AN ASSESSMENT OF THE HEALTH STATUS AND EDIBILITY OF FISH FROM THREE IMPOUNDMENTS IN THE NORTH WEST PROVINCE, SOUTH AFRICA

BY

BYRON M. BESTER

DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MAGISTER SCIENTIÆ

IN

AQUATIC HEALTH

IN THE

FACULTY OF SCIENCE

AT THE

UNIVERSITY OF JOHANNESBURG

SUPERVISOR: PROF. G. M. WAGENAAR CO-SUPERVISOR: DR J. C. VAN DYK

DECEMBER 2013

NOTE: The financial assistance of the National Research Foundation (NRF), University of Johannesburg (UJ) and Cancer Association of South Africa (CANSA) is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily attributed to the abovementioned institutions. M. Sc. – 2013

TABLE OF CONTENTS

TABLE OF CONTENTS ...... II ACKNOWLEDGEMENTS ...... VII LIST OF ABBREVIATIONS ...... VIII GLOSSARY OF TERMS ...... XI LIST OF TABLES ...... XIII LIST OF FIGURES ...... XVII ABSTRACT ...... XIX 1. PROJECT OVERVIEW ...... 1 1.1. INTRODUCTION ...... 2 1.2. STUDY MOTIVATION ...... 2 1.3. STUDY DIRECTIVE ...... 5 1.3.1. Aims and Objectives ...... 5 1.3.2. Hypothesis ...... 6 1.3.3. Study Approach ...... 6 1.4. SELECTED SITES ...... 7 1.4.1. Roodekopjes Dam (RD) ...... 7 1.4.2. Vaalkop Dam (VD) ...... 7 1.4.3. Marico-Bosveld Dam (MBD) ...... 8 1.5. SELECTED SPECIES ...... 8 1.5.1. Clarias gariepinus ...... 9 1.5.2. Cyprinus carpio ...... 9 1.6. SELECTED TARGET ORGANS ...... 9 1.6.1. Gills ...... 10 1.6.2. Liver ...... 10 1.6.3. Kidney ...... 10 1.6.4. Heart ...... 10 1.6.5. Gonads...... 11 1.6.6. Skin ...... 11 2. BACKGROUND INFORMATION...... 12 2.1. STATE OF AQUATIC ECOSYSTEMS ...... 13 2.1.1. Current Concerns ...... 13 2.1.1.1. Eutrophication ...... 13 2.1.1.2. Endocrine Disrupting Chemicals (EDCs) ...... 14 2.1.1.3. Cancer ...... 14 2.2. STUDY AREA ...... 14 2.2.1. Location ...... 15

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2.2.2. Catchment-specific Impacts ...... 15 2.2.3. Socio-economic Climate ...... 16 2.3. SUBSTRATE ANALYSIS ...... 16 2.3.1. Physico-chemical Parameters ...... 16 2.3.1.1. Temperature ...... 17 2.3.1.2. pH ...... 17 2.3.1.3. Electrical Conductivity & Total Dissolved Solids (TDS) ...... 17 2.3.1.4. Dissolved Oxygen ...... 18 2.3.2. Chemical Analysis...... 18 2.3.2.1. Organic ...... 18 2.3.2.1.a. Aldrin (HHDN) ...... 18 2.3.2.1.b. Chorpyriphos ...... 18 2.3.2.1.c. DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) ...... 19 2.3.2.1.d. Lindane (HCH) ...... 19 2.3.2.1.e. Nonylphenol (p-NP) ...... 19 2.3.2.1.f. Terbuthylazine ...... 19 2.3.2.2. Inorganic ...... 20 2.3.2.2.a. Arsenic (As) ...... 20 2.3.2.2.b. Beryllium (Be) ...... 20 2.4. SURVIVAL MECHANISMS OF FISH ...... 20 2.4.1. Fish as a Bioindicator Species ...... 21 2.4.2. Histopathology as a Biomarker ...... 22 2.5. NECROPSY-BASED HEALTH ASSESSMENT ...... 22 2.5.1. Haematological Assessment ...... 23 2.6. BIOMETRIC INDICES ...... 23 2.6.1. Condition Factor ...... 23 2.6.2. Organo-somatic Indices...... 23 2.6.2.1. Hepatosomatic Index (HSI) ...... 24 2.6.2.2. Splenosomatic Index (SSI) ...... 24 2.6.2.3. Gonadosomatic Index (GSI) ...... 24 2.7. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 24 2.8. AGE ESTIMATION ...... 25 2.8.1. Otoliths ...... 25 2.8.2. Scales ...... 25 2.9. EDIBILITY ...... 25 2.9.1. Muscle Analysis ...... 26 2.9.2. Human Health Risk Assessment (HHRA) ...... 26 2.9.2.1. Hazard Identification ...... 27 2.9.2.2. Dose – Response Assessment ...... 28 2.9.2.3. Exposure Assessment ...... 28

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2.9.2.4. Risk Characterisation ...... 28 3. MATERIALS & METHODS ...... 29 3.1. INTRODUCTION ...... 30 3.2. FIELD SURVEYS ...... 30 3.3. SUBSTRATE ANALYSIS ...... 31 3.3.1. Physico-chemical Parameters ...... 31 3.3.2. Chemical Analysis...... 33 3.4. SPECIMEN COLLECTION ...... 35 3.5. NECROPSY-BASED HEALTH ASSESSMENT ...... 36 3.5.1. Haematological Assessment ...... 37 3.6. BIOMETRIC INDICES ...... 37 3.6.1. Condition Factor ...... 37 3.6.2. Organo-somatic Indices...... 38 3.6.2.1. Hepatosomatic Index ...... 38 3.6.2.2. Splenosomatic Index ...... 38 3.6.2.3. Gonadosomatic Index ...... 38 3.7. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 38 3.7.1. Tissue Processing ...... 39 3.7.1.1. Fixation ...... 40 3.7.1.2. Dehydration ...... 40 3.7.1.3. Clearing, Infiltration and Embedding ...... 40 3.7.1.4. Sectioning, Mounting and Staining ...... 40 3.7.2. Qualitative Histological Assessment ...... 41 3.7.3. Quantitative Histological Assessment ...... 42 3.7.3.1. Score Value...... 42 3.7.3.2. Importance Factor ...... 43 3.7.3.3. Calculation of Indices ...... 43

3.7.3.3.a. Reaction Pattern Index (Irp) ...... 44

3.7.3.3.b. Organ Index (Iorg) ...... 44

3.7.3.3.c. Total Reaction Pattern Index (ITot rp) ...... 44

3.7.3.3.d. Fish Index (Ifish) ...... 45 3.8. AGE ESTIMATION ...... 45 3.8.1. Otoliths ...... 45 3.8.2. Scales ...... 46 3.9. EDIBILITY ...... 46 3.9.1. Muscle Analysis ...... 46 3.9.2. Human Health Risk Assessment (HHRA) ...... 47 3.9.2.1. Toxic Risk ...... 47 3.9.2.2. Cancer Risk ...... 48

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3.10. STATISTICAL ANALYSIS ...... 48 4. RESULTS ...... 50 4.1. INTRODUCTION ...... 51 4.2. SUBSTRATE ANALYSIS ...... 51 4.2.1. Physico-chemical Parameters ...... 51 4.2.2. Chemical Analysis...... 52 4.2.2.1. Organic ...... 52 4.2.2.2. Inorganic ...... 53 4.3. SPECIMEN COLLECTION ...... 54 4.4. NECROPSY-BASED HEALTH ASSESSMENT ...... 54 4.4.1. Haematological Assessment ...... 56 4.5. BIOMETRIC INDICES ...... 57 4.5.1. Condition Factor ...... 57 4.5.2. Organo-somatic Indices...... 57 4.6. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 59 4.6.1. Qualitative Histological Assessment ...... 59 4.6.2. Quantitative Histological Assessment ...... 76

4.6.2.1. Organ Indices (Iorg) ...... 76

4.6.2.2. Total Reaction Pattern Indices (ITot rp) ...... 77

4.6.2.3. Fish Index (Ifish) ...... 78 4.7. AGE ESTIMATION ...... 79 4.7.1. Otoliths ...... 79 4.7.2. Scales ...... 80 4.8. EDIBILITY ...... 80 4.8.1. Muscle Analysis ...... 80 4.8.2. Human Health Risk Assessment (HHRA) ...... 81 4.8.2.1. Hazard Identification ...... 82 4.8.2.2. Dose-Response Assessment ...... 82 4.8.2.3. Exposure Assessment ...... 83 4.8.2.4. Risk Characterisation ...... 84 5. DISCUSSION & CONCLUSION ...... 87 5.1. SUBSTRATE ANALYSIS ...... 88 5.1.1. Physico-chemical Parameters ...... 88 5.1.2. Chemical Analysis...... 89 5.1.2.1. Organic ...... 89 5.1.2.1.a. DDT & metabolites (DDE & DDD) ...... 89 5.1.2.1.b. Nonylphenol (p-NP) ...... 90 5.1.2.2. Inorganic ...... 91 5.1.2.2.a. Aluminium (Al) ...... 91

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5.1.2.2.b. Silver (Ag) ...... 92 5.2. NECROPSY-BASED HEALTH ASSESSMENT ...... 92 5.3. BIOMETRIC INDICES ...... 93 5.4. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 94 5.4.1. Gill Histology ...... 94 5.4.2. Liver Histology ...... 95 5.4.3. Kidney Histology ...... 97 5.4.4. Heart Histology ...... 97 5.4.5. Gonad Histology ...... 98 5.4.5.1. Testis Histology ...... 98 5.4.5.2. Ovary Histology ...... 99 5.4.6. Skin Histology ...... 100 5.4.7. Combined Histological Effects ...... 100 5.5. EDIBILITY ...... 102 5.5.1. Muscle Analysis ...... 102 5.5.2. Human Health Risk Assessment (HHRA) ...... 103 5.5.2.1. Risk Communication ...... 105 5.5.2.2. Uncertainty Analysis ...... 105 5.6. CONCLUSION ...... 106 5.7. RECOMMENDATIONS ...... 108 REFERENCES...... 109 APPENDICES ...... 125 Appendix 1 – Record & Assessment Sheets ...... 126 Appendix 2 – Substrate Analysis Data ...... 137 Appendix 3 – Haematological Assessment Data ...... 142 Appendix 4 - Biometric Indices Data ...... 144 Appendix 5 – Histology-Based Fish Health Assessment (HBFHA) Data ...... 154 Appendix 6 - Age Estimation Data ...... 158 Appendix 7 – Edibility Assessment Data ...... 159 Appendix 8 – List of Histopathological Alterations ...... 172 Appendix 9 – List of Organic Toxicants ...... 176

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ACKNOWLEDGEMENTS

 To my Creator, Lord and Saviour, for the abilities to persevere and partake in the amazing opportunities and experiences provided throughout my studies, and for the sacrifice and provision in my everyday life.  To Prof. G. M. Wagenaar, for her professional guidance, her high expectations and openness in discussion, her credibility, her ongoing encouragement and enduring patience – it was a privilege to have her as my Supervisor.  To Dr J. C. Van Dyk, for his professional guidance, his keen eye for detail, his incredible efficiency and work ethic, his remarkable organisational skills, and for his integrity in his approach to all aspects of his research.  To my amazing and beautiful wife, Shayna-Lee, for the much needed distractions throughout this process, for believing in me and pushing me to continue, and also for understanding the importance of this commitment.  To Bettina Genthe of the CSIR, for her expertise in the application of the human health risk assessment model and interpretation thereof.  To Juliana Van Straaten, at Statkon, for her help and expertise in applying the statistical comparisons within the data.  To Ms Amanda Mooney, Ms Bosupeng Motshegoa, Mr Mike Tresise, Mr Ngcebo Sikhakhane, and Mr Ruan Gerber for the help during the sampling trips, histological assessment and age estimation process.  To the Zoology Department at University of Johannesburg (UJ) for the use of the laboratories, vehicles, boats, equipment and services.  To the National Research Fund (NRF), Cancer Association of South Africa (CANSA) and the University of Johannesburg (UJ) for financial assistance towards this research.

vii M. Sc. – 2013

LIST OF ABBREVIATIONS

AUTHORITIES / INSTITUTIONS / PROGRAMMES: ATSDR - Agency Toxic Substance and Disease Registry BPD - Bojanala Platinum District CDEP - Connecticut Department of Environmental Protection DWAF - Department of Water Affairs and Forestry HEAST - Health Effects Assessment Summary Tables IRIS - Integrated Risk Information System ITER - International Toxicity Estimates of Risk NAS - National Academy of Sciences NEMP - National Eutrophication Monitoring Programme NJDEP - New Jersey Department of Environmental Protection NOAA - National Oceanic and Atmospheric Administration NWA - National Water Act NWDACE - North West Department of Agriculture, Conservation and Environment NWP - North West Province NWP&TB - North West Parks & Tourism Board RHP - River Health Programme STATS SA - Statistics South Africa TERA - Toxicology Excellence for Risk Assessment UJ - University of Johannesburg UN - United Nations USDA - United States Department of Agriculture USEPA - United States Environmental Protection Agency WHO - World Health Organization STUDY SITES: MBD - Marico-Bosveld Dam RD - Roodekopjes Dam VD - Vaalkop Dam WATER / SEDIMENT QUALITY: LEL - Lowest Effect Level SQuiRTs - Screening Quick Reference Tables (NOAA) RQOs - Resource Quality Objectives RWQOs - Resource Water Quality Objectives TWQR - Target Water Quality Range FISH HEALTH / HISTOPATHOLOGY: CF - Condition factor CD - Circulatory disturbances EDTA - Ethylene Diamine Triacetic Acid FCA - Focal cellular alteration GSI - Gonadosomatic index H&E - Hematoxylin and eosin HBFHA - Histology-based fish health assessment

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Hct - Haematocrit HSI - Hepatosomatic index I - Inflammation Ifish - Fish index Iorg - Organ index Irp - Reaction pattern index ITot rp - Total reaction pattern index IF - Importance factor IS - Intersex Lct - Leukocrit NBF - Neutral buffered formalin PC - Progressive changes RC - Regressive changes SSI - Splenosomatic index SV - Score value T - Tumour/Neoplasia HUMAN HEALTH RISK ASSESSMENT: ADD - Average daily dose Beta (휷) - Cancer slope factor or cancer potency BW - Average body weight Ctox - Concentration of bioaccumulated toxicant in fish muscle ED - Exposure duration HHRA - Human Health Risk Assessment HQ - Hazard quotient or hazard index IR - Intake rate LADD - Lifetime average daily dose LOAEL - Lowest-observed-adverse-effect level Lft - Average life expectancy NOAEL - No-observed-adverse-effect level RfD - Reference Dose TOXICANTS (INORGANIC): Ag - Silver Al - Aluminium As - Arsenic B - Boron Ba - Barium Be - Beryllium Bi - Bismuth Ca - Calcium Cd - Cadmium Co - Cobalt Cr - Chromium Cu - Copper Fe - Iron K - Potassium

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Li - Lithium Mg - Magnesium Mn - Manganese Mo - Molybdenum Na - Sodium Ni - Nickel P - Phosphorus Pb - Lead Sb - Antimony Se - Selenium Si - Silicon Sn - Tin Sr - Strontium Ti - Titanium V - Vanadium W - Tungsten Zn - Zinc Zr - Zirconium TOXICANTS (ORGANIC): op’-DDD - o,p’-dichlorodiphenyldichloroethane op’-DDE - o,p’-dichlorodiphenyldichloroethylene pp’-DDE - p,p’-dichlorodiphenyldichloroethylene op’-DDT - o,p’-dichlorodiphenyltrichloroethane EDCs - Endocrine Disrupting Chemicals HCH - Hexachlorocyclohexane (Lindane) - Dieldrin or (1R,4S,4aS,5R,6R,7S,8S,8aR)-1,2,3,4,10,10-hexachloro- HEOD 1,4,4a,5,6,7,8,8a-octahydro-6,7-epoxy-1,4:5,8-dimethanonaphthalene HHDN - Aldrin or (1R,4S,4aS,5S,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a- hexahydro-1,4:5,8-dimethanonaphthalene p-NP - p-nonylphenol (& derivatives) OCs - Organochlorines OPs - Organophosphates PCBs - Polycholinated biphenols MISCELLANEOUS: d.f. - Degree of freedom (Statistical term) GDP - Gross Domestic Product GGP - Gross Geographic Product ICP-MS - Inductively-coupled plasma mass spectrometry ICP-OES - Inductively-coupled plasma optical emission spectrometry TDS - Total dissolved solutes WMA - Water Management Area

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GLOSSARY OF TERMS

Acute - Single dosage of toxicant or an exposure to the toxicant over a short period of time, whereby the endpoint is usually survival. Annuli - Annuli are the darker bands on a scale, which are formed when a group of concentric rings (circuli) form close to each other during a winter season, or alternatively a difficult season. This is opposed to the wider spaced circuli that form during summer. Antagonistic - Interaction between two or more components that have an effect upon one another, such that the combined effect is less than the sum of the separate components. Bioaccumulation - Uptake via accumulation or sequestration of toxicants into living organism. Bioactivation - During the biotransformation process, the toxicant is transformed into a more toxic substance and causes damage within the organism. Bioassessment - Assessment of environmental health or biotic integrity through characterisation of health, prevalence or biodiversity of an indicator organism or a group of indicator organisms. *Similar to biomonitoring. Bioavailability - The form or structure of a toxicant that can be accumulated within an organism. This depends on exposure and persistence, whereby no exposure results in no risk i.e. toxicant is present in an environmental partition that is not in contact with the exposure medium, and the persistence of a toxicant in the environment depends it degradation rate. Bioconcentration - Bioaccumulation of toxicants into living organism directly from ambient environment, such as via gills or skin. Bioindicator - An organism (or group of organisms) that allow characterisation of the state of an ecosystem through detectable changes within biochemical, cytological, physiological, ethological or ecological tests. Biomagnification - Bioaccumulation of toxicants into living organism via bioconcentration as well as trophic transport, such as via ingestion. Concentration of toxicants increase as toxicant moves higher up in food web and trophic level. Biomarker - The measurable biological response in a living organism (or group of living organisms) during a biochemical, cytological, physiological, ethological or ecological test caused by exposure to toxicants. Biomonitoring - Use of living organisms in aquatic systems as biological indicators of ecosystem or environmental “health” or integrity. Flora and fauna provide a long-term outlook of water quality and quantity, habitat quality and other environmental conditions.

