard length. The colour of the adults is striking with a silvery body and head and a bluish sheen on the back [34]
and a series of parallel longitudinal black stripes. The adipose fin is black and the caudal fin varies from yellow to blood red at full intensity, with black trailing edges. The other fins are also yellow to red especially toward their bases. The tip and trailing edge of dorsal fin is black (Skelton, 2001). Hydrocynus vittatus has a remarkable distribution in Africa, from the rivers of West Africa through to the Zambezi, Okavango and Limpopo and Phongolo Rivers in the South (Jubb, 1967 cited by Gaigher, 1970). Tigerfish are inhabitants of open, well- oxgenated water (Pienaar, 1978). Spawning is thought to occur downstream in the floodplains of Mozambique (Gaigher, 1970) during the peak of the rainy season (Badenhuizen, 1966). A radio-telemetry was done by Økland et al. (2005) where it was shown that tigerfish mainly occupied the main stem of the stream but were also found in other habitats such as backwaters and floodplains, especially during rising water level. The numbers of this important predatory fish have decreased in recent years, especially in South Africa, due to water abstraction, pollution, obstructions such as dams and weirs, and fishing pressure (Steyn et al., 1996 and Skelton, 2001 cited by Smit et al., 2009). As a result the tigerfish has been placed on the protected species list by the South African Department of Environmental Affairs and Tourism (DEAT, 2007).
Previous research on this species includes an ecological study of H. vittatus in the Incomati River, a movement and habitat use study by Økland et al. (2005), an angling stress study (Smit et al., 2009) as well as an ageing study on the tigerfish (Gerber et al., 2009) from the Okavango Delta. Soekoe et al. (2010) found low allozyme variation in populations from the Okavango, Zambezi and Olifants Rivers. Other studies include artificial reproduction of the tigerfish (Steyn et al., 1996), cryopreservation (Steyn and Van Vuren, 1991) and physio- chemical characteristics of tigerfish semen (Steyn, 1993). A study on tooth replacement was also done (Gagiano et al., 1996). Studies of concentrations of metals in the tissue of tigerfish from the OR (Du Preez and Steyn, 1992) and Lake Kariba (Mhlanga et al., 2000) have also been done. There has however not been any studies done using histopathology as a biomarker in this species.
2.4.2 Labeobarbus marequensis Class: Actinopterygii
Order: Cypriniformes
Family: Cyprinidae
Species: Labeobarbus marequensis
[35]
Common name: Lowveld largescale yellowfish
Figure 2.4: Lowveld largescale yellowfish (Labeobarbus marequensis)
Labeobarbus marequensis is known by the common name of largescale yellowfish and is widely distributed from the middle and lower Zambezi south to the Phongolo system (Skelton, 2001). The colour of L. marequensis varies with water clarity from pale olive to bright golden yellow with juveniles being silvery with dark blotches (Skelton, 2001). The dorsal fin is in front of the pelvics with a primary ray of extremely variable height. The mouth is subterminal with extremely variable lips and two pairs of barbels. Both males and females develop small tubercules on top of and on the side of the head (Skelton, 2001). Labeobarbus marequensis has a streamlined body and broad V-shaped caudal fins and their body shape gives them the ability to accelerate rapidly and swim strongly (Fouché et al., 2009). Stomach content analysis and intestinal morphology data seemed to indicate that L. marequensis of size classes 31-110 mm are opportunistic feeders with a tendency to ingest plant and algal material. However, as the fish increase in size, larger volumes of insect residues are found in the stomach content, which indicates a possible shift towards a more insectivorous diet (Fouché et al., 2003). Labeobarbus marequensis breeds in summer and migrates upstream in rain-swollen rivers to spawn in rapids (Skelton, 2001). Although L. marequensis is generally adaptable and maintaining populations in moderately polluted rivers and impoundments, they are becoming less common in South Africa due to water extraction, flow regulation and water pollution (Fouché, 2007). Labeobarbus marequensis are sensitive to reduction in flow rates and increased levels of siltation, especially at breeding sites (Fouché and Gaigher 2001) while other key impacts include water pollution, in-stream dams and weirs that fragment populations, invasive alien fishes and plants, and illegal gill netting (Fouché, 2007).
A metal concentration study was carried out on the species in the lower OR, determining the levels of Mn, Pb and Sr. This study suggested that metal studies should be done using bony structures, gills, liver and muscle tissue as this was where the higher metal concentration [36]
was found (Seymore et al., 1995). A study done on the levels of Zn in this species from the OR suggested that there was no serious Zn pollution in the OR. At the time of the study the fish were possibly chronically exposed to Zn (Seymore et al., 1996).
2.4.3 Labeo cylindricus Class: Actinopterygii
Order: Cypriniformes
Family: Cyprinidae
Species: Labeo cylindricus
Common name: Redeye labeo
Figure 2.5: Redeye Labeo (Labeo cylindricus) adapted from Skelton (2001)
Labeo cylindricus is a relatively small species, attaining a maximum length of 25 cm standard length and a mass of 0.9 kg (Skelton 2001) which inhabits rocky habitats of small and large rivers and in lakes and dams (Weyl and Booth 1999) and feeds mainly by grazing on algae and ‗aufwuchs‘ from rocks, tree trunks and other firm surfaces (Gaigher 1973; Skelton, 2001).
This species is widespread from East African rivers South through the Zambezi system and East coastal drainages to the Phongolo system in Kwazulu-Natal. The body of this species is cylindrical; the head has a prominent stepped snout, usually with rough, star – shaped tubercules. The mouth is large with fleshy outer lips and inner rims with a horny sharp edge and the lower lip is papillose and there is a single pair of barbels. The gill openings are restricted to the sides of the head. Labeo cylindricus is olive to yellow-green in colour with a darker body band and larger specimens are darker olive grey, the eye of this species is distinctly red above. Labeo cylindricus migrates upstream in masses to breed, using the
[37]
mouth and broad pectoral fins to climb damp surfaces of barrier rocks and weirs (Skelton, 2001).
Not many studies have been done on the health of this species but a study on the levels of Hg, Pb, Cu and Fe in the species in a Nairobi river was done (Budumbula and Mwachiro, 2006) showing that bioaccumulation occurred particularly in the scales, kidney and heart in fish from Nairobi rivers.
2.4.4 Labeo rosae
Class: Actinopterygii
Order: Cypriniformes
Family: Cyprinidae
Species: Labeo rosae
Common name: Rednose labeo
Figure 2.6: Rednose Labeo (Labeo rosae)
Labeo rosae is an active fish which leaps barriers when migrating upstream and inhabits the lowveld reaches of the Limpopo River, Incomati and Phongolo systems. The species prefers sandy stretches of large perennial and intermittent rivers and feeds on detritus, algae and small invertebrates. Labeo rosae breeds in summer after migrating upstream. This species is moderately deep-bodied and compressed and the dorsal fin has a concave posterior edge. The head is small and the mouth has papillose lips and a single pair of barbels. The colour is variable with a golden green base colour and silvery pink scales. The eye is reddish above and the snout has red tubercules. The juveniles are silvery with a black caudal peduncle (Skelton, 2001). A study on the health assessment index (HAI) and parasite prevalence in L. rosae in the OR was done by Luus-Powell (1997) who found mean HAI values ranging from 44.5 to 95 from two sites and four sampling trips each.
[38]
2.5 Health Assessment Index The health assessment index (HAI) protocol of Adams et al. (1993) is an extension and refinement of a protocol by Goede and Barton (1990). This protocol involves necropsy, which is an external and internal macroscopic examination as well as blood constituents analysis (haematocrit, leukocrit and total plasma protein). A numerical score is assigned to each variable examined. The assigned numerical values for each fish studied are then summed to give an HAI score per fish. This allows for a meaningful statistical comparison of fish from different systems.
However, the HAI is not meant to be a diagnostic tool but rather to alert investigators to possible problems with fish health that need to be investigated with more specific tests (Pieterse et al., 2010a). The HAI was applied by the Tenessee Valley Authority (TVA) and the range of reservoir HAI values ranged from 17 (best) which was found in a relatively pristine system to 79 (worst) in a system that received contaminants from various sources, including pulp and paper mill effluent (Adams et al., 1993). The HAI was also applied to common carp and black bass in the Colorado River basin (Hinck et al., 2007). Carp in the more affected sites had HAI values of ≥100 and bass in the more affected sites had HAI values of >90 and these values were considered to be indicative of poor health in these fish (Hinck et al., 2007). HAI was also used by Olarinmoye et al. (2010) to assess the health of Chrysicthys nigrodigitatus in the Lagos lagoon complex. The lagoon with the highest HAI had values ranging from 0 to 399 with a mean HAI of 76.57 while the lagoon with the lowest HAI had values ranging from 2 to 34 with a mean HAI of 29.66. The HAI on its own does not give a complete picture of the health of the fish studied and further research is required. Therefore the biometric indices (Condition factor, Hepatosomatic index, Splenosomatic index and Gonadosomatic index) and histopathological assessment discussed below were also included in this study.
2.6 Blood constituents’ analysis Blood constituents tested were haematocrit (volume of red blood cells or red blood cells per unit volume of blood); leukocrit (gross measure of white blood cell abundance) and total plasma protein. Low haematocrit values may indicate diseases (Cardwell and Smith, 1971) or anaemia (Roche and Boge, 1996) and is a good indicator of deteriorating health (Chun- Yao et al., 2004) while increased haematocrit values is as a result of increased cell volume (Roche and Boge, 1996). Normal haematocrit values are 30-45% (Adams et al., 1993). Increased leukocrit values may be indicative of infection. Normal leukocrit values are less
[39]
than 4%. Increased total plasma protein concentrations may be indicative of liver impairment (Rehulka, 2003).
2.7 Biometric indices
2.7.1 Condition factor The condition factor (Cf) is a relation of mass and length (Carlander, 1969) (see section 3.6.4.1 for the formula to calculate Cf). A decline in the condition factor is usually interpreted as depletion of energy reserves such as stored liver glycogen or body fat. This decline could be a reflection of a change in feeding patterns, which could be in response to certain stressors (Brown et al., 1987). In laboratory bred fish, Van Dyk (2006) found a mean Cf value of 0.67 in Clarias gariepinus, which is a long, dorso-ventrally flattened fish, and a mean Cf value of 1.64 in Oreochromis mossambicus which has a more rounded body shape and that suggests that body shape plays a role in Cf values. In a study done in the Okavango panhandle, Van Dyk et al. (2009b) recorded Cf values of 0.8 in Clarias gariepinus, 0.7 in Clarias ngamensis, 1.8 in Oreochromis andersonii, and 1.6 in Seranochromis angusticeps which lends further evidence to the aforementioned statement regarding body shape affecting Cf values.
2.7.2 Hepatosomatic index The hepatosomatic index (HSI) is a ratio of liver weight to body weight (see section 3.6.4.2 for the formula to calculate HSI). It allows for identification of possible hyperplasia (increase in cell number), hypertrophy (increase in cell size) or atrophy (decrease in cell size) and a comparison of the index values of each specimen can be made (Van Dyk et al., 2007). The mean HSI value is species specific and is about 1-2% in Osteichyes (Munshi and Dutta, 1996). For Southern African fish, Van Dyk (2006) bred C. gariepinus and O. mossambicus as a baseline to compare further studies to. The mean HSI values recorded in that study were 1.08 for C. gariepinus and 1.30 for O. mossambicus. HSI values were higher than unexposed fish in Oreochromis niloticus specimens exposed to waterborne Cu (Figueirdo- Fernandes et al., 2007).
2.7.3 Splenosomatic index The splenosomatic index (SSI) is a ratio of spleen weight to body weight (see section 3.6.4.3 for the formula to calculate SSI). Spleen size is considered a useful diagnostic factor because the spleen is a haematopoietic organ and dysfunction could have effects on the whole-organism level. The SSI has not been as thoroughly investigated as the HSI but
[40]
certain endogenous and exogenous factors are known to affect it (Schmitt and Dethloff, 2000). Enlargement or swelling of the spleen is considered to be an indication of disease or immune system problems (Goede and Barton, 1990). Chemical contaminants can also affect the SSI, with higher SSI values shown in fish exposed to petroleum, Polychlorinated biphenyls, polycyclic aromatic hydrocarbons and metals (Schmitt and Dethloff, 2000). SSI values of unexposed specimens were 0.04% for C. gariepinus and 0.06 for O. mossambicus (Van Dyk, 2006). In a study on C. gariepinus from an urban nature reserve, the mean SSI value was 0.21 in the Marais Dam site and 0.19 in the Rietvlei Dam site (Marchand, 2006).
2.7.4 Gonadosomatic index The gonadosomatic index (GSI) is a ratio of gonad weight to body weight (see section 3.6.4.4 for the formula to calculate GSI). GSI is said to be the state of gonadal development and maturity (Schmitt and Dethloff, 2000). It has been used to assess the gonadal changes in response to environmental dynamics (seasonal changes) or exogenous stresses (contaminant exposure) (Schmitt and Dethloff, 2000). The GSI mean values for laboratory bred C. gariepinus specimens were 0.37 in males and 6.93 in females (Van Dyk, 2006). The GSI mean values for laboratory bred O. mossambicus specimens were 0.41 in males and 1.80 in females (Van Dyk, 2006). In a study on C. gariepinus from an urban nature reserve, the mean GSI value for males was 0.42 in the Marais Dam site and 0.28 in the Rietvlei dam site and the mean GSI value for females was 3.97 in the Marais Dam site and 3.01 in the Rietvlei Dam site (Marchand, 2006).
2.8 Histology and histopathology of selected target organs Histopathological alterations are biomarkers of effect exposure to environmental stressors, revealing prior alterations in physiological and or biochemical function (Hinton et al., 1992) and because they also incorporate biotic factors as well as water quality, they are able to give a more holistic view of fish health (Handy et al., 2002). The histological assessment would show changes before they manifest macroscopically and can therefore be used as an early warning system for pathological changes in the fish tested (Marchand et al., 2008).
2.8.1 Liver as a target organ The liver is the largest of the extramural (outside the alimentary canal) organs. Its functions include assimilation of nutrients, production of bile, detoxification, maintenance of body metabolic homeostasis that includes the processing of carbohydrates, proteins, lipids and vitamins. The liver also plays a key role in the synthesis of plasma proteins, like albumin,
[41]
fibrinogen, and complement factors (Genten et al., 2009). The liver is, by far, the most studied organ in terms of toxic effects (Blazer, 2000).
2.8.1.1 Histological description of the liver in teleosts
Takashima and Hibiya (1995) stated that the livers of higher vertebrates have lobular structures containing a small vein in the centre. The structure of fish liver varies between species (Takashima and Hibiya, 1995) but there are general features found in the majority of species. The parenchyma of the liver is contained within a thin capsule of fibrous connective tissue and is composed of polyhedral hepatocytes with central nuclei which are concentrically arranged around the sinusoids forming the cord-like structures known as hepatic cords (Groman, 1982; Takashima and Hibiya, 1995) but may demonstrate histological variability following extensive fat and glycogen storage (Woodhead, 1977).
Sinusoids are lined with endothelial cells, whose nuclei protrude into the sinusoidal lumen. The main cell type of the liver is the parenchymal hepatocyte, while endothelial cells, fat- storing cells, kupffer cells, mesothelial (serosa) cells, and fibrobalsts complement the basic liver architecture. Bile canaliculi originate between adjacent hepatocytes to produce ducts of increasing diameter which merge and always end in the gall bladder. They are lined by a pseudostratified epithelium (Groman, 1982; Van Dyk, 2006). Two basic types of fish livers exist: Those which contain pancreatic tissue, which are commonly called hepatopancreas, versus those which do not. An example of a Southern African fish with hepatopancreatic tissue is O. mossambicus and one without is C. gariepinus (Van Dyk, 2006). The exocrine pancreas consists of clusters of pyramidal cells mostly organised in acini (Genten et al., 2009) and surrounds all of the portal veins in mature striped bass (Kristal, 1946; Kendall and Hawkins 1975; Groman, 1982). The cells have a dark basophilic cytoplasm, distinct basal nuclei, and many large eosinophilic zymogen granules containing enzymes responsible for the digestion of proteins, carbohydrates, fats and nucleotides (Genten et al., 2009). Zymogen is released by stimulus provided by the passage of food in the alimentary canal. Thus, the accumulation of zymogen implies the short-term starvation of an otherwise well- fed individual (Takashima and Hibiya, 1995).
2.8.1.2 Description of liver histopathological alterations
Descriptions of liver histopathological alterations by Meyers and Hendricks (1985) and Takashima and Hibiya (1995) are summarised in table 2.1. Alterations are separated into circulatory disturbances, regressive changes and progressive changes.
[42]
2.8.1.3 Liver histopathological alterations literature
Table 2.2 is a summary of literature where studies found fish with liver histopathological alterations. The species as well as what these fish were exposed to and what alterations were found are given.
2.8.2 Kidney as a target organ The kidney has a varied function among fish species and filters large quantities of blood and produce urine, which is the major route of excretion for some xenobiotics (Blazer, 2000). The kidney of fishes receives the vast majority of postbranchial blood and therefore renal lesions might be expected to good indicators of environmental pollution (Hinton and Lauren, 1992). In freshwater teleosts the main function of the kidney is to exrete the large amounts of water which enter the fish body through the gills (Takashima and Hibiya, 1995). The lymphoid tissue of the head kidney and intertubular of body kidney are the haematopoietic (blood forming) tissue of teleosts (Takashima and Hibiya, 1995).
2.8.2.1 Histological description of the kidney in teleosts
The Teleostean kidney consists of head and body kidneys (Takashima and Hibiya, 1995). The head kidney is the anterior section of the kidney and is made up of of lymphoid tissue (Takashima and Hibiya, 1995) and is almost exclusively hematopoietic tissue with immature (blast cells) and mature blood cells (Van Dyk, 2006) and very few tubules (Genten et al., 2009). The posterior kidney contains of nephrons (renal corpuscles and tubules) with surrounding haematopoietic tissue (Groman, 1982; Van Dyk, 2006). The renal corpuscle consists of a large glomerulus and surrounding Bowman‘s capsule (Van Dyk, 2006).
[43]
Table 2.1: Description of common liver histopathological alterations
[44]
Table 2.2: Liver histopathological alterations found in literature
[45]
The glomerulus is a lobulated tuft of capillaries (Takashima and Hibiya, 1995) and just before entering the glomerulus, the afferent arteriole divides into several capillaries which diverge, converge and wind to make the capillary loop before reuniting to leave the glomerulus as the efferent arteriole (Takashima and Hibiya, 1995). Mesangial cells fill the space between the capillary loops (Takashima and Hibiya, 1995). The Bowman‘s space consists of an inner visceral epithelium of podocytes and an outer layer of parietal layer of squamous epithelium supported by a basement membrane which separates the renal corpuscle from the rest of the kidney (Patt and Patt, 1969) with the lumen between these layers known as the Bowman‘s space (Grizzle and Rogers, 1976; Groman, 1982; Van Dyk, 2006). The renal tubules of the nephron are divided into the following segments: neck segment, first proximal segment, second proximal segment, distal segment, and the collecting tube (Van Dyk, 2006). The proximal segment consists of cuboidal to columnar epithelium with a large centrally located nucleus and a distinct brush border within the lumen (Van Dyk, 2006). The distal tubules are smaller and consist of lower columnar epithelium with basally located nuclei and no brush border (Van Dyk, 2006).
2.8.2.2 Description of kidney histopathological alterations
Descriptions of kidney histopathological alterations by Takashima and Hibiya (1995) are summarised in table 2.3. Alterations are separated into circulatory disturbances, regressive changes and progressive changes.
2.8.2.3 Kidney histopathological alterations literature
Table 2.4 is a summary of literature where studies found fish with kidney histopathological alterations. The species as well as what these fish were exposed to and what alterations were found are given.
2.8.3 Gill as a target organ Gills are sensitive indicators of environmental stress, including exposure to harmful compounds present in aquatic ecosystems as a result of human activities (Hinton et al., 1992).
[46]
Table 2.3: Description of common kidney histopathological alterations
[47]
Table 2.4: Kidney histopathological alterations found in literature
[48]
2.8.3.1 Histological description of the gill in teleosts
The main function of the gills in teleosts is gas exchange between the water and the blood (Grizzle and Rogers, 1976; Groman, 1982). The gill is a system for bringing the blood haemoglobin into close contact with the water, so that oxygen can be absorbed and carbon dioxide resleased (Yasutuke and Wales, 1983). Teleosts have five pairs of gill arches, although the last pair transform into the pharyngeal bone and contributes minimally to respiration (Takashima and Hibiya, 1995). Each of the gill arches is supported by a cartilaginous and/or bony skeleton with associated striated abductor and adductor muscles which facilitate movement of gills to favourable respiratory positions (Genten et al., 2009). Each gill arch bears a number of gill filaments or holobranchs, which are each made up of two halves, called hemibranchs which bear many subdivisions called lamellae (Genten et al., 2009). The lamellae are subdivided into primary and secondary lamellae with numerous secondary lamellae lined up along both sides of the primary lamella (Takashima and Hibiya, 1995). The primary lamellae consist of cartilaginous support, a vascular system and multi- layered epithelium (Takashima and Hibiya, 1995). The secondary lamellae, which are semicircular in shape (Takashima and Hibiya, 1995), protrude along the entire length of the primary lamellae (Van Dyk, 2006). The secondary lamellae consist of blood arterioles supported by pillar cells and enclosed by an epithelial layer (Van Dyk, 2006). Chloride cells surrounded by flattened pavement cells can be observed at the junction between the primary and secondary lamellae (Genten et al., 2009) and generally exist in marine fish and rarely in freshwater fishes (Takashima and Hibiya, 1995). Mucous cells are a prominent feature of the gill epithelium (Genten et al., 2009) and are principally located on the afferent and efferent edges of the primary lamella (Takashima and Hibiya, 1995).
2.8.3.2 Description of gill histopathological alterations
Descriptions of gill histopathological alterations by Takashima and Hibiya (1995) are summarised in table 2.5. Alterations are separated into circulatory disturbances, regressive changes and progressive changes.
2.8.3.3 Gill histopathological alterations literature
Table 2.6 is a summary of literature where studies found fish with gill histopathological alterations. The species as well as what these fish were exposed to and what alterations were found are given.
[49]
Table 2.5: Description of common gill histopathological alterations
[50]
Table 2.6: Gill histopathological alterations found in literature
[51]
2.8.4 Testis as a target organ
2.8.4.1 Histological description of the testis in teleosts
Using a broad phylogenetic approach, Callard (1991), classified all vertebrate testes as either tubular (mammals, birds and reptiles) or lobular (amphibians and teleosts). In this classification, a tubule is an open-ended germinal compartment of the testes while a lobule is a blind-ended sac. Most teleosts testes would be of the lobular type despite differences in the patterns of spermatogonial distribution or the presence or absence of a lumen within the lobule (Van Dyk, 2006). The testes of C. gariepinus bred in controlled laboratory conditions has been described by Van Dyk and Pieterse (2008) who found that the testes showed a lobular organisation with a thin tunica albuginea surrounding it. The lobules consist of a basement membrane with accompanied myoid cells which encloses originating spermatogenic cysts of primary and secondary spermatogonia, primary and secondary spermatocytes and spermatids with free spermatozoa also visible in the lumen of the tubule (Van Dyk and Pieterse, 2008). Two other cells which were found in testis tissue are: 1.The Leydig cells which are found in the interstitial tissue, are round to oval in shape, and are responsible for secreting male hormones (Billard, 1986; Van Dyk, 2006). 2. Sertoli cells which are elongated to oval cells found in the basement membrane in close proximity to spermatogonia and serve as nutritive cells for developing sperm (Van Dyk, 2006).
2.8.4.2 Testis histopathological alterations literature
Table 2.7 is a summary of literature where studies found fish with testis histopathological alterations.
Table 2.7: Testis histopathological alterations found in literature
[52]
2.8.5 Ovary as a target organ
2.8.5.1 Histological description of the ovary in teleosts
Teleosts are unique among vertebrates in having hollow ovaries resulting from embryonic development of longitudinal ovarian folds that eventually fuse to enclose a coelomic cavity (Genten et al., 2009). A short oviduct releases eggs to the outside via an exit between the anus and the urinary pore (Genten et al., 2009). Histological examination of ovarian tissue commonly reveals eggs at all stages of development (Genten et al., 2009) and ovaries with this heterogenous population of follicles at different stages are called asynchrous ovaries (Takashima and Hibiya, 1995). Other developmental patterns include group-synchrous ovaries, in which clutches of follicles at different developmental stages exist in the same ovary, and synchrous ovaries, in which all follicles develop in unison (Takashima and Hibiya, 1995). The ovaries of teleosts do not commit all oogonia to meiosis during early development (Tokarz, 1978). Resting and proliferating oogonia can be found in the interstices between larger ovarian follicles in the gonads of most adult female teleosts. Oocytes undergo remarkable nuclear and cytoplasmic changes during growth. In general, the growth stages include the chromatin-nucleolus stage, peri-nucleolar stage, cortical alveoli stage, vittelogenic stage, maturation stage and ovulation stage (Wallace and Selman, 1990). The oocyte in a vertebrate ovary is commonly destined to either grow, mature, and ovulate and leave behind a corpus luteum (postovulatory follicle) or to undergo atresia (the degeneration and resorption of one or more ovarian follicles before a state of maturity has be reached) at some stage in its growth and development (Shanbag and Saidapur, 1996).