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Biotransformation A three phase process used to degrade a toxicant with a complex structure into a simpler form that can then be excreted naturally. Chronic - Multiple doses of toxicant or a prolonged exposure to a toxicant (usually 10% of test organism’s life span), whereby sub-lethal endpoints are measured such as growth or reproduction. Circuli - The concentric ring or polygon that is found on scales, often referred to as growth rings, which are dependent on seasonal changes in growth. Eutrophication - Excessive richness in nutrients in a water body caused by surface runoff from the land, and in turn causing dense growth of plant life. This can potentially deplete the oxygen supply available and, in the end, can lead to the death of various levels of life. Histology - The study of tissues. Inert - An environmental or organism partition where toxicants can be present or stored in a place where it has no toxic effect. In situ - In its original place, or in position. Intersex - Characterised by simultaneous presence of male and female gonadal tissue in the same gonad of individual gonochoristic organisms. In vivo - Taking place in living organism. Gonochoristic - Possessing only one set of reproductive apparatus of at least two distinct sexes in any individual organism i.e. male and female, as opposed to hermaphroditic. Lotic systems - A flowing body of freshwater such as a stream or river. Necropsy - (‘Necro’ Gr. dead, ‘opsis’ Gr. study) A critical analysis and/or inspection through dissection of an animal body after death. Pathology - The study of disease. Sentinel Species - A species whose presence, absence or relative well-being in a given environment is indicative of the health of its ecosystem, as a whole. Sequestration - A protective mechanism in animals that stores toxicants in inert tissues such as fat, teeth, hair and horns, which removes them from circulation and reduces their toxicity. Significant Risk - A hazard quotient greater than 1.0 (HQ≥1) for a toxic assessment and less than 1 in 100 000 people for a carcinogenic assessment. Synergistic - Interaction between two or more components to produce a combined effect greater than the sum of their separate effects. Teleost - Member of Teleostei or Teleostomi, a large group of fishes with bony skeletons, including most common fishes. They are distinct from the cartilaginous fishes such as sharks, rays, and skates. Toxicant - A toxic substance created or introduced into the environment by human beings e.g. pesticides and other pollutants.

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LIST OF TABLES

Table 3 - 1: Description of potential macroscopic alterations observed during the necropsy- based health assessment using Adams et al. (1993) as a guide...... 36 Table 3 - 2: Diagnostic histological features used to define developmental stages for testis (male) and ovaries (female) in fish. (McDonald et al., 2000) ...... 42 Table 3 - 3: Score values (SV) used to quantify the extent of severity of histological alterations observed...... 43 Table 3 - 4: Importance factors associated to each histological alteration (Bernet et al., 1999)...... 43 Table 3 - 5: Classification system by Zimmerli et al. (2007) adapted from Van Dyk et al. (2009b)...... 44 Table 4 - 1: Mean physico-chemical water parameters measured morning, noon and afternoon at each impoundment. Mean values in Red exceed the target range/objective (DWA, 2011ba; DWAF, 1996b)...... 51 Table 4 - 2: Incidence of selected organic toxicants detected in pooled water and sediment samples. Values that exceed the target/objective range are indicated in Red...... 53 Table 4 - 3: Incidence of selected metals detected in pooled water and sediment samples. Values that exceed the target/objective (Tar.) range are indicated in Red...... 53 Table 4 - 4: General characteristics of specimens collected per site per species...... 54 Table 4 - 5: Mean qualitative assessment of the gill alterations in both species at all sites...... 61 Table 4 - 6: Mean qualitative assessment of the liver alterations in both species at all sites...... 64 Table 4 - 7: Mean qualitative assessment of the kidney alterations in both species at all sites...... 67 Table 4 - 8: Mean qualitative assessment of the heart alterations in both species at all sites...... 70 Table 4 - 9: Percentage prevalence of each developmental stage present within the sample group...... 73 Table 4 - 10: Estimated ages (years) of each C. gariepinus, estimated using otoliths...... 79 Table 4 - 11: Estimated ages (years) of each C. carpio, estimated using scales...... 80 Table 4 - 12: Mean concentration of selected toxicants detected in pooled muscle samples across both species. Highest values between impoundments are indicated by Red...... 81 Table 4 - 13: Highest concentrations of selected toxicants detected in pooled muscle samples per species. Highest values between impoundments are indicated by Red...... 82 Table 4 - 14: Oral reference doses (RfD) and slope factors (β) sourced for the human health risk assessment. Refer to Chapter 2 – Literature Review for source...... 83

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Table 4 - 15: Highest and mean potential health risk through consumption of contaminated fish per species and between species. Significant toxic/cancer risks are indicated by Red...... 84 Table 4 - 16: Characterisation of toxicants with a significant risk...... 85 Table 4 - 17: Highest and mean overall toxic risk through consumption of contaminated fish per species and between species. Highest values between impoundments are indicated by Red...... 85 Table 4 - 18: Characterisation of other toxicants contributing to a high overall toxic risk. 86 Table A - 1: Physico-chemical water parameters recorded on site at Roodekopjes Dam. . 137 Table A - 2: Physico-chemical water parameters recorded on site at Vaalkop Dam...... 138 Table A - 3: Physico-chemical water parameters recorded on site at Marico-Bosveld Dam...... 139 Table A - 4: Incidence of organic toxicants measured in pooled water & sediment samples, from the outlets and inlets of each impoundment...... 140 Table A - 5: Inorganic toxicants found in the pooled water samples, from the outlets and inlets of each impoundment...... 141 Table A - 6: Raw data for the calculation of the haematocrit and leukocrit of each individual Clarias gariepinus at each assessment site...... 142 Table A - 7: Raw data for the calculation of the haematocrit and leukocrit of each individual Cyprinus carpio at each assessment site...... 143 Table A - 8: Body mass and total lengths are used to calculate the condition factor (CF) for each individual Clarias gariepinus, as per site...... 144 Table A - 9: Body mass and total lengths are used to calculate the condition factor (CF) for each individual Cyprinus carpio, as per site...... 145 Table A - 10: Hepatosomatic Index (HSI), calculated from body mass and liver mass, from Clarias gariepinus at each site...... 146 Table A - 11: Hepatosomatic Index (HSI), calculated from body mass and liver mass, from Cyprinus carpio at each site...... 147 Table A - 12: Splenosomatic Index (SSI), calculated from body mass and spleen mass, from Clarias gariepinus at each site...... 148 Table A - 13: Splenosomatic Index (SSI), calculated from body mass and spleen mass, from Cyprinus carpio at each site...... 149 Table A - 14: Male gonadosomatic index (GSI), calculated only from body mass and combined testes mass, from Clarias gariepinus...... 150 Table A - 15: Male gonadosomatic index (GSI), calculated only from body mass and combined testes mass, from Cyprinus carpio...... 151 Table A - 16: Female gonadosomatic index (GSI), calculated only from body mass and ovary mass, from Clarias gariepinus...... 152

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Table A - 17: Female gonadosomatic index (GSI), calculated only from body mass and ovary mass, from Cyprinus carpio...... 153

Table A - 18: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. gariepinus at Roodekopjes Dam...... 154

Table A - 19: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. gariepinus at Vaalkop Dam ...... 155

Table A - 20: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. gariepinus at Marico-Bosveld Dam...... 155

Table A - 21: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. carpio at Roodekopjes Dam...... 156

Table A - 22: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. carpio at Vaalkop Dam...... 156

Table A - 23: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. carpio at Marico- Bosveld Dam...... 157 Table A - 24: Ageing counts and calculated mode of each C. gariepinus at each impoundment...... 158 Table A - 25: Ageing counts and calculated mode of each C. carpio at each impoundment...... 158 Table A - 26: Incidence of EDCs measured in grouped muscle samples, evidence of bioaccumulation...... 159 Table A - 27: Incidence of metals/elements measured in grouped muscle samples from Roodekopjes Dam...... 160 Table A - 28: Incidence of metals/elements measured in grouped muscle samples from Vaalkop Dam...... 161 Table A - 29: Incidence of metals/elements measured in grouped muscle samples from Marico-Bosveld Dam...... 162 Table A – 30: Mean potential human health risk for consumption of C. gariepinus and/or C. carpio at Roodekopjes Dam...... 163 Table A - 31: Mean potential human health risk for consumption of C. gariepinus and/or C. carpio at Vaalkop Dam...... 164 Table A - 32: Mean potential human health risk for consumption of C. gariepinus and/or C. carpio at Marico-Bosveld Dam...... 165 Table A - 33: Highest potential human health risk for consumption of C. gariepinus at Roodekopjes Dam...... 166 Table A - 34: Highest potential human health risk for consumption of C. gariepinus at Vaalkop Dam...... 167 Table A - 35: Highest potential human health risk for consumption of C. gariepinus at Marico-Bosveld Dam...... 168

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Table A - 36: Highest potential human health risk for consumption of C. carpio at Roodekopjes Dam...... 169 Table A - 37: Highest potential human health risk for consumption of C. carpio at Vaalkop Dam...... 170 Table A - 38: Highest potential human health risk for consumption of C. carpio at Marico- Bosveld Dam...... 171 Table A - 39: Breakdown of alterations and associated importance factors (IFs) for each of the six assessed organs, as per reaction pattern...... 172 Table A - 40: List of PCB’s and pesticides screened for in the muscle samples, at FDA Labs...... 176

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LIST OF FIGURES

Figure 1 - 1: Diagrammatic overview of the study approach...... 6 Figure 2 - 1: Schematic diagram illustrating the risk assessment process (Heath et al., 2004)...... 27 Figure 3 - 1: (A) Overview of southern Africa, showing position of the North West Province (orange dashed box) (©SA-Venues.com). (B) An exploded view of the Bojanala Platinum Region (green shaded area), showing the selected sites (purple boxes) and the selected reference site (brown box) (©Linx.co.za)...... 30 Figure 3 - 2: Map of Roodekopjes Dam (RD) showing the sampling and measurement locations...... 32 Figure 3 - 3 Map of Vaalkop Dam (VD) showing the sampling and measurement locations...... 32 Figure 3 - 4: Map of Marico-Bosveld Dam (MBD) showing sampling and measurement locations...... 33 Figure 3 - 5: Photos demonstrating sampling during field surveys. (A) Measuring in situ physico-chemical water parameters, (B) water sample collection for chemical analysis, (C, D, E, F) gill netting and specimen sample collection, (G) weighing individual specimen, (H) mobile field laboratory for necropsy and assessment, and (I) late-evening sampling effort...... 34 Figure 3 - 6: The endemic Clarias gariepinus (Sharptooth Catfish) (A) and exotic Cyprinus carpio (Common Carp) (B) selected for the study...... 35 Figure 3 - 7: Some of the selected target organs, namely (A) the gill, (B) the liver, (C, D) the testis from C. gariepinus and C. carpio, respectively, (E) the bi-lobed kidney of the C. carpio, (F) the two-chamber heart, and (G) the ovary from C. carpio...... 39 Figure 3 - 8: Schematic process for the calculation of indices in the quantitative histological assessment, adapted from Bernet et al. (1999)...... 43 Figure 3 - 9: Counting the number of annuli on an otolith for age estimation...... 46 Figure 4 - 1: Substantial amounts of (A) Water Hyacinth (Eichhornia crassipes) and (B) green algae were observed at Roodekopjes Dam. (C) A dead fish was also blown to the shore together with foam and scum. (D) Clear water was observed on the shore of the reference site – Marico-Bosveld Dam...... 52 Figure 4 - 2: Abnormalities during necropsy included a (A) slightly discoloured appearance of the gills, the (B) presence of a cyst between gill filaments, (C) livers with fatty deposits and/or nodules and (D) livers that were vastly discoloured...... 55 Figure 4 - 3: Severe prevalence of nematode parasites in C. gariepinus at Vaalkop Dam (VD)...... 56

xvii M. Sc. – 2013

Figure 4 - 4: Mean (A) haematocrit and (B) leukocrit values from each site per species in relation to normal range (grey bars). Italic letters denote statistical differences: same letter = no significant difference...... 56 Figure 4 - 5: Graph to show difference in condition factor (CF) between fish species and sites. Italic letters denote statistical differences: same letter = no significant difference. 57 Figure 4 - 6: Mean organo-somatic indices calculated for both species per site: (A) Hepatosomatic index (HSI), (B) Splenosomatic index (SSI), and Gonadosomatic index for (C) male and (D) female. Italic letters denote statistical differences: same letter = no significant difference...... 58 Figure 4 - 7: Histological alterations in the gills ...... 62 Figure 4 - 8: Histological alterations in the liver ...... 65 Figure 4 - 9: Histological alterations in the kidney ...... 68 Figure 4 - 10: Histological alterations in the heart ...... 71 Figure 4 - 11: Histological alterations in the gonads (testes & ovaries) ...... 74 Figure 4 - 12: Histological alterations in the skin ...... 75

Figure 4 - 13: Graph to show differences in mean organ indices (Iorg) from quantitative assessment per species per impoundment: (A) gill index (Igill), (B) liver index (Iliver), (C) kidney index (Ikidney) & (D) heart index (Iheart). Italic letters denote statistical differences: same letter = no significant difference. Orange line denotes class category according to Van Dyk et al. (2009b)...... 76

Figure 4 - 14: Graph to show differences in mean total reaction pattern indices (ITot rp) from quantitative assessment per species per impoundment: (A) circulatory disturbance index

(Icd), (B) regressive changes index (Irc), (C) progressive changes index (Ipc), (D) inflammation index (Ii) & (E) focal cellular alterations index (Ifca). Italic letters denote statistical differences: same letter = no significant difference...... 77

Figure 4 - 15: Mean fish indices (Ifish) per species per impoundment. Italic letters denote statistical differences: same letter = no significant difference...... 79

xviii M. Sc. – 2013

ABSTRACT

Title: An assessment of the health status and edibility of fish from three impoundments in the North West Province, South Africa Author: B. M. Bester Supervisors: G. M. Wagenaar & J. C. Van Dyk Contact Details: Department of Zoology, University of Johannesburg, P. O. Box 524, Auckland Park, 2006, Johannesburg, Republic of South Africa [email protected] +27 11 559 2440 Keywords: Water quality, Fish health assessment, Fish edibility, Clarias gariepinus, Bojanala district, Human health risk, Cyprinus carpio, Carcinogenicity, Histopathology.

The Bojanala Platinum District (BPD) in North West Province (NWP) is a well-established mining and agricultural region of South Africa. These activities result in surface runoffs that are likely to pollute nearby freshwater impoundments, including the Roodekopjes (RD) and Vaalkop Dams (VD). These impoundments support subsistence fishing, where the fish caught, are often the sole source of dietary protein for local communities.

The aim of this study was two-fold: firstly, to assess the health status of the fish in these impoundments by (i) conducting a necropsy-based macroscopic evaluation, (ii) calculating appropriate biometric indices, and by (iii) performing a semi-quantitative histology-based fish health assessment (HBFHA) on selected target organs of two freshwater fish species, namely Clarias gariepinus (Sharptooth Catfish) and Cyprinus carpio (Common Carp). Secondly, the edibility (safe for human consumption) of these fish species was to be determined by (i) quantifying the bioaccumulation of selected organic and inorganic toxicants within the muscle of the fish collected and (ii) assessing the resultant potential health risk/s through consumption toward consumers of these fish.

In addition, in situ physico-chemical parameters were measured and samples of water and sediment were collected for laboratory analysis at each of the assessed impoundments. Otoliths and scales were also collected for age estimation. Tissue samples for histology were fixed in formalin (liver, kidney & heart) and Bouin’s (gills, gonads & skin) solution and processed for light microscopy analysis using standard histological techniques. Water, sediment and muscle samples were analysed for organic and inorganic toxicants by accredited laboratories using ICP-MS & ICP-OES. Results from the two assessment sites (RD & VD) were assessed against a reference site, the Marico-Bosveld Dam (MBD).

xix M. Sc. – 2013

The substrate analysis of each impoundment showed that the physico-chemical water quality was largely favourable to all life stages of aquatic organisms at all impoundments. However, given the hyper-eutrophic state of the assessment sites (RD & VD), the aquatic biota were expected to be under some stress from substantial diurnal fluctuations in pH and dissolved oxygen, as observed in other hyper-eutrophic systems. On the other hand, the chemical analysis documented the presence of numerous organic and inorganic toxicants in both the water and more so in the sediments of these systems. The high affinity of many of these toxicants to the ‘sinks’ of the aquatic ecosystem was a result of their molecular nature, as many of these toxicants eventually settle out of suspension and accumulate in the sediment of the impoundment. This could mean that all previous contaminants to the impoundment may be found trapped in some layer of the sediment. Hence, the potential danger to fish and other aquatic biota that feed within the sediments. They are likely to be directly exposed to these historic contaminants.

With regards to the health of C. gariepinus and C. carpio sample populations from each of the selected impoundments in this study, the necropsy-based macroscopic assessment indicated a naturally higher incidence of Contracaecum sp. larvae within the mesenteries of C. gariepinus compared to C. carpio. The gross body indices, including condition factor (CF) and selected organosomatic indices, helped to indicate the health condition and sexual maturity of collected specimens and potentially, their immunity levels with regards to their splenosomatic index (SSI). However, exposure studies that monitor changes in the SSI of fish were required to properly investigate the potential indicator strength of the SSI as an indicator of immunity. In conjunction with the age estimation and the histological staging of the gonads, it was further suggested that the male fish from this study become sexually mature at an earlier age than their female counterparts.

Furthermore, the microscopic HBFHA showed few to moderate structural alterations in four of the six target organs of both species at all sites, with regressive changes dominating the reaction pattern type. These types of alterations can be associated with a functional reduction, which is often a reversible alteration once the toxicant is neutralised, or loss of an organ in the cases of severe necrosis, which is largely irreversible. The overall health, as indicated by the fish index (Ifish), of the C. gariepinus population was determined to be lowest at the RD, whilst the C. carpio population was worst-affected at the MBD. These species differences were likely due to the increased biomagnification of toxicants in C. gariepinus through its piscivorous feeding habits, relative to the more herbivorous nature of C. carpio. Moreover, the increased biomagnification in C. gariepinus was likely to worse-affect the liver, kidney and heart, as shown by their respective organ indices, (Iliver,

Ikidney & Iheart). Conversely, the gill index (Igills) was observed to be worst-affected in the sample population of C. carpio at the reference site (MBD). This was potentially related to

xx M. Sc. – 2013 its sediment-modifying behaviour and the increased exposure to sediment-trapped toxicants.

Therefore, according to the significant difference determined between Ifish for C. carpio, the fish may have been significantly worse-affected by the toxicants trapped in the sediment at MBD relative to RD & VD. In contrast, the observed differences between impoundments shown for C. gariepinus were suspected only to indicate different levels of pollution, which indicated that RD was worse polluted of the impoundments assessed.

A significant toxic risk only exists when the average daily dose (ADD) for a specific toxicant exceeds the reference dose (RfD) for that specific toxicant, which can alternatively be expressed as a hazard quotient (HQ) greater than or equal to 1.0. It should be noted that RfDs have been conservatively estimated by applying numerous uncertainty factors and a number of safety buffer factors, and as a result the human health risk assessment (HHRA) process is perceived as a conservative estimate. Consequently, it was concluded that under worst-case scenario conditions, a slight toxic risk was posed through the consumption of either C. gariepinus from RD, VD and MBD or C. carpio from RD and VD as a result of the highest arsenic levels detected within each fish species. However, under realistic conditions, no significant toxic risk through arsenic exists at any of the sites through the consumption of either of the selected species, except at the MBD which marginally exceeded an HQ of 1.0. Alternatively, there were cumulative toxic risks posed from a combination of the detected toxicants present in the fish muscle in both species from each impoundment. However, synergistic and antagonistic interactions between the toxicants present were not determined and it is expected that any potential adverse effects from this ‘cocktail’ of toxicants can be controlled by eating less than 150 g of fish muscle per day for 350 days a year for 30 years.