2.8.5.2 Ovary histopathological alterations literature
Table 2.8 is a summary of literature where studies found fish with ovary histopathological alterations.
Table 2.8: Ovary histopathological alterations found in literature
[53]
2.8.6 Semi-quantitative histological assessment Bernet et al. (1999) saw the need to develop a standardised tool for the assessment of histological findings which could be applied to different organs, so as to assess the effects of sub-lethal and chronic effects of water pollution on fish. The methodology was based on the extent (score value) and pathological importance (importance factor) of the change observed. The sum of the multiplied score values and importance factors of all changes result in different indices which can be statistically analysed (Bernet et al., 1999). Zimmerli et al. (2007) developed a scoring system used in a study on brown trout in Swiss rivers which was based on the organ indices set out by Bernet et al. (1999). This scoring separated organ index values into 5 classes according to the pathological condition of the organs by using the organ index values of the specimens studied.
This scoring system was adapted by Van Dyk et al. (2009a) for Southern African fish species to include only four classes which ranged from tissue with slight alterations (Class 1) to severe alterations of organ tissue structure (Class 4) (See section 3.9 for further explanation of these classes). This semi-quantitative protocol has been successfully applied in a number of studies which include: A study on the testicular histology as part of a study on the reproductive health of C. gariepinus and O. mossambicus from a DDT sprayed area (Marchand et al., 2010) and a study on the histological alterations in the ovaries and gonads of C. gariepinus from an urban nature reserve in South Africa (Pieterse et al., 2010b) as well as a study on the histological changes in the gills of C. gariepinus from a South African urban aquatic system (Van Dyk et al., 2009b) and a study comparing the histology-based health of four commercially important species from the Okavango panhandle in Botswana (Van Dyk et al., 2009a). The scoring system allows for the results of semi-quantitative histological studies to be easily summarised and the severity of alterations within a specific organ to be compared to a Southern African standard.
2.9 Ageing Age information forms the basis for calculations of growth rate, mortality rate and productivity (Campana, 2001). Ageing of fish is important as proper management of fish populations require the knowledge of population specific age structure and growth rates. Counting bands or annuli on otoliths and circuli on scales are the most common methods for determining fish age. The deposition patterns found on these hard structures are most visible in fish that exist in temperate water zones, and thus, are directly equivalent to seasonal changes in environmental conditions. Generally, growth is faster during the warmer summer months and slow during the colder winter months. These bands are differentiated mainly by the amount
[54]
of material deposited during each seasonal event, with summer bands (translucent) being wider and winter bands (opaque) being thinner. The overall result of this type of growth pattern is an alternating series of light and dark bands. The combination of these two bands is referred to as the annual mark, or annulus. The convention of ageing is typically achieved by counting the thinner winter opaque zones (Wischniowsky, 1998). Within the sampling guidelines for the semi-quantitative histological assessment protocol of Bernet et al. (1999), it was stated that the age of fish sampled should be recorded if possible as the age of fish stocks will strongly determine the range and nature of pathologies found. The occurrence of neoplasms was said to be more likely to be found in older fish (Bernet et al., 1999).
[55]
Chapter 3: Materials and Methods
3.1 Introduction As mentioned in Chapter 1, this chapter is a description of the materials and methods used in this study. It includes a description of work done in the field and the laboratory. Field work done includes the taking of physical water quality parameters and the collection of water and sediment for chemical analysis. The collection of fish, calculation of biometric indices (condition factor (Cf), hepatosomatic index (HSI), splenosomatic index (SSI) and gonadosomatic index (GSI)), blood collection and analysis of constituents (haematocrit (Hct), leukocrit (Lct) and total plasma protein (TP)) and necropsy as well as collection of histological samples are described. Histological processing and quantitative and semi- quantitative assessments are described. The techniques to determine age are also described.
3.2 Study period Two rivers, namely the Olifants - and Luvuvhu Rivers, both located in the Kruger National Park (KNP), were sampled. The Olifants River (OR) was sampled in September 2009 and April 2010 at 5 sampling sites along the river, as shown in Figure 3.2 below. The Luvuvhu River (LR) was sampled in November 2009 and May 2010 on 5 sampling sites as shown in Figure 3.3 below.
The sites were selected as a good representation of the rivers sampled as well as being easily accessible.
The Olifants River sites and their GPS co-ordinates are given in Table 3.1 while the Luvuvhu River sites and their GPS co-ordinates are given in Table 3.2.
[56]
Table 3.1: Olifants River sites and GPS co-ordinates
Olifants river
Site no. Site name GPS co-ordinates
Site 1 Mamba S24 03 58.7 E31 14 35.2
Site 2 Tseri S24 05 07.2 E31 19 16.3
Site 3 Vyeboom S24 02 06.7 E31 33 55.9
Site 4 Balule S24 03 14.7 E31 43 50.5
Site 5 Gorge S23 59 25.2 E31 49 33.3
Table 3.2: Luvuvhu River sites and GPS co-ordinates
Luvuvhu river Site no. Site name GPS co-ordinates Site 1 Dongadziva S22 42 34.6 E30 53 19.6
Site 2 Shidzivani S22 38 05.3 E30 57 33.5
Site 3 Confluence S22 27 04.3 E31 04 47.7
Site 4 Bobomene S22 25 40.5 E31 12 34.0
[57]
Figure 3.1: Map of the Kruger National Park, highlighting the Olifants – and Luvuvhu Rivers in red blocks
[58]
Figure 3.2: Map of the Olifants River in the Kruger National Park, showing the five sampling sites
Figure 3.3: Map of the Luvuvhu River in the Kruger National Park, showing the four sampling sites
[59]
3.3 Water and sediment collection Water samples for metal analysis were collected using polypropylene bottles and frozen for further analysis. The water samples were filtered using 0.45μm filter paper. The filtrate was acidified with 2mL 65% suprapur nitric acid. The filter paper was dried and then digested before being read on the ICP-OES and ICP-MS.
Sediment for metal analysis was collected in polypropylene bottles and frozen for further analysis. The sediment samples were dried in a drying oven and underwent BCR extraction before being allowed to digest in a milestone ethos microwave, before 500μL indium, de- ionised water and 1% nitric acid were added to make up 500mL. The samples were then filtered using 0.45μm filter paper and analysed using ICP-OES and ICP-MS.
A broad range of metals were tested for and the following metals were present in at least trace amounts: Aluminium (Al), Chromium (Cr), Manganese (Mn), Iron (Fe), Copper (Cu), Zinc (Zn), Arsenic (As), Cadmium (Cd) and Lead (Pb).
The concentrations in the water were compared to the target water quality range (TWQR) stipulated by the South African water quality guidelines for aquatic ecosystems (DWAF, 1996).
3.4 Physical water quality parameters Physical water quality parameters were measured when sampling was done. Parameters were measured at the same time of day to ensure comparable results. The water quality parameters measured included temperature, pH, oxygen percentage, oxygen concentration (mg/L) total dissolved salts (TDS) and conductivity The pH was measured using a pH Scan 2 pH meter (Eutech Instruments) and the oxygen percentage, oxygen concentration, TDS and conductivity were measured using a Cyberscan CON 300, Conductivity/TDS/°C RS 232 (Eutech Instruments). All the meters were calibrated to ensure reliable results were obtained.
[60]
3.5 Field surveys Hydrocynus vittatus (n=16) and Labeobarbus marequensis (n=15) were caught from the OR in September 2009. Hydrocynus vittatus (n=6), L. marequensis (n=15) and Labeo rosae (n=7) were caught from the OR in May 2010, H. vittatus (n=16) and Labeo cylindricus (n=10) were caught from the LR in November 2009 and H. vittatus (n=2) were caught from the LR in April 2010. The H. vittatus were caught using rod and reel (Fig. 3.4a; Fig. 3.4b) while the other species were caught using castnetting (Fig. 3.4c), electroshocking and seine netting. The specimens were then taken to the mobile field lab (Fig. 3.4d) for dissection.
All fish were weighed in grams (g) using a lip grip scale, and their total and standard lengths measured in millimetres (mm) (Fig. 3.4e). Blood was drawn using 22 gauge needles into cold heparin-containing vacutainers (Fig. 3.5a). The blood was placed on ice until further analysis. The fish were examined externally (Fig. 3.5b) and any bodily abnormalities noted. The fish were then sacrificed by severing the spinal cord.
During the necropsy the liver, spleen and gonad (ovaries or testes) were removed from each fish and weighed so as to calculate the hepatosomatic index, splenosomatic index and gonadosomatic index respectively (Fig. 3.5c). The second pair of gills (Fig. 3.5d), a piece of liver, a piece of kidney from the mid section of the kidney and a piece from the middle of the gonad (ovaries or testes) were removed and placed in fixative for processing as described in section 3.7 below. An internal and external necropsy (Fig. 3.5e) was done to determine macroscopic abnormalities as described in section 3.6.2.
[61]
Figure 3.4: Fish sampling and measurement a. and b. Sampling tigerfish using rod and reel; c. Castnetting; d. Field laboratory; e. Measuring fish length
[62]
Figure 3.5: Necropsy and blood collection a. Blood drawn using 22 gauge needle; b. ―Black spots‖ in the fin; c. Measuring testes length; d. Fish dissection; e. Necropsy
[63]
3.6 Histology-based health assessment protocol
3.6.1 Blood constituents analysis Each blood sample was centrifuged at 3000 rpm for 10 minutes. The haematocrit (Hct = RBC/Total blood volume) was measured and recorded. Leukocrit (Lct = WBC/Total blood volume) was also measured and recorded. Both haematocrit and leukocrit are expressed as a percentage. The top layer of plasma was pipetted into eppendorph tubes and frozen for further analysis. The plasma was used to determine total plasma protein (TP) using a Total Protein Kit (Roche) with absorbance was read on a universal microplate reader.
3.6.2 Necropsy A necropsy was performed in the field by examining the fish externally (fins, skin, eyes, opercula, external parasites) and internally (gills, liver, kidney, spleen, hindgut, internal parasites) following the protocol of Adams et al. (1993). These examinations were used to fill in the HAI form, as described below.
3.6.3 HAI The HAI, which is a method for rapid evaluation of fish condition in the field, (Adams et al., 1993) was calculated using the Hct, Lct, TP, and necropsy data. All necropsy and blood variables mentioned above were scored in the field with a numerical value given to each according to the HAI protocol. The HAI of a single fish was calculated by the summation of all the numerical values for each of the variables. The HAI for a sample population (mean HAI value) was calculated by summing all the individual HAI values and dividing that number by the number of fish in that sample population. The sum HAI value for a population is the sum of HAI values of each fish in the population. See Appendix A for an example of a HAI score sheet.
3.6.4 Biometric indices
3.6.4.1 Condition factor
The condition factor, which is a relation of length and weight, for each fish was determined using the formula seen below, as described by Carlander (1969).
Condition factor = Mass (g) x 105 / Standard length³ mm
[64]
3.6.4.2 Hepatosomatic index (HSI)
The HSI is an organosomatic index which gives an indication of liver weight in relation to total body weight and has been used in stress-related studies. The HSI was determined using the liver weight and total body weight.
HSI = liver weight / body weight x 100
3.6.4.3 Splenosomatic index (SSI)
Spleen size is important as the spleen is a haematopoietic organ. The SSI is a ratio of spleen weight in relation to body weight and is determined using the spleen weight as well as the total body weight.
SSI = spleen weight / body weight x 100
3.6.4.4 Gonadosomatic index (GSI)
GSI can give an indication of gonadal development as well as sexual maturity. It is a ratio of gonad weight in relation to total body weight and is determined using the gonad weight as well as the total body weight.
GSI = gonad weight / body weight x 100
3.7 Tissue processing All histological samples, except for gonad samples, were immediately placed into 10% neutrally buffered formalin (NBF). Gonad samples were placed in Bouin‘s fixative. Tissue is fixed so as to prevent post-mortem changes in the tissue. Gonad samples were removed from the fixative after 24 hours before being washed with water. Samples in BNF were removed from the fixative after 72 hours and then washed with water, then dehydrated (Fig. 3.6a) by placing samples in rising concentrations of ethanol (30%; 50%; 70%; 80%; 90%; 100%) and cleared using xylene. Samples were then infiltrated with Paramat wax (Merck) in a 60°C oven (Fig. 3.6b) (Humason, 1979).
Once fully infiltrated, the samples were placed in wax which was moulded into blocks and solidified in a refrigerator overnight before being sectioned at 5 μM using a wax microtome (Leica) (Fig. 3.6c). The resulting sections were placed onto microscope slides and stretched using a water and albumin solution (Humason, 1979). The slides were dried over night in an
[65]
oven, and stained using Haemotoxylin and Eosin (H&E) (Humason, 1979). The slides were mounted with Entellan and left to dry.
Figure 3.6: Histological processing and microscope slide assessment a. Tissue sample dehydration; b. Wax oven; c. Microtome; d. Discussion microscope; e. Using discussion microscope for histological assessment
[66]
3.8 Qualitative histological assessment The slides of gill, liver, gonad and kidney histology of the H. vittatus, L. marequensis, L. cylindricus and L. rosae were examined under light microscopy (Leica) (Figure 3.6 d an e) and the structures within the different organs were identified and photomicrographs were taken and measured using IM50 Image Manager Software.
3.8.1 Gonadal reproductive stage determination: The gonadal development staging was done by identifying the histological characteristics defined by McDonald et al. (2000) (Table 3.3) with oocyte diameter being measured using the IM50 Image Manager Software (Pixel IT).
[67]
Table 3.3: Gonadal reproductive stage determination (McDonald et al., 2000)
Stage Testis characteristics (male) Ovary characteristics (female)
0 Undeveloped (immature): Undeveloped: Little or no spermatogenic activity Pre-vitellogenic oocytes observed exclusively; oocyte in germinal epithelium; immature diameter <250 μm: cytoplasm stains basophilic with states of spermatogenesis (largely H&E. spermatocytes); no spermatozoa observed. 1 Early spermatogenic: Early development: Mostly thin germinal epithelium with >90% of oocytes pre-vitellogenic, remaining oocytes scattered spermatogenic activity; early to mid-vitellogenic; oocytes slightly larger (up to spermatocytes to spermatids 300 μm); late perinucleolus through cortical alveolar predominate; few spermatozoa stages. observed. 2 Mid-spermatogenic: Mid-development: Germinal epithelia of moderate Majority of observed follicles early and mid- thickness; moderate proliferation vitellogenic; oocytes > 300-600 μm diameter, and and maturation of spermatozoa and containing peripheral yolk vesicles; globular and equal mix of spermatocytes, uniformly thick chorion (5-10 μm in black basses, 10- spermatids and spermatozoa 20 μm in common carp); cytoplasm basophilic, yolk present globules eosinophilic. 3 Late spermatogenic: Late development: Thick germinal epithelium; diffuse Majority of developing follicles late vitellogenic; oocyte regions of proliferation and diameter 600-1000 μm, eosinophilic yolk globules maturation of spermatozoa; all distributed throughout the cytoplasm; chorion stages of development thickness 10-30 μm in black basses, 40-50 μm in represented, spermatozoa common carp. predominate. 4 N/A Late development/hydrated: Majority of developing follicles late vitellogenic; follicles much larger (>1000 μm). 5 N/A Post ovulatory: Spent follicles, remnants of the theca externa and granulosa present.
[68]
3.9 Semi-quantitative histological assessment A standard semi-quantitative histological assessment according to the modified protocol of Bernet et al. (1999) modified by Van Dyk et al. (2009) was done to quantify the histopathological alterations observed in the gill, liver, gonad and kidney histology slides. This assessment is useful because it can be applied to any organ as well as allowing for standardised quantification of results. This assessment also allows for comparisons between different studies and with restrictions, between different organs as well. (See Appendix B for examples of score sheets).
The semi-quantitative histological assessment was carried out by at least three histologists using a multi-headed light microscope to ensure unbiased results.
There are five reaction patterns described by Bernet et al. (1999) with another reaction pattern added by Pieterse et al. (2010b) according to which each organ was assessed. The reaction patterns are as follows:
3.9.1 Histopathological alterations
Reaction pattern 1 (rp1): circulatory disturbances (CD)
Circulatory disturbances come from a pathological condition of blood and tissue fluid flow. Fluid content alterations in tissues related to inflammatory processes (e.g. exudate) are considered in reaction pattern 4. The alterations included here are: (1) haemorrhage/ hyperaemia/ aneurysm and (2) intercellular oedema.
Reaction pattern 2 (rp2): regressive changes (RC)
Regressive changes are processes which terminate in a functional reduction or loss of an organ. These involve atrophy, degeneration (malformation or dysfunction of cellular structures as a result of cell damage) and necrosis. The following reaction patterns are involved: (1) architectural and structural alterations; (2) plasma alterations; (3) deposits; (4) nuclear alterations; (5) atrophy; and (6) necrosis.
Reaction pattern 3 (rp3): progressive changes (PC)
Progressive changes are processes which lead to an increase in the activity of cells or tissues. Characteristic lesions are: (1) hypertrophy and (2) hyperplasia.
[69]
Reaction pattern 4 (rp4): inflammation (I)
Inflammation is often associated with processes belonging to other reaction patterns (e.g. oedema). That is why it is often difficult to attribute inflammatory changes to one solitary reaction pattern. Thus, Bernet et al. (1999) used the term ‗inflammation‘ in a very strict sense, namely: (1) exudates; (2) activation of the reticuloendothelial system (RES); and (3) infiltration.
Reaction pattern 5 (rp5): tumour (T)
A tumour is an uncontrolled cell and tissue proliferation (autonomous proliferation). Tumours are separated into two classes: (1) benign tumours and (2) malignant tumours.
Reaction pattern 6 (rp6): intersex (IS)
Marchand et al. (2010) and Pieterse et al. (2010b) described a sixth reaction pattern due to its presence in testis tissue which was assessed using a modified protocol of Bernet et al. (1999). Intersex (IS) was described as a condition in which individuals appear as one sex but whose gonads contain cells that are normally typical of the opposite sex (testis-ova or ovo- testis).
3.9.2 Importance factor (w)
An importance factor ranging from 1 to 3 was assigned to each alteration according to Bernet et al. (1999). The significance of a lesion depends on its pathological importance, i.e. how it affects organs and the ability of the fish to survive (Bernet et al., 1999). The three importance factors are differentiated as follows: (1) Minimal pathological importance, the lesion is easily reversible as exposure to irritant ends; (2) Moderate pathological importance, the lesion is reversible in most cases if the stressor is neutralised; and (3) Marked pathological importance, the lesion is generally irreversible, leading to partial or total loss of organ function.
3.9.3 Score value (a)
Each alteration is assigned a score value from 0 to 6 by the histologists performing the assessment, depending on the degree and extent of the alteration: (0) unchanged; (2) mild occurrence or a focal alteration; (4) moderate occurrence or present in 50% of tissue assessed; and (6) severe occurrence or an alteration present throughout the tissue assessed.
[70]
3.9.4 Calculation
Using importance factors and score values, four different indices can be calculated. If the lesions within one organ only are studied, the two following indices are applicable:
3.9.4.1 Organ Index (Iorg)
This index represents the degree of damage to an organ. This calculation allows a comparison between the degree of damage of the same organ in different individuals.
It is calculated as follows:
Iorg = Σrp Σalt (aorg rp alt x w org rp alt)
Where: org = organ (constant); rp = reaction pattern; alt = alteration; a = score value; w = importance factor. This index is the sum of the multiplied importance factors and score values of all changes found within the assessed organ.
3.9.4.2 Reaction index of an organ (Iorg rp)
The reaction index expresses the quality of lesions within an organ. Respective reaction indices of an organ (Iorg rp) from different individuals can be compared. The reaction index is calculated as follows:
Iorg rp = Σalt (aorg rp alt x w org rp alt)
Where: org, rp = constant (for abbreviations see organ index formula). This index is calculated by the sum of the multiplied importance factors and score values of the alterations of the corresponding reaction pattern.
The organ index results were separated into four classes according to the protocol of Van Dyk et al. (2009a) which were modified from Zimmerli et al. (2007).
[71]
The classes are as follows:
Class 1 (index< 10) –tissue with slight histological alterations
Class 2 (index 10-25) - tissue with moderate histological alterations
Class 3 (index 26-35) – pronounced alterations of organ tissue
Class 4 (index >35) – severe alterations of organ tissue
3.9.4.3 Fish Index (Ifish)
The fish index represents a measure of the overall health status of the fish based on the occurrence of histological alterations observed in all of the organs. As this index is calculated in the same way for each fish, a comparison between individuals is possible. The fish index is calculated as follows:
Ifish = ΣorgΣrp Σalt (a org rp alt x w org rp alt)
3.10 Ageing Otolith sections were used for ageing, according to the methods of Gerber et al. (2009). Left and right lapillus otoliths were removed from all H. vittatus, cleaned; air dried and stored in 25mL McCartney bottles. Otoliths were prepared for sectioning following standard techniques (Wischniowski and Bobko, 1998) and then sliced using a double-bladed diamond-edged otolith saw. Cut sections were mounted on microscope slides using DPX mountant to enhance the of the clarity of the sections. The sections were then viewed under transmitted and growth rings were counted. The second lateral line scale was taken from L. marequensis, and L. rosae specimens and then dried between two clean microscope slides. The scales were viewed using Nikon Profile Projector model 6CT2 at 20x magnification and a 30 cm diameter viewing screen and the growth rings were counted (Gerber et al., 2009).
[72]
3.11 Statistical analysis Statistical analysis was done using SPSS 18 software. Normality of data was tested using the Kolmogorov-Smirnov test. If the data was normally distributed, the t-test for equality of means was used. If the data was not normally distributed, the Mann-Whitney U test was used. These tests were used to determine whether there was a significant difference between biometric indices, blood parameters and histological organ indices between the following groups: H. vittatus specimens were grouped into OR and LR groups as the OR 2010 and LR 2010 groups were too small for individual statistical comparison; L. marequensis compared between sampling trips (OR 2009 and OR 2010); and L. cylindricus specimens from LR 2009 were compared to L. rosae specimens from OR 2010. Significance was set at 5%. Age results were correlated with liver index and fish index values in H. vittatus and L. marequensis using Spearman Rank Order Correlation (rho). Rho is a measure of the strength of the association between the two variables. Where rho = -1, the data lie on a perfect straight line with a negative slope; where rho= 0, there is no linear relationship between the variables; and where rho = +1, the data lie on a perfect straight line with a positive slope (Pallant, 2010). For rho values between 0 and 1 the following guidelines were suggested by Cohen (1988) (as cited by Pallant, 2010):
Small rho=.10 to .29
Medium rho = .30 to .49
Large - rho = .50 to 1.0.
[73]
Chapter 4: Results
4.1 Introduction As mentioned in Chapter 1, this chapter includes the results obtained in this study. The results include physical and chemical water quality, biometric indices, blood constituents‘ analysis, health assessment index, qualitative and semi-quantitative histological assessments. The normal histological structure of the liver, kidney, gill, testis and ovary of H. vittatus; L. marequensis; L. cylindricus and L. rosae are described. Furthermore, results on ageing are reported on and an age-histopathology correlation is also included.
4.2 Physical water quality parameters The physical water quality parameters include the temperature (°C), pH, oxygen percentage (%), oxygen concentration (mg/L), conductivity (µS), and total dissolved salts (mg/L) and will be provided for both trips of each of the OR and LR. The results for each sampling trip were pooled to give a value for each sampling trip.
4.2.1 Temperature The higher temperature was recorded in the LR in November 2009 while the LR‘s April 2010 temperature was the second highest temperature, showing that the LR had higher temperatures (Table 4.1).
4.2.2 pH The highest pH was from OR in September 2009 at 8.41 and the second highest pH being also at OR but from the May 2010 trip. The LR November 2009 trip had the lowest pH at 7.18 (Table 4.1).
4.2.3 Oxygen percentage All oxygen percentages were above the saturation level, with the highest oxygen concentration in the Olifants 2010 trip (141.72%) and the lowest from the Olifants 2009 trip (107.10%) (Table 4.1).
4.2.4 Conductivity The OR had higher conductivity values than the LR. The 2009 trips had higher conductivity values than the 2010 trips for both rivers. The highest conductivity value was found in the [74]
2009 Olifants trip at 1047µS while the lowest conductivity was found in the LR at 92.18µS (Table 4.1).
4.2.5 Total dissolved salts The OR had higher total dissolved salts (TDS) values than the LR. The 2009 trips had higher total dissolved salts values than the 2010 trips for both rivers. The highest TDS value was found in the 2009 OR trip at 397.5 mg/L and the lowest TDS value was found at the 2010 LR trip at 46.1 mg/L (Table 4.1).
Table 4.1: Physical water quality parameters results
Physical water quality parameters OR 2009 OR 2010 LR 2009 LR 2010 Temperature (°C) 23.12 23.80 28.40 24.09 pH 8.41 7.94 7.18 7.84 Oxygen percentage (%) 107.10 141.72 131.25 125.75 Oxygen concentration (mg/L) 9.09 10.35 10.21 10.28 Conductivity (µS) 1047.00 284.40 167.45 92.28 Total dissolved salts (mg/L) 397.50 141.80 83.58 46.10
4.3 Metal concentrations The metal concentrations found in both rivers were compared to the Target water quality requirement (TWQR) as set out by DWAF (1996). The metal concentrations per site were pooled to give the results summarised in Table 4.2 and Table 4.3.