Finally, with regards to the carcinogenic potential of the toxicants present in the fish, a cancer risk was evident due to the arsenic levels detected, as well as cumulatively with the carcinogenic effects of beryllium (Be) and β-HCH. The risk was defined that 1 in 5 000 fish consumers consuming only 150 g of fish per day over an extended period have a high probability of acquiring some form of cancer. Therefore, in order to avoid any carcinogenic effects, it is advised that consumers eat less than 150 g per day or eat the collected fish less frequently.

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1. PROJECT OVERVIEW

CHAPTER 1: IN THIS CHAPTER PROJECT OVERVIEW 1.1. INTRODUCTION ...... 2 1.2. STUDY MOTIVATION ...... 2 1.3. STUDY DIRECTIVE ...... 5 CHAPTER 2: 1.3.1. Aims and Objectives ...... 5 BACKGROUND 1.3.2. Hypothesis ...... 6 INFORMATION 1.3.3. Study Approach ...... 6 1.4. SELECTED SITES ...... 7 CHAPTER 3: 1.4.1. Roodekopjes Dam (RD) ...... 7 MATERIALS & 1.4.2. Vaalkop Dam (VD) ...... 7 METHODS 1.4.3. Marico-Bosveld Dam (MBD) ...... 8 1.5. SELECTED SPECIES ...... 8 1.5.1. Clarias gariepinus ...... 9 CHAPTER 4: 1.5.2. Cyprinus carpio ...... 9 RESULTS 1.6. SELECTED TARGET ORGANS...... 9 1.6.1. Gills ...... 10 1.6.2. Liver ...... 10 CHAPTER 5: 1.6.3. Kidney ...... 10 DISCUSSION & 1.6.4. Heart ...... 10 CONCLUSION 1.6.5. Gonads ...... 11 1.6.6. Skin ...... 11

APPENDICES

Ephesians 3: 20 – “Now unto him that is able to do exceedingly abundantly above all that we ask or think, according to the power that worketh in us.” CHAPTER 1 – PROJECT OVERVIEW

1.1. INTRODUCTION

Water’s movement down the landscape from source to sea used to be a natural and unhindered journey driven by gravity and topography (Allanson, 1995; Whitfield, 2009). Through this journey of unidirectional flow along the downward gradient of the landscape, an incredibly diverse and unique longitudinal ecosystem called the riverine ecosystem was formed (Allanson, 1995; Dudgeon et al., 2006; Darwall et al., 2009; Ollis et al., 2013). Despite general flow-driven changes along its longitudinal continuum, it was inherently a well- buffered system capable of maintaining a balanced water quality and a rich associated biodiversity (Allanson, 1995; Dallas & Day, 2004). However, due to the unique properties of water, which are exploited for the necessity of human beings and the intimate contact these systems have with their surrounding catchment, the innate buffering function of these systems has largely been impaired and its effectiveness has diminished (Allan & Flecker, 1993; Dallas & Day, 2004). Consequently, these lotic (or flowing) systems are possibly the most endangered and most impacted ecosystems in the world today (Sala et al., 2000; Malmqvist & Rundle, 2002).

As a consequence of the scarceness of water in South Africa and as a result of its population’s extensive reliance on rivers and aquifers for basic human needs, the importance of preserving these limited resources was realised (Allanson, 1995; Dallas & Day, 2004) and the revolutionary National Water Act (NWA) [Act No.36 of 1998] was written into law. This act aimed to ensure the sustainability of the nation’s water resources in the interests of all water users, by setting aside a ‘reserve’ of water within each significant water resource or catchment (Government Gazette of South Africa, 1998). This ‘reserve’ is the quantity and quality of the water required (i) to satisfy basic human needs and (ii) to protect aquatic ecosystems (Dallas & Day, 2004). Therefore, it is implied that a poor quality and/or a lack of sufficient water can adversely affect both human health and the state of affected aquatic ecosystems.

This study explores the present state of selected systems in the North West Province (NWP) by examining the condition of the associated fish populations and potential health risk/s to consumers of these fish in the communities surrounding these systems.

1.2. STUDY MOTIVATION

Aquatic ecosystems are known to be very susceptible to pollution, as the rivers essentially act as ‘drains’ for the landscape and the impoundments and/or wetlands act as ‘sinks.’ Materials brought by wind, water and humans from all surrounding land use activities

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CHAPTER 1 – PROJECT OVERVIEW accumulate within these systems, especially within impoundments and wetlands (Dallas & Day, 2004). The Bojanala Platinum District (BPD) in the NWP, in which the study was carried out, supports many of these anthropogenic activities including substantial mining and agriculture (Urban-Econ, 2007; NWDACE, 2008). Therefore, the potential presence of toxicants originating from these activities are likely to affect the integrity of the surrounding freshwater environments such as Roodekopjes Dam (RD) and Vaalkop Dam (VD), as well as the fauna therein (van der Oost et al., 2003; Dallas & Day, 2004; Bornman et al., 2010).

Toxicants in polluted aquatic systems, if bioavailable are bioaccumulated by aquatic biota through many mechanisms including bioconcentration, via the gills or skin, and biomagnification, via ingestion of contaminated food (van der Oost et al., 2003; Heath et al., 2004; Cunningham & Cunningham, 2012). Of these aquatic biota, fish have a relatively long life span and they are biologically sensitive to changes in their environment (Harris, 1995). Fish are exposed to the surrounding water and its constituents including dissolved toxicants. This allows for a continuous uptake of potential toxicants throughout their life span (Harris, 1995; Streit, 1998). Assessing their general health condition has been shown to reflect the condition of the system they inhabit and represent a historical snapshot of potential changes within the system (Roberts, 1989; Kleynhans, 1999; DWAF & Rand Water, 2013).

Previous studies on Clarias gariepinus (Sharptooth Catfish) have shown histological alterations in the testes at the Hartbeespoort Dam (Wagenaar et al., 2012), as well as in the gills (Van Dyk et al., 2009a), the liver (Marchand et al., 2008b) and the gonads (Pieterse et al., 2010) at Rietvlei Dam in the Rietvlei Nature Reserve. These studies were particularly relevant to this study as the impoundments assessed (RD & VD) as well as the reference site, the Marico-Bosveld Dam (MBD) occur within the same catchment area and similar impacts were expected. Furthermore, intersex was als0 found in the estrogen-polluted Rietvlei and Marais dams and was suspected to be as a result of elevated levels of p- nonylphenol (p-NP) detected in the water and sediment (Barnhoorm et al., 2004). Finally, a baseline study by Marchand et al. (2012) on Oreochromis mossambicus (Mozambique Tilapia) and C. gariepinus at Roodeplaat Dam within the Roodeplaat Nature Reserve showed histological alterations in various target organs (gills, liver, kidney, heart and gonads) of both species, but especially in the liver. The health of these fish within the abovementioned impoundments were a cause for concern and the rationale behind this study was to determine if these concerns were widespread within other fish populations, namely C. gariepinus and Cyprinus carpio (Common Carp) from other nearby impoundments.

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CHAPTER 1 – PROJECT OVERVIEW

Many impoundments in the NWP serve as recreational tourist attractions to both local and foreign tourists (Freeman et al., 1996). However, the North West Department of Agriculture, Conservation and Environment (NWDACE) has recognised that high volumes of sewage and high nutrient run-off lead to eutrophication in these ecosystems, which can cause odours, fish kills and potential human health risks (NWDACE, 2008). Although eutrophication was likely to be a contributing factor to the adverse effects observed within the fish from the abovementioned studies and in other nearby impoundments, it is more likely that the fish are affected by a complex mixture of toxicants and contaminants, as well as indirect effects from changes in the aquatic environment such as eutrophication (van der Oost et al., 2003; Barnhoorn et al., 2013).

On the other hand, fish is a primary protein source for 21% of the population in Africa (Zabik et al., 1995; Jackson, 2009). Therefore, a further cause for potential concern was the potential health risks through consumption of the fish caught within these systems, as the bioaccumulation of toxicants as a result of the surrounding pollution may potentially pose adverse health risks to their consumers. Most communities with access to freshwater systems practice recreational fishing especially within the NWP, but these fishermen most often fish for sport and seldom consume their catch (Bevelhimer, 1995; Weyl et al., 2007). However, driven by the socio-economic state of the province, subsistence fishing is known to be an important source of dietary protein in areas where communities are vulnerable to food shortages (Rouhani, 2004; NWDACE, 2008; Weyl et al., 2007).

The provincial government has further recognized the need for developing freshwater fisheries in the NWP due to its severe underutilisation, given the diversity and vast number of impoundments within the province (Weyl et al., 2007). It has been recognized as an alternative to traditional methods of securing food and economic independence within rural communities (Walmsley et al., 2002; Weyl et al., 2007). Therefore, the safe consumption (no significant health risk) of fish with from these impoundments may soon become a necessity and not only feed some subsistence fisherman and their families, but protect their livelihoods and potential economic independence.

Numerous studies have assessed the presence of elevated levels of mercury in human cohorts or communities, as a result of eating wild and/or canned fish and associated products (Harnly, et al., 1997; Xue et al., 2007). However, an alternative approach to investigating and defining health risks related to the consumption of fish have been applied through human health risk assessment (HHRA) models (MacIntosh et al., 1996; Heath & Claassen, 1999; NJDEP, 2002; Burger et al., 2005; Ginsberg & Toal, 2009; Cardoso et al., 2010b; Ruelas-Izunza et al., 2011; Dai et al., 2011). This alternative method was the preferred method used for the current study as the toxicant concentrations found within

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CHAPTER 1 – PROJECT OVERVIEW aquatic biota, such as fish was the only major input required to apply these HHRA models. The outcome from these models was also an accurate and conservative health risk or probability/likelihood toward the consumers of these fish acquiring disease and/or cancer from consuming these fish (Heath et al., 2004).

A pilot study on the Rietvlei Dam, by Barnhoorn et al. (2013), found a number of potentially damaging toxicants in the fat and muscle of fish, but determined that the indicator species C. gariepinus was safe for human consumption due to low concentrations. However, another HHRA performed at the Roodeplaat Dam showed that the fish were unsafe for human consumption due to high concentrations of potentially dangerous toxicants (Marchand, 2009). Consequently, it was concluded that these fish had the potential to be adversely affected by toxicants present in the impoundments and the determination of potential health risks to these realistic scenarios was required for health reasons.

1.3. STUDY DIRECTIVE 1.3.1. AIMS AND OBJECTIVES

This study aimed to assess the health status of C. gariepinus and C. carpio at the RD and VD, and to compare the condition of these fish populations with the same species at a reference site, the MBD. Furthermore, the study also proposed calculating the risk of human beings contracting cancer and/or disease from consuming these fish in the BPD, of the NWP.

In order to achieve these study aims, the following objectives were defined: (i) To measure selected in situ physico-chemical parameters of the water within each impoundment, (ii) To measure the levels of selected organic and inorganic toxicants present in the water and sediment at each impoundment, (iii) To conduct a necropsy-based macroscopic evaluation of the external and internal condition of fish collected, (iv) To calculate the appropriate biometric indices for the fish collected, specifically the condition factor and three organosomatic indices (hepato-, spleno-, and gonadosomatic indices), (v) To perform a semi-quantitative histology-based fish health assessment using six selected target organs (gills, liver, kidney, heart, gonads & skin) on representative samples of each fish species, (vi) To estimate the age of the fish collected using otoliths and scales, (vii) To quantify the bioaccumulation of particular toxicants in the muscle of the fish collected, and

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CHAPTER 1 – PROJECT OVERVIEW

(viii) To assess the potential human health risk/s through the consumption of these fish by the surrounding communities and/or subsistence fishermen.

1.3.2. HYPOTHESIS

The hypothesis for the study is formulated as follows:

“C. gariepinus and C. carpio from the assessment sites (RD & VD) will show adverse health effects at a macroscopic and histological level, whilst the consumption of muscle tissue of these fish will pose a potential health risk/s to the local people consuming these fish.”

1.3.3. STUDY APPROACH

Figure 1 – 1 summarises the approach to the study and outlines the process of each portion of the study, namely the substrate analysis, fish health assessment, age estimation and edibility assessment.

SUBSTRATE ANALYSIS FISH HEALTH ASSESSMENT

Water Sediment Necropsy Biometric Indices Histopathology

Chemical Chemical Macroscopic Condition Factor Organ Index (Iorg) •Organic •Organic Observation (CF) •Gills • Liver •External •Inorganic •Inorganic •Kidney • Heart •Internal Organosomatic •Gonads • Skin Physico-chemical Indices Total Reaction •Temperature Haematology •Liver (HSI) Pattern Index •pH •Hematocrit •Spleen (SSI) (I ) •Conductivity (Hct) •Gonad (GSI) Tot rp •Total Dissolved Solids (TDS) •Leukocrit •Testes Fish Index (I ) •Dissolved Oxygen (DO) (Lct) •Ovaries fish

AGE ESTIMATION EDIBILITY ASSESSMENT

Otoliths Scales Muscle Human Health Risk Analysis Assessment (HHRA)

Organic Toxic Risk •Pesticides Clarias gariepinus Cyprinus carpio •Hazard Quotient (HQ) (Sharptooth Catfish) (Common Carp) •EDC's

Inorganic Cancer Risk •Metals

Figure 1 - 1: Diagrammatic overview of the study approach.

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CHAPTER 1 – PROJECT OVERVIEW

1.4. SELECTED SITES

The Crocodile West – Marico WMA supports the largest proportionate contribution to the national economy, generating almost a third of the country’s Gross Domestic Product (GDP) (NWDACE, 2008). This statistic further highlights the high potential presence of toxicants in the BPD. Therefore, for the purposes of this study, two of the larger impoundments in the BPD (Weyl et al., 2007), namely the RD and VD were selected.

A pristine, unpolluted or unaffected reference site within South Africa is difficult, if not impossible to locate (Malmqvist & Rundle, 2002). However, for the purposes of good comparison, the MBD was selected as a reference site largely because it was located within the same Water Management Area (WMA) as the abovementioned assessment sites (RD & VD). Having each of the sites located within the same WMA hoped to limit variability and allow for a reliable comparison between all sites (Dallas & Day, 2004; RHP, 2005).

1.4.1. ROODEKOPJES DAM (RD)

This impoundment is situated 30 km north-west of the town of Brits, it covers an area of 1 571 ha and has a capacity of 102.3 million m3 with a mean depth of about 6.5 m (Weyl et al., 2007). The impoundment has two tributaries, namely the Crocodile River, which meanders through some densely populated areas into the Hartbeespoort Dam and then into the south east side of the impoundment, and the Sterkstroom, which is a smaller stream that winds through various settlement areas and enters the impoundment from the south west (RHP, 2005; MapStudio, 2011). It should be noted that the Crocodile River is known to be high in nutrients and to have an elevated salinity as a result of the agricultural and urban runoff from Johannesburg and Pretoria, whilst some mining impacts on the Sterkstroom River also exhibits high salinities (DWA, 2011a). The outlet from the RD remains the Crocodile River, and later converges with the outlet of the VD, which is shortly known as the Elands River. The RD was selected as an assessment site because the research would be of a novel nature and there would be a likely potential for adverse effects on fish and humans given the surrounding activities.

1.4.2. VAALKOP DAM (VD)

This impoundment is 54 km north of the town of Brits in the Vaalkop Nature Reserve, it covers an area of 1 111 ha and holds approximately 56 million m3 with an average depth of 5.0 m (Weyl et al., 2007). VD is recognized to exhibit fairly good water quality as a result of its current and known land uses which are classified largely as tribal areas. However, upstream activities are causing a substantial deterioration with occasional algal blooms

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CHAPTER 1 – PROJECT OVERVIEW occurring and in addition, there is an increasing mining interest in platinum and associated platina group metals within the catchment area. Any significant impacts at this impoundment are particularly important as Magalies Water treats the water and provides drinking water to the surrounding areas. The impoundment also has two main tributaries, namely the Hex River, which flows directly from the Bospoort Dam and is known to be substantially impacted upon by agriculture and mining. This had been likely to cause an increase in the nutrient load, salinity and turbidity. Secondly, the Elands River, which is suspected to originate from a cleaner source, as it exhibits good quality water in its upper reaches, but more recently has been recognised to be deteriorating as a result of slate mining within the catchment (RHP, 2005; DWA, 2011a). Extensive slate mining adds additional fine sediment to the system and increases the turbidity, which can in turn potentially affect the spawning success of some fish species (RHP, 2005). Similarly, the VD was also selected as an assessment site due to the novel nature of the study and the presence of similar upstream impacts.

1.4.3. MARICO-BOSVELD DAM (MBD)

The MBD is located directly north (approximately 12 km) of the town of Groot Marico, in the same eco-region and WMA as the assessment sites (RD & VD) (Kleynhans et al., 2005). It was the smallest of the three assessed sites, only covering an area of 438 ha and estimated to hold approximately 27.8 million m3. A single inlet tributary called the Groot Marico River fills the impoundment through a number of natural springs within the Groot Marico dolomite aquifer compartment, such as the Molemane Eye and the Marico Eye (RHP, 2005; NWDACE, 2008). It has a short, small, sparsely populated catchment area where significant agricultural activities are limited by the geology and geographical land available for cultivating and as such, potential impacts on the system are limited. However, it should be mentioned that localised active agricultural activities are known to occur in the adjacent and surrounding areas causing slightly elevated nutrient loads, which is possibly the most significant impact to the impoundment (DWA, 2011a).

1.5. SELECTED SPECIES

According to the study by Weyl et al. (2007), the species composition within 10 dams in the NWP, ranging in size from 35 – 1571 ha, which was dominated by C. gariepinus, O. mossambicus and C. carpio. Therefore, for the purposes of this study C. gariepinus and C. carpio were selected for the fish health and edibility assessment, since they were most likely to be well-established in RD, VD and MBD (Skelton, 2001; Weyl et al., 2007).

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CHAPTER 1 – PROJECT OVERVIEW

1.5.1. Clarias gariepinus

The endemic C. gariepinus is an important angling and food fish species, it is also probably the most widely distributed fish in Africa (Skelton, 2001; Darwall et al., 2009). It occurs in almost any habitat and it can endure harsh conditions such as high turbidity or desiccation, which often makes it the last or the only inhabitant of diminishing pools of drying rivers or lakes (Skelton, 2001). A specific adaptation that attributes to its hardiness is its ability to crawl over land in damp conditions, with the aid of its long pectoral spines, as well as the presence of an accessory respiratory organ (Skelton, 2001). It is also completely omnivorous, in that it will prey, scavenge or grub on any available organic food source (Skelton, 2001).

The species was selected as one of the bioindicator species because it has been widely used as an indicator organism in previous studies (Barnhoorn, et al., 2004; Marchand et al., 2008b; Van Dyk & Pieterse, 2008; Van Dyk et al., 2009a; Van Dyk et al., 2009b; Abalaka et al., 2010; Pieterse et al., 2010; Van Dyk et al., 2012; Marchand et al., 2012; Mokae et al., 2013) and there is baseline histological data from a laboratory-bred population for determination of histological alterations (Van Dyk, 2006; Van Dyk & Pieterse, 2008).