4.3.1 Water metal concentrations The OR water had concentrations higher than the TWQR in aluminium (Al), copper (Cu), zinc (Zn), and lead (Pb) (Table 4.2).
The LR water had concentrations higher than the TWQR in aluminium (Al), copper (Cu), zinc (Zn), and lead (Pb) (Table 4.3).
[75]
Table 4.2: Metal concentrations in Olifants River (OR) water
OR Metal Highest Conc. (µg/L) Lowest conc. (µg/L) Mean conc. (µg/L) Al 64.7 40.79 51.19 Cr 1.4 0.39 0.71 Mn 1.54 0.83 1.29 Fe 39.2 13.48 26.48 Cu 3.17 0.6 1.53 Zn 6.95 3.02 4.71 As 1.43 0.49 0.95 Cd 0.32 0.03 0.14 Pb 1.93 0.89 3.14
Table 4.3: Metal concentrations in Luvuvhu River (LR) water
LR Metal Highest Conc. (µg/L) Lowest conc. (µg/L) Mean conc. (µg/L) Al 89.87 25.63 50.85 Cr 2.06 0.29 1.04 Mn 2.36 0.55 1.23 Fe 114.9 15.81 65.25 Cu 2.43 1.19 1.68 Zn 7.39 2.3 3.84 As 0.37 0.32 0.34 Cd 0.28 0.03 0.12 Pb 1.93 0.77 1.22
[76]
4.3.2 Sediment metal concentrations The concentrations of metals in sediment from the different sites of the OR and LR were also pooled and the results are summarised in Table 4.4 and Table 4.5
Table 4.4: Metal concentrations in Olifants River (OR) sediment
Olifants river Metal Highest Conc. (µg/L) Lowest conc. (µg/L) Mean conc. (µg/L) TWQR (µg/L) Al 94.38 52.24 78.35 5.00 Cr 36.67 14.50 20.35 12.00 Mn 1.67 0.78 1.18 180.00 Fe 20.25 9.76 15.63 N/A Cu 7.06 2.57 4.58 0.30 Zn 0.00 0.00 0.00 2.00 As 6.91 3.43 4.64 10.00 Cd 95.46 4.36 23.51 0.15 Pb 48.83 4.61 14.41 0.20
Levels of Al, Cr, Cu, Cd and Pb in OR sediment were all higher than the TWQR values and also higher than the levels found in OR water.
Table 4.5: Metal concentrations in Luvuvhu River (LR) sediment
Luvuvhu river Metal Highest Conc. (µg/L) Lowest conc. (µg/L) Mean conc. (µg/L) TWQR (µg/L) Al 61.11 45.15 55.12 5.00 Cr 62.90 13.85 39.51 12.00 Mn 0.90 0.64 0.80 180.00 Fe 11.14 7.63 8.64 N/A Cu 3.95 1.42 2.35 0.30 Zn 0.00 0.00 0.00 2.00 As 4.30 3.02 3.57 10.00 Cd 38.38 9.81 21.48 0.15 Pb 22.33 9.70 15.27 0.20
Levels of Al, Cr, Cu, Cd and Pb in LR sediment were all higher than the TWQR values and also higher than the levels found in LR water.
[77]
4.4 Biometric indices The biometric indices include the condition factor, hepatosomatic index, splenosomatic index and gonadosomatic index (male and female split).
4.4.1 Condition factor (Cf) Condition factor was only compared between fish of the same genus or species. The Condition factor results were all within a similar range with no significant differences when comparing the different groups (OR H. vittatus compared to LR H. vittatus (p=0.524); OR 2009 L. marequensis compared to OR 2010 L. marequensis (p=0.144); and LR 2009 L. cylindricus compared to OR 2010 L. rosae (p=0.345)).
Table 4.6: Summary of the mean values for mass, length and Cf for the different fish species at the corresponding sites
Species Site Date Male Female Mass (g) Length (mm) Cf OR H. vittatus 2009 9 7 320.00 ± 211.79 348.63 ± 47.76 0.70 ± 0.27 OR H. vittatus 2010 5 1 490.00 ± 194.63 388.63 ± 36.15 0.80 ± 0.09 LR H. vittatus 2009 9 7 708.28 ± 866.70 362.06 ± 144.60 0.95 ± 0.23 LR H. vittatus 2010 1 1 830.00 ± 692.96 474.50 ± 130.81 0.68 ± 0.08 OR L. marequensis 2009 9 6 237.33 ± 151.54 272.67 ± 37.22 1.08 ± 0.41 OR L. marequensis 2010 10 5 136.00 ± 42.22 247.00 ± 37.22 0.88 ± 0.11 LR L. cylindricus 2009 5 5 104.22 ± 86.88 208.70 ± 39.55 1.14 ± 0.90 OR L. rosae 2010 2 5 228.57 ± 188.28 279.86 ± 39.60 0.90 ± 0.35
4.4.2 Hepatosomatic index (HSI) The LR H. vittatus had higher HSI values than those H. vittatus specimens from the OR (Table 4.7), but these differences were not significant (p=0.052). The L. marequensis from OR 2009 had higher HSI values than specimens from OR 2010 (Table 4.7) but these differences were not significant (p=0.783). The L. cylindricus specimens had higher HSI values than L. rosae specimens (Table 4.7) but these results were not significant (p=0.232).
4.4.3 Splenosomatic index (SSI) The H. vittatus from the OR had higher SSI values than specimens from the LR (Table 4.7), but these results were not significant (p=0.065). The L. marequensis from OR 2010 had higher SSI values than specimens from OR 2009 (Table 4.7) but not significantly so (p=0.49). The L. cylindricus specimens had higher (Table 4.7) SSI values than L. rosae specimens.
[78]
4.4.4 Male Gonadosomatic index (GSI) GSI results of male fish showed significantly higher (p=0.020) values in LR H. vittatus when compared to OR H. vittatus specimens (Table 4.7). OR 2010 L. marequensis male GSI values were significantly higher (p=0.010) than OR 2009 L. marequensis specimens (Table 4.7). Labeo cylindricus specimens had higher male GSI values than those of L. rosae specimens (Table 4.7) but these results were not significant (p=0.209).
4.4.5 Female Gonadosomatic index (GSI) When comparing GSI values of female fish, the H. vittatus specimens from LR had significantly higher (p=0.000) GSI values (Table 4.7) of those from OR. There were no significant differences when comparing L. marequensis specimens, there were no significant differences (p=0.072) with GSI values from OR 2010 values being higher than those from OR 2009 (Table 4.7). There were significantly higher (p=0.032) GSI values of female L. cylindricus specimens from LR 2009 than L. rosae specimens from OR 2010 (Table 4.7).
Table 4.7: Summary of biometric indices of all specimens (mean values for HSI, SSI, GSI)
Species Site Date Male Female HSI ± SD SSI ± SD GSI (male) ± SD GSI (female) ± SD H. vittatus Olifants 2009 9 7 0.54 ± 0.12 0.06 ± 0.05 0.56 ± 1.02 0.38 ± 0.22 H. vittatus Olifants 2010 5 1 0.49 ± 0.08 0.06 ± 0.02 0.83 ± 0.73 1.02 H. vittatus Luvuvhu 2009 9 7 0.46 ± 0.19 0.03 ± 0.01 4.28 ± 1.32 4.41 ± 2.70 H. vittatus Luvuvhu 2010 1 1 0.27 ± 0.29 0.06 ± 0.01 1.02 0.54 L. marequensis Olifants 2009 9 6 0.70 ± 0.17 0.08 ± 0.03 0.62 ± 0.66 0.72 ± 0.70 L. marequensis Olifants 2010 10 5 0.54 ± 0.16 0.09 ± 0.02 2.78 ± 3.31 1.53 ± 2.44 L. cylindricus Luvuvhu 2009 5 5 0.65 ± 0.34 0.17 ± 0.11 2.15 ± 0.70 6.58 ± 9.06 L. rosae Olifants 2010 2 5 0.49 ± 0.13 < 0.01 0.13 ± 0.04 0.82 ± 0.48
4.5 Blood parameters The blood parameters tested were haematocrit, leukocrit, and total plasma protein. These paramaters formed part of the HAI calculations dealt with later in this chapter (Section 4.6).
4.5.1 Haematocrit (Hct) There were no significant differences in comparisons of haematocrit (Hct). See figure 4.1 for a summary of the mean Hct values from all species and all sites. The H. vittatus Hct results from OR were higher than those from LR (p=0.756). The L. marequensis specimens from OR 2010 had higher Hct results than specimens from OR 2009 (p=0.113). The L. cylindricus specimens had higher hct results than those of L. rosae (p=0.645).
[79]
Figure 4.1: Mean Haematocrit values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
4.5.2 Leukocrit (Lct) There were no significant differences in the leukocrit (Lct) results other than the OR 2009 L. marequensis specimens having significantly higher L. cylindricus values than specimens from OR 2010 (p=0.004). See figure 4.2 for a summary of the mean Lct values from all species and all sites.
[80]
Figure 4.2: Mean Leukocrit values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
4.5.3 Total plasma protein (TP) There were no significant differences between the groups when total plasma protein (TP) was compared. The H. vittatus specimens from LR had higher TP values than specimens from OR (p=0.668). See figure 4.3 for a summary of the mean TP values from all species and all sites. The L. cylindricus values were higher than L. rosae values (p=0.797).
[81]
Figure 4.3: Mean Total Protein values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
[82]
4.6 Health assessment index When comparing H. vittatus health assessment index (HAI), the values of fish from the OR were significantly higher (p=0.034) than those from LR. The L. marequensis specimens from OR 2010 had significantly higher (p=0.016) HAI values than specimens from OR 2009. The L. rosae specimens from OR 2010 had significantly higher (p=0.000) than L. cylindricus specimens from LR 2009. See figure 4.4 for a summary of the mean HAI values from all species and all sites.
Anomalies found in H. vittatus specimens included: liver discolouration; parasitic infection; lower than normal Hct values; higher than normal Lct values; and lower than normal TP values. Hydrocynus vittatus specimens from OR 2009 (n=16) had a sum HAI value of 380 and a mean HAI value of 23.75. The H. vittatus specimens from OR 2010 (n=6) had a sum HAI value of 190 and a mean HAI value of 31.67. The H. vittatus specimens from LR 2009 (n=16) had a sum HAI value of 160 and mean HAI value of 10. The H. vittatus specimens from LR 2010 (n=2) had a sum HAI value of 60 and a mean HAI value of 30 (see Appendix C for full HAI score sheets).
Anomalies found in L. marequensis specimens included: inflamed hindgut; inflamed kidney; liver discolouration; pale gills; parasitic infections; higher than normal Hct and Lct values; and lower than normal TP values. Labeobarbus marequensis from OR 2009 (n=15) had a sum HAI value of 530 and a mean HAI value of 35.33. L. marequensis from OR 2010 (n=15) had a sum HAI value of 740 and a mean HAI value of 49.33 (see Appendix C for detailed HAI score sheets).
The only anomaly found in L. cylindricus specimens was a higher than normal TP value. L. cylindricus from LR 2009 (n=10) had a sum HAI value of 10 and a mean HAI value of 1 (see Appendix C for full HAI score sheets).
Anomalies found in L. rosae specimens were: inflamed kidney; higher and lower than normal hct values; and lower than normal TP values. L. rosae specimens from OR 2010 (n=7) had a sum HAI value of 280 and a mean HAI value of 40 (see Appendix C for full HAI score sheets).
[83]
Figure 4.4: Mean Health Assessment Index values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
[84]
4.7 Normal histology The normal histology of liver, kidney, gill, testis and ovary tissue of specimens of H. vittatus, L. marequensis, L. cylindricus and L. rosae will be briefly described. The structures of liver, kidney, gill, testis and gonad will be dealt with for all species by organ.
4.7.1 Normal liver histology The normal histological structure of the liver of H. vittatus, L.marequensis, L.cylindricus and L. rosae specimens sampled in this study all consisted of hepatocytes arranged in hepatic cords. The hepatocytes (Fig. 4.5 - 1) were observed as hexagonal to oval-shaped cells with a central nucleus (Fig. 4.5 - 2) and a distinct cell membrane (Fig. 4.5 - 3). The hepatic cords consist of ―flower-like‖ arrangements of hepatocytes surrounding a blood sinusoid (Fig. 4.5 - 4). The sinusoids are lined with elongated endothelial cells and were identified as unstained areas between the hepatocytes and red blood cells (Fig. 4.5 - 5) were frequently found within them.
Hepatopancreatic tissue (Fig. 4.6 - 1) was found in H. vittatus, L. cylindricus and L. rosae specimens sampled but not in L. marequensis specimens. The hepatopancreatic tissue was identified as dark purple stained (H&E) pancreatic acini (Fig. 4.6 - 2) with a large nucleus (Fig. 4.6 - 3) and pink stained zymogen granules (Fig. 4.6 - 4).
Portal veins (Fig. 4.6 - 5) were found in all species. Bile ducts (Fig. 4.6 - 6) of various sizes were found in the liver tissue of all species. The inside of the bile ducts were lined with columnar epithelium (Fig. 4.6 - 7) surrounded by a thin layer of connective tissue (Fig. 4.6 - 8).
[85]
Figure 4.5: Normal liver histology showing hepatic parenchyma. A. Detail of hepatic parenchyma (a) (100X); B. Detail of hepatic parenchyma (b) (100X); C. Detail of hepatic parenchyma (c) (100X); D. Hepatic parenchyma (40X); E. Hepatic parenchyma showing sinusoids(40x); F. Hepatic parenchyma showing red blood cells (40x). 1.Hepatocyte; 2. Nucleus; 3.Cell membrane; 4.Blood sinusoid; 5.Red blood cell
[86]
Figure 4.6: Normal liver histology showing hepatopancreatic tissue and bile ducts. A. Hepatopancreatic tissue (1) (40X); B. Hepatopancreatic tissue (2) (40X); C. Detail of hepatopancreatic tissue (100x); D. Hepatic portal vein (40X) E. Bile duct (a) (40x); F. Bile duct (b) (40x). 1. Hepatopancreatic tissue; 2. Pancreatic acini; 3. Nucleus; 4. Zymogen granules; 5. Hepatic portal vein 6. Bile duct; 7. Columnar epithelium; 8. Connective tissue.
[87]
4.7.2 Normal kidney tissue The normal histological structure of the kidney of H. vittatus, L.marequensis, L.cylindricus and L. rosae specimens sampled in this study consisted of nephrons surrounded by haematopoietic tissue (Fig 4.7 - 1). The nephrons were made up of renal corpuscles (Fig. 4.7 - 2) and renal tubules (Fig. 4.7 - 3).
The renal corpuscle is made up of a glomerulus (Fig. 4.7 - 4) surrounded by Bowman‘s capsule. The glomerulus consists of a cluster of blood capillaries (Fig. 4.7 - 5) filled with red blood cells (Fig. 4.7 - 6). The capillaries are lined with capillary endothelial cells (Fig. 4.7 - 7). Mesangial cells (Fig. 4.7 - 8) make up the rest of the glomerulus. Bowman‘s capsule is made up of an inner visceral epithelium of podocytes (Fig. 4.7 - 9) and an outer parietal layer of squamous epithelium supported by a basement membrane (Fig. 4.7 - 10). The two layers described above form a lumen which is known as Bowman‘s space (Fig. 4.7 - 11).
Two types of renal tubules were identified in the specimens sampled, namely the proximal and distal tubules. The proximal tubules (Fig. 4.8 - 2) consisted of columnar and cuboidal epithelium (Fig. 4.8 -3) with a large centrally located nucleus (Fig. 4.8 - 4) and had a distinct brush border (Fig. 4.8 - 5) lining their lumen. The distal renal tubules (Fig. 4.8 - 6) were distinguished from the proximal tubules by being generally smaller and lacking a brush border. These tubules consisted of cuboidal epithelium and a centrally located nucleus surrounding a lumen. Intercalated cells (Fig. 4.8 - 7) were also identified within the renal tubules in all species. Thyroid follicles (Fig. 4.8 - 9) were observed as circular pink stained structures surrounded by a thin layer of epithelium. Thyroid follicles were only found in L. marequensis specimens.
[88]
Figure 4.7: Normal kidney histology showing renal corpuscles. A. Kidney tissue (40X); B. Renal corpuscle (a) (100X); C. Renal corpuscle (b) (100X); D. Renal corpuscle (c) (100X); E. Renal corpuscle (d) (40x); F. Renal corpuscles (40x). 1.Haematopoietic tissue; 2. Renal corpuscles; 3. Renal tubules; 4. Glomerulus; 5. Blood capillaries; 6. Red blood cells; 7. Endothelial cells; 8. Mesangial cells; 9. Podocytes; 10. Basement membrane; 11. Bowman‘s space. [89]
Figure 4.8: Normal kidney histology showing tubules and thyroid follicles. A. Proximal tubules (a) (100X); B. Distal tubules (a) (100X); C. Proximal tubules (b) (100X); D. Proximal tubules (c) (100x); E Distal tubules (b) (40x); F. Thyroid follicle (100X). 1. Haematopoietic tissue; 2. Proximal tubule; 3. Tubular epithelium; 4. Nucleus; 5. Brush border; 6. Distal tubule; 7. Intercalated cells; 8. Tubule lumen; 9. Thyroid follicle.
[90]
4.7.3 Normal gill tissue The normal histological structure of the gill of H. vittatus, L. marequensis, L. cylindricus and L. rosae specimens sampled in this study consisted of a gill arch and associated striated muscles (Fig. 4.9 - 1) from which primary lamellae (Fig. 4.9 - 2) protruded. The primary lamellae were supported by chondrocytes (Fig. 4.9 - 3) and calcified cartilage (Fig.4.9 - 4) and were lined with epithelium and contained a vascular system. The outer epithelial layer consisted of epithelial cells (Fig. 4.9 - 5) and mucous cells.
The primary lamellae (Fig. 4.9 - 6) were lined with secondary lamellae (Fig. 4.9 - 7). The secondary lamellae had a semicircular shape and consisted of blood arterioles in which red blood cells (Fig. 4.9 - 8) were often found. The capillary lumens (Fig. 4.9 - 9) were supported by darkly stained (H&E) pillar cells (Fig. 4.9 - 10) and were surrounded by a layer of epithelial cells.
[91]
Figure 4.9: Normal gill histology. A. Striated muscle (40X); B. Primary lamella support (a) (40X); C. Primary lamella support (b) (100X); D.Primary lamella support (c) (40X); E. Secondary lamellae (a) (100x); F. Secondary lamellae (b) (100x). 1. Striated muscle; 2. Primary lamella; 3. Chondrocytes; 4. Calcified cartilage; 5. Epithelium ; 6. Primary lamella; 7. Secondary lamella; 8. Red blood cell; 9. Capillary lumen; 10. Pillar cell.
[92]
4.7.4 Normal testis tissue The normal histological structure of the testis of H. vittatus, L. marequensis, L. cylindricus and L. rosae specimens sampled in this study consisted of seminiferous lobules (Fig. 4.10 - 1) as well as interstitial tissue (Fig. 4.10 - 2) enclosed in a tunica albuginea (Fig. 4.10 - 3). Primary and secondary spermatogonia; primary and secondary spermatocytes were found in the lobules.
Primary spermatogonia (Fig. 4.10 - 4) were found on the periphery of the seminiferous lobules. Secondary spermatogonia (Fig. 4.10 - 5) were found to be generally smaller than primary spermatogonia and were generally found in groups of two or more cells. Sertoli cells (Fig. 4.10 - 6) were found as round to oval-shaped cells in close association to primary spermatogonia. Leydig cells (Fig. 4.10 - 7) were also found in the interstitial tissue.
Primary spermatocytes (Fig. 4.11 - 1) were found in cysts (Fig. 4.11 - 2) of many cells with little nuclear detail or cell membranes observed. Secondary spermatocytes (Fig. 4.11 - 3) were smaller and were stained darker (H&E) than the primary spermatocytes as a result of compressed nuclear material. Spermatids (Fig. 4.11 - 4) were found to be smaller than the secondary spermatocytes and were found near to the secondary spermatocytes. Spermatozoa (Fig. 4.11 - 5) were smaller and darker than the spermatids and were found within the seminiferous lobules.
[93]
Figure 4.10: Normal testis histology showing seminiferous lobules and interstitial tissue. A. Semiferous lobules (a) (40X); B. Semiferous lobules (b) (10X); C. Semiferous lobules (c) (40X); Semiferous lobules (d) (100X); E. Semiferous lobules (e) (40x); F. Seminiferous lobules (f) (100x). 1. Semiferous lobules; 2. Interstitial tissue; 3. Tunica albuginea. ; 4. Primary Spermatogonium; 5. Secondary Spermatogonium; 6. Sertoli cell; 7. Leydig cell.
[94]
Figure 4.11: Normal testis histology showing spermatocytes, spermatogonia and spermatids. A. Primary and Secondary Spermatocytes (a) (100X); B. Primary and Secondary Spermatocytes (b) (100X); C. Primary and Secondary Spermatocytes (c) (100X); D. Primary and Secondary Spermatocytes (d) (100X); E. Secondary Spermatogonia and Spermatids (40X); F. Spermatozoa (100X). 1. Primary Spermatocytes; 2. Primary Spermatocyte cyst; 3. Secondary Spermatocytes; 4. Spermatids; 5. Spermatozoa.
[95]
4.7.5 Normal ovary tissue The normal histological structure of the ovary of H. vittatus, L. marequensis, L. cylindricus and L. rosae specimens sampled in this study consisted of ovarian follicles enclosed within a tunicia albuginea (Fig. 4.12 - 1). Oogonial nests (Fig. 4.12 - 2) were found to have clusters of oogonia (Fig. 4.12 - 3) which were found to be oval-shaped cells with a large nucleus.
The peri-nucleolar oocytes (Fig. 4.12 - 4) were found to have multiple nucleoli (Fig. 4.12 - 5) in the peripheral region of the nucleus with a purple stained cytoplasm (H&E).
The cortical alveolar oocytes (Fig. 4.13 - 1) had a cytoplasm which stained pink with H&E and many lipid droplets (Fig. 4.13 - 2).
The vittelogenic oocytes (Fig. 4.13 - 3) had more yolk globules in their cytoplasm than the cortical alveolar oocytes had and were enclosed by an oocyte wall (Fig. 4.13 - 4). Maturation oocytes (Fig. 4.13 - 5) were filled with yolk globules (Fig. 4.13 - 6) which stained pink with H&E. These oocytes were visibly larger than the other oocytes identified.
[96]
Figure 4.12: Normal ovary histology showing oogonia and peri-nucleolar oocytes. A. Oogonial nest (a) (100X); B. Oogonial nest (b) (100X); Peri-nucleolar oocyte (a) (100X); Peri-nucleolar oocyte (b) (100X); Peri-nucleolar oocyte (c) (100X); Peri-nucleolar oocyte (d) (10X). 1. Tunical albuginea; 2. Oogonial nest; 3. Oognium; 4. Peri-nucleolar oocyte; 5. Nucleolus.
[97]
Figure 4.13: Normal ovary histology showing cortical alveolar, vittelogenic and maturation oocytes. A. Cortical alveolar oocytes (a) (40X); B. Cortical alveolar oocytes (b) (40X); C Cortical alveolar oocytes (c) (40X); C. Vittelogenic oocyte (40X); D. Cortical alveolar oocyte detail (100X); E. Maturation oocytes (10X); F. Detail of maturation oocyte (40X). 1. Cortical alveolar oocyte; 2. Lipid droplets; 3. Vittelogenic oocytes; 4. Oocyte wall; 5. Maturation oocytes; 6. Yolk globules.
[98]
4.8 Gonadal staging The gonads were staged using the methods described in Section 3.8.1.
Tables 4.8 and 4.9 summarise the percentage of testis and ovary stages respectively.
4.8.1 Testis staging Table 4.8: Testis stagimg percentages
Species Site Date Number (n=) Stage 0 % Stage 1 % Stage 2 % Stage 3 % H.vittatus OR 2009 6 50 50 0 0 H.vittatus OR 2010 5 0 80 20 0 H.vittatus LR 2009 7 0 0 14.28 85.72 H.vittatus LR 2010 1 0 100 0 0 L. marequensis OR 2009 9 77.78 11.11 11.11 0 L. marequensis OR 2010 10 0 20 60 20 L. cylindricus LR 2009 3 0 0 0 100 L. rosae OR 2010 4 100 0 0 0
4.8.2 Ovary staging Table 4.9: Ovary staging percentages
Species Site Date Number (n=) Stage 0 % Stage 1 % Stage 2 % Stage 3 % H.vittatus OR 2009 10 100 0 0 0 H.vittatus OR 2010 1 0 100 0 0 H.vittatus LR 2009 9 42.86 0 57.14 0 H.vittatus LR 2010 1 100 0 0 0 L. marequensis OR 2009 5 80 0 20 0 L. marequensis OR 2010 5 20 60 20 0 L. cylindricus LR 2009 4 50 0 50 0 L. rosae OR 2010 3 100 0 0 0
See Appendix C for full staging tables
4.9 Histopathology Histopathological alterations of liver, kidney, gill, testis and ovary respectively for all species were noted and the percentage prevalences of those alterations were also be noted. Mean organ indices for all organs were compared to each other by species and sampling trip as well as to the classes discussed in section 3.9. The mean fish index (see section 3.9) of each species is also compared to each other.
[99]
4.9.1 Liver histopathology The following alterations were identified in the liver tissue assessed and the percentage prevalence for each species is given in Table 4.10 and Table 4.11 for the OR and LR respectively.
Intracellular deposits (Fig. 4.14 A) were identified as small brown deposits visible within the cytoplasm of the affected hepatocytes.
Glycogen vacuoles were identified as very small rounded open spaces within the cytoplasm of hepatocytes and were visibly smaller than the other vacuoles.
Vacuolation (Fig. 4.14 B) was identified as round, empty spaces within the cell cytoplasm and the contents of the cells were most probably dissolved as a result of the H&E staining technique.
Nuclear pleomorphism (Fig. 4.14 C) was identified as nuclei being of different sizes and shapes in a specific area. Nuclei were smaller and larger than a normal nucleus and some nuclei had an irregular shape. Chromatin clearing was associated with this alteration where the nuclei affected are disintegrated, leaving an empty nucleus.
Nuclear pyknosis was identified as nuclei which had densely compacted chromatin. The nuclei were smaller and the nuclei also stained very darkly.
Necrosis of hepatocytes (Fig. 4.14 D) was identified as having a darkly stained eosinophilic cytoplasm and usually having a pyknotic nucleus.
Parasites (Fig. 4.14 E) were identified in L. marequensis livers only. The parasites were encysted and commonly found in groups of three or more cysts packed tightly together with a surrounding membrane.
Melanomacrophage centres (MMCs) (Fig. 4.14 F) were identified as groups of deposits with yellow-brown pigmentation within the L. rosae liver tissue.
[100]
Figure 4.14: Liver histopathology. A. Intracellular deposits (100X); B. Vacuolation (100X); C. Nuclear pleomorphism (100X); D. Necrosis (100X); E. Parasite (100X); F. MMCs (40X).
[101]
Table 4.10: Percentage prevalence of liver alterations (Olifants River)
Percentage prevalence Olifants 2009 Olifants 2010 H. vittatus L. marequensis H. vittatus L. marequensis L. rosae
Histological alteration n=16 n=15 n=6 n=15 n=7 Liver Intracellular deposits 75.00 – 66.67 53.33 42.86 Glycogen vacuoles – 13.33 – 6.67 – Vacuolation 68.75 46.67 16.67 26.67 57.14 Pleomorphism 50.00 73.33 – 80.00 28.57 Pyknosis 25.00 – – 13.33 28.57 Necrosis 75.00 26.67 – 26.67 – MMCs – – – – 100.00 Intracellular oedema – – – – –
Table 4.11: Percentage prevalence of liver alterations (Luvuvhu River)
Percentage prevalence Luvuvhu 2009 Luvuvhu 2010 H. vittatus L. cylindricus H. vittatus
Histological alteration n=16 n=10 n=10 Liver Intracellular deposits 81.25 30.00 50.00 Glycogen vacuoles – – – Vacuolation 75.00 90.00 50.00 Pleomorphism 25.00 80.00 50.00 Pyknosis 6.25 – – Necrosis 25.00 – – MMCs – – – Intracellular oedema 6.25 – –
See figure 4.15 for a summary of the mean liver index values from all species and all sites.
Hydrocynus vittatus specimens from OR 2009 had liver index values ranging from 8.00 to 26.00 and a mean liver index of 13.00 ± 4.56. The H. vittatus specimens from OR 2010 had liver index values ranging from 8.00 to 14.00 with a mean liver index of 10.33 ± 2.94. The H. vittatus specimens from LR 2009 had liver index results ranging from 2.00 to 16.00 with a mean liver index of 9.75 ± 4.12. The H. vittatus specimens from LR 2010 had liver index values ranging from 6.00 to 10.00 with a mean liver index of 8.00 ± 2.83. Labeobarbus marequensis specimens from OR 2009 had liver index results ranging 0.00 to 16.00 with a
[102]
mean liver index 9.33 ± 5.22. The L. marequensis from OR 2010 had liver index results ranging from 0.00 to 14.00 with a mean liver index of 8.00 ± 3.70. Labeo cylindricus specimens from LR 2009 had liver index values ranging from 4.00 to 14.00 with a mean liver index of 8.20 ± 2.57. Labeo rosae specimens from OR 2010 had liver index results ranging from 6.00 to 12.00 with a mean liver index value of 8.29 ± 2.43. There were no significant differences between the liver index results of H. vittatus from OR and LR (p=0.966) or L. marequensis from OR 2009 and 2010 (p=0.193) or L. cylindricus from LR 2009 and L. rosae from OR 2010 (p=0.062).
Class 2
Class 1
Figure 4.15: Mean liver index values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
[103]
4.9.2 Kidney histopathology The following histopathological alterations were found in the kidney tissue assessed and the percentage prevalence are given in Table 4.12 and 4.13 for the OR and LR respectively.
Vacuolation of the renal tubule epithelium (Fig. 4.16 A) was identified as round vacuoles in the renal tubule epithelial cells
Nuclear alterations (chromatin clearing) (Fig. 4.16 B) of the renal tubule epithelial cells were identified as having nuclei with less or absent chromatin
Melanomacrophage centres (Fig. 4.16 C and D) were identified in the haematopoietic tissue
Hyaline droplet degeneration (Fig 4.16 E) was identified in the renal tubule epithelial cells as the cytoplasm degenerated into eosinophilic droplets of varying size
Table 4.12: Percentage prevalence of kidney alterations (Olifants River)
Percentage prevalence Olifants 2009 Olifants 2010 Histological alteration H. vittatus L. marequensis H. vittatus L. marequensis L. rosae n=16 n=15 n=6 n=15 n=7 Kidney Tubular vacuolation 68.75 81.25 100.00 20.00 – Hyaline droplet degeneration – – – 20.00 – Nuclear alterations 12.50 – 16.67 – – MMCs – 6.25 – 20.00 71.43 Eosinophilic degeneration – – – 20.00 –
[104]
Table 4.13: Percentage prevalence of kidney alterations (Luvuvhu River)
Luvuvhu 2009 Luvuvhu 2010 Histological alteration H. vittatus L. cylindricus H. vittatus n=16 n=10 n=2 Kidney Tubular vacuolation 68.75 30.00 100.00 Hyaline droplet degeneration 25.00 – – Nuclear alterations – 20.00 – MMCs – 20.00 – Eosinophilic degeneration – – –
See figure 4.17 for a summary of the mean kidney index values from all species and all sites.
Hydrocynus vittatus specimens from OR 2009 had kidney index values ranging from 0.00 to 8.00 and a mean kidney index of 2.25 ± 2.52. The H. vittatus specimens from OR 2010 had kidney index values ranging from 2.00 to 8.00 with a mean kidney index of 4.00 ± 2.19. The H. vittatus specimens from LR 2009 had kidney index values ranging from 0.00 to 12.00 with a mean kidney index of 5.00 ± 3.65. The H. vittatus specimens from LR 2010 had kidney index values ranging from 2.00 to 4.00 with a mean kidney index value of 3.00 ± 1.41. Labeobarbus marequensis specimens from OR 2009 had kidney index values ranging from 0.00 to 4.00 with a mean kidney index value of 1.07 ± 1.28. The L. marequensis specimens from OR 2010 had kidney index values ranging from 0.00 to 6.00 with a mean kidney index value of 2.40 ± 2.03. Labeo cylindricus specimens from LR 2009 had kidney index values of 0.00 to 6.00 with a mean kidney index value of 1.80 ± 2.39. Labeo rosae specimens from OR 2010 had kidney index values ranging from 0.00 to 8.00 with a mean kidney index value 3.43 ± 2.99. There were notably high standard deviations of kidney index values but no significant differences between kidney index values between H. vittatus specimens from OR and LR (p=0.164). The L. marequensis specimens from OR 2009 had significantly higher values than specimens from OR 2009 (p=0.026). There were no significant differences between L. cylindricus specimens from LR 2009 and L. rosae specimens from OR 2010 (0.717)
[105]
Figure 4.16: Kidney histopathology. A. Vacuolation (100X); B. Chromatin clearing (10X); C. MMCs (40X); D. Detail of MMCs (100X); E. Hyaline droplet degeneration (40X).
[106]
Figure 4.17: Mean kidney index values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
[107]
4.9.3 Gill histopathology There were no histopathological alterations observed in any of the specimens studied. However, parasites (Fig. 4.18) were observed in the gill tissue of H. vittatus specimens from OR 2009 (31.25%) and LR 2009 (25%) and 2010 (50%) as well as L. marequensis from OR 2009 (6.67%) and L. cylindricus from LR 2009 (10%). No parasites were observed in gill tissue studied from the OR in 2010.
Figure 4.18: Monogean parasite on the gills of L. marequensis (40X).
[108]
4.9.4 Testis histopathology The only histopathological alteration was melanomacrophage centres (Fig. 4.19) which were observed in L. marequensis (18.18 %) and L. rosae (50%) specimens from OR 2010.
Figure 4.19: Melanomacrophage centres in testis tissue of L. rosae (40X).
Table 4.14: Percentage prevalence of testis alterations
Percentage prevalence Olifants 2009 Olifants 2010 Histological alteration H. vittatus L. marequensis H. vittatus L. marequensis L. rosae n=16 n=15 n=6 n=15 n=7 Testes – – – – – MMCs – – – 18.18 50.00
[109]
See figure 4.20 for a summary of the mean testis index values from all species and all sites.
The differences between testis index values of male H. vittatus specimens from OR and LR were not significant (0.066). The differences between testis index values of L. marequensis specimens from OR 2009 and OR 2010 (p=0.002) and those of L. cylindricus specimens from LR 2009 and L. rosae specimens from OR 2010 (p=0.000) were significant.
Figure 4.20: Mean testis index values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
[110]
4.9.5 Ovary histopathology The only histopathological alterations observed in the ovarian tissue studied were melanomacrophage centres (Fig. 4.21) which were observed in ovarian tissue of H. vittatus and L. rosae specimens from the OR 2010 sampling trip.
Figure 4.21: Melanomacrophage centres in ovary tissue of H. vittatus (40X)
[111]
Table 4.15: Percentage prevalence of ovary alterations (Olifants River)
Percentage prevalence Olifants 2009 Olifants 2010 Histological alteration H. vittatus L. marequensis H. vittatus L. marequensis L. rosae n=16 n=15 n=6 n=15 n=7 Ovaries – – 16.67 – 66.67 MMCs – – – – –
Table 4.16: Percentage prevalence of ovary alterations (Luvuvhu River)
Percentage prevalence Luvuvhu 2009 Luvuvhu 2010 Histological alteration H. vittatus L. cylindricus H. vittatus n=16 n=10 n=10 Ovaries – – – MMCs – – –
Differences in ovary index values of female H. vittatus specimens from OR and LR were not significant (0.066). Differences in ovary index values of L. marequensis specimens from OR 2009 and OR 2010 (p=0.002). Differences in ovary index values of L. cylindricus specimens from LR 2009 and L. rosae specimens from OR 2010 were also significant (p=0.000).
See figure 4.22 for a summary of the mean ovary index values from all species and all sites.
[112]
Figure 4.22: Mean Ovary index values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
4.9.6 Fish index Hydrocynus vittatus specimens from OR 2009 had fish index values ranging from 8.00 to 26.00 with a mean fish index value of 15.25 ± 4.30. The H. vittatus specimens from OR 2010 had fish index values ranging from 12.00 to 18.00 with a mean fish index value of 14.33 ± 2.66. The H. vittatus specimens from LR 2009 had fish index values ranging from 2.00 to 20.00 with a mean fish index of 14.75 ± 5.65. The H. vittatus specimens from LR 2010 had fish index values ranging from 8.00 to 14.00 with a mean fish index value of 11.00 ± 4.24. Labeobarbus marequensis specimens from OR 2009 had fish index values ranging from 2.00 to 18.00 with a mean fish index value of 10.40 ± 5.62. The L. marequensis specimens from OR 2010 had fish index values ranging from 4.00 to 18.00 with a mean fish index value of 10.67 ± 3.98. Labeo cylindricus from LR 2009 had fish index values ranging from 8.00 to 14.00 with a mean fish index value of 10.00 ± 2.11. Labeo rosae specimens from OR 2010 had fish index values ranging from 6.00 to 14.00 with a mean fish index value of 12.86 ± [113]
3.98. The fish index results were generally higher in the H. vittatus specimens with no significant differences between values observed in fish specimens from the OR and LR (p=0.175). There were no significant differences between fish index results of L. marequensis specimens obtained from OR 2009 and 2010 (p=0.105). There were higher fish index values in L. rosae specimens when compared to L. rosae specimens but these differences were not statistically significant (p=0.118).
See figure 4.23 for a summary of the mean fish index values from all species and all sites.
Figure 4.23: Mean fish index values for all species from all sampling trips
(OR = Olifants River; LR = Luvuvhu River; HV = H. vittatus; LMar = L. marequensis; LC = L. cylindricus; Lros = L. rosae)
[114]
4.10 Age The length, weight, sex and age of H. vittatus and L. marequensis specimens from both rivers are shown in tables 4.17 to 4.22 below.
Table 4.17: Hydrocynus vittatus Olifants 2009 Age
Age Fish number Species Length (mm) Mass (g) Sex (months) 1 Hydrocynus vittatus 360.00 720.00 Female 81.00 2 Hydrocynus vittatus 380.00 480.00 Female 69.00 3 Hydrocynus vittatus 410.00 480.00 Female 45.00 4 Hydrocynus vittatus 340.00 240.00 Male 57.00 5 Hydrocynus vittatus 319.00 220.00 Female 57.00 6 Hydrocynus vittatus 318.00 220.00 Male 33.00 7 Hydrocynus vittatus 479.00 860.00 Male 33.00 8 Hydrocynus vittatus 377.00 380.00 Female 33.00 9 Hydrocynus vittatus 328.00 180.00 Male 21.00 10 Hydrocynus vittatus 330.00 220.00 Male 21.00 11 Hydrocynus vittatus 270.00 200.00 Male 57.00 12 Hydrocynus vittatus 323.00 180.00 Male 45.00 13 Hydrocynus vittatus 321.00 200.00 Female 33.00 14 Hydrocynus vittatus 368.00 220.00 Male 45.00 15 Hydrocynus vittatus 327.00 140.00 Male 9.00 16 Hydrocynus vittatus 328.00 180.00 Female 81.00 Mean 348.63 320.00 n/a 45.00 Std Dev. 47.76 211.79 n/a 21.01
Table 4.18: Hydrocynus vittatus Olifants 2010 Age
Age Fish number Species Length (mm) Mass (g) Sex (months) 1 Hydrocynus vittatus 450.00 860.00 Male 53.00 2 Hydrocynus vittatus 385.00 420.00 Male 29.00 3 Hydrocynus vittatus 380.00 460.00 Male 41.00 4 Hydrocynus vittatus 400.00 480.00 Male 29.00 5 Hydrocynus vittatus 375.00 440.00 Female 29.00 6 Hydrocynus vittatus 340.00 280.00 Male 53.00 Mean 388.33 490.00 n/a 39.00 Std Dev. 36.15 194.63 n/a 11.80
[115]
Table 4.19: Hydrocynus vittatus Luvuvhu 2009 Age
Age Fish number Species Length (mm) Mass (g) Sex (months) 1 Hydrocynus vittatus 414.00 560.00 Male 47.00 2 Hydrocynus vittatus 418.00 820.00 Female 53.00 3 Hydrocynus vittatus 358.00 360.00 Male 47.00 4 Hydrocynus vittatus 360.00 340.00 Male 59.00 5 Hydrocynus vittatus 145.00 25.38 Male 35.00 6 Hydrocynus vittatus 234.00 77.48 Male 59.00 7 Hydrocynus vittatus 450.00 800.00 Female 83.00 8 Hydrocynus vittatus 174.00 37.30 Male 95.00 9 Hydrocynus vittatus 668.00 3360.00 Female 95.00 10 Hydrocynus vittatus 622.00 2260.00 Female 95.00 11 Hydrocynus vittatus 322.00 280.00 Male 35.00 12 Hydrocynus vittatus 336.00 560.00 Male 47.00 13 Hydrocynus vittatus 438.00 980.00 Female 59.00 14 Hydrocynus vittatus 402.00 740.00 Female 59.00 15 Hydrocynus vittatus 318.00 280.00 Male 47.00 16 Hydrocynus vittatus 356.00 540.00 Female 59.00 Mean 375.94 751.26 n/a 60.88 Std Dev. 137.16 876.21 n/a 20.28
Table 4.20: Hydrocynus vittatus Luvuvhu 2010 Age
Age Fish number Species Length (mm) Mass (g) Sex (months) 1 Hydrocynus vittatus 565.00 1320.00 Female 76.00 2 Hydrocynus vittatus 380.00 340.00 Male 64.00 Mean 472.50 830.00 n/a 70.00 Std Dev. 130.81 692.96 n/a 8.49
[116]
Table 4.21: Labeobarbus marequensis Olifants 2009 Age
Age Fish number Species Length (mm) Mass (g) Sex (months) 1 Labeobarbus marequensis 235.00 140.00 Male 93.00 2 Labeobarbus marequensis 255.00 140.00 Male – 3 Labeobarbus marequensis 240.00 140.00 Male – 4 Labeobarbus marequensis 250.00 140.00 Male 81.00 5 Labeobarbus marequensis 200.00 80.00 Male 117.00 6 Labeobarbus marequensis 275.00 200.00 Male 69.00 7 Labeobarbus marequensis 265.00 200.00 Female 69.00 8 Labeobarbus marequensis 265.00 180.00 Female 57.00 9 Labeobarbus marequensis 335.00 480.00 Female 9.00 10 Labeobarbus marequensis 295.00 640.00 Female 93.00 11 Labeobarbus marequensis 280.00 220.00 Male 93.00 12 Labeobarbus marequensis 285.00 220.00 Female 93.00 13 Labeobarbus marequensis 280.00 180.00 Male 105.00 14 Labeobarbus marequensis 280.00 200.00 Male 93.00 15 Labeobarbus marequensis 350.00 400.00 Female 105.00 Mean 272.67 237.33 n/a 82.00 Std Dev. 37.22 151.54 n/a 27.65
Table 4.22: Labeobarbus marequensis Olifants 2010 Age
Age Fish number Species Length (mm) Mass (g) Sex (months) 1 Labeobarbus marequensis 325.00 220.00 Female 77.00 2 Labeobarbus marequensis 245.00 120.00 Male 53.00 3 Labeobarbus marequensis 265.00 180.00 Male 77.00 4 Labeobarbus marequensis 250.00 140.00 Female 53.00 5 Labeobarbus marequensis 155.00 40.00 Male 65.00 6 Labeobarbus marequensis 270.00 160.00 Female 41.00 7 Labeobarbus marequensis 260.00 160.00 Female 65.00 8 Labeobarbus marequensis 255.00 180.00 Male 53.00 9 Labeobarbus marequensis 240.00 120.00 Male 53.00 10 Labeobarbus marequensis 250.00 140.00 Male 41.00 11 Labeobarbus marequensis 240.00 120.00 Male 41.00 12 Labeobarbus marequensis 230.00 100.00 Female 53.00 13 Labeobarbus marequensis 235.00 120.00 Male 41.00 14 Labeobarbus marequensis 260.00 140.00 Male 53.00 15 Labeobarbus marequensis 225.00 100.00 Male 77.00 Mean 247.00 136.00 n/a 56.20 Std Dev. 34.68 42.22 n/a 13.20 [117]
4.11 Age – Histopathology correlations
4.11.1 Age – Liver Index Correlations The Spearman‘s correlation coefficient value when testing for correlation between age and liver index for H. vittatus specimens was -0.045. This value indicates a small negative correlation for these variables. The p-value was 0.783 and this indicates that this correlation is not significant.
The Spearman‘s correlation coefficient value when testing for correlation between age and liver index for L. marequensis specimens was 0.142. This value indicates a positive correlation for these variables. The p-value was 0.470 and this indicates that this correlation is not significant.
Thus there was no significant correlation between age and liver index results for H. vittatus (see Fig. 4.24) or L. marequensis (Fig. 4.25) specimens sampled in this study.
4.11.2 Age – Fish Index Correlations The Spearman‘s correlation coefficient value when testing for correlation between age and liver index for H. vittatus specimens was 0.088. This value indicates a very small correlation for these variables. The p-value was 0.590 and this indicates that this correlation is not significant.
The Spearman‘s correlation coefficient value when testing for correlation between age and liver index for L. marequensis specimens was -0.014. This value indicates a very small negative correlation for these variables. The p-value was 0.942 and this indicates that this correlation is not significant.
Thus there was no significant correlation between age and fish index results for H. vittatus (see Fig. 4.26) or L. marequensis (see Fig. 4.27) specimens sampled in this study. Therefore the histopathological changes observed were not as a result of age and instead due to environmental changes.
[118]
Figure 4.24: Age – liver index correlation - Hydrocynus vittatus
Figure 4.25: Age – liver index correlation - Labeobarbus marequensis [119]
Figure 4.26: Age – fish index correlation - Hydrocynus vittatus
Figure 4.27: Age – fish index correlation - Labeobarbus marequensis [120]
Chapter 5: Discussion
5.1 Introduction This chapter provides a discussion of the results obtained. All of the results are discussed and compared to relevant literature to give a clear picture of the health of the fish studied according to the parameters studied. Possible causes and reasons for the findings are also discussed.
5.2 Physical water quality parameters
5.2.1 Temperature The water temperatures recorded for all sites and sampling trips fell within the normal range of South African rivers of 5-30 °C (DWAF, 1996) (OR 2009= 23.12 °C; OR 2010=23.80 °C; LR 2009=28.4 °C; LR 2010=24.09 °C). Therefore the temperature was not likely to have had an effect on the health of the fish sampled. Temperature was higher in LR 2009, but not outside of the normal range.
5.2.2 pH The highest pH of 8.41 was recorded in the OR in the 2009 sampling trip. The lowest pH of 7.18 was recorded in the LR in the 2009 sampling trip. The OR had overall higher pH values and this is probably as a result of the higher sediment loads in the river (WRC, 2001a). The pH values recorded for the OR in this study correspond with historical data of sampling points of the Mamba Weir (site 1 for this study) and Balule (site 4 of this study) water quality monitoring points (Heath et al., 2010). The median pH values for both Mamba and Balule sites were between 8.1 and 8.3 in a study which incorporated water quality data gathered from 1983 to 2008 (Heath et al., 2010). The pH values recorded for sites in the LR were lower than pH values recorded in sites upstream by Bornman et al. (2010). The sites sampled by Bornman et al., (2010) were Albasini Dam; Nandoni Dam and Xikundu Weir and were sampled in October 2006; March 2007; October 2007 and February 2008. Albasini Dam had pH values from 7.62 to 9.72; Nandoni Dam had pH values of 7.41 to 10.20 and Xikundu Weir had pH values ranging from 6.96 to 8.54 (Bornman et al., 2010). The pH values were not much different to the results of studies in the same systems as discussed above. The pH values were within the normal range for South African rivers (DWAF, 1996)
[121]
and fell within the optimal pH range which is 6.5 to 8.5 (Svobodová et al., 1993) and thus the pH values would not have had an effect on fish health.
5.2.3 Dissolved oxygen The highest dissolved oxygen concentration was recorded in the OR in the 2010 sampling trip at (141.72%) with the lowest dissolved oxygen being recorded in the OR in the 2009 sampling trip (107.10%). Dissolved oxygen was higher than the normal range of 80-120% (DWAF, 1996) in all sampling trips except the OR 2009 sampling trip. Oxygen concentrations below 5 mg/L are considered to be stressful to aquatic organisms (IDA, 1997) but mean oxygen concentrations recorded in this study for all sites and sampling trips were all above that concentration. The lowest mean oxygen concentration recorded in this study was recorded in OR in 2009 and was 9.09 mg/L. Therefore the fish sampled in this study would not have been under stress due to a lack of dissolved oxygen at the time of sampling.
5.2.4 TDS/Conductivity The highest total dissolved salts concentration was recorded in the OR in the 2009 sampling trip (397.5 µS). The OR had overall higher TDS values than the LR. Electrical conductivity results in the OR were found to be higher than historical data (1983-2008) of sampling points of the Mamba weir (site 1 for this study) and Balule (site 4 of this study) water quality monitoring points (Heath et al., 2010). The increase in TDS in the OR over the years may have been as a result of sediment from mining and industrial activities accumulating in the Phalaborwa barrage, which is flushed from time to time and results in large quantities of sediment being released into the OR, resulting in a higher TDS (WRC, 2001a).