1.5.2. Cyprinus carpio

The exotic C. carpio, which is naturally endemic to Asia and Europe, has become very well established and widely distributed in the warmer tropical areas throughout southern Africa since its introduction in the 1700’s (Skelton, 2001, Darwall et al., 2009). It is also an omnivorous, hardy, tolerant species that can survive in a wide variety of conditions taking in a wide range of plant and animal matter by destructively grubbing within the sediments (Skelton, 2001). Finally, C. carpio is recognised around the world as a valued aquaculture and angling species and to local communities, a form of dietary protein (Skelton, 2001).

This species was selected as the second bioindicator species because it was comparable with similar studies at the nearby Hartbeespoort Dam (DWAF & Rand Water, 2013) and also at other sites around the world, such as Turkey (Ayas et al., 2007).

1.6. SELECTED TARGET ORGANS

Numerous target organs have previously been selected for histopathological assessments on fish. For the purposes of this study, six target organs were selected per individual specimen collected. These target organs were recognised to be good indicators of existing

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CHAPTER 1 – PROJECT OVERVIEW pollution and significant pathology was likely to be evident in severe cases (Bernet et al., 1999; Van Dyk et al., 2009a; Van Dyk et al., 2012; Marchand et al., 2012).

1.6.1. GILLS

The gills are the primary respiratory organ in fish and specially adapted to facilitate gaseous exchange, as well as regulate osmotic concentration and excrete nitrogenous wastes (Schmidt-Nielsen, 1997). Due to the constant contact with the aquatic medium and potential presence of toxicants, this organ is known to be a sensitive indicator of environmental stress especially in systems impacted by human activities (Mumford et al., 2007; Van Dyk et al., 2009a).

1.6.2. LIVER

A large organ in fish and within some species such as C. carpio, it exists as a compound organ in the form of a hepatopancreas. It plays key roles in metabolism, detoxification, excretion, digestion and storage (Mader, 2007). Although only 10% of the liver tissue is required to maintain normal liver function, the liver remains very susceptible to damage from toxicant exposure, since the system metabolises substances that are toxic or become toxic through bioactivation (Mumford et al., 2007; Trujillo-Jimenez et al., 2011). Liver histopathology has been proven to indicate exposure to environmental pollution with hepatocytes showing toxicant-induced histological alterations (Hinton & Laurén, 1990; Marchand et al., 2008b; Van Dyk et al., 2012).

1.6.3. KIDNEY

A mixed organ comprised of hematopoietic, reticulo-endothelial, endocrine and excretory elements (Camargo & Martinez, 2007). Its primary function is not excretion of nitrogenous waste, as in mammals, but rather that of osmotic regulation of water and salts (Mumford et al., 2007). Kidneys consist of a large number of functional units called nephrons, which actively excrete waste to accommodate higher glomerular filtration and re-absorption to limit any losses of salts within fish (Schmidt-Nielsen, 1997). The kidney has also been proven to be indicative of pollution, especially heavy metals (Borges et al., 2003; Bernet et al., 2004; Camargo & Martinez, 2007).

1.6.4. HEART

The heart is a vital organ that is responsible for circulation and it is the central hub for the main transport system. It consists of two chambers in series, namely an atrium with a sinus

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CHAPTER 1 – PROJECT OVERVIEW venosus and a ventricle with a bulbous arteriosus (Schmidt-Nielsen, 1997). The heart is not well regarded as a target organ for exposure to toxicants. However there are at least two previously recorded alterations found within the histology of previous literature, especially with regard to metal pollution, namely muscular necrosis and inflammation (Poppe & Taksdal, 2000; Borges et al., 2003).

1.6.5. GONADS

Gonads are the primary sex organs that produce germ cells for reproduction: the male gonad produces sperm and is called the testis, whereas the female gonad forms eggs and is referred to as the ovary (Hickman et al., 2006). Sexually mature specimens can easily be affected by increased sequestration of toxicants within the inert gonadal tissue. These may lead to alterations within the (i) actual germ cells, which will affect the success of future generations of the species, or the (ii) actual organs directly, which may later lead to alterations within the germ cells (Elskus, 2001; Pieterse et al., 2010). Endocrine Disrupting Chemicals (EDCs) can also severely and directly affect the gonads through the feminisation of fish, a form of intersex. Other effects include skewed sex ratios and gonadal malformations (Van Dyk & Pieterse, 2008; Van Vuuren, 2008).

1.6.6. SKIN

The skin of fish provides physical protection and serves as an impermeable wall between the body fluids and the external medium. Mucous is often secreted in order to provide a smooth, friction-reducing layer, for protection against diseases and parasites, and for initial healing of wounds (Skelton, 2001). Therefore, the skin of fish is in permanent contact with the aquatic surrounding and is often the first level of defence against toxicants. However, it is also largely responsible for the absorption and excretion of important compounds, which can allow toxicants such as dermatotoxins to mimic the action and shape of important compounds to enter through the skin/scale barrier (Mader, 2007; Cunningham & Cunningham, 2012). The most common alteration observed in similar studies is leukocyte infiltration and mucous cell hyperplasia (Bernet et al., 2004; Abalaka et al., 2010).

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2. BACKGROUND INFORMATION

IN THIS CHAPTER CHAPTER 1: PROJECT 2.1. STATE OF AQUATIC ECOSYSTEMS ...... 13 OVERVIEW 2.1.1. Current Concerns ...... 13 2.2. STUDY AREA ...... 14 CHAPTER 2: 2.2.1. Location ...... 15 BACKGROUND 2.2.2. Catchment-specific Impacts...... 15 INFORMATION 2.2.3. Socio-economic Climate ...... 16 2.3. SUBSTRATE ANALYSIS ...... 16 2.3.1. Physico-chemical Parameters ...... 16 CHAPTER 3: 2.3.2. Chemical Analysis ...... 18 MATERIALS & 2.4. SURVIVAL MECHANISMS OF FISH ...... 20 METHODS 2.4.1. Fish as a Bioindicator Species ...... 21 2.4.2. Histopathology as a Biomarker ...... 22 CHAPTER 4: 2.5. NECROPSY-BASED HEALTH ASSESSMENT ...... 22 2.5.1. Haematological Assessment ...... 23 RESULTS 2.6. BIOMETRIC INDICES ...... 23 2.6.1. Condition Factor ...... 23 2.6.2. Organo-somatic Indices ...... 23 CHAPTER 5: 2.7. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 24 DISCUSSION & ONCLUSION 2.8. AGE ESTIMATION ...... 25 C 2.8.1. Otoliths...... 25 2.8.2. Scales ...... 25

2.9. EDIBILITY ...... 25 APPENDICES 2.9.1. Muscle Analysis ...... 26 2.9.2. Human Health Risk Assessment (HHRA) ...... 26

1 Peter 1: 8 – Whom having not seen, ye love; in whom, though now ye see him not, yet believing, ye rejoice with joy unspeakable and full of glory. CHAPTER 2 – BACKGROUND INFORMATION

2.1. STATE OF AQUATIC ECOSYSTEMS

According to a review of published literature, South Africa’s aquatic ecosystems (oceans, lakes, dams, pans, wetlands, estuaries, rivers and groundwater or karst systems) are in a severely polluted state (Heath et al., 2004). This can be linked directly to: (i) point source discharges, which include sewage discharges from treatment works, direct industrial effluent or decant/overflow/spillage from holding tanks with potential toxicants into aquatic systems (Morrison et al., 2001), and (ii) surface runoff, which drains the surrounding land surface after rains and dissolves numerous solutes before entering aquatic watercourses. As a result, there are almost no pristine or unpolluted water bodies left South Africa (Malmqvist & Rundle, 2002).

2.1.1. CURRENT CONCERNS

Although the National Water Act (NWA) and associated policies aims to satisfy the country’s water supply to users, numerous current well-known issues of concern have arisen such as increased eutrophication, increased salinisation, and changes to ecological habitat including water quality and the prevalence of EDCs and carcinogens (Walmsley, 2000; Marchand, 2008; Oberholster & Ashton, 2008).

2.1.1.1. Eutrophication In recent years, South African impoundments have been noted as the most eutrophic systems in the world (Walmsley, 2000). Eutrophication is an increase in nutrient levels and biological productivity. Cases can result from a number of different factors, from higher temperatures to more sunlight reaching the water surface. However, most often, extreme cases result from elevated phosphorus and nitrogen levels that stimulate ‘blooms’ of algae and cyanobacteria (blue green algae) or thick growths of aquatic plants. These elevated levels of nutrients can occur suddenly and cause almost-instant ‘blooms’ after heavy rain passes collecting fertilizer runoff or sewage decant. The process can also often be much slower as nutrients can accumulate over time from various tributaries, and resulting algae and aquatic vegetation gradually grow in abundance. In addition, bacterial populations increase due to greater abundance of organic matter for feeding. Over a sustained period, as plants and algae die and sink to the bottom of the system, these bacterial decomposers can deplete available oxygen, which sometimes causes unfavourable in the ecosystem (Walmsley, 2000; Oberholster & Ashton, 2008; Cunningham & Cunningham, 2012).

Furthermore, many of these cyanobacteria species produce severe and potent toxins called dermatotoxins, neurotoxins and hepatotoxins, each of which has the capacity to kill within minutes to a couple of days or weeks. These toxins within the infected water

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CHAPTER 2 – BACKGROUND INFORMATION systems can also be potentially detrimental to the fauna using the water (Fleming & Stephan, 2001; Gupta et al., 1998).

2.1.1.2. Endocrine Disrupting Chemicals (EDCs) Endocrine Disrupting Chemicals represent a diverse range of man-made chemicals discharged into the environment that mimic or antagonize the function of hormones (Bornman et al., 2010). Numerous EDCs are commonly used on a day-to-day basis in households, in the form of pharmaceuticals, chemicals and natural hormones including estrogens, as well as in agriculture, in the form of pesticide or as a constituent of livestock feeds. These chemicals contaminate aquatic watercourses through sewage effluent, decant, or runoffs from livestock feedlots. The chemicals can be dangerous as they interact with physiological systems and the implications thereof have been proven to skew sex ratios, reduce biodiversity and cause gonadal malformations, which ultimately compromises development, growth and reproduction in wildlife, and especially fish (Van Vuuren, 2008; Bornman et al., 2009; Bornman et al., 2010).

Furthermore, the condition of intersex in naturally gonochoristic species is defined as the presence of both male and female reproductive features within the same individual. Often, its occurrence is a result of embryonic exposure to EDCs (Barnhoorn et al., 2010). Recently, a couple of individuals with the condition were found at the nearby Rietvlei Dam (Barnhoorn et al., 2004).

2.1.1.3. Cancer According to the Registry of Tumours in Lower Animals, there have been 4000 specimens of cancer in fish, amphibians, reptiles and invertebrates collected by the Smithsonian and the National Cancer Institute (Armstrong et al., 2008). More specifically, there have been a number of liver cancer epidemics in over 16 species of fish in 25 different polluted freshwater and saltwater locations around the world (Armstrong et al., 2008).

There has been recent evidence of testicular growths (Wagenaar et al., 2012), ovarian growths (Marchand 2008; Pieterse et al., 2008; Mooney, 2013) and liver neoplasia (Van Dyk et al. 2012) from sites within the same catchment area of this study, such as Rietvlei Dam (Marchand et al., 2008b), Roodeplaat Dam (Marchand et al., 2012) and Hartbeespoort Dam (Wagenaar et al., 2012; DWAF & Rand Water, 2013).

2.2. STUDY AREA

Howard et al. (2002) listed 28 major dams within the province which have a combined surface area of more than 11 000 ha. These are used primarily for the maintenance of water

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CHAPTER 2 – BACKGROUND INFORMATION supply to the human population and for irrigation of crops. These dams have a considerable tourism value to the rich Gauteng Province, which is in close proximity, particularly in the areas that have formed nature reserves or game parks around them, as well as the additional recreational value that goes together with most water bodies and its surrounding environment, such as boating, bird watching and recreational angling (Howard et al., 2002; Weyl et al., 2007; NWP&TB, 2012).

There are numerous impoundments located within the BPD, but in order to assess the state of the selected sites, it is beneficial to understand the surrounding area and associated impacts at other nearby impoundments. Furthermore, in an effort to better understand the motivation for the study the current socio-economic status in the region is further explored. The following will briefly describe the location of the study region and associated socio-economic conditions that are potentially forcing communities to use these resources.

2.2.1. LOCATION

The NWP of South Africa is situated north of the country between the Northern Cape and the Limpopo Province, it is also bordered by Botswana to the north. It is the sixth largest province of the country and covers 129 821 km2, which is about 11% of total surface area of South Africa. It is largely a grassland and savannah biome, however areas of desert occur to the west of the province and areas of varied bushveld occur to the east, especially the north-east in the BPD, where the Magaliesberg and Pilanesberg Mountain ranges occur (NWDACE, 2008). The climate is characterised by clear hot summers and cool sunny winters and the rainfall is varied across the province, but concentrated in the east and lessened toward the westerly desert area (NWP&TB, 2012).

2.2.2. CATCHMENT-SPECIFIC IMPACTS

The NWP is known as the Platinum province, and mining forms the back-bone of the provincial economy, contributing 42% to the GGP and 39% to the employment. The mining sector is dominated by large platinum mines and smelters (Anglo Platinum, Impala Platinum and Lonmin Platinum), all concentrated in the BPD, whilst gold mines occur in Orkney and Klerksdorp areas. There are also various other mines situated across most of the eastern part of the province, that specialize in chrome and other minerals. Agriculture is the second-most important sector, with 13% of the GGP and 18% of employment – maize and sunflowers are the most important crops grown, while cattle and game farming is also well-established (Walmsley et al., 2002; NWDACE, 2008).

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CHAPTER 2 – BACKGROUND INFORMATION

2.2.3. SOCIO-ECONOMIC CLIMATE

The total population consists of about 3.3 million (6.43% of the national total), of which 65% live in the rural areas (NWDACE, 2008; STATS SA, 2011). The province is one of the poorest in the country, with a gross geographic product (GGP) of R3, 964 per person, as opposed to the country’s average of R6, 490 per person. There is an estimated 38% of the population that are unemployed and 30% of the adult population are illiterate, which is the highest figure in the country (Walmsley et al., 2002; NWDACE, 2008). As such, particularly within this region, there is a need to provide food for a family in another way as opposed to making a purchase at the supermarket. Subsistence fishing is one such method and it may provide the sole source of protein for some of these communities.

Therefore, given the necessity to provide for a family, in the form of dietary protein and the current economic state of the province, there is a direct link between the local communities surrounding the impoundments and the toxicants in these systems as well as their biota.

2.3. SUBSTRATE ANALYSIS 2.3.1. PHYSICO-CHEMICAL PARAMETERS

Water quality is broadly used to describe the health and state of different watercourses, in order to define its variable state of integrity according to various guidelines, as determined by the Department of Water Affairs and Forestry (DWAF), or in the World Health Organization (WHO). Each set of guidelines is specific to its intended use. Guidelines will differ vastly for drinking water relative to domestic use in irrigation, or recreational use in swimming and boating (DWAF, 1996; WHO, 2008).

Similarly, as water quality varies slightly with climate, geomorphology, geology and the biotic composition, water quality will differ from region to region, irrelevant of proximity of separate water courses. Each of these drivers will be specific at each site and time of assessment. Thus each site will show diverse differences in their driver variables, as a watercourse progresses through its horizontal profile to the sea – these factors can potentially amount to a considerable difference in water quality (Dallas & Day, 2004).

According to the South African Water Quality Guidelines for Aquatic Ecosystems, the target water quality range (TWQR) is a management objective derived from quantitative and qualitative criteria. It is a range of concentrations or levels that shows no measurable adverse effects on the health of the aquatic ecosystem (DWAF, 1996). Another important management objective is the Resource Water Quality Objectives (RWQOs) that function

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CHAPTER 2 – BACKGROUND INFORMATION as the water quality components of the Resource Quality Objectives (RQOs), which are defined by the National Water Act as ‘clear goals relating to the quality of the relevant water resources’ (DWA, 2011a).

On a practical level, the TWQR describes the ‘fitness for use’ of a water resource, while the RWQOs define ‘what management action is required’ for a water resource. The generic RWQOs, developed as part of the national water quality assessment study were used in the assessment of the catchment water quality (DWA, 2011a).

2.3.1.1. Temperature Aquatic organisms have upper and lower thermal tolerance limits, optimal temperatures for growth, and temperature limitations for migration, spawning and egg incubation. Therefore, any disturbance that might cause a change in temperature within a system may have substantial effect on the aquatic community. The temperatures of inland waters in South Africa generally range from 5-30oC, but if these levels become elevated: metabolic rates will increase, including respiration and thus oxygen demand. Furthermore, as the oxygen demand increases, the dissolved oxygen supply within the system is slowly depleted and may have detrimental effects on other the survivability of the aquatic community (DWAF, 1996).

2.3.1.2. pH Most fresh waters in South Africa are relatively well buffered with pH ranges between 6 and 8. Diurnal fluctuations are known to occur in productive systems where the relative rates of photosynthesis and respiration vary over a 24-hour period. Photosynthesis alters the carbonate/bicarbonate equilibrium by removing CO2 from the water (DWAF, 1996).

Extreme rates of photosynthesis, whether natural or as a result of eutrophication, commonly result in very high pH values in standing waters. High rates of consumption of

CO2 during photosynthesis drive the carbonate species equilibrium toward carbonic acid and hence to extremely high pH values (> 10). This process occurs only in the light during the day. At night, the major biotic processes of respiration and decomposition release CO2, resulting in a decrease in pH (DWAF, 1996).

2.3.1.3. Electrical Conductivity & Total Dissolved Solids (TDS) Electrical conductivity (EC) is a measure of the ability of water to conduct an electrical current. This ability is a result of the presence of ions such as carbonate, bicarbonate, chloride, sulphate, nitrate, sodium, potassium, calcium and magnesium in the water – all of which carry an electrical charge. Many organic compounds dissolved in water do not dissociate into ions (ionise), and consequently they do not affect the EC. Similarly, Total

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CHAPTER 2 – BACKGROUND INFORMATION

Dissolved Solids (TDS) is a measure of the quantity of all the compounds dissolved in the water, irrespective of electrical charge (DWAF, 1996).

2.3.1.4. Dissolved Oxygen The maintenance of adequate dissolved oxygen concentrations is critical for the survival and functioning of the aquatic biota because it is required for the respiration of all aerobic organisms. There is a natural diel variation (24 hour cycle) in dissolved oxygen associated with the 24-hour cycle of photosynthesis and respiration by aquatic biota. Concentrations decline through the night to a minimum near dawn, then rise to a maximum by mid- afternoon. Seasonal variations arise from changes in temperature and biological productivity (DWAF, 1996).

2.3.2. CHEMICAL ANALYSIS

Analysis of water and sediment samples for organic and inorganic toxicants documents the presence/absence of these toxicants as well as the level of contamination within each substrate (Adams, 2005). However, it is imperative to interpret these values relative to their associated water quality. In this study, selected organic and inorganic toxicants were screened, some of which are described below.