5.3 Metal concentrations in water and sediment Aluminium (Al), copper (Cu), zinc (Zn) and lead (Pb) levels in the water of both the OR and LR were higher than the target water quality range (TWQR) (DWAF, 1996) (see Table 4.2 and Table 4.3). The TWQR is defined as a management objective which has been derived from quantitative and qualitative criteria and not a water quality criterion (DWAF, 1996). The TWQR is the range of concentrations or levels within which no measurable adverse effects are expected on the health of aquatic ecosystems, and should therefore ensure their protection, assuming life-long exposure (DWAF, 1996). The concentrations of Al from both rivers were much higher than the TWQR of 10 µg/L at a mean concentration of 51.19 µg/L in the OR and a mean concentration of 50.85µg/L in the LR. Al is partially soluble at intermediate pHs and thus would have been so at the pH values measured in the two rivers (Table 4.1). Aluminium is known to cause interference with ionic [122]
and osmotic balance and with respiratory problems resulting from the coagulation of mucous on the gills (DWAF, 1996) and has been found to cause severe fusion of the lamella and filaments in fish (Abdel-moneim et al., 2008) According to Correia et al. (2010) Al is an endocrine disrupting chemical in female O. niloticus females. High levels of Al were found in sediment collected from sites in the LR (Lotonyanda, Albasini Dam, Hasani, Nandoni Dam, Tshikonela, Xikundu Weir and Mhinga) upstream of the sites for this study in a study undertaken during four sampling trips (October 2006; March 2007; October 2007 and February 2008). However Al levels in the water at those same sites were found to be low with the highest level recorded being 21.1 in the Lotonyanda site (Bornman et al., 2010). These high levels of Al may have contributed to the histopathological alterations found. Aluminium has been related to interference with ionic and osmotic balance and exposure to Al has been been found to result in respiratory problems (Abdel-Latif, 2008). According to Alwan et al., (2009) fish exposed to Al showed higher Hct values. The higher than normal Hct values in H. vittatus from OR 2009 (64.96 %); L. marequensis from OR 2010 (58.96 %) and L. cylindricus from LR 2009 (72.15 %) may be due to higher Al levels in OR (40.79- 64.70 %) and LR (25.63-89.97 %).
The TWQR for Cu is 0.30 µg/L and the levels detected in the OR (1.53 µg/L) and the LR (1.68 µg/L) were both above that level. Copper is generally more soluble in acidic waters and the Cu toxicity increases with a decrease in dissolved oxygen (DWAF, 1996). The neutral pH and relatively high dissolved oxygen levels (Table 4.1) mean that the toxicity of the Cu may have been reduced. Copper exposure has been shown to cause vacuolation and necrosis in the livers of O. niloticus (Figueiredo-Fernandes et al., 2007). Vacuolation was found in the livers of specimens of H. vittatus and L. marequensis from OR 2009; L. marequensis from OR 2010 and H. vittatus from LR 2009 (Table 4.10 and 4.11) These alterations may have been as a result of Cu levels in the areas sampled. Oncorhynchus mykiss exposed to Cu sulphate for 28 days was found to have histopathological alterations (non-homogenous regions; congestion of the central vein; dark stained hepatocytes; increasing number of Kupffer cells) in the liver (Atamanalp et al., 2008) which were not found in fish sampled in this study. Copper exposure has been found to cause histopathological alterations (oedema, vasodilations of the lamellar axis) in the gills of O. niloticus (Figueiredo- Fernandes et al., 2007). None of these gill alterations were found in fish sampled in this study.
[123]
Lead (Pb) has a TWQR of 0.20 µg/L (DWAF, 1996) and the mean concentration values of Pb in the OR (3.14 µg/L) and the LR (1.22 µg/L) were both higher than the TWQR. The major sources of Pb in the aquatic environment are anthropogenic, these include: precipitation, fallout of Pb dust and street runoff (associated with Pb emissions from gasoline-powered motor vehicles); industrial and municipal wastewater discharge; mining, milling and smelting of Pb and metals associated with Pb, e.g. Zn, Cu, silver, As and antimony; and combustion of fossil fuels (DWAF, 1996). Lead interacts metabolically with Fe and thus interferes with haemoglobin synthesis and also affects membrane permeability by displacing Ca at functional sites, and inhibits some of the enzymes involved in energy metabolism (DWAF, 1996). Clarias gariepinus exposed to sublethal Pb had gill histopathological alterations (occlusion of interlamellar spaces; shrinkage of cartilaginous supporting mass; epithelial cell lysis; intracellular vacuolation; oedema; reduced pillar cell size and epithelial attachment) (Olojo et al., 2005) which were not observed in fish sampled in this study. Clarias gariepinus exposed to sublethal concentrations of Pb had liver histopathological alterations (hepatic cirrhosis; detached bile connective tissue; parenchyma degeneration; increase of fibro-connective tissue; blood sinusoid congestion and necrosis) (Olojo et al., 2005) not observed in any fish sampled in this study.
TWQR for Zn is 2 µg/L and the mean concentrations of Zn were higher than the TWQR in both the OR (4.71 µg/L) and the LR (3.84 µg/L). Zn occurs in two oxidation states in aquatic ecosystems, namely as the metal, and as Zn (II) (DWAF, 1996). Zinc is a trace metal which is also an essential micronutrient in all organisms (DWAF, 1996). The requirement for trace elements frequently varies substantially between species, but the optimal concentration range is generally narrow. Severe imbalances can cause death, whereas marginal imbalances contribute to reduced fitness (DWAF, 1996). Zinc exposure has been shown to induce histopathological alterations in the kidney (dilated renal tubules; separation of renal tubule epithelial lining; oedema of renal tubule; hypertrophied nuclei; glomerulus vacuolation; disorganised blood capillaries of glomerulus; necrosis and pyknotic nuclei in mesenchymal tissue) (Gupta and Srivasta, 2006) which were not observed in any fish sampled in this study. Liver alterations found to be induced by Zn but not observed in this study include haemorrhage and distended sinusoids (Loganathan et al., 2006) as well as hyalinisation; cellular swelling and congestion of blood vessels (Van Dyk et al., 2007). Degeneration and hyperaemia were found in ovarian tissue of fish exposed to Zn (Carino and Cruz, 1990) however neither of these alterations was observed in any specimens sampled in this study. Necrosis was found to be induced by Zn exposure (Loganathan et al., 2006) and this [124]
alteration was found in H. vittatus specimens from OR 2009 and L. marequensis specimens from OR 2009 and 2010. Vacuolation was found in C. gariepinus exposed to Cd and Zn (Van Dyk et al., 2007) and this alteration was found in specimens from all species and all sampling trips. It is therefore possible that the Zn levels recorded in this study may have resulted in the necrosis and vacuolation found in this study
5.4 Biometric indices
5.4.1 Condition factor (Cf) Cf has been used extensively in fish health and population assessments and the calculation used for this study, namely Fulton‘s condition factor described by Carlander (1969) can be indicative of the overall condition and nutritional status of an individual fish (Schmitt and Dethloff, 2000). According to Bolger and Connolly (1989) in studies based on length-weight data, the heavier fish will be in the better condition. There are many factors which affect fish weight including food availability, metabolic rate as dependant on temperature and seasonal changes in terms of breeding activity (Marchand, 2006) and may increase or decrease in response to chemical contaminants (Schmitt and Dethloff, 2000). Because fish condition factors vary between species due to their differing architecture (Schmitt and Dethloff, 2000) the species were not compared to each other but rather to fish of the same species from different rivers and/or seasons.
Hydrocynus vittatus specimens‘ Cf varied between 0.70 and 1. The highest mean Cf value was from the LR in 2009. Since this trip was taken in November it is possible that these higher values are because of seasonality. The higher Cf results could be because the fish are closer to breeding and thus their body mass is increased as a result of increased gonad mass, these results are reflected in the GSI values.
Labeobarbus marequensis values were between 0.9 and 1.1 with the higher values found in the 2009 trip and it is possible that these differences are also due to seasonality.
Labeo cylindricus and L. rosae Cf values were compared to each other as these species are from the same genus. Of the two species L. cylindricus had the higher Cf. The L. rosae from the OR could be compared to a previous study done by Luus-Powell (1997) on the same species and in the same river. The fish from the current study showed a lower Cf value and may indicate that the fish from the current study are in a worse condition but this may be because these fish were caught during a period of heavy rains which may have cause the fish to stress (Abujanra et al., 2009).
[125]
5.4.2 Hepatosomatic index (HSI) As mentioned before, the hepatosomatic index (HSI) is a ratio of liver weight to body weight and can be affected by contaminant exposure (Schmitt and Dethloff, 2000). The normal value for HSI ranges from 1-2% for Osteichthyes (Munshi and Dutta, 1996) although the range is species specific. A baseline laboratory-based study of two Southern African fish species showed mean HSI values of 1.08% for C. gariepinus specimens and 1.30% for O. mossambicus (Van Dyk, 2006). However, a study done in the Okavango panhandle showed HSI values of 0.50% for C. gariepinus specimens; 0.60% for C. ngamensis specimens; 1.00% for O. andersonii specimens; and 0.80% for S. angusticeps specimens (Van Dyk et al., 2009a). The HSI values of these specimens from a supposed pristine area were all below the supposedly normal discussed above. The fish specimens from the Okavango panhandle were affected by parasitic infections and showed moderate histological alterations (Van Dyk et al., 2009a) and the lower than expected HSI values may be indicative that the fish were under stress and the same may be true of the fish specimens in this study. There is a negative correlation between body condition and flood conditions (Abujanra et al., 2009) and it is possible that the higher rainfall during the OR and LR 2010 sampling trips may have stressed the fish and caused them to have lower HSI values. Oreochromis niloticus exposed to Cu had higher HSI values (Figueiredo-Fernandes et al., 2007).
5.4.3 Splenosomatic index (SSI) The splenosomatic index (SSI) is a ratio of spleen weight and body weight. The SSI is important due to the spleen being a haematopoietic organ and thus playing a role in the immune system (Schmitt and Dethloff, 2000) consequently enlargement or swelling of the spleen is considered to be indicative of disease or immune system problems (Goede and Barton, 1990). The differences of spleen morphology between species (Schmitt and Dethloff, 2000) are probably the cause of the L. cylindricus and L. rosae SSI values being lower. Although there are no standard SSI values, the values obtained appear to be normal which is evidenced by the fact that no enlarged spleens were observed during the necropsy. SSI values from a Southern African laboratory-based baseline study showed SSI values of 0.04% in C. gariepinus specimens and 0.06% in O. mossambicus specimens (Van Dyk, 2006) and the SSI results from this study are similar to those results.
5.4.4 Gonadosomatic index (GSI) The gonadosomatic index (GSI) is an indicator of gonadal development and maturity and has been used to assess gonadal changes in response to environmental dynamics [126]
(seasonal changes) or exogenous stresses (contaminant exposure) (Schmitt and Dethloff, 2000). Since the histological alterations in the gonads were minimal, it is probable that the differences in GSI between the sites is most probably due to seasonality as the higher GSI results were found closer to the summer breeding season of the fish sampled.
5.5 Blood parameters
5.5.1 Haematocrit (Hct) The normal Hct range for fish is 30-45% (Adams et al., 1993) H. vittatus from Olifants 2009 and Luvuvhu 2010 as well as L. marequensis from Olifants 2009 and L. rosae specimens had mean Hct values which fell into the normal range. Hydrocynus vittatus and L. marequensis from Olifants 2010 and L. cylindricus from Luvuvhu had mean Hct values higher than the normal range. The H. vittatus from Luvuvhu 2009 had a mean Hct value lower than the normal range. Low Hct values indicate disease (Cardwell and Smith, 1971) or anaemia (Roche and Boge, 1996) and a low Hct value is a good indicator of deteriorating health of fishes (Chun-Yao et al., 2004)
5.5.2 Leukocrit (Lct) The normal Lct range for fish is less than 4% (Adams et al., 1993). All fish groups sampled had mean Lct values in the normal range apart from the L. marequensis specimens sampled from the OR in the 2009 sampling trip which has a mean Lct value of 6.07 but there was not external evidence of disease or infection in those specimens.
5.5.3 Total plasma protein (TP) The normal total plasma protein range for fish is 30-69 mg/dL (Adams et al., 1993). All fish groups sampled had mean TP values in the normal range, apart from L. marequensis specimens from the OR in both the 2009 (22.17) and 2010 (21.75) trips and L. rosae specimens from the OR from the 2010 sampling trip (11.02), which were lower than the normal range. TP values may vary depending on the sex, size and state of maturity of the fish and are affected by factors such as temperature and food availability (Miller et al., 1983). Since the OR in 2010 was in high flow, the lower TP values may be due to environmental stress. However, according to Mackenzie (1995) fish exposed to pollutants are likely to have a lower TP than fish that are not and it is possible that the lower TP values may be due to pollution.
[127]
5.6 Health assessment index (HAI) The L. marequensis specimens had higher HAI values than fish from other species and L. marequensis from the OR 2010 trip had higher HAI values than specimens from the OR 2009 trip but these differences were not significant (p=0.016). HAI values of H. vittatus were significantly higher in the OR than the LR (p=0.034). L. rosae specimens had significantly higher HAI values than L. cylindricus specimens (p=0.000). Parasites did not contribute greatly to the HAI scores in this study. Parasites were not found in L. cylindricus from LR 2009 or L. rosae from OR 2010. Low percentages of parastites were found in L. marequensis specimens sampled (13.21% of specimens from OR 2009 and 27.03% from OR 2010. Higher percentages of parasites were found in H. vittatus specimens when compared to the other species in this study (OR 2009= 47.31%; OR 2010=21.06%; LR 2009=50.00%; LR 2010=66.67%). HAI values of L. marequensis were lower than those found in a study on the same species by Watson (2001). The HAI values of L. rosae were lower than HAI values found in a study done on the same species by Luus-Powell (1997). HAI values were generally quite low for all species and sampling trips when comparing to similar studies mentioned below. All HAI values in this study were lower than the worst HAI value (79) found by the Tenessee Valley Authority (TVA) (Adams et al., 1993). Hinck et al. (2007) documented high HAI values in Carp (≥100) and Bass (>90) which were considered to be indicative of poor health in those fish. The HAI values in this study were lower than those values. Olarinmoye et al. (2010) studied the HAI of Chrysicthys nigrodigitatus in a lagoon complex in Nigeria and all mean HAI values in this study were lower than the lagoon with the highest mean HAI value (76.57) but compared favourably with the mean HAI value of the lagoon with the lowest mean HAI value (29.66). This indicates that according to the HAI, the fish specimens sampled were in a good state of health based on the macroscopic assessment of the organs. However, a laboratory study on C. gariepinus and O. mossambicus found no macroscopic abnormalities (Van Dyk, 2006) which means that the macroscopic health of the fish studied in this study were not as good as laboratory - bred specimens.
It is notable that there were anomalies in the blood (see section 5.5) which were contributors to the HAI values (see Appendix C). These blood anomalies could ultimately result in histopathological lesions if the affected fish continue to be exposed to the stressors causing these anomalies (Hinton and Laurèn, 1990).
[128]
5.7 Normal histology
5.7.1 Liver histology The general histological structure of the livers of all species sampled in this study had similar structures to what was described by Takashima and Hibiya (1995); Van Dyk (2006) and Genten et al. (2009). Hepatocytes were hexagonal to oval-shaped and arranged in a hepatic cord structure around blood sinusoids. The main difference in the liver tissue between the species sampled is the presence of hepatopancreatic tissue in H. vittatus, L. cylindricus and L. rosae specimens which was also found in O. mossambicus specimens by Van Dyk (2006) while none of this tissue was observed in L. marequensis specimens.
5.7.2 Kidney histology The histological structure of the kidney of specimens from species sampled in this study corresponded with the structures described by Takashima and Hibiya (1995); Van Dyk (2006) and Genten et al. (2009). The anterior kidney in all four species is made up of nephrons surrounded by haematopoietic tissue. The nephron structure was made up of renal corpuscles and tubules as described by Van Dyk (2006) for C. gariepinus and O. mossambicus. Similarly, the renal corpuscles consisted of a glomerulus with a surrounding Bowman‘s space. Within the renal tubules of both species, intercalated cells were observed. + - - + These cells are known to mediate H and HCO3 secretion and Cl and K reabsorption (Schuster, 1993). Thyroid follicles were only observed in L. marequensis kidney tissue. This means that the thyroid gland of this species is similar to carp and goldfish by having some thyroid follicles visible in the kidney (Takashima and Hibiya, 1995). Carp and goldfish also have thyroid follicles present in the heart, head kidney, spleen and blood canal (Takashima and Hibiya, 1995).
5.7.3 Gill histology The normal gill structure of specimens from all four species sampled in this study was similar to what was described by Van Dyk (2006), particularly the O. mossambicus gills, which did not have shorter secondary lamellae. Gill arches with with epithelial cells, mucous cells, connective tissue and supporting tissue were observed in all four species. Primary lamellae with supporting chondrocytes were also observed. Semi-circular secondary lamellae, which correspond to those described by Takashima and Hibiya (1995) and Van Dyk (2006) were also observed and had a capillary lumen and supporting pillar cells in both species. There were no obvious differences in the histological structure of the gills of the four species studied.
[129]
5.7.4 Testis histology The testis histology of specimens from species sampled in this study corresponded with that of C. gariepinus and O. mossambicus described by Van Dyk (2006) and the general teleost testis structure described by Takashima and Hibiya (1995) and Genten et al. (2009). The testes consist of lobules with cysts which contain spermatogenic cells in different stages of development. Inside the cysts of the lobules primary and secondary spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa were all observed in all species. Sertoli cells were found at the periphery of the lobule in all species and were in association with primary spermatogonia as described by Van Dyk (2006) for C. gariepinus and O. mossambicus. Leydig cells were observed in the interstitial tissue between the lobules and have a function of secreting male hormones (Billard, 1986; Van Dyk, 2006). Distinct lobules were also observed in all four species, with H. vittatus specimens in particular having highly organised lobules.
5.7.5 Ovary histology The ovary histology of specimens from species sampled in this study corresponded with that of C. gariepinus and O. mossambicus described by Van Dyk (2006) and the general teleost ovary structure described by Takashima and Hibiya (1995) and Genten et al. (2009) and showed an asynchrous structure found by Van Dyk (2006) in C. gariepinus and O. mossambicus. An asynchrous ovary structure is a structure where the mature female contains oogonia and oocytes in several stages of maturation prior to spawning (Takashima and Hibiya, 1995).
5.7.6 Gonadal staging The stage of development could potentially affect the histological appearance of certain organs (Bernet et al., 1999). This is why the sampling was done in two seasons and the gonadal staging of each fish was noted.
5.7.6.1 Testis staging
Gonadal staging showed that male H. vittatus specimens from the OR 2009 were in the immature (50%) and early spermatogenic (50%) stages of development, these fish were either not sexually mature or the sampling was not carried out during their breeding season. Male H. vittatus specimens from OR 2010 were in the early spermatogenic (80%) and mid spermatogenic (20%) stages of development, this means that these fish were either not sexually mature or the sampling was not carried out during their breeding season. All but
[130]
one of the male H. vittatus specimens from LR 2009 were in the late spermatogenic stage of development, which corresponds to the higher GSI values for these specimens. Male L. marequensis specimens from OR 2009 were in the immature (77.78%) and mid spermatogenic (11.11%) stages of development, this means that these fish were either not sexually mature or the sampling was not carried out during their breeding season. Male L. marequensis specimens from OR 2010 were in the early spermatogenic (20%), mid spermatogenic (60%) and late spermatogenic (20%) stages of development. Male L. cylindricus specimens from LR 2009 were all in the late spermatogenic stage of development, this means that most of these fish were either not sexually mature or the sampling was not carried out during their breeding season. All male L. rosae specimens from from OR 2010 were in the immature stage of development (McDonald et al., 2000), meaning that these fish were either not sexually mature or the sampling was not carried out during their breeding season.
5.7.6.2 Ovary staging
All female H. vittatus specimens from OR 2009 were in the immature stage of gonadal development. Female H. vittatus specimens from LR 2009 were in the immature (42.86%) and mid development (57.14%) stages of gonadal development, this means that these fish were either not sexually mature as the sampling was carried out during their breeding season (summer) (Skelton, 2001). The more developed ovaries in LR 2009 were reflected by higher GSI values. Female L. marequensis specimens from OR 2009 were in the immature (80%) and mid development (20%) stages of development. Female L. marequensis specimens from OR 2010 were in the immature (20%), early development (60%) and mid development (20%) stages of development, this means that these fish were either not sexually mature or the sampling was not carried out during their breeding season. Female L. cylindricus specimens from LR 2009 were in the immature (50%) and mid development (50%) stages of development, this means that these fish were either not sexually mature or the sampling was not carried out during their breeding season. Female L. rosae specimens from LR 2010 were all in the immature stage of development (McDonald et al., 2000), this means that these fish were either not sexually mature or the sampling was not carried out during their breeding season.
[131]
5.8 Histopathology
5.8.1 Liver histopathology The H. vittatus specimens from the OR 2009 and 2010 sampling trips had mean liver index values which fell into Class 2 which means that the liver tissue of those specimens had tissue with moderate histological alterations (Van Dyk et al., 2009a). The H. vittatus specimens from LR 2009 and 2010; L. marequensis from OR 2009 and 2010; L. cylindricus from LR 2009; and L. rosae from OR 2010 had mean liver index values which fell into Class 1 which means that the liver tissue from those specimens had tissue with slight histological alterations (Van Dyk et al., 2009a). All liver alterations observed were regressive changes (RC) with parasites only observed in L. marequensis specimens and melanomacrophage centres only observed in L. rosae specimens. The liver organ index values were higher than all other organ indices in all of the species from both of the rivers and all of the sampling trips. This was not an unexpected result as the liver is a major detoxification organ and is involved in the metabolism and excretion of heavy metals and xenobiotics. Since the pathway of blood vessels that transport substances from the digestive system, it is the first organ exposed to ingested toxicants (Ross et al., 1989).
Alterations may be indicative of prior exposure to environmental stressors (Hinton and Laurèn, 1990) and this may be the case with the specimens assessed during this study as there seemed to be very little acute stressors with the gills in particular being unaffected while the liver tissue showed higher organ index values than the other organs assessed. Nevertheless, even the organ index values of H. vittatus from the OR in 2009 and 2010 which fell into Class 2 meaning the tissue had moderate histological alterations (Van Dyk et al., 2009a).
The liver index results are higher than the liver index results found by Van Dyk (2006) in lab- bred baseline specimens. The C. gariepinus specimens had a mean liver index value of 9.0 and the O. mossambicus specimens had a mean liver index value of 8.2. In a study on C. gariepinus from the Rietvlei Nature Reserve found mean liver index values of 26.1 in the Marais Dam site and 25.3 in the Rietvlei Dam site (Marchand, 2006). Alterations found in the livers of specimens in this study were hepatic cord structure disarray; granular degeneration; inter - and intracellular deposits; fatty degeneration; increase in connective tissue surrounding central veins; infiltration of mononuclear leukocytes; bile duct wall proliferation; bile duct structural alterations; bile duct granular degeneration; bile duct nuclear alterations (Marchand, 2006). All of the mean liver index values in this study were lower than those found in the Rietvlei study (see Fig. 4.15). [132]
In a study on fish from the Okavango Delta panhandle found mean liver index values of 12.0 (10-24) in C. gariepinus; 12.7 (6-22) in C. ngamensis; 23.9 (16-30) in O. andersonii and 6.7 (0-18) in S. angusticeps (Van Dyk et al., 2009a). Alterations found in the liver tissue of specimens in the Okavango study included: intracellular deposits; macrovesicular steatosis; nuclear alterations; necrosis; inflammatory response; increase in melanomacrophage centres. Mean liver index values in the present study were lower than the values in the Okavango study with the exception of S. angusticeps. All of the mean liver index values in this study were lower than those found in the Okavango study (see Fig. 4.15).
In a study on Clarias gariepinus from the Hartbeespoort Dam (a dam known to be polluted) Botha (2011) found the following alterations in the liver tissue: Fatty degeneration; glycogen vacuoles; MMCs; necrosis; nuclear alterations; structural alterations; vacuolation; intracellular deposits; hypertrophy and infiltrations of leukocytes. Mean liver index values for C. gariepinus from the above-mentioned study were 18.2 in the low flow sampling trip and 13.6 in the high flow sampling trip, both of these liver index values were in Class 2, like the mean liver index values of H. vittatus from OR 2009 and 2010. This means that the liver tissue is still functional but the present results may be an early warning sign to future health risks of these fish in these rivers, especially those in the OR.
5.8.2 Kidney histopathology The kidney tissue assessed had the second highest number of histopathological alterations of the target organs assessed in all species from both rivers and all sampling trips. This shows that the kidneys are also under stress. However, the mean kidney index values of all species from both rivers in and all sampling trips were under 10 and thus fell into Class 1 (Van Dyk et al., 2009a). Renal lesions are likely to be indicators of environmental pollution, as the kidney receives the largest amount of postbranchial blood (Hinton and Laurèn, 1992).
All of the changes observed in the kidney tissue were regressive changes. The H. vittatus specimens did have higher mean kidney index values which fell into Class 1 (slight alterations) (Van Dyk et al., 2009a). Based on the histopathology, the kidneys of all fish specimens from all species, both rivers and all sampling trips were in good health. A study on a laboratory bred reference group showed kidney index results of 3.2 for C. gariepinus and 3.0 for O. mossambicus specimens (Van Dyk, 2006). In a study on Clarias gariepinus from the Hartbeespoort Dam (Botha, 2011) found the following alterations in the kidney tissue: Dilations of glomerulus capillaries; hyaline droplet degeneration; MMCs and vacuolation. Mean kidney index values for C. gariepinus from the above-mentioned study were 4.1 in the low flow sampling trip and 2.6 in the high flow sampling trip. Both of these [133]
kidney index values were in Class 1. The kidney index results of H. vittatus specimens from OR in 2009, 2010 and LR in 2009 were all higher than the reference specimens and although the results all being in Class 1 mean there is not much immediate concern over the histopathological health of the kidneys of the fish species sampled in the OR and LR, there may be future cause for concern if pollution in the area increases.
5.8.3 Gill histopathology Histological assessment of the gills of all species from both rivers showed no histological alterations. This is particularly significant as the gills are vulnerable to any dissolved or suspended irritants in the water due to their external location and intimate contact with the water (Roberts, 2001). According to Takashima and Hibiya (1995), low levels of external irritants can cause permeability changes in the membranes which can lead to mucous cell death and increased mucous secretion in primary lamellae, with telangiectasia, lamellar oedema, hyperplasia and fusion occurring if the irritant stimulus is more severe.
Some of these changes and others were found in C. gariepinus specimens from a DDT- sprayed area in the LR catchment, namely: aneurysms, intercellular oedema, epithelial structural alterations, rupture of pillar cells, supporting tissue alterations, epithelial hypertrophy, supporting hyperplasia and MNL (mono leukocyte) infiltration (Pieterse et al., 2010a). In a study on C gariepinus from the Rietvlei Nature Reserve mean gill index values of 9.8 in the Marais Dam site and 9.9 in the Rietvlei Dam site were found (Marchand, 2006). Alterations found in the gills of fish in this study were telangiectasia and oedema of the secondary lamellae; fusion of the branching of the primary lamellae; vacuolation; hyperplasia of the epithelium (Marchand, 2006). In a study of fish from the Okavango Delta panhandle found mean gill index values of 8.4 (4-18) in C. gariepinus; 11.1 (4-20) in C. ngamensis; 22.3 (12-32) in O. andersonii and 8.8 (2-20) in S. angusticeps (Van Dyk et al., 2009a). Alterations found in the gill tissue of specimens in the Okavango study included: telangiectasia; epithelial lifting; hyperplasia; increase in mucous cells; hyaline droplet degeneration (Van Dyk et al., 2009a).
In a study on Clarias gariepinus from the Hartbeespoort Dam (Botha, 2011) found the following alterations in the gill tissue: Telangiectasia; epithelial lifting; congestion; branching of primary lamellae; branching of secondary lamellae; plasma alterations; rupture of pillar cells and hyperplasia. Mean gill index values for C. gariepinus from the above-mentioned study were 3.6 in the low flow season and 1.4 in the high flow season. Both of these kidney index values were in Class 1. The gills of the specimens assessed were not affected in the same way as those specimens. Although parasites were observed in gill histological slides of [134]
some specimens, the semi-quantitative histological assessment protocol used in this study does not recognise the presence of parasites as scoreable but rather any alterations in the gill tissue cause would be scored. According to Van Dyk (2006), it is particularly important when assessing gill tissue for histopathological alterations that artifactual alterations in the gill tissue are not mistaken for histopathology and care was taken when assessing the gills for this study to not make that mistake. When comparing to other studies in Southern Africa, the gills of the specimens sampled are in good health based on histological observation.
5.8.4 Testis histopathogy The testis tissue assessed had minimal pathological alterations with only MMCs found in L. marequensis and L. rosae specimens from OR 2010. Since this alteration was only found in specimens collected from OR and only in the 2010 sampling trip, where there were higher flow conditions, these alterations may have been a result of environmental conditions experienced by those fish rather than pollution. Pieterse et al. (2010a) found intersex and other histopathological alterations in testes tissue of C. gariepinus sampled in a DDT- sprayed area of the LR catchment. These alterations were not found in testes tissue of male specimens in this study as this means that the male fish from this study were not exposed to the same pollutants as those sampled by Pieterse et al. (2010a).
All mean testes index values were below 10 and thus fell into Class 1 which indicates normal tissue structure with slight histological alterations (Van Dyk et al., 2009a). Testes index values in a laboratory baseline study showed values of 0.4 for C. gariepinus specimens and 0.0 for O. mossambicus specimens (Van Dyk, 2006). A study on C gariepinus from the Rietvlei Nature Reserve found mean testis index values of 15.7 in the Marais Dam site and 10.8 in the Rietvlei Dam (Marchand, 2006). Alterations found in the testis tissue of fish in this study were telangiectasia disorganisation of lobules; possible inhibition of spermatogenesis; vacuolation and nuclear alterations of spermatogenic stages; wall proliferation and detachment of basal membrane; infiltration of mononuclear leukocytes; leukocytes (Marchand, 2006). In a study of fish from the Okavango Delta panhandle mean gill index values of 3.5 (2-4) in C. gariepinus; 4.6 (2-8) in C. ngamensis and 2.4 (0-8) in S. angusticeps (Van Dyk et al., 2009a) were observed. Alterations found in the testis tissue of specimens in the Okavango were testicular oocytes and melanomacrophage centres (Van Dyk et al., 2009a).
All of the mean testis index values in this study were lower than those found in the Rietvlei and Okavango studies. In a study on Clarias gariepinus from the Hartbeespoort Dam (a dam known to be polluted) Botha (2011) found vacuolation and MMCs in testis tissue. Mean testis [135]
index values for C. gariepinus from the above-mentioned study were 0.7 in the low flow sampling trip and 0.3 in the high flow sampling trip. Both of these kidney index values were in Class 1. When comparing to other studies in Southern Africa, the testes of the specimens sampled are in good health based on histological observation.
5.8.5 Ovary histopathology The ovarian tissue assessed had minimal histopathological alterations. MMCs were the only alterations observed in the ovarian tissue assessed, which was found in H. vittatus and L. rosae specimens from the OR 2010. Similarly to the alterations found in testes sampled, this alteration was only found in specimens collected from OR and only in the 2010 sampling trip, where there were much higher flow conditions, which means that these alterations may have been a result of environmental conditions experienced by those fish rather than pollution.
MMCs were also found in the ovaries of C. gariepinus in a DDT- sprayed area, but other alterations such as structural alterations of oocytes and cholesterol granulomas were also found in that study. In a study on C. gariepinus from the Rietvlei Nature Reserve found mean ovary index values of 6.1 in the Marais Dam site and 3.5 in the Rietvlei Dam site (Marchand, 2006). Alterations found in the ovary tissue of fish in this study were misformed oocytes; hyperplasia of interstitial tissue; increase in melanomacrophage centres; unidentified tissue growth (possible tumour) and intersex (Marchand, 2006).
In a study of fish from the Okavango Delta panhandle mean gill index values of 3.7 (2-6) in C. gariepinus; 3.6 (2-6) in C. ngamensis and 10.2 (4-16) in S. angusticeps (Van Dyk et al., 2009a) were found. Alterations found in the ovary tissue of specimens in the Okavango were plasma alterations; melanomacrophage centres and inflammatory response (Van Dyk et al., 2009a). All of the mean ovary index values in this study were lower than those found in the Rietvlei and Okavango studies. A study on Clarias gariepinus from the Hartbeespoort Dam by Botha (2011) found MMCs in ovary tissue. Mean ovary index values for C. gariepinus from the above-mentioned study were 2.0 in the low flow sampling trip and 0.8 in the high flow sampling trip. Both of these kidney index values were in Class 1. All mean ovary index values were under 10 and thus fell into Class 1 which indicates tissue with slight histological alterations. When comparing to other studies in Southern Africa, the ovaries of the specimens sampled are in good health based on histological observation.
5.8.6 Fish index (Ifish) values The mean fish index values were higher in H. vittatus specimens, with the liver index values contributing to most of the score of the fish indices in all species. The H. vittatus specimens [136]
had higher fish index values than specimens of other species (see Fig 4.23). Hydrocynus vittatus specimens from OR had higher fish index values than H. vittatus specimens from LR (see Fig. 4.23). The mean fish index values for all species were lower than values found in studies on C. gariepinus from the a DDT-sprayed area in the LR catchment (Pieterse et al., 2010a) and a study on four species (C. gariepinus, C. ngamensis, O. andersonii, and S. angusticeps) from the Okavango panhandle in Botswana (Van Dyk et al., 2009a). With the study by Pieterse et al. (2010a) not having included kidney tissue in their assessment, shows that the severity of the histopathological alterations observed in this study were low and thus it can be concluded that according to the histology of the selected target organs (liver, kidney, gill, gonads) the fish specimens were in good health at the time of sampling. The presence of moderate histopathological alterations in some of the liver tissue (Class 2) may mean that the health of these fish may be at risk at a later stage if the water is continually polluted.
5.9 Age The fish specimens sampled in this study were relatively young. According to Gerber et al. (2009) H. vittatus can live up to 20 years for males and 16 years for females .The oldest H. vittatus sampled in this study was not four years old (95 months). All fish sampled in this study were relatively young (see Table 4.17 to 4.22).
5.9.1 Age – Liver index correlation There were no significant correlations between liver index values and age in H. vittatus and L. marequensis specimens sampled. Thus histological alterations observed in the liver in this study were not likely to have been age-related. As mentioned above, fish sampled in this study were all relatively young and this is likely why there was not a correlation between age and liver index.
5.9.2 Age – Fish index correlation There were no significant correlations between fish index values and age in H. vittatus and L. marequensis specimens. Thus the histological alterations observed in this study were not likely to have been age-related. As mentioned above, fish sampled in this study were all relatively young and this is likely why there was not a correlation between age and fish index.
[137]
Chapter 6: Conclusion and recommendations
Although both the OR and LR are known to be polluted by anthropogenic activities, the HAI and semi-quantitative histological assessment results indicate that the fish sampled in this study were in good health based on macroscopic and microscopic observations respectively. The H. vittatus specimens did have higher histopathological organ and fish index values when compared to L. marequensis, L. cylindricus and L. rosae specimens. However, these values were within a normal range and were lower than values found in polluted systems where the fish were affected by heavy metals and EDC pollution.
The age of fish did not have an effect on histopathological alterations in the fish sampled in this study but this may be because the fish sampled in this study were all relatively young.
HAI values showed significant differences between H. vittatus specimens from OR and LR (p=0.034); L. marequensis from OR 2009 and 2010; and L. cylindricus from LR 2009 and L. rosae from OR 2010. According to the HAI, all fish sampled were in good health and did not seem to be affected by the anthropogenic activities in the two rivers. The relatively good health may be due to the fish being chronically exposed to the pollutants in their environments and possibly building up resistance to them.
Despite there being no immediate cause for concern with regards to the fish health in terms of the HAI and semi-quantitative histological assessment, there were histopathological alterations found in this study. The mean liver index values of H. vittatus from OR were already in Class 2 (moderate histological alterations) which indicates that the livers of those fish in the OR were affected.
Another consideration is that histological alterations serve as an early warning system. Although the alterations observed in this study were mild in terms of severity, they were nevertheless present. The presence of necrotic hepatocytes in some fish indicates that pollution has affected the livers of these fish to the point where liver cells have started to die. In the specimens where necrotics were observed, they were only observed in focal areas, which is why the liver index values are at the highest in Class 2 (tissue with moderate alterations). The presence of tubule degeneration (vacuolation and hyaline droplet degeneration) but no necrosis in the kidneys of specimens sampled in this study, means that the fish may have been exposed for a short time before necrosis could set in (Camargo and
[138]
Martinez, 2008). Tubular degeneration can lead to necrosis in severe cases (Takashim and Hibiya, 1995). Therefore fish from these two rivers may show gross histopathological and external lesions in the future if the pollution in the area continues.
The hypothesis of fish from OR being more affected than fish from LR was accepted. Hydrocynus vittatus from specimens OR had significantly higher HAI values than H. vittatus from LR. There were higher mean liver index and fish index in H. vittatus from OR when compared to H. vittatus from LR but these were not significant.
The hypothesis of H. vittatus specimens being more affected than the other specimens was partially accepted. Hydrocynus vittatus specimens had higher mean liver index and fish index values than other species but L. marequensis had higher HAI values than H. vittatus specimens.
This study is the first fish histology-based health study on H. vittatus, L. marequensis, L. cylindricus and L. rosae in the Olifants - and Luvuvhu Rivers within the Kruger National Park. This data can be used as a baseline to compare future studies to and determine trends in fish health in these two rivers in the Kruger National Park.
This study is also the first description of the normal histological structures of H. vittatus, L. marequensis, L. cylindricus and L. rosae. This can also be used as a baseline for future studies.
Recommendations:
A continued study on the histology-based health of H. vittatus, L. marequensis, L. cylindricus and L. rosae specimens from the Olifants – and Luvuvhu Rivers to increase the overall sample size and determine whether there is a trend of declining health of these fish in these two rivers.
Include C. gariepinus in future semi-quantitative histological assessments on the Olifants and Luvuvhu Rivers as there is baseline histological data to compare them to.
Studies on the correlation between age and histopathological alterations can be done on future semi-quantitative histological assessments where age of fish will also be determined. This should give a sample of fish with varying ages and if there is a correlation between age and histopathology it will then be seen.
[139]
Chapter 7: References
Aarab N, Pampanin DM, Ævdal N, Øysæd, Gastadi L, Bechmann RK. 2008. Histopathology alterations and histochemistry measurements in mussel, Mytilus edulis collected offshore from an aluminium smelter industry (Norway). Mar. Poll. Bull. 57: 569-574.
Abdel-Latif HA. 2008. The influence of calcium and sodium on aluminum toxicity in Nile tilapia (Oreochromis niloticus). Aust. J. Basic and Appl. Sci., 2: 747-751.
Abdel-Moneim AM, Abou Shabana AM, Khadre SEM and Abdel-kader HH. 2008. Physiological and histopathological effects in catfish (Clarias lazera) exposed to dyestuff and chemical wastewater. Int. J. Zool. Res., 4: 189-202.
Abujanra F, Agostinho AA and Hahn NS. 2009. Effects of the flood regime on the body condition of fish of different trophic guilds in the Upper Paraná River floodplain, Brazil. Braz. J. Biol., 69: 469-479.
Ackermann C. 2008. A quantitative and qualitative histological assessment of selected organs of Oreochromis mossambicus after acute exposure to cadmium, chromium and nickel. MSc dissertation. University of Johannesburg: Johannesburg.
Adams SM, Brown AM and Goede RW. 1993. A quantitative health assessment index for rapid evaluation of fish condition in the field. T. Am. Fish. Soc., 122:63-73.
Adams DH, Sonne C, Basu N, Dietz R, Nam DH, Leifsson PS and Jensen AL. 2010. Mercury contamination in spotted seatrout, Cynoscion nebulosus: An assessment of liver, kidney, blood, and nervous system health. Sci. Tot. Env., 23: 5808-5816.
Adonalyo VN, Oteiza PI. 1999. Pb2+ promotes lipid peroxidation and alterations in membrane physical properties. Toxicol., 132: 19-32.
Agency for Toxic Substances and Disease Registry (ATDSR). 2006. Draft toxicological profile for aluminium. Atlanta, U.S. Department of Health and Human Services, Public Health Sevice, Agency for Toxic Substances and Disease Registry.
[140]
Agency for Toxic Substances and Disease Registry (ATDSR). 2008. ATDSR medical fact sheet; Division of Toxicology and Environmental Medicine ToxFAQs; Accessed 9 March 2011. http://www.atdsr.cdc.gov/tfacts5.pdf
Ahmed K, Akhand AA, Hasan M, Islam M and Hasan A. 2008. Toxicity of arsenic (sodium arsenite) to freshwater snakehead Channa punctatus (Bloch) on cellular death and DNA content. Amer.-Eurasian J. Agri. Environ. Sci., 4: 18-22.
Altinok I and Capkin E. 2007. Histopathology of rainbow trout exposed to sublethal concentrations of methiocarb or endosulfan. Toxico. Path., 35: 405 - 410.
Alwan SF, Hadi AA and Shokr AE. 2009. Alterations in haematological parameters of freshwater fish, Tilapia zilli, exposed to aluminium. J. Sci. Appl., 3: 9-12.
Anderson RO and Gutreuter SJ. 1983. Length, weight, and associated structural indices. In Fisheries techniques. Edited by Nielsen, L.A. and Johnson, D.L. American Fisheries Society Bethesda: Maryland.
Ashton PJ, Love D, Mahachi H, and Dirks PGH. 2001. Impacts of mining and mineral processing activities on water resources and water quality in the Zambezi, Limpopo and Olifants Basins in Southern Africa. Mining, Minerals and Sustainable Development Project Report. IIED, Johannesburg.
Ashton PJ. 2007. Riverine biodiversity conservation in South Africa: current situation and future prospects. Aquatic Conserv. Freshw. Ecosyst., 17: 441-445.
Ashton PJ. 2010. The demise of the Nile crocodile (Crocodylus niloticus) as a keystone species for aquatic ecosystem conservation in South Africa: The case of the Olifants River. Aquatic Conserv. Mar. Freshw. Ecosyst., 20: 489–493.
Atamanalp M, Sisman T, Geyikoglu F and Topal A. 2008. The histopathological effects of Copper sulphate on Rainbow trout liver (Oncorhynchus mykiss). J. Fish. Aquat. Sci., 3: 291-297.
Avenant-Oldewage A and Marx HM. 2000. Bioaccumulation of chromium, copper and iron in organs and tissues of Clarias gariepinus in the Olifants River, Kruger National Park. Water SA, 26: 569-582.
Badenhuizen TR. 1966. Some notes on the population dynamics of Hydrocynus vittatus (Castelnau) in Lake Kariba. Limnol. Soc. S. A. News. 19: 527-540. [141]
Baker RTM, Martin P and Davies SJ. 1997. Ingestion of sublethal levels of iron sulphate by African catfish affects growth and tissue lipid peroxidation. Aquat. Toxicol., 40: 51-61.
Ballatori N. 2002. Transport of toxic metals by molecular mimicry. Environ. Health Perspect. 110:689-694.
Barnhoorn IEJ, Bornman MS, Pieterse GM and van Vuren JHJ. 2004. Histological evidence of intersex in feral Sharptooth Catfish (Clarias gariepinus) from an Estrogen-polluted water source in Gauteng, South Africa. Environ. Tox., 19: 603-608.
Barnhoorn IEJ, Bornman MS, Jansen van Rensburg, C Bouwman H. 2009. DDT residues in water, sediment, domestic and indigenous biota from a currently DDT-sprayed area. Chemosphere, 77: 1236-1241.
Bears H, Richards JG and Schulte PM. 2006. Arsenic exposure alters hepatic arsenic species composition and stress-mediated expression in the common killfish (Fundulus heteroclitus). Aquat. Toxicol., 77: 257-266.
Benli ACK, Koksal G and Ozkul A. 2008. Sublethal ammonia exposure of Nile Tilapia (Oreochromis niloticus): effects on gill, liver and kidney histology. Chem., 72: 1355- 1358. Bernet D Schmidt H Meier W Burkhaldt-Holm and P Wahli T. 1999. Histopathology in fish: proposal for a protocol to assess aquatic pollution. J. Fish Dis., 22: 25-34. Billard R. 1986. Spermatogenesis and spermatology of some teleost species. Reprod. Nutr. Develop., 26: 877-920.
Bjerregaard LB, Korsgaard B and Bjerregaard P. 2005. Intersex in wild roach (Rutilus rutilus) from Danish sewage effluent-receiving streams. Ecotox. Environ. Safe., 64: 321-328.
Blazer VS 2000. Histopathologic assessment in: Schmitt CJ and Dethloff GM (editors). Biomonitoring of Environmental Status and Trends (BEST) program: selected methods for monitoring chemical contaminants and their effects in aquatic ecosystems. U.S. Geological Survey, Biological Resources Division, Columbia, (MO): Information and Technology Report USGS/BRD-2000—2005. pp. 22-25.
Bolger T and Connolly PL. 1989. The selection of suitable indices for the measurement and analysis of fish condition. J. Fish. Biol., 34: 171-182
[142]
Botha TL. 2011. A histology-based fish health assessment to determine the health and reproductive status of Clarias gariepinus in the Hartbeespoort Dam in South Africa. MSc Dissertation. University of Johannesburg: Johannesburg.
Boran H, Capkin E, Altinok and Terzi E. 2010. Assessment of acute toxicity and histopathology of the fungicide captan in rainbow trout. Exp. Toxicol. Pathol., Article in press. Borgmann U, Couillar Y, Doyle P and Dixon DG. 2005. Toxicity of sixty three metals and metalloids to Hyalella azteca at two levels of water hardness. Environ. Toxicol. Chem., 24: 641-652.
Bornman MS, Barnhoorn IEJ and Swemmer A. 2010. Analyses of water and sediment. In: Bornman MS, Barnhoorn IEJ, Ghenthe B. DDT for malaria control: effects and indicators in health risk. WRC Report No. 1674/1/09. Chapter 3.
Brown JA, Johansen PH, Colgan PW and Mathers RA. 1987. Impairment of early feeding behaviour of largemouth bass by pentachlorophenol exposure: a preliminary assessment. T. Am. Fish. Soc., 116: 71-78.
Budambula NLM and Mwachiro EC. 2006. Metal Status of Nairobi River waters and their bioaccumulation in Labeo cylindricus. Water Air Soil poll., 169: 275–291.
Buermann Y, Du Preez HH, Steyn GJ, Harmse JT and Deacon A. 1995. Suspended silt concentrations in the lower Olifants river (Mpumalanga) and the impact of silt releases from the Phalaborwa barrage on water quality and fish survival. Koedoe., 38: 11-34. Bury NR, Walker PA and Glover CN. 2003. Nutritive metal uptake in teleost fish. J. Exp. Biol., 206, 11–23. Callard GV. 1991. Spermatogenesis. In: Vertebrate Endocrinology: Fundamentals and Biomedical Implications Vol. 4A. Academic Press, San Diego. pp. 303-341.
Camargo MMP and Martinez CBR. 2007. Histopathology of gills, kidney and liver of a Neotropical fish caged in an urban stream. Neotrop. Ichth., 5: 327-336.
Campana SE. 2001. REVIEW PAPER Accuracy, precision and quality control in age determination, including a review of the use and abuse of age validation methods. J. Fish Biol., 59: 197-242.
[143]
Cardwell RD and Smith LS. 1971. Hematological manifestations of vibriosis upon juvenile Chinook Salmon. Prog. Fish-Cult., 33: 232-235.
Carino VS and Cruz NC. Effects of low levels of zinc on ovarian development Tilapia nilotica Linneaus. 1990. Sci. Dil., 3: 34-39.
Carlander KD. 1969. Handbook of freshwater fishery biology. Iowa State University Press: Iowa.
Cengiz EI. 2006. Gill and kidney histopathology in the freshwater fish Cyprinus carpio after acute exposure to Deltamethrin. Environ. Toxicol. Pharm., 22: 200-204.
Chowdury MJ, McDonald DG and Wood CM. 2004. Gastrointestinal uptake and fate of cadmium in rainbow trout acclimated to sublethal dietary cadmium. Aquat. Toxicol., 69: 149-163.
Chun-Yao C, Wooster GA and Bowser PR. 2004. Comparative blood chemistry and histopathology of Tilapia infected with Vibrio vulnificus or Streptococcus iniae or exposed to carbon tetrachloride, gentamicin or Copper sulphate. Aquaculture. 239:421-443.
Coetzee L, du Preez HH and van Vuren JHJ. 2002. Metal concentrations in Clarias gariepinus and Labeo umbratus from the Olifants and Klein Olifants River, Mpumalanga, South Africa: Zinc, copper, manganese, lead, chromium, nickel, aluminium and iron. Water SA., 28: 433-438.
Cohen JW. 1988. Statistical power analysis for the behavioural sciences. (2nd ed.). Hillsdale, NJ: Lawrence Erlbaum Associates.
Correia TG, Narcizo AM, Bianchini A and Moreira RG. 2010. Aluminium as an endocrine disruptor in female Nile Tilapia (Oreochromis niloticus). Comp. Biochem. Phys. Part C., 151: 461-466.
Dallinger R, Egg M, Ginther KK and Hofer R. 1997. The role of metallotheionen in cadmium accumulation of Arctic char (Salvinus alpines) from high alpine lakes. Aquat. Toxicol., 69: 149-163.
Datta S, Ghosh D, Saha DR, Bhattacharaya S and Mazumder S. 2009. Chronic exposure to low concentration of arsenic is immunotoxic to fish: Role of head kidney
[144]
macrophages as biomarkers of arsenic toxicity to Clarias batrachus. Aquat. Toxicol., 92: 86–94.
De Villiers S and Mkwelo ST. 2009. Has monitoring failed the Olifants River, Mpumalanga? WRC., 35: 671-676.
DEAT (Department of Environmental Affairs and Tourism). 2007. National Environmental Management Biodiversity Act, 2004 (Act 10 of 2004): publication of lists of critically endangered, endangered, vulnerable and protected species. Government Gazette, Republic of South Africa. No. 29657: 4-11.
Driescher AC. 2008. A water quality study of Loskop Dam and the upper catchment of the Olifants River. Unpublished MSc thesis. University of the Free State: Bloemfontein, South Africa.
Du Preez HH and Steyn GJ. 1992. A preliminary investigation of the concentration of selected metals in the tissues and organs of Tigerfish (Hydrocynus vittatus) from the Olifants river, Kruger National Park, South Africa. Water SA., 18: 131-136.