2.3.2.1. Organic 2.3.2.1.a. Aldrin (HHDN) Aldrin (HHDN or (1R,4S,4aS,5S,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro- 1,4:5,8-dimethanonaphthalene) is an insecticide that was previously used to kill termites. It slowly evaporates in the air and often breaks down to dieldrin (HEOD or (1R,4S,4aS,5R,6R,7S,8S,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7- epoxy-1,4:5,8-dimethanonaphthalene), which is also an insecticide and further breaks down through sunlight and bacterial activity. Dieldrin can travel large distance by attaching to dust particles that get transported great distances by wind. It also has a high affinity for soil or sediments where it can remain unchanged for many years, whilst it does not dissolve well in water and only low concentrations are usually found (ATSDR, 2002a).

2.3.2.1.b. Chorpyriphos Chorpyriphos is an organophosphorus insecticide that has been used both as a household pesticide for cockroaches, fleas and termites, and an agricultural pesticide for ticks and crop pests. Chlorpyriphos enters the environment through direct application, volatilisation, spills or disposal of waste, which then binds tightly to the soil making difficult for it to be washed into local watercourses. However, if it enters the watercourse, it does not dissolve easily and small amounts remain at or near the surface and will evaporate. In

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CHAPTER 2 – BACKGROUND INFORMATION addition, it is broken down by sunlight, bacteria, and other chemical processes within the environment (ATSDR, 1997).

2.3.2.1.c. DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) is a pesticide that was once widely used in South Africa between the 1940’s through 1995 and reintroduced in 2000. It was used to control insects on agricultural crops and insects that carry diseases like malaria and typhus (Biscoe et al., 2005). DDT breaks down very slowly through the action of microorganisms into DDE (dichlorodiphenyldichloroethylene) and DDD (dichlorodiphenyldichloroethane), and they all persist in the environment for potential centuries. They have strong bond to soil and sediments, but can bind to particles in the water before settling (ATSDR, 2002c). Therefore, the presence of all forms of DDT within the water as opposed to within the sediment could be a potential indication of recent (last 10 years) spraying of DDT within the catchment area.

2.3.2.1.d. Lindane (HCH) Lindane (HCH or hexachlorocyclohexane) is a synthetic insecticide used for fruit, vegetables, and animals that exists in eight different chemical forms (isomers). The gamma- (γ) form holds the majority of the insecticidal properties. In soil, sediments and water, HCH is broken down to less toxic substances by algae, fungi, and bacteria over a long period of time (ATSDR, 2005a).

2.3.2.1.e. Nonylphenol (p-NP) Nonylphenol is manufactured from cyclic intermediates in the refinement of petroleum and coal-tar crudes resulting in a mixture of various isomers of nonylphenol. These are largely used as non-ionic surfactants in detergents, wetting agents, emulsifiers, etc. Nonylphenol and its ethoxylates have been shown to be quite volatile, whereby it is easily lost to the atmosphere when dissolved in water, whilst it has been shown to have an affinity for the sediment within natural system, but can be broken down within a couple of weeks (USEPA, 2005). Nonylphenol technical is sometimes used as a standard when measuring the occurrence of nonylphenol and/or its isomers in either water, sediment or muscle and if detected, it usually indicates the presence of numerous nonylphenol isomers in the substrate.

2.3.2.1.f. Terbuthylazine Terbuthylazine is an algicide, microbicide and microbiostat used to control slime forming algae, fungi and bacteria. It is a soluble liquid concentrate with a low acute toxicity that degrades very slowly under aerobic aquatic conditions (USEPA, 1995).

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CHAPTER 2 – BACKGROUND INFORMATION

2.3.2.2. Inorganic 2.3.2.2.a. Arsenic (As) Arsenic is a naturally occurring metalloid (having properties of both a non-metal and metal) in soil and in many kinds of rock, which may enter the air, water and land by wind- blown dust and/or leaching from rocks. Although its inorganic and organic forms were previously used in pesticides and lead-acid batteries for automobiles, its current commercial use is in wood preservatives. Arsenic cannot be destroyed or degraded, but only its form can be changed, or become attached to or separated from another particle. Although fish and other biota often take up As, which may accumulate in the tissue, most of this As is in an organic form called arsenobetaine (often called “fish arsenic”), which is a much less harmful form (ATSDR, 2007a).

2.3.2.2.b. Beryllium (Be) Although Be occurs naturally in a variety of materials such as rocks, coal, oil, soil and volcanic dust, it also mined and converted into alloys used widely in electrical parts and construction materials. It is the lightest metal and many of its compounds are easily soluble within water, which poses a greater human health risk than its insoluble forms. Most Be enters the watercourse through the weathering of rocks that the water runs over, whilst a small part is settled out from dust in the air or point source discharges. Fish do not accumulate a substantial amount of Be directly from water (ATSDR, 2002b).

2.4. SURVIVAL MECHANISMS OF FISH

All fauna and flora is adapted to, and specifically able to, survive in optimum conditions suited to a specific environment (Schmidt-Nielsen, 1997). These environmental conditions are largely dictated by the climate, nature of the surrounding land uses, natural vegetative cover, geology and geo-morphology of the area (Kleynhans et al., 2005). However the species-specific adaptations are largely inherited from previous generations, and slowly modified over time one generation to the next based on previous environmental factors (Schmidt-Nielsen, 1997; Kleynhans et al., 2005).

Aquatic fauna, especially fish, have numerous mechanisms in place that afford the specimen the ability to survive comfortably and energy efficiently within an optimum range of conditions (Schmidt-Nielsen, 1997). Should the conditions of the environment become unfavourable and fall outside of their optimum range, whether it is temperature, solute concentration or oxygen availability. The fish usually responds in one of two ways: either in (i) tolerance or by (ii) regulation. If the species is unable to perform either, there is a tendency for disease to set in (Roberts, 1989). They are able to do this through a number of different mechanisms that may include behavioural changes, physiological

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CHAPTER 2 – BACKGROUND INFORMATION changes, or biochemical changes which are often subtle changes at a molecular level (Schmidt-Nielsen, 1997).

These mechanisms employed by aquatic fauna are effective over short periods of time, but if conditions worsen or remain impaired over chronic periods, these mechanisms slowly fail and become less effective. These biota begin to show symptoms of stress in the form of various biomarkers, and most often, these symptoms are quantifiable in some form, hence the preferred approach to bioassessment of natural systems.

2.4.1. FISH AS A BIOINDICATOR SPECIES

Toxicants are easily dissolved within the water column and show a diversity of effects (van der Oost et al., 2003). Most if not all, cause a decline in the water quality and in turn the health of certain fish communities are affected (Gimenez Casalduero, 2001; van der Oost et al., 2003; Adams, 2005). It is widely known that long-term exposure to toxicants can be detrimental to important fish homeostatic mechanisms such as metabolism, growth, disease, health condition and survival of the species (van der Oost et al., 2003; Van Dyk, 2006). These toxic effects depend on the bioavailability and persistence of the toxicants, the capacity of the organism to accumulate them and the interference of such compounds with biochemical, physiological, and/or ecological processes (Connell, 1999; Trujillo- Jimenez et al., 2011; Cunningham & Cunningham, 2012). Fish are key aquatic organisms that are able to accumulate such toxicants through multiple pathways, including (i) direct osmosis through skin and anus, (ii) oral ingestion into the alimentary canal, and (iii) air- intake via oral intake or gills (Schmidt-Nielsen, 1997).

Fish are a bio-indicator species as they live, eat and breathe within the aquatic system and integrate the effects of many abiotic and biotic variables acting on the system (Adams et al., 1993). Fish are permanently exposed to the ecosystem, and are potentially vulnerable to the accumulation of solutes within the environment and habitat (Hickman et al., 2006). Fish are also large and easily identified to species level, which helps to eliminate confusion in identification. Fish fall into a high trophic level, which adds further to their value as a biomarker species, as toxicants would bioconcentrate through various levels of the food web and in turn, biomagnified in the local communities through ingestion (Harris, 1995; Streit, 1998; Connell, 1999).

Due to the key position of these organisms within the trophic chain, fish act as sentinel organisms for humans and other fauna that are exposed to similar concentrations of toxicants within the environment (Wester et al., 2002; Viarengo et al., 2007).

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CHAPTER 2 – BACKGROUND INFORMATION

2.4.2. HISTOPATHOLOGY AS A BIOMARKER

Histopathology has become an enhancement of aquatic toxicology and its advantage as a biomarker is due its intermediate location in the hierarchy of biological organisation (Wester et al., 2002; Zimmerli, et al., 2007). Therefore, it is able to integrate the effect of both abiotic factors and fish health, whereby the results are often interpreted as an underestimation of realistic circumstances (Hinton & Lauren, 1990; Lang et al., 2006; Zimmerli, et al., 2007). It is regarded as a reliable tool to assess and measure relative levels or disturbances within specific target organs like the gills (Stentiford et al., 2003; Van Dyk et al., 2009a), livers (Lang et al., 2006; Camargo & Martinez, 2007; Van Dyk et al., 2012), kidneys (Ayas et al., 2007), heart (Borges et al., 2003; Marchand et al., 2012), gonads (Marchand et al., 2008a; Louiz et al., 2009; Marchand et al., 2012; Pieterse et al., 2010) and skin (Bernet et al., 1999; Vogelbein et al., 2001) The application of this tool can be further applied holistically when the number of alterations can be quantified per target organ and used to calculate an overall fish condition, as applied in this study (Bernet et al., 1999; Zimmerli et al., 2007).

2.5. NECROPSY-BASED HEALTH ASSESSMENT

The assessment of living organisms in biomonitoring procedures in addition to the water and sediment analysis has been well accepted by numerous disciplines for a number of years, whereby the biotic integrity of the ecological system is often reflected in the health of the organisms that reside in it (Adams et al., 1993; Gimenez Casalduero, 2001).

The necropsy-based health assessment is an extension and refinement of a previously published field necropsy system by Goede & Barton (1990). It is a rapid and inexpensive quantitative index that has been proven to accurately characterise fish health, especially relative to other more sophisticated health assessment methods and in addition, it is a rapid and inexpensive alternative (Adams et al., 1993). For the purposes of this study, the qualitative aspects of the protocol were used as a guide, as the quantitative index was not calculated. The fins, spleen, hindgut, kidney, skin, liver, eyes, gills and the presence of parasites is macroscopically assessed, whilst the haematological assessment is performed separately, and the colour of the bile and the amount of mesenteric fat is noted (Schmitt & Dethloff, 2000).

It should be recognized that this method is not diagnostic, but rather a systematic method for gross macroscopic observations of external and internal abnormalities or lesions, whereby cumulative stress can be estimated and further assessment can then be performed (Schmitt & Dethloff, 2000).

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CHAPTER 2 – BACKGROUND INFORMATION

2.5.1. HAEMATOLOGICAL ASSESSMENT

Haematological techniques have proven valuable for fisheries biologists in assessing environmental health and monitoring normal and pathological processes in fish (Elahee & Bhagwant, 2007). Basic haematocrit and leukocrit levels are classic stress indicators that can be indicators to the type of stress, such as organic or metallic intoxication (Adams et al., 1993; Roche & Boge, 1996).

2.6. BIOMETRIC INDICES

Since toxicants are usually present in natural environments as complex mixtures from multiple sources, there is no single biomarker that can provide a complete diagnosis of environmental degradation or fish health. A set of complementary biomarkers is often useful in evaluating organism responses to stress and environmental conditions (Adams, 2005; Trujillo-Jimenez et al., 2011). Measurements of condition factor and organosomatic indices are standard procedures in fish physiology studies and fish biology because they integrate many levels or sub-organismal processes (Schmitt & Dethloff, 2000).

2.6.1. CONDITION FACTOR

The condition factor is an organism-level response that relates weight to length and take into account numerous factors such as nutritional status, pathogen effects and toxicant exposure causing greater-than-normal or less-than-normal weights (Schmitt & Dethloff, 2000). The selected index is a Fulton-type condition factor that reflects the overall condition and nutritional state of an individual fish (Carlander, 1969). This approach should be used with caution because the condition factors could vary between species, allowing only same species comparisons (Anderson & Neumann, 1996).

2.6.2. ORGANO-SOMATIC INDICES

Organo-somatic indices are quantitative values that relate a specific organ to the overall body size of the sampled specimen. They are useful tools within a thorough assessment of fish health, as various contaminants affect different areas or organs of each specific organism in differing degrees of severity, dependent on the nature of each toxicant. Therefore, these indices reflect the status of specific organ systems and/or organs as the organism’s weight is affected by any changes in organ mass. These indices should be interpreted with caution as these indices may change as a result of seasonal variation, breeding, metabolic activities, size, gender, age, gonadal development, etc. (Schmitt & Dethloff, 2000).

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CHAPTER 2 – BACKGROUND INFORMATION

2.6.2.1. Hepatosomatic Index (HSI) Fish store energy in the muscle, but they also accummulate energy in the liver during periods of high energy intake, which is mostly in the form of glycogen. The HSI value should correlate directly with the nutritional state and growth rate of each fish. This index is also often used in studies that assess seasonal and yearly changes in growth (Busacker et al., 1990) and is most often associated with toxicant exposure, whereby relative enlargement or reduction of liver can suggest toxicant exposure (Schmitt & Dethloff, 2000).

2.6.2.2. Splenosomatic Index (SSI) The size of the spleen is also considered a useful diagnostic factor due to its haematopoietic function, in that its dysfunction will potentially affect the whole organism, especially at an immunological level (Schmitt & Dethloff, 2000; Hadidi et al., 2008). Larger spleen sizes have been shown to indicate increased resistance to pathogens in Rainbow Trout (Oncorhynchus mykiss) (Hadidi et al., 2008).

2.6.2.3. Gonadosomatic Index (GSI) The gonadosomatic index (GSI) is another ponderal index that provides structural information about gonadal health and maturation stage, especially in assessing changes in response to environmental dynamic (natural cyclic changes) and/or environmental stresses (McDonald et al., 2000). The GSI provides structural information concerning the gonads that are advantageous for identifying effects of long-term toxicant exposure, whereby chronic exposure to toxicants has been shown to result in gonadal alterations, often indicated by a reduced GSI (Marchand et al., 2008a). It should also be noted that GSI varies between males and females during the reproductive cycle and it is influences by seasonal changes of abiotic parameters such as temperature and photoperiod (Louiz et al., 2009).

2.7. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA)

Cellular biomarkers act as early warning signals of stress suffered by organisms exposed to some form of contamination. However, only when changes are as severe as to alter the tissue/organ function(s) can the development of a stress-induced condition be confirmed (Viarengo et al., 2007). Histopathology has been considered and promoted as an effective and rapid environmental health assessment technique used for bio-monitoring of aquatic environments. It is the study of lesions or abnormalities at a cellular level by assessing a selected sentinel organism and histopathologically assessing each selected target organ for any changes from the normal histology (Schmitt & Dethloff, 2000; Wester et al., 2002).

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CHAPTER 2 – BACKGROUND INFORMATION

Numerous studies have been able to characterise the health condition of fish and their associated environments using histopathology (Paul & Banerjee, 1997; The et al., 1997 Bernet et al., 2000; Stentiford et al., 2003; Lang et al., 2006; Ayas et al., 2007; Camargo & Martinez, 2007; Van Dyk et al., 2009a; Abalaka et al., 2010; Jin et al., 2010; Pieterse et al., 2010; Salazar-Lugo et al., 2011; Van Dyk et al., 2012; Marchand et al., 2012). For the purposes of this study, the assessment was largely adapted from Bernet et al., (1999).

2.8. AGE ESTIMATION

As recommended in previous studies by Bernet et al. (1999) and Marchand (2008), age is an important biological variable that can add valuable information to the state of health of an individual and the population. Age is known to contribute to an increased number of neoplasms, as evident in humans when age makes individuals more susceptable to various stress factors (Bernet et al., 1999; Campana, 2001; Marchand, 2008; Gerber et al., 2009).

2.8.1. OTOLITHS

Since, C. gariepinus have no scales, and are sometimes referred to as being ‘naked,’ another calcified structure other than the commonly used scales needs to be used for age estimation. As a result, an alternative structure called the otolith is used, which often yields more accurate results. There are usually three otoliths found in the auditory organs of fish, namely the sagitta (usually the largest), astericus and lapillus (Jones & Hynes, 1950).

Otoliths show various alternating bands when examined through a light microscope; each band is either light and dark, or opaque and transparent. However, these bands were only defined and counted as growth zones, if the bands were complete and continuous. Additionally, the outer zone was excluded as otoliths growth over time, and the outermost zone shows incomplete growth (Jones & Hynes, 1950; Gerber, 2010).

2.8.2. SCALES

Conversely, C. carpio do have scales, and simply due to the simplicity in collecting two to three scales per individual from the same location, this method was preferred with respect to the C. carpio as opposed to the C. gariepinus ageing protocol (Jones & Hynes, 1950, Crenshaw, 2009; Gerber 2010).

2.9. EDIBILITY

Edibility is defined as any food being safe for human consumption. In essence, this would PAGE | 25

CHAPTER 2 – BACKGROUND INFORMATION exclude the presence of any potentially unsafe toxicants that might be bio-available to humans through various levels of bioaccumulation and bioconcentration through each level of the food web. As said by Moriarty, “the task of ecotoxicology is to assess, monitor and predict the fate of foreign substances in the environment.” Therefore, the edibility of fish and other organisms i.e. the final partition of dangerous toxicants can be tracked, measured and interpreted as a potential health risk. This implies the analyses of the content of the organism’s muscle, liver, fat and/or gonadal tissue, in order to determine the effects of bioconcentration and sequestration of any and all toxicants, including pre- carcinogenic forms (Connell, 1999).

2.9.1. MUSCLE ANALYSIS

The detection of specific toxicants in a biomarker species is evidence of the presence those toxicants within the environment (Connell, 1999). If the biomarker species is as high in the food web as is fish, it remains possible that the concentration of the detected toxicant remains low and potentially manageable of a system level (Connell, 1999). However, the presence of the toxicant at relatively higher concentration presents a potential risk to consumers, as shown in various studies (Burger et al, 2005; Ginsberg & Toal, 2009; Cardoso et al., 2010a; Ruelas-Inzunza et al., 2011). Therefore, a complete analysis of the content of the muscle is important in defining human health risk through consumption.

2.9.2. HUMAN HEALTH RISK ASSESSMENT (HHRA)

A human health risk assessment (HHRA) is the probability of an adverse health effect occurring due to associated measured levels of a hazardous agent (Heath et al., 2004). In South Africa, there is a limited number of assessments that use the data from bioaccumulation studies and input this data into a risk-based assessment (Heath & Claassen, 1999). However, there are a couple of international studies that calculate human health risks through consumption, especially with regards to the mercury content of sea- dwelling fish (NJDEP, 2002; Cardoso et al., 2010a; Cardoso et al., 2010b; Dai et al., 2011; Ruelas-Inzunza et al., 2011). Many of these studies defined a significant health risk to elevated consumption of some fish species.