Dumitrescu G, Petculescu Ciochină L, Voia S, Dronca D and Boca L. 2010. Histological Changes Induced in Gonads, Liver and Kidney of Zebra Fish (Danio Rerio) Under the Effect Octylphenol (OP). Scientific Papers: Anim. Sci. Biotechnol. 43: 485 - 489.
DWAF (Department of Water Affairs and Forestry). 1996. South African Water Quality Guidelines. Volume 7: Aquatic ecosystems. Pretoria: DWAF.
Egusa S. 1970. Branchionephritis prevailed among eel populations in farm ponds in the winter of 1969-1970. Fish pathol., 5: 51-66.
Eisler R. 1985. Cadmium hazards to fish, wildlife and invertebrates: a synoptic review. U.S. Fish and Wildlife Service Biological Report 85 (1.2), Contaminant Reviews Report No. 2 44 pp.
Eisler R. 2000. Handbook of chemical risk assessment: health hazards to humans, plants and animals. Volume 1. Boca Ranton: Lewis publishers.
Fanta E, Sant‘Anna Rios R, Romao S, Casagrande Vianna AC and Freiberger S. 2003. Histopathology of the fish Corydoras paleatus contaminated with sublethal levels oforganophosphorous in water and food. Ecotox. Environ. Safe., 54: 119-130.
[145]
Farag AM, May T, Marty GD, Easton M, Harper DD, Little EE and Cleaveland L. 2006. The effect of chronic chromium exposure on the health of Chinook salmon (Oncorynchus tshawytscha). Aquat. Toxicol., 76: 246-257.
Figueiredo-Fernandes A, Ferreira-Cardoso JV, Garcia-Santos S, Monteiro SM, Carrola J, Matos P and António Fontaínhas- Fernandes A. 2007. Histopathological changes in liver and gill epithelium of Nile tilapia, Oreochromis niloticus, exposed to waterborne copper. Pesq. Vet. Bras., 27: 103-109.
Fouché PSO and Gaigher IG. 2001. Niche differentiation in the rheophilic fishes of the Mutshindini river. In: IG Gaigher (editor). A socio-biological study of aquatic resources and their utilisation in an underdeveloped rural region, the Mutshindini river catchment. WRC project report 714/3/01. Water Research Commission, Pretoria. 52- 69.
Fouché P, Vlok W and Angliss M. 2003. The habitat preferences and food selection of Labeobarbus marequensis in the Luvuvhu and Mutale Rivers. pp. 19-27. In: Arderne P. (editor) The Federation of Southern African Flyfishers (FOSAF): Proceedings of the 7th yellowfish working group. Elgro Lodge, Potchefstroom. 22-24 May 2003.
Fouché PSO. 2007. Status of the Lowveld largescale yellowfish Labeobarbus marequensis (A. Smith, 1841) in: Impson ND, Bills IR, Wolhuter L (editors). Technical report on the state of yellowfish in South Africa. WRC report No. KV 212/08.
Fouché PSO, Vlok W and Jooste A. 2009. Body shape and accompanying habitat shifts: observations in the lifecycle of Loweveld Largescale Yellowfish, Labeobarbus marequensis, in the Luvuvhu River catchment. pp. 48-62. In: Arderne P. (editor). The Federation of Southern African Flyfishers (FOSAF): Proceedings of the 13th yellowfish working group conference. Sterkfontein Dam, Harrismith. 6-8 March 2009.
Gagiano CL, Steyn GJ and du Preez HH. 1996. Tooth replacement of the tigerfish Hydrocynus vittatus from the Kruger National Park. Koedoe., 39: 117-122.
Gaigher IG. 1970. Ecology of the tigerfish (Hydrocynus vittatus) in the Incomati river system, South Africa. Zool. Afr., 5: 211-227
Gaigher IG. 1973. The habitat preferences of fishes from the Limpopo river system, Transvaal and Mocambique. Koedoe., 16: 103-116.
Galvin RM. 1996. Occurrence of metals in waters: An overview. Water SA., 22: 7-18. [146]
Genten F, Terwinghe E and Danguy A. 2009. Atlas of fish histology. Science Publishers, Enfield, NH, USA.
Gerber R, Smit NJ, Pieterse GM and Durholtz D. 2009. Age estimation, growth rate and size at sexual maturity of tigerfish Hydrocynus vittatus from the Okovango Delta, Botswana. Afr. J. Aquat., 34: 239-247.
Ghosh D, Datta S, Bhattacharya S and Mazumder S. 2007. Long-term exposure to arsenic affects head kidney and impairs humoral immune responses of Clarias batrachus. Aquat. Toxicol. 81: 79–89.
Goede RW and Barton BA. 1990. Organismic Indices and an Aotopsy-Based Assessment as Indicators of Health and Condition of Fish. Am. Fish. Soc.Sym., 8: 93-108.
Gonzalez HO, Roling JA, Baldwin WS and Bain LJ. 2006. Physiological changes and differential gene expression in mummichogs (Fundulus heteroclitus) exposed to arsenic. Aquat. Toxicol., 77: 43-52.
Griscom HR, Miller SN, Gyedo-Ababio T and Sivanpillai R. 2010. Mapping land cover change of the Luvuvhu River catchment, South Africa for environmental modelling. Geojournal, 75: 163-173.
Grizzle JM and Rogers WA. 1976. Anatomy and histology of the channel catfish. Auburn University Agricultural Experiment Station, Auburn, Alabama, USA.
Groman DB. 1982. Histology of the striped bass. American Fisheries Society. Bethesda, Maryland.
Gül S, Belge-Kurutas E, Yildiz E, Sahan A and Doran F. 2004. Pollution correlated modifications of liver antioxidant systems and histopathology of fish (Cyprinidae) living in the Seyhan Dam Lake, Turkey. Environ. Int., 30: 605-609.
Gupta P and Srivasta N. 2006. Effects of sub-lethal concentrations of zinc on histological changes and bioaccumulation of zinc by kidney of fish Channa punctatus (Bloch). J. Environ. Biol., 27: 211-215.
Handy RD, Runnalis T and Russell PM. 2002. Histopathologic biomarkers in three spined sticklebacks, Gasterotus aculeatus, from several rivers in Southern England that meet the freshwater fisheries directive. Ecotoxicology, 11: 467-479.
[147]
Heath R, Coleman T and Engelbrecht J. 2010. Water quality overview and literature review of the ecology of the Olifants River. WRC Report No. TT 452/10.
Hinck JO, Blazer VS, Denslow ND, Echols KR, Gross TS, May TW, Anderson PJ, Coyle JJ and Tillitt DE. 2007. Chemical contaminants, health indicators, and reproductive biomarker responses in fish from the Colorado River and its tributaries. Sci. Tot. Env., 378: 376–402.
Hinton DE and Laurèn DJ. 1990. Integrative histopathological approaches to detecting environmental stressors on fishes. Am. Fish. Soc. Sym., 81: 51-66.
Hinton DE, Baumann PC, Gardner GR, Hawkins WE, Hendricks JD and Murchelano RA. 1992. Histopathologic biomarkers. In: Huggett RJ, Kimerle KA, Mehrle PM, Bergman HL editors. Biomarkers, Biochemical, Physiological and Histological Markers of Anthropogenic Stress. Lewis Publishers, Boca Raton, Florida; pp.155-209.
Hirata Y. 2002. Manganese-induced apoptosis in PC12 cells. Neurotoxicol. Teratol., 24: 639- 653.
Hogstrand C, Wilson RW, Polgar D and Wood CM. 1994. Effects of zinc on the kinetics of branchial calcium uptake in freshwater rainbow trout during adaptation to waterborne zinc. J. Exp. Biol., 186: 55-73.
Hogstrand C and Wood CM. 1995. Mechanisms for zinc acclimation in freshwater rainbow trout. Mar. Environ. Res., 39: 131-135.
Hogstrand C, Verboost PM, Bonga SEW and Wood CM. 1996. Mechanisms of zinc uptake in gills of freshwater rainbow trout: Interplay with calcium transport. Am. J. Physiol. Regul. Integr. Comp. Physiol., 39: R1141-R1147.
Humason GL. 1979. Animal tissue techniques. 4th edition. United States of America: Freeman and Company.
IDA. 1997. Water quality parameters: An interpretation of test results. Available from: http://imc.lisd.kl2.mi.us/tests.html#anchor494734
Ishaque AB, Tchounwou PB and Wilson BA. 2004. Developmental arrest in Japanese medaka (Oryzias latipes) embryos exposed to sublethal concentrations of atrazine and arsenic trioxide. J. Environ. Biol., 25:1-6.
[148]
Jewitt GPW, Garatt JA, Calder IR and Fuller L. 2004. Water resources planning and modelling tools for the assessment of land use change in the Luvuvhu catchment, South Africa. Phys. Chem. Earth., 29: 1233-1241.
Jia X, Zhang H and Liu X. 2010. Low levels of cadmium exposure induce DNA damage and oxidative stress in the liver of Oujiang coloured common carp Cyprinus carpio var, colour. Fish Physiol. Biochem. In press, corrected proof.
Jing J, Qi R, Yuling G, Xingya C and Dayong W. 2009. Prolonged manganese exposure induces severe defecits in lifespan, development and reproduction possibly by altering oxidative stress response in Caenorhabditis elegans. J. Environ Sci., 21: 842-848.
Jubb RA. 1967. Freshwater fishes of Southern Africa. A.A. Balkema, Cape Town, pg. 248.
Kendall MW and Hawkins WE. 1975. Hepatic morphology and acid phosphatise localisation in the channel catfish. J. Fish. Res. B. Can., 32: 1459-1464.
Kitazawa M, Anantharam V, Yang Y, Hirata Y, Kanthasamy A and Kanthasamy AG. 2005. Activation of protein kinase Cᵹ by proteolytic cleavages contributes to manganese- induced apoptosis in dopaminergic cells: protective role Bcl-2. Biochem. Pharmacol., 69: 133-146.
Kleynhans, CJ. 1991. Voorlopige riglyne vir die klassifisering van die Transvaalse vissoorte in sensitiwiteitsklasse. Transvaalse Hoofdirektoraat Natuur en Omgewingsbewaring, Werkswinkel, Skukuza.
Kleynhans CJ. 1996. A qualitative procedure for the assessment of the habitat integrity status of the Luvuvhu River (Limpopo system, South Africa). J. Aquat. Ecosys. Health. 5: 41-54.
Kotze P, du Preez HH, van Vuren JHJ. 1999. Bioaccumulation of copper and zinc in Oreochromis mossambicus and Clarias gariepinus, from the Olifants river, Mpumalanga, South Africa. Water SA., 25: 99-110.
Kristal J. 1946. The exocrine pancreatic tissue of seven teleosts. S.A. J. Med. Sci., 11: 79- 87.
Lin Y, Hoang H, Hsieh SI, Ranger N, Foster AL, Sampayo JN, Lithgrow GJ and Srinivasan C. 2006. Manganous ion supplementation accelerates wild type development,
[149]
enhances stress resistance and rescues the lifespan of a short-lived Caenorhabditis elegans mutant. Free Rad. Biol. Med., 40: 1185-1193.
Loganathan K, Velmurugan B, Howrelia JH, Selvanayagam M and Patnaik BB. 2006. Zinc induced histological changes in brain and liver of Labeo rohita (Ham.). J. Environ. Biol., 27(1): 107-110.
Lushchak VI. 2011. Environmentally induced oxidative stress in aquatic animals. Aquat. Toxicol., 101: 13-30.
Luus-Powell WJ. 1997. Evaluation of a health assessment index with reference to bioaccumulation of metals in Labeo species and aspects of the morphology of Chonopeltis victori. MSc Dissertation. Rand Afrikaans University: Johannesburg.
Marchand MJ. 2006. Histopathological assessment of selected organs as a bio-monitoring tool to assess the health status of Clarias gariepinus in two dams in the Rietvlei Nature Reserve. MSc dissertation. Johannesburg: University of Johannesburg.
Marchand MJ, Van Dyk JC, Pieterse GM, Barnhoon IEJ and Bornman MS. 2008. Histopathological alterations in the liver of the sharptooth catfish Clarias gariepinus from polluted aquatic systems in South Africa. Environ. Toxicol., 24:133-147.
Marchand MJ, Pieterse GM and IEJ Barnhoorn. 2010. Sperm motility and testicular histology as reproductive indicators of fish health of two feral fish species from a currently DDT sprayed area, South Africa. J. Appl. Ichthyol., 26: 707–714.
McCartney MP and Arranz R. 2007. Evaluation of the historic, current and future demand in the Olifants River catchment, South Africa. IWMI Research Report No. 118.
McDonald KK, Gross TS, Denslow ND, Densmore C and Blazer VS. 2000. Reproductive indicators in: Biomonitoring of Environmental Status and Trends (BEST) Program: Selected methods for monitoring chemical contaminants and their effects in aquatic ecosystems. Columbia: U.S. Geological Survey, Biological Resources Division, (MO): Information and Technology Report USGS BRD-2000—0005. 30-42
Meyers TR and Hendricks. 1985. Histopathology. In: Fundamentals of aquatic toxicology. p. 58-95. Edited by Rand GM and Petrocelli SR. Hemisphere, Washington, D.C.
[150]
Mhlanga W. 2000. Mercury in tigerfish (Hydrocynus vittatus, Castlenau), green happy (Sargochromis codringtoniii, Boulenger) and kapenta (Limnothrissa miodon, Boulenger) from Lake Kariba, Zimbabwe. Afr. J. Ecol., 38: 224-229.
Mishra AK and Mohanty B. 2008. Acute toxicity impacts of hexavalent chromium on behavior and histopathology of Channa punctatus (Bloch). Environ. Toxicol. Pharm., 26: 136- 141.
Monette MY, Yada T, Matey V and McCormick SD. 2010. Physiological, molecular and cellular mechanisms of impaired seawater tolerance following exposure of Atlantic Salmon Salmo salmar, smolts to acid and aluminium. Aquat. Toxicol., 99:17-32.
Montaser M, Mahfou ME, El-Shazly SAM, Abdel-Rahman GH and Bakry S. 2010. Toxicity of heavy metals on fish at Jeddah coast KSA: metallothionein expression as a biomarker and histopathological study on liver and gills. World J. Fish Mar. Sci., 2: 174-185.
Monteiro SM, Rocha E, Fontainhas-fernandes A and Sousa M. 2008. Quantitative histopathology of Oreochromis niloticus gills after exposure J. Fish Biol., 73: 1376- 1392.
Munshi JSD and Dutta HM. (editors) 1996. Fish morphology: a horizon of new research. USA: Science publishers inc.
Mussagy A. 2008. Massingir Dam and Smallholder Agricultural Rehabilitation Project Plankton Monitoring Programme MDSAR DP04 – Annual Report (September 2006 to August 2007). Ministry of Public Works and Housing, National Directorate of Water, Maputo, Mozambique.
Nath K and Kumar N. 1990. Gonadal histopathology following nickel intoxication in the giant gaurami Colisia fasciatus (Bloch and Schneider), a freshwater tropical perch. Bull. Environ. Contam. Toxicol., 45: 299-304.
Nel JL, Roux DJ, Maree G, Kleynhans CJ, Moolman J, Reyers B, Rouget M and Cowling M. 2007. Rivers in peril inside and outside of protected areas: a systematic approach to conservation assessment of river ecosystems. Diversity Distrib., 13: 341-352.
Nordberg GF. 2007. Historical perspectives on cadmium toxicology. Toxciol. Appl. Pharmacol., 238: 192-200.
[151]
Oberholster PJ. 2009. Impact of ecotourism by water pollution in the Olifants River catchment, South Africa. SIL News., 55: 8-9.
Økland F, Thorstad EB, Hay CJ, Naesje TF and Chanda B. 2005. Patterns of movement and habitat use by tigerfish (Hydrocynus vittatus) in the upper Zambezi River (Namibia). Ecol. Freshw. Fish., 14: 79-86.
Olarinmoye OM, Clarke EO, Kumolu-Johnson CA and Aderinola OJ. 2010. A preliminary assessment of the health status of feral populations of Chrysichthys nigrodigitatus in Lagos lagoon complex, Nigeria, using a modified Health Assessment Index protocol. Afr. J. Aquat. Sci., 33: 77-82.
Olojo EAA, Olurin KB, Mbaka G and Oluwemino AD. 2005. Histopathology of the gill and liver tissue of the African catfish Clarias gariepinus exposed to lead. Afr. J. Biotechnol. 4: 117-122.
Oweson CAM, Baden SP and Hernroth BE. 2006. Manganese induced apoptosis in haematopoietic cells of Nephrops norvegicus. Aquat. Toxicol., 77: 322-328.
Pandey S, Parves S, Ansari RA, Ali M, Kaur M, Hayat F, Ahmad F and Raisuddin S. 2008. Effects of exposure to multiple trace metals on biochemical, histological and ultrastructrural features of gills of a freshwater fish, Channa punctata Bloch. Chemico-Biol. Interact. 174: 183-192.
Pallant J. 2010. SPSS survival manual: A step by step guide to data analysis using SPSS. (4th ed.). McGraw Hill: England.
Patt DI and Patt GR. 1969. Comparative vertebrate histology. Harper and Row, New York, New York, USA.
Pienaar U de V. 1978. The freshwater fishes of the Kruger National Park. National Parks Board of Trustees, Pretoria.
Pieterse GM. 2004. Histological changes in the testis of Oreochromis mossambicus (Cichlidae) as a biomarker of heavy metal pollution. PhD thesis. Rand Afrikaans University: Johannesburg.
Pieterse GM, Van Dyk JC and Marchand MJ. 2010a. A histology-based fish health assessment (HBFHA) in a DDT sprayed area. In: Bornman MS, Barnhoorn IEJ,
[152]
Genthe B. DDT for malaria control: Effects in indicators and health risk. WRC Report No. 1674/1/09.
Pieterse GM, Marchand MJ, Van Dyk JC and Barnhoorn IEJ. 2010b. Histological alterations in the testes and ovaries of the sharptooth catfish (Clarias gariepinus) from an urban nature reserve in South Africa. J. Appl. Ichthyol., 26: 789–793.
Radhakrishnan MV and Hemalatha S. 2010. Sublethal Toxic Effects of Cadmium Chloride to Liver of Freshwater Fish Channa striatus (Bloch.). Amer.-Eurasian J. Toxicol. Sci., 2: 54-56.
Rehulka J. 2003. Haematological parameters in Rainbow trout Oncorhynchus mykiss affected by viral haemorrhagic septicaemia (VHS). Dis. Aquat. Organ., 56: 185-193.
Resh VH. 2008. Which group is best? Attributes of different biological assemblages used in freshwater biomonitoring programs. Environ. Monit. Assess., 138: 131–138.
Roberts RJ. 2001. Fish Pathology. W. B. Saunders: Harcourt Publishers Ltd.
Robinson J and Avenant-Oldewage A. 1997. Chromium, copper, iron and manganese bioaccumulation in some organs and tissues of Oreochromis mossambicus from the lower Olifants River, inside the Kruger National Park. Water SA., 23: 387-403.
Roche H and Boge H. 1996. Fish blood parameters as a potential tool for indentification of stress caused by environmental factors and chemical intoxication. Mar. Environ. Res., 41: 27-43.
Rodmilans M, Torra M, To-Figueras J, Cobella J, Lopez B, Sauchez C and Mazzara R. 1996. Effects of the reduction of petrol lead on blood levels of the population of Barcelona. Bull. Environ. Contam. Toxicol., 56: 717-721.
Ross MH, Reith EJ and Rombell LJ. 1989. Histology: A text and atlas. 2nd edition. Williams and Wilkins.
Roth JA and Garrick MD. 2003. Iron interactions and other biological reactions mediating the physiological and toxic actions of manganese. Biochem. Pharmacol., 66: 1-13.
Saenphet S, Thaworn W and Saenphet K. 2009. Histopathological alterations of the gills, liver and kidneys in Anabas testudineus (Bloch) fish living in an unused Lignite mine,
[153]
Li district, Lamphun province, Thailand. SE As. J. Trop. Med. Public Health., 40: 1121-1126.
Schmidt H, Bernet D, Wahli T, Meier W and Burkhardt-Holm P. 1999. Active biomonitoring with brown trout in diluted sewage effluents. J. Fish Biol., 54: 585-596.
Schmitt CJ and Dethloff GM (editors) 2000. Biomonitoring of Environmental Status and Trends (BEST) Program: Selected methods for monitoring chemical contaminants and their effects in aquatic ecosystems. Columbia: U.S. Geological Survey, Biological Resources Division, (MO): Information and Technology Report USGS BRD-2000— 0005.
Schmitt CJ, Dethloff GM, Hinck JE, Blazer VS, Coyle JJ, Denslow ND and Tillitt DE. 2004. Biomonitoring of Environmental Status and Trends (BEST) Program: environmental contaminants and their effects on fish in the Rio Grande Basin. Columbia, Missouri. U.S. Geological Survey, Columbia Environmental Research Center, Scientific Investigations Report 2004-5108.
Schuster VL. 1993. Function and regulation of collecting duct intercalated cells. Annu. Rev. Physiol., 55: 267-288.
Sesha SV, Rao BM. 1998. Chromium induced alterations in the gill of freshwater teleost fish, Labeo rohita. Ind. J. Compar. Physiol., 17: 31-33.
Seymore T, du Preez HH and van Vuren JHJ. 1995. Manganese, lead, strontium bioaccumulation in the tissues of the yellowfish Barbus marequensis from the lower Olifants River, Eastern Transvaal. Water SA., 21: 159-172.
Seymore T, du Preez HH and van Vuren JHJ. 1996. Concentrations of zinc in Barbus marequensis from the lower Olifants river, Mpumalanga, South Africa. Hydrobiologica., 332: 141-150.
Shanbag BA and Saidapur K. 1996. Atretic follicles and Corpora lutea in the ovaries of fishes: Structure-function correlations and significance. In Fish morphology: Horizon of new research. Edited by Munshi JSD, Dutta HM. Science publishers, Inc. USA.
Silva AG and Martinez CBR. 2007. Morphological changes in the kidney of fish living in an urban stream. Environ. Toxicol. Pharm., 23: 185-192.
[154]
Skelton P. 1990. Scientific nomenclature of tigerfish – rehabilitation of Hydrocynus vittatus. Ichthos., 26: 7
Skelton P. 2001. A complete guide to the freshwater fishes of Southern Africa. Struik publishers: South Africa.
Smit NJ, Howatson G and Greenfield R. 2009. Blood lactate levels as a biomarker for angling-induced stress in tigerfish Hydrocynus vittatus from the Okovango Delta, Botswana. Afr. J. Aquat. Sci., 34: 255-259.
Soekoe M, Smit NJ and van der Bank H. 2010. Low allozyme variation in tigerfish Hydrocynus vittatus (Teleostei: Alestidae) from the Okavango panhandle, with notes on the selection of candidates for artificial breeding. Afr. J. Aquat. Sci., 34: 249–254.
Spry DJ and Wood CM. 1985. Ion flux rates, acid-base status, and blood gases in rainbow trout, Salmo gardneri, exposed to toxic zinc in natural soft water. Can. J. Fish. Aquat. Sci., 42: 1332-1341.
Steyn GJ and van Vuren JHJ. 1991. Cryopreservation of the spermatozoa of two African freshwater fishes (Characidae). S. – Afr. Tydskr. van Natuurnav., 21: 76-81.
Steyn GJ. 1993. Physico-chemical characteristics of tigerfish semen. S. – Afr. Tydskr. van Natuurnav., 23: 44-47.
Steyn GJ, Gagiano CL, Deacon AR & Du Preez HH. 1996. Notes on the induced reproduction and development of the tigerfish, Hydrocynus vittatus (Characidae), embryos and larvae. Environ. Biol. Fish., 47: 387-398.
Svobodová Z, Lloyd R, Máchová J and Vykusová. 1993. Water quality and fish health. EIFAC Technical Paper. No. 54. Rome. FAO.
Takashima F and Hibiya T. 1995. An atlas of fish histology: Normal and pathological features, 2nd Ed. Gustav Fischer Verlag, Stuttgart, New York.
Tempelhoff E. 2010. April 26. Olifants se geelvis het ook krokodille se siekte. Beeld.
Tokarz RR. 1978. Oogonial proliferation, oogenesis, and folliculogenesis in nonmammalian vertebrates. In The vertebrate ovary. Plenum press, New York. pp.145-179.
[155]
Turpie J and Joubert A. 2001. Estimating potential impacts of a change in river quality on the tourism value of Kruger National Park: An application of travel cost, contingent and conjoint valuation methods. Water SA., 27: 387-398.
United States Environmental Protection Agency (USEPA). 2006. National recommended water quality criteria. Office of water, Washington D.C.
Van Dyk JC. 2006. A qualitative and quantitative assessment of the normal histology of selected target organs of Clarias gariepinus and Oreochromis mossambicus. PhD Thesis. University of Johannesburg. Johannesburg.
Van Dyk JC, Pieterse GM and van Vuren JHJ. 2007. Histological changes in the liver of Oreochromis mossambicus (Cichlidae) after exposure to cadmium and zinc. Ecotox. Environ. Safe., 66: 432-440.
Van Dyk JC and Pieterse GM. 2008. A histo-morphological study on the testis of the sharptooth catfish (Clarias gariepinus) as reference for future toxicological assessments. J. Appl. Icthyol., 24: 415-422.
Van Dyk JC, Marchand MJ, Smit NJ and Pieterse GM. 2009a. A histology-based fish health assessment of four commercially and ecologically important species from the Okavango Delta panhandle, Botswana. Afr. J. Aquat. Sci., 34: 273-282.
Van Dyk JC, Marchand MJ, Pieterse GM, Barnhoorn IEJ and Bornman MS. 2009b. Histological changes in the gills of Clarias gariepinus (Teleostei: Clariidae) from a polluted South African urban aquatic system. Afr. J. Aquat. Sci., 34: 283-291.
Van Vuuren L. 2009. Experts unite to save abused river from extinction. Water Wheel., 8: 14-17. Velasco-Santamaría YM, Bjerregaard P and Korsgaard B. 2010. Gonadal alterations in male eelpout (Zoarces viviparus) exposed to ethinylestradiol and trenbolone separately or in combination. Mar.Environ. Res. 69: S67-S69.
Velma V and Tchounwou VPB. 2010. Chromium-induced biochemical, genotoxic and histopathologic effects in liver and kidney of goldfish, carassius auratus. Mut. Res., 698: 43–51.
Velmurugan B, Selvanayagam M, Cengiz IE and Unlu E. 2007. Histopathology of lambda- cyhalothrin on tissues (gill, kidney, liver and intestine) of Cirrhinus mrigala. Environ.Toxicol. Pharm., 24: 286-291. [156]
Vetillard A and Bailhache T. 2004. Cadmium: an endocrine disrupter that affects gene expression in the liver and brain of juvenile rainbow trout. Biol. Repr., 72: 119-126.
Wallace RA and Selman K. 1990. Ultrastructural aspects of oogenesis and oocyte growth in fish and amphibians. J. Electron Microscop. Tech., 16: 175-201.
Walmsey RD, Walmsey JJ, Mzuri Consultants and Silberbauer M. 1999. National State of the Environment Report – South Africa. Department of Water Affairs and Forestry.
Water Research Commission. 2001a. State of the Rivers Report: Crocodile, Sabie-Sand & Olifants River Systems. WRC Report No. TT 147/01. Water Research Commission, Pretoria, South Africa.
Water Research Commission. 2001b. State of the Rivers Report: Letaba and Luvuvhu River Systems. WRC Report No. TT 165/01. Water Research Commission, Pretoria, South Africa.
Weyl OLF and Booth AJ. 1999. On the life history of a cyprinid fish, Labeo cylindricus. Environ. Biol. Fish., 55: 215-225. Wischniowski S and Bobko S. 1998. Age and growth laboratory manual. Norfolk, USA: Old Dominion University, Virginia Marine Resources Commission.
Woodhead AD. 1977. Ageing changes in the liver of two poecilid fishes. Exper. Geront. 13: 37-45. Yasutake WT and Wales JH. 1983. Microscopic anatomy of salmonids: An atlas. Fish and Wildlife Services, US Department of Interior. Washington, D.C.
Yonkos LT, Kane AS and Reimschuessel R. 2000. Fathead minnow histology atlas: worldwide web outreach and utilisation. Mar. Env. Res., 50: 307-312.
Zimmerli S, Bernet D, Burkhadt-Holm P, Schmidt-Posthaus H, Vonlanthen P, Wahli T and Segner H. 2007. Assessment of fish health status in four Swiss rivers showing a decline of brown trout catches. Aquat. Sci., 69: 11-25.
[157]
Appendix A: HAI score sheet
HEALTH ASSESSMENT INDEX (HAI) VARIABLES
Eyes Skin Fins Opercula Gills Bile Mesenteric fat Liver Spleen Hindgut Kidney Parasites Comments Fish no Fish Gender
Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal No Exopthal Aberration Erosion Shortening Frayed D Straw < 50% Slight disc Granular Inflammation Swollen Few Heamo Moderate Severe Severe Clubbed L Green 50% Fatty Nodular Mild inflam Mottled Moderate Blind Severe Discolour D Green >50% Nodules/cysts Enlarged Severe Granular Severe Missing Pale 100% Focal disc Other Urolithiasis Other Other Discolouration Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal No Exopthal Aberration Erosion Shortening Frayed D Straw < 50% Slight disc Granular Inflammation Swollen Few Heamo Moderate Severe Severe Clubbed L Green 50% Fatty Nodular Mild inflam Mottled Moderate Blind Severe Discolour D Green >50% Nodules/cysts Enlarged Severe Granular Severe Missing Pale 100% Focal disc Other Urolithiasis Other Other Discolouration Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal No Exopthal Aberration Erosion Shortening Frayed D Straw < 50% Slight disc Granular Inflammation Swollen Few Heamo Moderate Severe Severe Clubbed L Green 50% Fatty Nodular Mild inflam Mottled Moderate Blind Severe Discolour D Green >50% Nodules/cysts Enlarged Severe Granular Severe Missing Pale 100% Focal disc Other Urolithiasis Other Other Discolouration Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal No Exopthal Aberration Erosion Shortening Frayed D Straw < 50% Slight disc Granular Inflammation Swollen Few Heamo Moderate Severe Severe Clubbed L Green 50% Fatty Nodular Mild inflam Mottled Moderate Blind Severe Discolour D Green >50% Nodules/cysts Enlarged Severe Granular Severe Missing Pale 100% Focal disc Other Urolithiasis Other Other Discolouration Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal No Exopthal Aberration Erosion Shortening Frayed D Straw < 50% Slight disc Granular Inflammation Swollen Few Heamo Moderate Severe Severe Clubbed L Green 50% Fatty Nodular Mild inflam Mottled Moderate Blind Severe Discolour D Green >50% Nodules/cysts Enlarged Severe Granular Severe Missing Pale 100% Focal disc Other Urolithiasis Other Other Discolouration Other Other
[158]
Appendix B: Histological assessment sheets
Liver Specimen no: n RP Functional Unit Alterations a w a x w Aneurysm/Hyperaemia/ CD Haemorrhage e.g. induce congestion 0 1 0 Intercellular oedema 0 1 0
RP INDEX 0
RC Liver tissue Structural alterations e.g. cord disarray & cell structure 0 1 0
Hepatocytes Plasma alterations Granular degeneration / intra cellular deposits 0 1 0
Fatty degeneration (e.g. fatty change) 0 1 0
Glycogen vacuoles 0 1 0
Vacuolation (content unknown) 0 1 0
Inter cellular deposits 0 1 0
Increase in MMC 0 1 0
Nuclear alterations Pleomorphism / chromatin clearing 0 2 0
Pyknosis 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
Interstitial tissue Structural alterations 0 1 0
Plasma alterations Granular degeneration / intra cellular deposits 0 1 0
Vacuolation 0 1 0
Intercellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
Bile ducts Structural alterations 0 1 0
Plasma alterations Granular degeneration / intra cellular deposits 0 1 0
Vacuolation 0 1 0
Intercellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
RP INDEX 0
PC Liver tissue Hypertrophy 0 1 0
Hyperplasia 0 2 0
Wall proliferation e.g. blood vessels 0 1 0
Interstitial tissue Hypertrophy 0 1 0
Hyperplasia e.g. cirrhosis 0 2 0
Bile ducts Hypertrophy 0 1 0
Hyperplasia 0 2 0
Wall proliferation 0 2 0
RP INDEX 0
I Exudate 0 1 0
Activation of RES 0 1 0
Infiltration e.g. leucocytes (MNL) - lymphocytes 0 2 0
e.g. granulocytes 0 2 0
RP INDEX 0
T Benign 0 2 0 Malignant 0 3 0 RP INDEX 0 [159]
Kidney Specimen no: n RP Functional Unit Alterations a w a x w Aneurysm/Hyperaemia/Haemor CD rhage e.g. induce congestion 0 1 0 Intercellular oedema 0 1 0 Dilation of glomerulus capillaries 0 1 0
RP INDEX 0
RC Tubule Structural alterations 0 1 0
Plasma alterations Vacuolation 0 1 0
Hyaline droplet degeneration 0 1 0
Basophilic cytoplasm 0 1 0
Granular degeneration 0 1 0
Inter cellular deposits 0 1 0
Nuclear alterations Pleomorphism / chromatin clearing 0 2 0
Pyknosis 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
Glomerulus Structural alterations 0 1 0
Plasma alterations Granular degeneration / intra cellular deposits 0 1 0
Intercellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
Interstitial tissue Structural alterations 0 1 0
Plasma alterations Granular degeneration / intra cellular deposits / vacuolation 0 1 0
Intercellular deposits MMC 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
RP INDEX 0
PC Tubule Hypertrophy e.g. albuminous degeneration (reversible) (cloudy swelling) 0 1 0
Hyperplasia 0 2 0
Glomerulus Thickening of BC membrane 0 1 0
Hypertrophy 0 1 0
Hyperplasia 0 2 0
Interstitial tissue Hypertrophy 0 1 0
Hyperplasia e.g. cirrhosis 0 2 0
RP INDEX 0
I Exudate 0 1 0
Activation of RES 0 1 0
Infiltration e.g. leucocytes (MNL) - lymphocytes 0 2 0
e.g. granulocytes 0 2 0
RP INDEX 0
T Benign 0 2 0 Malignant 0 3 0 RP INDEX 0
[160]
Gill Specimen no: n RP Functional Unit Alterations a w a x w Aneurysm/Hyperaemia/ CD Haemorrhage e.g. telangiecstasia 0 1 0 e.g. congestion 0 1 0 Intercellular oedema 0 1 0
RP INDEX 0
RC Epithelium Structural alterations e.g. sec lam branching 0 1 0
Plasma alterations e.g. vacuolation 0 1 0
Inter cellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
Rupture of pilar cells 0 2 0
Supporting tissue Structural alterations e.g. prim lam branching 0 1 0
Plasma alterations 0 1 0
Intercellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
RP INDEX 0
PC Epithelium Hypertrophy 0 1 0
Hyperplasia 0 2 0
Mucous cells Hypertrophy 0 1 0
Hyperplasia 0 2 0
RP INDEX 0
I Exudate 0 1 0
Activation of RES 0 1 0
Infiltration Lymphocytes 0 2 0
Granulocytes 0 2 0
RP INDEX 0
T Benign 0 2 0 Malignant 0 3 0
[161]
Testes Specimen no: n RP Functional Unit Alterations a w a x w Aneurysm/Hyperaemia/Haemorrha CD ge e.g. induce congestion 0 1 0 Intercellular oedema 0 1 0
RP INDEX 0
RC Lobule cysts Disorganization of lobules 0 1 0
Detachment of basal membrane 0 1 0
Inhibition of spermatogenesis 0 3 0
Degeneration of Sertoli cells 0 2 0
Interstitial tissue Structural alterations 0 1 0
Plasma alteration in leydig cells 0 1 0
Deposits MMC 0 1 0
Vacuolation 0 1 0
Nuclear alterations in leydig cells 0 2 0 Atrophy 0 2 0 Necrosis 0 3 0 Developmental stages
(1) Spermatogonia Structural alterations 0 1 0
Plasma alterations e.g. intra cellular deposits 0 1 0
Vacuolation 0 1 0
Inter cellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
(2) Spermatocytes Structural alterations 0 1 0
Plasma alterations e.g. intra cellular deposits 0 1 0
Vacuolation 0 1 0
Inter cellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
(3) Spermatids Structural alterations 0 1 0
Plasma alterations e.g. intra cellular deposits 0 1 0
Vacuolation 0 1 0
Inter celllular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
(4) Spermatozoa Structural alterations 0 1 0
Plasma alterations e.g. intra cellular deposits 0 1 0
Vacuolation 0 1 0
Inter celllular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
RP INDEX 0
PC Lobule cysts Wall proliferation e.g. basal membrane / Tunica A 0 1 0
Wall proliferation e.g. blood vessels 0 1 0
Interstitial tissue Hypertrophy 0 1 0
Hyperplasia 0 2 0 Developmental stages
(1) Spermatogonia Hypertrophy 0 1 0
Hyperplasia 0 2 0
(2) Spermatocytes Hypertrophy 0 1 0
Hyperplasia 0 2 0
(3) Spermatids Hypertrophy 0 1 0
Hyperplasia 0 2 0
(4) Spermatozoa Hypertrophy 0 1 0
Hyperplasia 0 2 0
RP INDEX 0
I Exudate 0 1 0
Activation of RES 0 1 0
Infiltration Leucocytes (MNL) 0 2 0
RP INDEX 0
T Benign 0 2 0 Malignant 0 3 0
RP INDEX 0
IS Intersex 0 3 0 RP INDEX 0 [162]
Ovaries Specimen no: n RP Functional Unit Alterations a w a x w Aneurysm/Hyperaemia/H CD aemorrhage e.g. induce congestion 0 1 0 Intercellular oedema 0 1 0
RP INDEX 0
RC Ovary Inhibition of oogenesis 0 3 0 Develop. stages
(1) Oogonia Structural alterations 0 1 0
Plasma alterations 0 1 0
Inter cellular deposits 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
(2) Oocytes Structural alterations 0 1 0
Plasma alterations 0 1 0
Intercellular deposits 0 1 0
Nuclear alterations e.g. germinal vesicle 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
Interstitial tissue Structural alterations 0 1 0
Plasma alterations e.g. vacuolation 0 1 0
Intercellular deposits MMC 0 1 0
Nuclear alterations 0 2 0
Atrophy 0 2 0
Necrosis 0 3 0
RP INDEX 0 PC Develop. stages
(1) Oogonia Hypertrophy 0 1 0
Hyperplasia 0 2 0
(2) Oocytes Hypertrophy 0 1 0
Hyperplasia 0 2 0
Interstitial tissue Hypertrophy 0 1 0
Hyperplasia 0 2 0
Tunica Thickening 0 2 0
RP INDEX 0
I Exudate 0 1 0
Activation of RES 0 1 0
Infiltration Leucocytes (MNL) - lymphocytes 0 2 0
Granulocytes 0 2 0
RP INDEX 0
T Benign 0 2 0 Malignant 0 3 0
RP INDEX 0
IS Intersex 0 3 0
RP INDEX 0
[163]
Appendix C: HAI score sheet results
HAI Calculations SITE: Olifants September 2009 SPECIES: Hydrocynus vittatus Fish No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Gender Fins 0000000000000000 Spleen 0000000000000 30 0 0 Hindgut 0000000000000000 Kidney 0000000000000000 Skin 0000000000000000 Liver 0 0 0 30 0 0 30 0 0 0 0 0 0 0 0 0 Eyes 0000000000000000 Gills 0000000000000000 Pseudobranchs 0000000000000000 Opercula 0000000000000000 Parasites 20 20 0 10 10 10 20 20 10 10 0 10 20 10 10 0 Hematocrit 0 0 0 0 0 0 0 0 0 0 20 20 0 20 0 0 Leukocrit 0 30 00000000000000 Plasma protein 0 0 0 0 0 0 0 0 0 0 10 0 0 10 0 0 Total 20 50 0 40 10 10 50 20 10 10 30 30 20 70 10 0
SUM HAI 380 MEAN HAI 23.75
[164]
HAI Calculations SITE: Olifants May 2010 SPECIES: H vittatus Fish No. 1 2 3 4 5 6 Gender Fins 0 0 0 0 0 0 Spleen 0 0 0 0 0 0 Hindgut 0 0 0 0 0 0 Kidney 0 0 0 0 0 0 Skin 0 0 0 0 0 0 Liver 0 0 0 0 0 0 Eyes 0 0 0 0 0 0 Gills 0 0 0 0 0 0 Pseudobranchs 0 0 0 0 0 0 Opercula 0 0 0 0 0 0 Parasites 0 10 0 10 10 10 Hematocrit 10 10 10 10 10 10 Leukocrit 0 0 0 0 0 0 Plasma protein 30 0 0 30 0 30 Total 40 20 10 50 20 50
SUM HAI 190 MEAN HAI 31.67
[165]
HAI Calculations SITE: Luvuvhu November 2009 SPECIES: Hydrocynus vittatus Fish No. 1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 Gender Fins 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Spleen 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Hindgut 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Kidney 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Skin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Liver 0 0 0 0 0 0 0 0 30 30 0 0 0 0 0 Eyes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Gills 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pseudobranchs 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Opercula 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Parasites 10 0 10 10 10 0 0 0 0 10 0 0 10 10 10 Hematocrit 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 Leukocrit 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Plasma protein 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 Total 10 10 10 10 20 0 0 0 30 40 0 0 10 10 10
SUM HAI 160 MEAN HAI 10.67
[166]
HAI Calculations SITE: Luvuvhu April 2010 SPECIES: Hydrocynus vittatus Fish No. 1 2 Gender Fins 0 0 Spleen 0 0 Hindgut 0 0 Kidney 0 0 Skin 0 0 Liver 0 0 Eyes 0 0 Gills 0 0 Pseudobranchs 0 0 Opercula 0 0 Parasites 20 20 Hematocrit 0 0 Leukocrit 0 0 Plasma protein 10 10 Total 30 30
SUM HAI 60 MEAN HAI 30
[167]
HAI Calculations SITE: Olifants Sept 09 SPECIES: Labeobarbus marequensis Fish No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Gender Fins 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 Spleen 000000000000000 Hindgut 0 10 0 0 0 10 0 0 0 0 0 0 0 0 0 Kidney 0 0 0 0 30 0 30 30 0 0 0 0 0 0 0 Skin 0 0 0 0 0 0 0 0 Liver 30 0 0 0 0 30 30 30 0 0 0 0 0 0 0 Eyes 000000000000000 Gills 0 30 30 30 30 0 0 0 0 0 0 0 0 0 0 Pseudobranchs 000000000000000 Opercula 000000000000000 Parasites 0 0 0 0 0 0 0 0 0 0 10 10 30 0 20 Hematocrit 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 Leukocrit 0 30 30 0 0 0 0 0 30 0 0 0 0 0 0 Plasma protein 000000000000000 Total 30 70 60 30 60 40 60 70 40 0 10 10 30 0 20
SUM HAI 530 MEAN HAI 35.33
[168]
HAI Calculations SITE: Olifants May 2010 SPECIES: Labeobarbus marequensis Fish No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Gender Fins 000000000000000 Spleen 000000000000000 Hindgut 000000000000000 Kidney 000000000000000 Skin 000000000000000 Liver 000000000000000 Eyes 000000000000000 Gills 000000000000000 Pseudobranchs 000000000000000 Opercula 000000000000000 Parasites 0 10 10 10 10 20 20 20 10 20 20 10 10 10 20 Hematocrit 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Leukocrit 000000000000000 Plasma protein 0 30 30 30 30 30 30 30 30 30 30 30 30 0 30 Total 10 50 50 50 50 60 60 60 50 60 60 50 50 20 60
SUM HAI 740 MEAN HAI 49.33
[169]
HAI Calculations SITE: Luvuvhu November 2009 SPECIES: Labeo cylindricus Fish No. 1 2 3 4 5 6 7 8 9 10 Gender Fins 0 0 0 0 0 0 0 0 0 0 Spleen 0 0 0 0 0 0 0 0 0 0 Hindgut 0 0 0 0 0 0 0 0 0 0 Kidney 0 0 0 0 0 0 0 0 0 0 Skin 0 0 0 0 0 0 0 0 0 0 Liver 0 0 0 0 0 0 0 0 0 0 Eyes 0 0 0 0 0 0 0 0 0 0 Gills 0 0 0 0 0 0 0 0 0 0 Pseudobranchs 0 0 0 0 0 0 0 0 0 0 Opercula 0 0 0 0 0 0 0 0 0 0 Parasites 0 0 0 0 0 0 0 0 0 0 Hematocrit 0 0 0 0 0 0 0 0 0 0 Leukocrit 0 0 0 0 0 0 0 0 0 0 Plasma protein 0 0 10 0 0 0 0 0 0 0 Total 0 0 10 0 0 0 0 0 0 0
SUM HAI 10 MEAN HAI 1
[170]
[171]
Appendix D: Gonadal staging sheets
Testis gonadal staging Trip Species Fish number Stage Olifants September 2009 Hydrocynus vittatus 4 1 Olifants September 2009 Hydrocynus vittatus 6 1 Olifants September 2009 Hydrocynus vittatus 9 0 Olifants September 2009 Hydrocynus vittatus 10 0 Olifants September 2009 Hydrocynus vittatus 12 0 Olifants September 2009 Hydrocynus vittatus 14 1 Olifants June 2010 Hydrocynus vittatus 1 2 Olifants June 2010 Hydrocynus vittatus 2 1 Olifants June 2010 Hydrocynus vittatus 3 1 Olifants June 2010 Hydrocynus vittatus 4 1 Olifants June 2010 Hydrocynus vittatus 6 1 Luvuvhu November 2009 Hydrocynus vittatus 1 3 Luvuvhu November 2009 Hydrocynus vittatus 3 3 Luvuvhu November 2009 Hydrocynus vittatus 4 3 Luvuvhu November 2009 Hydrocynus vittatus 11 3 Luvuvhu November 2009 Hydrocynus vittatus 12 3 Luvuvhu November 2009 Hydrocynus vittatus 13 3 Luvuvhu November 2009 Hydrocynus vittatus 15 2 Luvuvhu May 2010 Hydrocynus vittatus 2 1 Olifants September 2009 Laebeobarbus marequensis 1 2 Olifants September 2009 Laebeobarbus marequensis 2 0 Olifants September 2009 Laebeobarbus marequensis 3 0 Olifants September 2009 Laebeobarbus marequensis 4 0 Olifants September 2009 Laebeobarbus marequensis 5 0 Olifants September 2009 Laebeobarbus marequensis 6 1 Olifants September 2009 Laebeobarbus marequensis 11 0 Olifants September 2009 Laebeobarbus marequensis 13 0 Olifants September 2009 Laebeobarbus marequensis 14 0 Olifants June 2010 Laebeobarbus marequensis 2 2 Olifants June 2010 Laebeobarbus marequensis 3 2 Olifants June 2010 Laebeobarbus marequensis 5 1 Olifants June 2010 Laebeobarbus marequensis 8 2 Olifants June 2010 Laebeobarbus marequensis 9 3 Olifants June 2010 Laebeobarbus marequensis 10 2 Olifants June 2010 Laebeobarbus marequensis 11 3 Olifants June 2010 Laebeobarbus marequensis 13 1 Olifants June 2010 Laebeobarbus marequensis 14 2 Olifants June 2010 Laebeobarbus marequensis 15 2 Luvuvhu November 2009 Labeo cylindricus 2 3 Luvuvhu November 2009 Labeo cylindricus 8 3 Luvuvhu November 2009 Labeo cylindricus 9 3 Olifants June 2010 Labeo rosae 1 0 Olifants June 2010 Labeo rosae 2 0 Olifants June 2010 Labeo rosae 6 0 Olifants June 2010 Labeo rosae 7 0 [172]
Ovary gonadal staging Trip Species Fish number Stage Olifants September 2009 Hydrocynus vittatus 1 0 Olifants September 2009 Hydrocynus vittatus 2 0 Olifants September 2009 Hydrocynus vittatus 3 0 Olifants September 2009 Hydrocynus vittatus 5 0 Olifants September 2009 Hydrocynus vittatus 7 0 Olifants September 2009 Hydrocynus vittatus 8 0 Olifants September 2009 Hydrocynus vittatus 13 0 Olifants September 2009 Hydrocynus vittatus 16 0 Olifants June 2010 Hydrocynus vittatus 5 1 Luvuvhu November 2009 Hydrocynus vittatus 2 2 Luvuvhu November 2009 Hydrocynus vittatus 6 0 Luvuvhu November 2009 Hydrocynus vittatus 8 0 Luvuvhu November 2009 Hydrocynus vittatus 9 2 Luvuvhu November 2009 Hydrocynus vittatus 10 2 Luvuvhu November 2009 Hydrocynus vittatus 14 2 Luvuvhu November 2009 Hydrocynus vittatus 16 0 Luvuvhu May 2010 Hydrocynus vittatus 1 0 Olifants September 2009 Laebeobarbus marequensis 5 0 Olifants September 2009 Laebeobarbus marequensis 7 0 Olifants September 2009 Laebeobarbus marequensis 8 0 Olifants September 2009 Laebeobarbus marequensis 9 2 Olifants September 2009 Laebeobarbus marequensis 10 2 Olifants September 2009 Laebeobarbus marequensis 11 0 Olifants September 2009 Laebeobarbus marequensis 12 0 Olifants September 2009 Laebeobarbus marequensis 13 0 Olifants September 2009 Laebeobarbus marequensis 14 0 Olifants September 2009 Laebeobarbus marequensis 15 0 Olifants June 2010 Laebeobarbus marequensis 1 2 Olifants June 2010 Laebeobarbus marequensis 4 1 Olifants June 2010 Laebeobarbus marequensis 6 1 Olifants June 2010 Laebeobarbus marequensis 7 1 Olifants June 2010 Laebeobarbus marequensis 12 0 Luvuvhu November 2009 Labeo cylindricus 3 0 Luvuvhu November 2009 Labeo cylindricus 4 2 Luvuvhu November 2009 Labeo cylindricus 6 2 Luvuvhu November 2009 Labeo cylindricus 10 0 Olifants June 2010 Labeo rosae 3 0 Olifants June 2010 Labeo rosae 4 0 Olifants June 2010 Labeo rosae 5 0
[173]