The risk assessment process is defined by the National Academy of Sciences of America (NAS; 1983) and recommended by the United States of America Environmental Protection Agency (USEPA; 1997) consists of four distinguishable, but interacting steps, namely (i) hazard identification, (ii) dose – response assessment, (iii) exposure assessment, and (iv) risk characterisation (Figure 2 – 1).

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CHAPTER 2 – BACKGROUND INFORMATION

2.9.2.1. Hazard Identification This step assesses the potential that exposure to a chemical under specific conditions will pose a threat to human health (NAS, 1983; USEPA, 1997). General information is found on each of the various toxicants that have hazardous properties, such as physical and chemical properties, route and patterns of exposure, metabolic and pharmacokinetic properties, toxicological effects, etc. This information is available from the following toxicity databases:  Health Effects Assessment Summary Tables (HEAST) (United States Environmental Protection Agency (USEPA), 1997b)  Agency Toxic Substance and Disease Registry (Agency for Toxic Substances and Disease Registry (ATSDR), 2012)  Integrated Risk Information System (IRIS) (United States Environmental Protection Agency (USEPA), 2012)  Toxicology Excellence for Risk Assessment (Toxicology Excellence for Risk Assessment (TERA), 2012)  Toxicology Data Network (TOXNET) (U. S. National Library of Medicine, 2008)  Risk Assessment Information System (RAIS) (University of Tenessee, 2013)

Figure 2 - 1: Schematic diagram illustrating the risk assessment process (Heath et al., 2004).

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CHAPTER 2 – BACKGROUND INFORMATION

2.9.2.2. Dose – Response Assessment The relationship between the dose of a hazardous chemical and the incidence of an adverse health effect in the exposed population is characterised in this step. The dose- response dynamics of a specific chemical and functional relationship between the exposure and the observed human and/or animal effects is evaluated. There are two distinct groups of hazardous chemicals, namely carcinogenic and non-carcinogenic, and each group is assessed differently (Heath et al., 2004).

An oral reference dose (RfD) is calculated for the protection against chronic toxicity resulting from exposure to toxicants. It is calculated by identifying the most appropriate no-observed-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level (LOAEL) and applying the relevant uncertainty and modifying/safety factors. This RfD is used to calculate the toxic and cancer risk of a particular toxicant. However, since it is generally assumed that carcinogens do not have a safe threshold of exposure and any exposure may cause cancer, an additional variable is require to define the cancer risk (USEPA, 1997). Data obtained from one or more epidemiological studies and/or bioassays are used in cancer risk extrapolation models to calculate the cancer slope factor (β) (Heath et al., 2004).

2.9.2.3. Exposure Assessment This step in the process measures or estimates the intensity, frequency and duration of human exposure to a specific toxicant in potentially exposed populations. A complete exposure assessment deals with the (i) source of the health hazard, the (ii) exposure pathways via various media or routes, and the (iii) measured or estimated concentrations and exposure duration (Heath et al., 2004). In essence, this step calculates the total dose of a specific toxicant that a subject was exposed to, whereby in a health risk assessment of consumption of fish – only the concentration present within the edible part of the fish is necessary.

2.9.2.4. Risk Characterisation This step uses all information gathered in the previous three steps to characterise and describe the extent of the overall individual or population risk. The quantitative and qualitative aspect of the risk assessment, the assumptions used and identification of uncertainties are assessed and discussed to provide an overall health risk (Heath et al., 2004).

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3. MATERIALS & METHODS

CHAPTER 1: IN THIS CHAPTER PROJECT OVERVIEW 3.1. INTRODUCTION ...... 30 3.2. FIELD SURVEYS ...... 30 3.3. SUBSTRATE ANALYSIS ...... 31 CHAPTER 2: 3.3.1. Physico-chemical Parameters ...... 31 BACKGROUND 3.3.2. Chemical Analysis ...... 33 INFORMATION 3.4. SPECIMEN COLLECTION ...... 35 3.5. NECROPSY-BASED HEALTH ASSESSMENT ...... 36 CHAPTER 3: 3.5.1. Haematological Assessment ...... 37 MATERIALS & 3.6. BIOMETRIC INDICES ...... 37 METHODS 3.6.1. Condition Factor ...... 37 3.6.2. Organo-somatic Indices ...... 38 3.7. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 38 CHAPTER 4:

3.7.1. Tissue Processing ...... 39 RESULTS 3.7.2. Qualitative Histological Assessment ...... 41 3.7.3. Quantitative Histological Assessment ...... 42 3.8. AGE ESTIMATION ...... 45 CHAPTER 5: 3.8.1. Otoliths...... 45 DISCUSSION & 3.8.2. Scales ...... 46 CONCLUSION 3.9. EDIBILITY ...... 46 3.9.1. Muscle Analysis ...... 46 3.9.2. Human Health Risk Assessment (HHRA) ...... 47 3.10. STATISTICAL ANALYSIS ...... 48 APPENDICES

1 Peter 1: 8 – Whom having not seen, ye love; in whom, though now ye see him not, yet believing, ye rejoice with joy unspeakable and full of glory. CHAPTER 3 –MATERIALS & METHODS

3.1. INTRODUCTION

This chapter will describe the methodology followed during the sampling procedure, lab and tissue analysis, and stepwise assessment protocols for the fish health assessment. Furthermore, the stepwise calculation of various indices used in the study will be defined and the risk assessment approach will be elaborated upon.

3.2. FIELD SURVEYS

The three sites chosen for the study were the two assessment sites, namely RD (S25o 25.607' E027o 35.648') and VD (S25o 19.063' E027o 29.005') and the reference site, the MBD (S25o 28.310' E026o 23.914'). All sites were well-established impoundments located within the Bojanala Platinum region (Figure 3 – 1) of the NWP in the Crocodile (West) and Marico WMA (RHP, 2005).

B

A Vaalkop Dam

Marico-Bosveld Dam Roodekopjes Dam

Figure 3 - 1: (A) Overview of southern Africa, showing position of the North West Province (orange dashed box) (©SA-Venues.com). (B) An exploded view of the Bojanala Platinum Region (green shaded area), showing the selected sites (purple boxes) and the selected reference site (brown box) (©Linx.co.za).

Two field surveys were undertaken for specimen collection. The two assessment sites (RD & VD) were sampled from the 30th January 2011 to the 10th February 2011, whilst the reference site (MBD) was sampled between the 6th March 2011 and the 18th March 2011. Both collection efforts occurred during the wet season when temperatures are warmer

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CHAPTER 3 –MATERIALS & METHODS and the seasonal rains increase flow, thereby bringing additional food and potential toxicants into each respective system. As a result, the fish communities were expected to be more active at a surface level, as opposed to remaining at depth during the winter period. This would better the probability of acquiring an optimal sample size using gill nets.

3.3. SUBSTRATE ANALYSIS 3.3.1. PHYSICO-CHEMICAL PARAMETERS

The following physico-chemical water parameters were measured in situ using the following pre-calibrated Eutech Instruments (Figure 3 – 5A): a Cyberscan pH110 for the pH, a Cyberscan CON110 for the conductivity (mS/m), Total Dissolved Solutes (TDS; ppm) and temperature (oC) and a Cyberscan DO100 for the dissolved oxygen (mg/L and % saturation).

These parameters were recorded at specific locations within each of the impoundments where water and sediment samples were collected (inlets and outlets), where fish were collected, and in the middle of the dam. These parameters were measured at three different times on the same day, namely morning (±09h00), noon (±12h00) and late afternoon (±17h00) in order to define potential daily trends.

A total of seven sites were selected for the RD (Figure 3 - 2): . Site 1 – a fish sampling site east of release point in the dam wall, . Site 2 – a fish sampling site and a substrate collection point at the outlet of impoundment, . Site 3 – the middle of the impoundment, . Site 4 – a fish sampling site south of north-west bay, . Site 5 – fish sampling site north of north-west bay, . Site 6 – a substrate collection point on the Crocodile River inlet, and . Site 7 – a substrate collection point on the Sterkstroom River inlet.

A total of six sites were chosen at the VD (Figure 3 - 3): . Site 1 – a fish sampling site on the eastern portion of impoundment, . Site 2 – a fish sampling site east of dam wall, . Site 3 – middle of eastern portion of impoundment, . Site 4 – a substrate collection point at the outlet of impoundment, . Site 5 – a substrate collection point on the Hex River inlet, and . Site 6 – a substrate collection point on the Elands River inlet.

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CHAPTER 3 –MATERIALS & METHODS

N

2000m

Figure 3 - 2: Map of Roodekopjes Dam (RD) showing the sampling and measurement locations.

N

2000m

Figure 3 - 3 Map of Vaalkop Dam (VD) showing the sampling and measurement locations.

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CHAPTER 3 –MATERIALS & METHODS

A total of four sites were chosen at the reference site (MBD) (Figure 3 - 4): . Site 1 – a fish sampling site on the eastern shoreline, . Site 2 – fish sampling sites in middle of the impoundment, . Site 3 – a fish sampling site and a substrate collection point at the outlet of impoundment, and . Site 4 – a substrate collection point on the Groot Marico River inlet

N

1000m

Figure 3 - 4: Map of Marico-Bosveld Dam (MBD) showing sampling and measurement locations.

3.3.2. CHEMICAL ANALYSIS

Water samples for organic and inorganic analysis were taken at 30cm below the surface, in pre-acid-washed 2L glass jars and plastic bottles, respectively (Figure 3 – 5B). These samples were collected at the main inlet/s and the outlet of each of the impoundments. Samples for inorganic analysis were treated with 1-2 mL of 65% nitric acid for preservation of the metal content. Each impoundment’s samples were later pooled together by thoroughly mixing all samples (inlet/s & outlet) per impoundment to make a single representative sample per impoundment for the lab analysis. Similarly, the sediment samples for organic and inorganic analysis were collected using pre-acid-washed 300mL

PAGE | 33

CHAPTER 3 –MATERIALS & METHODS glass honey jars and plastic jars, respectively. These samples were also collected at the inlet/s and near/at the outlet of each of the impoundments. The water depths at the outlets were too deep for sediment collection, and a sample was taken on the shoreline adjacent to the dam wall. These samples were also later combined to form a pooled representative sample as per impoundment.

A B C

D E F

G H I

Figure 3 - 5: Photos demonstrating sampling during field surveys. (A) Measuring in situ physico-chemical water parameters, (B) water sample collection for chemical analysis, (C, D, E, F) gill netting and specimen sample collection, (G) weighing individual specimen, (H) mobile field laboratory for necropsy and assessment, and (I) late-evening sampling effort.

Both the water and sediment samples were kept in a cooler box with ice, and/or in the refrigerator at about 4oC until further processing. Samples were sent to an accredited ISO 17025 laboratory to be analysed and/or screened for selected organic and inorganic toxicants at Food and Drug Assurance (FDA) Laboratories (Pty) Ltd and WaterLab (Pty) Ltd, respectively. FDA Labs screened for pesticides, organochlorines (OCs), polychlorinated biphenols (PCBs), other phenols, and hormones, such as estrone and estradiol (Appendix 9: Table A – 40). WaterLab performed a semi-quantitative 32 metal/element scan (screening) on representative water and sediment samples.

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CHAPTER 3 –MATERIALS & METHODS

3.4. SPECIMEN COLLECTION

Fish were sampled using multi-filamentous twine and mono-filamentous nylon gill nets, of varying mesh sizes (9 - 18cm). These nets were deployed, with the aid of a boat (Figure 3 – 5C, D), from early morning (±07h30), in areas suited to the selected species’ (Figure 3 – 6) habitat preferences and behavioural patterns (Skelton, 2001). Since the study required live specimens, each net was serviced at two hour intervals and removed just before sunset in an effort to limit unnecessary casualties and limit the struggling time of each specimen sampled (Figure 3 – 5E, F). This practice aimed to reduce any external imposed stress or injury to the specimen, in order to reduce or prevent false or inaccurate observations caused by sampling effort rather than the condition of the impoundment. All other fish species that were not required for the study were quickly released to reduce any unnecessary stress to the fish communities at each impoundment.

A

B

Figure 3 - 6: The endemic Clarias gariepinus (Sharptooth Catfish) (A) and exotic Cyprinus carpio (Common Carp) (B) selected for the study.

A total sample size of thirty (30) fish per site was set, of which fifteen (n = 15) were C. gariepinus and fifteen (n = 15) were C. carpio. Once caught, each specimen was removed from the net and kept alive in 100L cooler boxes (live wells). These live wells were filled with water from the impoundment to prevent imposing additional stress on the organism

PAGE | 35

CHAPTER 3 –MATERIALS & METHODS through re-acclimatisation and oxygenated using portable oxygen pumps to avoid further stress from an anoxic environment. The live wells were then transported back to the field laboratory and collected specimens were immediately assessed.

3.5. NECROPSY-BASED HEALTH ASSESSMENT

A qualitative fish necropsy was performed on each individual specimen using Adams et al. (1993) as a guide. Table 3 – 1 describes the condition of potential alteration in the fish.

Each specimen was sexed, weighed (g; total weight including viscera) with a lip grip scale (Figure 3 – 5G) and measured for length (cm) - both the standard (tip of snout to mid-base of caudal fin) and total (tip of snout to the end of the longest caudal ray) lengths. Each specimen was placed on a clean dissection board and the eyes of the fish were then covered with a cloth to reduce stress. Blood was collected from the posterior region of the dorsal aorta along lateral line using a plastic hub or ‘bulldog,’ sterilized needles and vacutainers, coated with EDTA for C. gariepinus samples and heparin for C. carpio samples to prevent clotting, and kept on ice. Vacutainers containing heparin are the conventional anti-clotting agents used when drawing blood. However, heparin is not effective with blood from C. gariepinus, hence the necessity for EDTA coated vacutainers. Following the blood collection, fish were sacrificed by severing the spinal cord anterior to the dorsal fin using a sharp scalpel and a sharp pair of bone scissors. Each specimen was then carefully dissected and macroscopically examined for any external (eyes, skin, fins and opercula) or internal (gills, liver, spleen, hindgut and kidney) abnormalities and the presence or absence of parasites was noted (Adams et al., 1993).

Table 3 - 1: Description of potential macroscopic alterations observed during the necropsy-based health assessment using Adams et al. (1993) as a guide. EYES SKIN FINS OPERCULA PARASITES Normal Normal Normal Normal None Haemorrhage Aberrations: Fins: Shortening Few Missing - Mild - Mild Moderate Other - Moderate - Moderate Severe - Severe - Severe GILLS LIVER SPLEEN HINDGUT KIDNEY Normal Normal Normal Normal Normal Frayed Fatty Granular Inflamed: Swollen Clubbed Nodules Nodular - Mild Mottled Discoloured Focal Discolour. Enlarged - Moderate Granular Pale Discolouration Other - Severe Urolithiasis Other Other Other

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CHAPTER 3 –MATERIALS & METHODS

The eyes, skin, fins and opercula were macroscopically assessed for abnormalities and described according to Table 3 – 1 (Adams et al., 1993). Similarly, the internal organs, namely the gills, liver, kidney, spleen and hindgut were also macroscopically observed and classed according to Table 3 – 1. In addition, the bile colour, mesenteric fat levels and haematological assessment followed and observations were noted. Finally, the number of macroscopically observable parasites were recorded both externally and internally (Adams et al., 1993; Van Dyk et al., 2009a).

3.5.1. HAEMATOLOGICAL ASSESSMENT

Blood was later transferred in duplicate per specimen into capillary tubes from the vacutainers and centrifuged individually at 30 000rpm for 10 minutes and analysed using the Hawksley micro-haematocrit reader. The haematocrit (Hct) and leukocrit (Lct) values were measured and calculated, as shown below, as a percentage portion of the total blood sample.

RBC∗ F fffff WBC∗ Hct = X 100 Lct = X 100 Total Blood Volume Total Blood Volume

*RBC = Red blood cell volume *WBC = White blood cell volume

3.6. BIOMETRIC INDICES

With the lengths and the weights measured during the fish necropsy, selected biometric indices were calculated as additional indicators of the general well-being of each fish as well as the condition of certain functional organs within each specimen.

3.6.1. CONDITION FACTOR

The condition factor (CF), also referred to as an index of well-being, was calculated from the total length and weight of each individual specimen of the sample according to the formula below, as described by Carlander (1969).

Weight (g) X 105 CF = Total Length (mm)3

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CHAPTER 3 –MATERIALS & METHODS

3.6.2. ORGANO-SOMATIC INDICES

Organo-somatic indices are quantitative values that relate a specific organ to the overall body size of the sampled specimen. They are useful tools within a thorough assessment of fish health that indicate the size of specific organ relative to total body weight (Schmitt & Dethloff, 2000). The liver, spleen and gonads were weighed for the calculation of their respective organo-somatic indices and in addition, the gonad’s length was also measured for general comparison.

3.6.2.1. Hepatosomatic Index The hepatosomatic index (HSI) is an index that describes the liver percentage composition of the whole organism, as previously described by Busacker et al. (1990).

Liver Weight (g) HSI (%) = X 100 Body Weight (g)

3.6.2.2. Splenosomatic Index The splenosomatic index (SSI) gives the proportion of the size of the spleen relative to the whole body weight, as shown below (Busacker et al., 1990).

Spleen Weight (g) SSI (%) = X 100 Body Weight (g)

3.6.2.3. Gonadosomatic Index The gonadosomatic index (GSI) is calculated as a weight percentage of the body weight, as described by McDonald et al. (2000).

Gonad Weight (g) GSI (%) = X 100 Body Weight (g)

3.7. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA)

A semi-quantitative microscopic assessment of the structural alterations within each one of the selected target organs (Figure 3 – 7) within each specimen was performed through

PAGE | 38

CHAPTER 3 –MATERIALS & METHODS histopathology (Bernet et al., 1999). In order to identify these alterations, a thorough knowledge of normal fish histology is required which was previously described by Van Dyk et al. (2009b) and Van Dyk (2006) under controlled captive conditions. This approach indicates the prevalence of alterations at a cellular level within each organ and infers the general health of each individual specimen (Bernet et al., 1999; Van Dyk et al., 2009b).

A B C D

E F G

Figure 3 - 7: Some of the selected target organs, namely (A) the gill, (B) the liver, (C, D) the testis from C. gariepinus and C. carpio, respectively, (E) the bi-lobed kidney of the C. carpio, (F) the two-chamber heart, and (G) the ovary from C. carpio.

3.7.1. TISSUE PROCESSING

During the fish necropsy, a portion of each one of the six selected target organs – namely gills, kidney, liver, heart, gonads (testes or ovaries) and skin were sampled. Sampling locations were standardised per organ, whereby a medial sample was taken in addition to sample taken at any macroscopically observed abnormalities (discolouration, lesions or cysts/lobules).

The gills were sampled immediately after each specimen’s spinal cord was severed, in order to limit post-mortem changes. Each of the remaining organs was sampled hereafter PAGE | 39

CHAPTER 3 –MATERIALS & METHODS in relation to the ease of access during the dissection. However, briskness of this sampling procedure remained a priority to best represent in vivo conditions, which in turn represented structural alterations within the tissue caused by environmental factors rather than factors relating to the sampling procedure or putrefaction (Van Dyk, 2006; Van Dyk et al., 2009b).

3.7.1.1. Fixation Each tissue sample was fixed in pre-prepared 10% neutral buffered formalin (NBF) (liver, kidney, heart and brain) and Bouin’s fixative (gills, gonads and skin/muscle) and standard histological procedures were followed hereafter (Humason, 1979). Samples were left in 10% neutrally buffered formalin (NBF) for 48 hours, whilst samples in Bouin’s fixative were left for 24 hours.

3.7.1.2. Dehydration All samples were washed in tap water for 1 hour to remove remaining fixative and dehydrated in increasing concentrations of ethanol 30% (1 hour), 50% (1 hour) and then stored in 70% until further processing at the laboratory. All samples were further dehydrated in increasing concentrations of ethanol (80%, 90%, 96%, and 100%) for 1 hour in each concentration to rid the tissues of excess water retention, which would prepare the tissue to take up wax during infiltration (Humason, 1979).

3.7.1.3. Clearing, Infiltration and Embedding The samples were cleared using xylene for 30 – 35 minutes, followed by infiltration through an inter-medium compatible pre-melted histosec/xylene (50:50) and a series of pre-melted histosec wax beakers in an oven at 60oC for 12 to 24 hours. Following infiltration, samples were orientated according to intended sectional result (gills – longitudinal section; all remaining organs – transverse sections) and embedded in histosec wax blocks, using L- shaped metal pieces and flat metal plates, and finally, stored in the refrigerator for at least 48 hours until sectioning (Humason, 1979).

3.7.1.4. Sectioning, Mounting and Staining Samples were sectioned, using a Leica wax rotary microtome, to a thickness of 5 μm and a short series of wax ‘ribbon’ was mounted onto pre-marked glass microscope slides, and stretched using an albumin-glutamine solution and a hot plate set at approximately 45ºC. The mounted slides were allowed to pre-dry on racks above the hot plate and further dried in a drying oven overnight. Once dried, the slides were stained with a rapid Hematoxylin and Eosin (H&E) stain (Van Dyk & Pieterse, 2008). Stained slides were immediately mounted with cover slips for preservation using an Entellan mounting medium and allowed to dry in a horizontal position for 24 – 48 hours.

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CHAPTER 3 –MATERIALS & METHODS

3.7.2. QUALITATIVE HISTOLOGICAL ASSESSMENT

All prepared slides for each particular organ were screened using the Olympus BH2 light microscope for qualitative histological assessment. This included the identification and grading of severity (none, mild, moderate, severe) of histological alterations present within each organ.

The histological alterations observed within each of the target organs were grouped together into the five reaction patterns as stipulated by Bernet et al. (1999), but adapted for this study with the addition two more reaction patterns, namely focal cellular alterations (FCAs) and intersex (IS) (Appendix 8):

- Reaction Pattern 1 – Circulatory Disturbances (CD): A specific pathology related to the changes in either blood or tissue fluid flow. - Reaction Pattern 2 – Regressive Changes (RC): This reaction pattern represents naturally occurring processes that have terminated in a functional reduction or loss in an organ or organ functional unit. In most cases, these alterations are reversible, given the right circumstances or environmental conditions. However, alterations can also be severe and irreversible in some cases, such as necrosis. - Reaction Pattern 3 – Progressive Changes (PC): Progressive changes relate to alterations within the tissues or cells that develop or increase the activity of specific functional processes. These alterations are less likely to be reversible, and as a result, they are considered to be more serious signs of adverse effects occurring within a system. - Reaction Pattern 4 – Inflammation (I) Numerous process can be associated with inflammatory responses, so for the purposes of this assessment, the presence of following alterations indicate inflammation: exudates, activation of reticulo-endothelial system and infiltration. - Reaction Pattern 5 – Tumour/Neoplasia (T): This reaction pattern represents the occurrence of lesions that have resulted from abnormal, uncontrollable growth, in which the cellular mechanism that used to operate normal cells no longer functions, correctly. - Reaction Pattern 6 – Focal Cellular Alterations (FCAs) An additional reaction pattern called a focal cellular alteration (FCA) is a reaction where a group of cells within the tissue exhibit structural differences to the surrounding tissue. These alterations can be quite severe and they are often precursor alterations to tumours. - Reaction Pattern 7 – Intersex (IS): This condition exists when individual specimens contain both male and female gonads.

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CHAPTER 3 –MATERIALS & METHODS

On the other hand, the gonads of male (testis) and female (ovaries) specimens were also staged according to their maturity and developmental stages (Table 3 – 2).

Table 3 - 2: Diagnostic histological features used to define developmental stages for testis (male) and ovaries (female) in fish. (McDonald et al., 2000) STAGE TESTIS CHARACTERISTICS OVARY CHARACTERISTICS Undeveloped: Undeveloped: Little or no spermatogenic activity in Pre-vitellogenic oocytes observed exclusively; 0 germinal epithelium; immature states of oocyte diameter <250 µm: cytoplasm stains spermatogenesis (largely spermatocytes); basophilic with H&E. no spermatozoa observed. Early spermatogenic: Early development: Mostly thin germinal epithelium with >90% pre-vitellogenic, remaining oocytes early to 1 scattered spermatogenic activity; mid-vitellogenic; oocytes slightly larger (up to spermatocytes to spermatids predominate; 300 µm); late perinucleolus through cortical few spermatozoa observed. alveolar stages. Mid-spermatogenic: Mid-development: Germinal epithelia are of moderate Majority of observed follicles are early and mid- thickness; moderate proliferation and vitellogenic; oocytes larger 300-600 µm 2 maturation of spermatozoa and equal mix diameter, and containing peripheral yolk of spermatocytes, spermatids and vesicles; globular and uniformly thick chorion; spermatozoa present. cytoplasm is basophilic, yolk globules eosinophilic. Late spermatogenic: Late development: Thick germinal epithelium; diffuse regions Majority of developing follicles are late 3 of proliferation and maturation of vitellogenic; oocyte diameter is 600-1000 µm; spermatozoa; all stages of development eosinophilic yolk globules distributed are represented, but spermatozoa throughout the cytoplasm; thicker chorion. predominate. N/A Late developmental/hydrated: 4 Majority of developing follicles are late vitellogenic; follicle are much larger (>1000 µm) N/A Post-ovulatory: 5 Spent follicles, remnants of theca externa and granulosa.

3.7.3. QUANTITATIVE HISTOLOGICAL ASSESSMENT

Following the above-mentioned qualitative assessment, for each alteration identified, the degree of pathology/severity throughout the tissue was given a ‘score value’ and a pathological importance factor was allocated an ‘importance value.’ With these values an organ index (Iorg), total reaction pattern index (ITot rp) and fish index (Ifish) was calculated per individual specimen collected, adapted from Bernet et al. (1999).

3.7.3.1. Score Value In the qualitative assessment, each histological alteration was observed and described

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CHAPTER 3 –MATERIALS & METHODS according to varying degrees of severity. These severities were quantified to a score value (SV) between zero and six (0, 2, 4 & 6) (Table 3 – 3):

3.7.3.2. Importance Factor Each histological alteration also carries an assigned Importance Factor (IF), which represents a degree of pathological importance (Appendix 8). Each alteration is scored between one and three (1, 2 & 3) depending on the reversibility of the specific alteration (Table 3 – 4):

Table 3 - 3: Score values (SV) used to quantify the extent of severity of histological alterations observed. SV DESCRIPTION 0 No occurrence of the alteration. 2 Mild occurrence of the alteration. (Focal area) 4 Moderate occurrence of the alteration. (<50% of tissue) 6 Severe occurrence of the alteration (>50% of tissue) Table 3 - 4: Importance factors associated to each histological alteration (Bernet et al., 1999). IF DESCRIPTION Minimal pathological importance: 1 The alteration is easily reversible, when the stress factor is removed or ends. Moderate pathological importance: 2 The alteration is reversible in most cases, when the stress factor is removed or ends. Marked/High pathological importance: 3 The alteration is usually irreversible, leading to partial or total loss of organ function

3.7.3.3. Calculation of Indices Four different indices were calculated for relative comparison, namely the reaction pattern index (Irp), organ index (Iorg), total reaction pattern index (ITot rp), and finally, the fish index

(Ifish) (Figure 3 – 8). The higher each index value, the more severe the organs were affected and as a result the less healthy respective individuals were estimated to be.

Figure 3 - 8: Schematic process for the calculation of indices in the quantitative histological assessment, adapted from Bernet et al. (1999). PAGE | 43

CHAPTER 3 –MATERIALS & METHODS

3.7.3.3.a. Reaction Pattern Index (Irp) This index represents the degree of damage within a single reaction pattern to a single organ of the individual fish species. It is calculated by the sum of multiplied SVs and IFs of all alterations identified within a single reaction pattern of a single specific organ per individual specimen. The following equation illustrates the calculation of this index:

푦 Irp = ∑푥=1(푆푉표푟푔(푎) 푟푝(푏) 푎푙푡(푥) × 퐼퐹표푟푔(푎) 푟푝(푏)푎푙푡(푥))

Irp = Reaction pattern index; SV = Score value; IF = Importance factor; org(a) = organ a; rp(b) = reaction pattern (b); alt(x) = alteration x; *Note: a = selected target organ, b = selected reaction pattern, x = number of alterations , y = last alteration listed during assessment.

3.7.3.3.b. Organ Index (Iorg) This index represents the degree of damage to a specific target organ across all reaction patterns. It is calculated by summing each of the reaction pattern indices (Irp) within the examined organ of each individual specimen. This index allows for relative comparisons of severity between the same organs of individual examined specimens across species and/or impoundments.

푦 Iorg = ∑푥=1(퐼푟푝(푥))

Iorg = Organ index; Irp = Reaction pattern index; *Note: x = number of rp, y = last rp within organ,

These responses were further classified according to their overall prevalence and severity of histological alterations observed, this classification system is based on Zimmerli, et al. (2007) and adapted from Van Dyk et al. (2009b) (Table 3 – 5). It should be noted that this classification system is specific to the organ indices only.

Table 3 - 5: Classification system by Zimmerli et al. (2007) adapted from Van Dyk et al. (2009b). CLASS INDEX RANGE PREVALENCE & SEVERITY 1 <10 Few histological alterations 2 10 – 25 Moderate histological alterations

3 26 - 35 Pronounced alterations of organ tissue

4 >35 Severe alterations of organ tissue

3.7.3.3.c. Total Reaction Pattern Index (ITot rp) This index represents the degree of damage of the histological alterations in all selected target organs of an individual fish specimen. It is the sum of the all the reaction pattern indices (Irp), across each of the assessed target organs, within the observed reaction

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CHAPTER 3 –MATERIALS & METHODS pattern of each individual specimen. This index allows for a comparison between the same reaction patterns observed within each individual specimen across each selected organ.

푦 ITot rp = ∑푥=1(퐼푟푝(푥))

ITot rp = Total reaction pattern index; Irp = Reaction pattern index; *Note: x = number of org, y = last org assessed.

3.7.3.3.d. Fish Index (Ifish) This index represents a measure of the overall health status of the individual specimen, based on the assessment of its observed histological alterations. It can be calculated in two ways, by summing all of the total reaction pattern indices of an individual fish, or by summing all the organ indices of an individual fish. An overall individual fish health can be compared as per individual using this index.

푦 푦 Ifish = ∑푥=1(퐼푇표푡 푟푝(푥)) Ifish = ∑푥=1(퐼표푟푔(푥))

Ifish = Fish index; ITot rp = Total reaction pattern index; Ifish = Fish index; Iorg = Organ index; *Note: x = number of rp, y = last rp assessed. *Note: x = number of org, y = last org assessed.

3.8. AGE ESTIMATION

Determining age in fish can be calculated using numerous calcified structures within each individual, such as the pectoral fins, the otoliths (middle ear bones), the scales, etc. However, a rapid and reliable approach was preferred and therefore otoliths were chosen for C. gariepinus as scales were absent and scales were used for the C. carpio.

3.8.1. OTOLITHS

Two otoliths were removed from the braincase of C. gariepinus and placed in labelled eppendorfs for freezing, storage and transportation. Samples were thawed, cleaned and air-dried at the laboratory before processing. Single otoliths per individual were then embedded in rods of industrial resin and transversely sliced through at the nucleus (central point) with a double-bladed diamond-edged otolith saw. In the instance of a lost, damaged or dirty otolith section, the second otolith was embedded and sliced for replacement. Sections were mounted on microscope slides using entellan and viewed through a light microscope (Figure 3 -9).

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CHAPTER 3 –MATERIALS & METHODS

Figure 3 - 9: Counting the number of annuli on an otolith for age estimation.

3.8.2. SCALES

Two to three scales were taken from each C. carpio specimen along the lateral line and directly below the anterior end of the dorsal fin, they were placed between two microscope slides and held together with sellotape to allow drying. Each scales’ annuli were counted and recorded on three separate occasions, at least seven days apart. Again, these values were recorded and the mode was selected at the end of the triplicate reading (Jones & Hynes, 1950; Gerber et al., 2009; Gerber, 2010).

3.9. EDIBILITY 3.9.1. MUSCLE ANALYSIS

Two samples of skeletal muscle fillets were collected during the necropsy – one was stored frozen in foil for detection of organic toxicants, whilst the other was stored frozen in plastic Zip-loc bags for inorganic toxicant analysis. Frozen samples were transported back to the laboratory and sent for chemical analysis at the same accredited ISO 17025 laboratories used for the water and sediment analysis: (i) FDA Labs (Pty) Ltd for organic analysis and (ii) Waterlab (Pty) Ltd for inorganic analysis. Due to the financial constraints of the study, the muscle samples were pooled into groups of five (5) specimens of the same species per site i.e. three (3) groups of five specimens per sample of 15 fish of the same species. Therefore, from a full quota sample of 15 specimens per species per impoundment, three (3) representative samples per species per impoundment were submitted to the laboratories for analysis.

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CHAPTER 3 –MATERIALS & METHODS

3.9.2. HUMAN HEALTH RISK ASSESSMENT (HHRA)

The model used was based on a CSIR risk analysis model. The model is used to characterise a toxic risk and a cancer risk from consuming these fish and any toxicants with a potential of causing adverse effects in the form of disease and/or cancer.

A total of 9 separate HHRA’s were calculated during the assessment in order to consider all scenarios. Firstly, 3 HHRA’s were calculated as per impoundment using the mean concentrations of the bioaccumulated toxicants detected within the muscle of both fish together i.e. the realistic risk posed from consuming one or both species over a chronic period. Secondly, 6 HHRA’s were calculated as per species per impoundment using the highest concentrations of the bioaccumulated toxicants detected within the muscle of each fish species separately i.e. risk posed during a ‘worst-case’ scenario through the consumption of only C. gariepinus or C. carpio from each impoundment over a chronic period.

3.9.2.1. Toxic Risk The calculation of the total dose (mg) is a standard calculation and consistent in both the toxic risk and cancer risk, it is calculated by multiplying the concentration of the toxicant within the fish muscle (Ctox; mg/kg) (obtained from bioaccumulation studies), the daily intake rate (IR) which was assumed to be 0.150 kg/day of fish muscle, and the exposure duration (ED) which was assumed to be 10 958 days (or 30 years):

Total Dose (푚푔) = Ctox (푚푔/푘푔) × IR (푘푔/푑푎푦) × ED (푑푎푦푠)

From the total dose, the average daily dose (ADD; mg/kg/day) can be determined by dividing the total dose by both the body weight (70 kg) of the consumer (BW; kg) and the abovementioned ED:

Total Dose (푚푔) ADD (푚푔/푘푔/푑푎푦) = BW (푘푔) × ED (푑푎푦푠)

Finally, the HQ or the toxic risk can be calculated and represents the comparison between the conservatively estimated exposures (ADD) and the RfD (mg/kg/day), which is an extrapolated exposure value with no associated toxic effect. Therefore, the HQ is a factor

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CHAPTER 3 –MATERIALS & METHODS of the RfD, whereby if it is greater than one (퐻푄 ≥1) – it is greater than or equal to the given RfD and potentially toxic, whereas if the value is between zero and one (0< 퐻푄 <1) – it is representative of only a fraction of the RfD and risk is considered to be very low and safe for consumption within parameters given. It is calculated as follows:

ADD (푚푔/푘푔/푑푎푦) HQ = RfD (푚푔/푘푔/푑푎푦)

3.9.2.2. Cancer Risk Calculating the cancer risk follows the same steps and calculation as shown above for the toxic risk, except that the ADD is used to calculate the lifetime average daily dose (LADD) rather than the HQ and thereafter the ADD is multiplied by a factor of the ED (10 958 days) against the average expected lifetime (Lft) for the consumer in the study area:

ED (days) LADD (푚푔/푘푔/푑푎푦) = ADD (푚푔/푘푔/푑푎푦) × Lft (days)

Finally, the cancer slope factor or cancer potency (훽; mg/kg/day), which represents the upper bound of the lifetime cancer risk for each particular toxicant, needs to be determined and multiplied by the LADD to give the resultant lifetime cancer risk for oral ingestion of contaminated fish:

Lifetime Cancer Risk = 훽 (푚푔/푘푔/푑푎푦) × LADD (푚푔/푘푔/푑푎푦)

3.10. STATISTICAL ANALYSIS

All data was carefully collated and accurately recorded, descriptive statistics were applied where necessary, in order to give a general condition of each species of the fish population at each impoundment respective to the index used.

All results from each respective index were compared by site/impoundment at Statkon (UJ’s Statistics Consultancy). Given the lack of normality within the samples according to the Kolmogorov-Smirov and/or Shapiro-Wilk tests, in addition to irregular skewness and kurtosis, non-parametric tests were used for comparison. The H-test after Kruskal & Wallis

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CHAPTER 3 –MATERIALS & METHODS was applied to each index to determine any significant difference between sites (2-tailed, p<0.05), after which a pair-wise U-test after Mann-Whitney was applied with a Bonferroni- correction (Dunn-Sidak procedure) to identify significant differences from the other sites (p<0.0167).

Reported results included the test statistic (H), degrees of freedom (d.f.) and a p-value for the Kruskal & Wallis H-test and the test statistics (U & z-value) and a p-value for the Mann & Whitney U-test.

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4. RESULTS

CHAPTER 1: IN THIS CHAPTER PROJECT 4.1. INTRODUCTION ...... 51 OVERVIEW 4.2. SUBSTRATE ANALYSIS ...... 51 4.2.1. Physico-chemical Parameters ...... 51 CHAPTER 2: 4.2.2. Chemical Analysis ...... 52 BACKGROUND PECIMEN OLLECTION 4.3. S C ...... 54 INFORMATION 4.4. NECROPSY-BASED HEALTH ASSESSMENT ...... 54 4.4.1. Haematological Assessment ...... 56 4.5. BIOMETRIC INDICES ...... 57 CHAPTER 3: 4.5.1. Condition Factor ...... 57 MATERIALS & 4.5.2. Organo-somatic Indices ...... 57 METHODS 4.6. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 59 4.6.1. Qualitative Histological Assessment ...... 59 CHAPTER 4: 4.6.2. Quantitative Histological Assessment ...... 76 4.7. AGE ESTIMATION ...... 79 RESULTS 4.7.1. Otoliths...... 79 4.7.2. Scales ...... 80 4.8. EDIBILITY ...... 80 CHAPTER 5: 4.8.1. Muscle Analysis ...... 80 DISCUSSION & 4.8.2. Human Health Risk Assessment (HHRA) ...... 81 CONCLUSION

APPENDICES

Romans 8: 24-25 – For we are saved by hope: but hope that is seen is not hope: for what a man seeth, why doth he yet hope for? But if we hope for that we see not, then do we with patience wait for it. CHAPTER 4 – RESULTS

4.1. INTRODUCTION

The results chapter will present and illustrate all the data and results recorded from the protocols mentioned in Chapter 3 – Materials & Methods. This data was compared to reference conditions from the reference site and/or normal or expected ranges, from previous studies and current legislature.

4.2. SUBSTRATE ANALYSIS 4.2.1. PHYSICO-CHEMICAL PARAMETERS

Table 4 – 1 summarises the mean in situ water quality parameters measured at various sites within each impoundment (Refer Chapter 3, Section 3.3.1) and representative of the water quality throughout the impoundment at the time of the assessment (Appendix 2).

Table 4 - 1: Mean physico-chemical water parameters measured morning, noon and afternoon at each impoundment. Mean values in Red exceed the target range/objective (DWA, 2011ba; DWAF, 1996b). TEMPERATURE CONDUCTIVITY TDS DISSOLVED OXYGEN TIME PH (OC) (mS/m) (mg/L) (mg/L) (% saturation) TARGET RANGE/OBJECTIVE 6.5 - 8.0a - <30 a <200 a - 80 – 120 b RD 09h00 9.35 25.90 45.12 225.20 17.11 212.04 12h00 9.18 28.30 45.34 226.86 17.66 226.27 17h00 9.43 27.70 46.08 229.80 19.80 233.88 MEAN 9.32 27.30 45.51 227.29 18.19 224.06 STDEV 0.13 1.25 0.50 2.33 1.42 11.09 VD 09h00 9.30 26.73 36.50 206.75 17.51 218.28 12h00 8.90 30.10 42.98 213.00 14.46 192.78 16h00 9.33 31.18 41.98 210.25 18.00 243.63 MEAN 9.18 29.33 40.49 210.00 16.65 218.23 STDEV 0.24 2.32 3.49 3.13 1.92 25.42 MBD 09h00 7.83 23.00 18.93 94.93 * * 12h00 7.71 23.75 18.80 93.90 * * 17h00 7.90 23.33 18.81 94.00 * * MEAN 7.81 23.36 18.84 94.28 * * STDEV 0.10 0.38 0.08 0.57 * * *Equipment malfunction – equipment was serviced and checked before field surveys.

With regards to the aesthetic condition of the impoundments, substantial amounts of Water Hyacinth (Eichhornia crassipes) (Figure 4 – 1A) and green algae (Figure 4 – 1B) were present at the RD. In addition, the wind had blown significant amounts of scum and foam as well as a dead fish onto the shore of RD at the time of the survey (Figure 4 – 1C). Conversely, the conditions observed at VD, which is located within a nature reserve and to a degree at MBD, which is associated with a bird sanctuary were different relative to the

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CHAPTER 4 – RESULTS

RD. The water was observed to be clear with relatively little turbidity or green algae present (Figure 4 – 1D).

A B

C D

Figure 4 - 1: Substantial amounts of (A) Water Hyacinth (Eichhornia crassipes) and (B) green algae were observed at Roodekopjes Dam. (C) A dead fish was also blown to the shore together with foam and scum. (D) Clear water was observed on the shore of the reference site – Marico-Bosveld Dam.

4.2.2. CHEMICAL ANALYSIS

All detected toxicants were compared to the TWQR for Aquatic Ecosystems (DWAF, 1996) or the Lowest Effect Level (LEL) values recorded by the National Oceanic and Atmospheric Administration (NOAA) Screening Quick Reference Tables (SQuiRTs) (Buchman, 2008), dependent on the lower value.

4.2.2.1. Organic Organic toxicants detected in the pooled water and sediment samples were selected from the raw data in Appendix 2 (Table A – 4) and shown in Table 4 – 2. The impoundment with the highest detected concentration is indicated in red.

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CHAPTER 4 – RESULTS

Table 4 - 2: Incidence of selected organic toxicants detected in pooled water and sediment samples. Values that exceed the target/objective range are indicated in Red. CONCENTRATION (ppb) TOXICANT WATER SEDIMENT Target* RD VD MBD Target* RD VD MBD Terbuthylazine ------Aldrin 1.50c - - 0.59 2.00b - - - op'-DDD 0.19c 0.11 - 0.62 8.00b - - - op'-DDE 1050c 0.12 0.15 - 4.00b - - - op'-DDT 0.55c - 0.58 1.41 8.00b - - - p-NP Technical 30000.00a - 3000.00 - 1400000.00d 64800.00 69900.00 75500.00 * Target/Objective: a - TWQR for Aquatic Ecosystems (DWAF, 1996), b – LEL for NOAA SQuiRTs, c – Acute value for NOAA SQuiRTs, d – TEL value for NOAA SQuiRTs (Buchman, 2008)

4.2.2.2. Inorganic Inorganic toxicants detected in the pooled water and sediment samples were selected from the raw data in Appendix 2 (Table A – 5) and shown in Table 4 – 3. The impoundment with the highest detected concentration is indicated in red. The highest inorganic concentrations in water were observed in the RD and to a lesser degree in VD, whilst with regards to the sediment composition, there was no clear trend observed between the sites.

Table 4 - 3: Incidence of selected metals detected in pooled water and sediment samples. Values that exceed the target/objective (Tar.) range are indicated in Red. CONCENTRATION (ppb) TOXICANT WATER SEDIMENT Tar.* RD VD MBD Tar.* RD VD MBD Ag 1.6c - 0.073 - 500b 493.014 255.949 760.697 Al 10a 19.560 5.454 1.540 - 29 520.958 40 511.898 44 096.672 As 10a 0.031 0.010 - 6000b 131.737 107.978 130.745 B 30c 1.295 0.161 0.152 - 3 067.864 3 331.334 3 199.287 Ba 110c 0.105 0.086 0.044 - 4 900.200 5 560.888 5 225.832 Be 35c ------Ca - 84.910 42.760 22.920 - 21 556.886 48 850.230 20 839.937 Cd 2c - - - 600b - 3.999 - Cr 12a - 0.002 - 26000b 389.222 405.919 657.686 K 373000c 6.846 6.459 1.305 - 8 321.357 8 530.294 13 801.506 Mg - 18.360 18.680 12.770 - 8 351.297 17 504.499 17 373.217 Mn 180a 0.353 0.171 0.045 460000b 1 876.248 2 541.492 1 555.071 Mo 16000c ------Na - 57.000 31.690 5.995 - 17 704.591 18 734.253 20 740.887 Ni 470c - - - 16000b 119.760 183.963 190.174 P - 1.361 2.009 1.055 - 2 497.006 - 774.564 Pb 0.2a 0.033 - 0.017 31000b 109.780 63.987 87.163 Sb 88c - - 0.001 - 1.996 - - Se 2a 0.176 0.180 0.136 - 299.401 269.946 283.281 Sr 15000c 0.496 0.257 0.059 - 83.832 149.970 65.372 V 280c 0.019 0.014 0.001 - 259.481 311.938 289.223 Zn 2a 0.007 0.001 - 120000b 3 465.070 3 731.254 3 664.818 * Target/Objective (Tar.): a - TWQR for Aquatic Ecosystems (DWAF, 1996), b – LEL for NOAA SQuiRTs, c – Acute value for NOAA SQuiRTs, d – TEL value for NOAA SQuiRTs (Buchman, 2008) PAGE | 53

CHAPTER 4 – RESULTS

4.3. SPECIMEN COLLECTION

The following table describes the catch composition at each impoundment (Table 4 – 4). All specimens were separated by sex as per sampled site, and totalled to give a full sample size and sex ratio (males: females). A full target sample size was not attained at VD and MBD for C. carpio.

Table 4 - 4: General characteristics of specimens collected per site per species.

SPECIES GENDER NUMBER OF SPECIMENS TOTAL

RD VD MBD Clarias gariepinus Male 9 10 11 30 Female 6 5 4 15 Sample Size (n) 15 15 15 45 Sex Ratio (M: F) 3 : 2 2 : 1 11 : 4 2 : 1 Cyprinus carpio Male 7 1 6 14 Female 8 13 3 24 Sample Size (n) 15 14 9 38 Sex Ratio (M: F) 1 : 1 1 : 13 2 : 1 7: 12

4.4. NECROPSY-BASED HEALTH ASSESSMENT

Each collected specimen was macroscopically assessed according to the abnormalities mentioned in Table 3 – 1 using the necropsy-based health assessment data sheet (Appendix 1). Generally, all the external organs and tissues (eyes, skin, fins, and opercula) were found to be normal with no abnormalities in either species at all impoundments. However, a single skin lesion was found on a single C. carpio specimen which was likely to be associated with a bacterial infection.

With regards to the internal organs and tissues (gills, liver, spleen, hindgut and kidney), all observations were considered normal, with the exception of a few abnormalities found in the gills and in the liver (Figure 4 – 2). Gills were observed to be slightly pale at times, most likely, as a result of an extended struggling time in the gill nets. With this exception, there were only two other occasions where abnormalities were observed within the gills: a (i) discolouration was observed on the surface of the gills of C. carpio at MBD, and a (ii) cyst was found within the gill filaments of C. gariepinus at the RD, which was sampled and fixed for the histology-based fish health assessment (HBFHA). Other abnormalities within the liver included vast discolouration, from light green to a ‘coffee and cream’ colour between species across all impoundments, as well as the presence of fatty nodules, and a nematode within the liver of a single C. gariepinus at the MBD. Furthermore, there was also some variation observed within the size of the gonads between species and sites, as some were

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CHAPTER 4 – RESULTS seemingly under-developed and on one occasion, even absent on the one side within a female C. carpio at RD.

A B

C D

Figure 4 - 2: Abnormalities during necropsy included a (A) slightly discoloured appearance of the gills, the (B) presence of a cyst between gill filaments, (C) livers with fatty deposits and/or nodules and (D) livers that were vastly discoloured.

Finally, the presence of parasites (Figure 4 – 3) was potentially the most common observation during the necropsy-based assessment, whereby nematodes were observed within the visceral cavity at varying loads in both species and at all impoundments. However, in overview, the parasite load in C. carpio was relatively low or absent, whilst in most observations, the parasite load in C. gariepinus was always higher.

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CHAPTER 4 – RESULTS

Figure 4 - 3: Severe prevalence of nematode parasites in C. gariepinus at Vaalkop Dam (VD).

4.4.1. HAEMATOLOGICAL ASSESSMENT

The haematological assessment of each specimen included the haematocrit (Hct) and leukocrit (Lct), the mean values are illustrated in Figure 4 – 4 below (Appendix 3). The mean Hct and Lct for both species across all sites was observed to be within the normal ranges between 30-45% for the Hct and less than 4% for the Lct (Adams et al., 1993).

A B

Figure 4 - 4: Mean (A) haematocrit and (B) leukocrit values from each site per species in relation to normal range (grey bars). Italic letters denote statistical differences: same letter = no significant difference.

The mean Hct values showed no significant differences (Figure 4 – 4A) between sites for C. gariepinus (H=3.675, d.f.=2, p=0.159) and C. carpio (H=1.494, d.f.=2, p=0.474), whereas the mean Lct values showed some significant differences (Figure 4 – 4B). The white blood

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CHAPTER 4 – RESULTS cell volume seemed significantly lower for C. gariepinus at RD relative to the reference site (U=53.0, z=-2.472, p<0.05), whilst a significant difference was evident for the C. carpio at RD relative to the VD (U=26.0, z=-3.453, p<0.01).

4.5. BIOMETRIC INDICES 4.5.1. CONDITION FACTOR

Figure 4 – 5 illustrates the mean condition factor of C. gariepinus and C. carpio in each of the assessed sites, taken from the individual data shown in Appendix 4.

Figure 4 - 5: Graph to show difference in condition factor (CF) between fish species and sites. Italic letters denote statistical differences: same letter = no significant difference.

The mean condition factors for the C. gariepinus ranged from 0.68 ± 0.09 at MBD to 0.80 ± 0.16 at RD, whilst the C. carpio ranged from 1.19 ± 0.13 to 1.47 ± 0.11 at MBD and RD, respectively. With regards to the catfish, both RD (U=37.0, z=-3.132, p<0.01) and VD (U=52.5, z=-2.489, p<0.05) were found to be significantly different from MBD. This indicates that the catfish from the assessment sites may have been better nourished than those at the reference site. However, it is more likely that these specimens were heavier relative to their length because of the increased biomass, algae and/or sediment that is indirectly ingested during feeding. Furthermore, only the condition factors for C. carpio from RD showed a significant difference from VD (U=27.5, z=-3.383, p<0.01) and MBD (U=6.0, z=-3.667, p<0.01).

4.5.2. ORGANO-SOMATIC INDICES

All the masses for each respective organ taken during the field surveys were used to calculate their respective organo-somatic index (Appendix 4). Figure 4 – 6 illustrates the

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CHAPTER 4 – RESULTS mean hepatosomatic index (HSI), splenosomatic index (SSI), and separate gonadosomatic indices (GSI).

The mean HSI values (Figure 4 – 6A) indicated the livers of the C. gariepinus at RD (U=10.0, z=-4.338, p<0.01) and VD (U=49.0, z=-2.712, p<0.05) to be significantly higher than those at MBD. This may indicate a pathology-induced enlargement of the detoxification organ. Whereas, there were no significant differences observed between sites within the mean HSI values of C. carpio (H=0.536, d.f.=2, p=0.765).

The mean SSI values (Figure 4 – 6B) showed no significant differences between sites for the C. gariepinus (H=3.562, d.f.=2, p=0.168), whilst there was a significantly higher mean SSI value observed at RD within the C. carpio relative to both VD (U=19.0, z=--3.955, p<0.01) and MBD (U=9.0, z=-3.681, p<0.01) . A B

C D

Figure 4 - 6: Mean organo-somatic indices calculated for both species per site: (A) Hepatosomatic index (HSI), (B) Splenosomatic index (SSI), and Gonadosomatic index for (C) male and (D) female. Italic letters denote statistical differences: same letter = no significant difference.

The mean male GSI values (Figure 4 – 6C) for C. gariepinus showed significantly higher values at RD relative to the reference site (U=10.0, p<0.05), but no significant difference was observed between VD and the reference site (U=44.5, p=0.704). Whereas no significant differences (H=0.103, d.f.=2, p=0.950) were observed for the male C. carpio between sites. The mean female C. gariepinus GSI values (Figure 4 – 6D) showed no significant differences between all the sites (H=5.618, d.f.=2, p=0.0603). However, the mean female GSI values for C. carpio showed that values from VD were significantly higher than RD (U=18.0, p<0.05), but not significantly different from MBD (U=9.0, p=0.189).

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4.6. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) 4.6.1. QUALITATIVE HISTOLOGICAL ASSESSMENT

Each of the target organs were assessed for histological alterations. The prevalence of these alterations was calculated for each individual sample population. The severity of the alterations were defined by the amount of tissue affected within the tissue sample and conservatively graded according to Table 3 -3.

The following tables represent the percentage prevalence and severity of respective histological alterations observed during the qualitative assessment of each species, as per site and per target organ: (i) gills (Table 4 - 5), (ii) liver (Table 4 - 6), (iii) kidney (Table 4 - 7), and (iv) heart (Table 4 - 8). No histological alterations were observed within the gonads (testes and ovaries) or the skin. However, Table 4 – 9 shows the developmental stages observed within the gonads of each sample group and the observed normal structure for the gonads (testes and ovaries). Accompanying these tables, are figure plates, which are representative of the observed histological alterations within each target organ.

The gills showed a number of histological alterations, but these were largely focal areas of change. Figure 4 – 7 shows the normal structure of the gills from C. gariepinus (A) and C. carpio (B). The following micrographs represent some of the more prominent histological alterations observed during the assessment such as congestion (C), secondary lamellae branching (D), epithelial hyperplasia (E), and in extreme cases, fusion (G), aneurysms or telangiectasia (F) and epithelial lifting (H). In addition, the presence of at least two different parasites attached between the secondary lamellae (I-J) were an interesting finding. However, there was no clear histological alterations observed as a direct result of attachment of these parasites.

The liver showed the most severe histological alterations of all of the assessed target organs, especially for C. gariepinus specimens, where areas affected were usually evenly spread through the majority of the tissue (Figure 4 – 8). Relative to the normal histological liver structure of both species (A-B), numerous alterations were observed at low to medium prevalence, namely steatosis (C), the presence of cellular deposits (D), leukocyte infiltration (E) and focal necrosis (F) of hepatocytes (Table 4 – 6; Figure 4 – 8). The presence of melano-macrophage centers (MMCs) was the most prevalent alteration observed (G), especially within the C. gariepinus. On the other hand, the occurrences of various forms of pre-carcinogenic FCAs (G-H) in C. gariepinus at VD and MBD were a potential cause for concern.

The kidney (Figure 4 – 9) exhibited a largely mild histological response to the pollution at

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CHAPTER 4 – RESULTS the assessment sites, as alterations were present at low prevalences and in focal areas of the tissue. The presence of some MMCs in both species and thyroid follicles, as well as Corpuscle of Stannius, in C. carpio were considered normal for kidney tissue (A-C). The most prevalent alterations observed within the kidney in both species was eosinophilic cytoplasm of the tubules (G) and glomerular atrophy (H). Whilst intracellular oedema (D) and infiltration of lymphocytes (E) was only observed in a few cases. Furthermore, there was also evidence of a mild inflammatory response in the kidney of the C. gariepinus at RD, as indicated by leukocyte infiltration (F).

Relative to the normal observed structure of the heart (Figure 4 – 10 A-E), only two general alterations were observed within the heart during the assessment, namely a mild form of vacuolation (E) and an inflammatory response indicated by leukocyte infiltration (F). The low prevalence exhibited suggested that the heart was potentially less vulnerable to pollution relative to other organs. However, it is also possible that the toxicants accumulated within these fish did not affect the heart to the same degree as the liver or kidney.

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Table 4 - 5: Mean qualitative assessment of the gill alterations in both species at all sites. PREVALENCE (%) AND DESCRIPTION OF SEVERITY (Des.) GILLS Clarias gariepinus Cyprinus carpio RD (n = 13) VD (n = 14) MBD (n = 15) RD (n = 13) VD (n = 14) MBD (n = 9)

ALTERATION % Des. * % Des. * % Des. * % Des. * % Des. * % Des. *

Telangiectasia (CD) 46 MILD 57 MILD 7 MILD 15 MILD 50 MILD 78 MILD

Congestion (CD) 8 MILD 21 MILD 7 MILD 46 MILD 64 MILD 56 MILD

Primary lamellae branching (RC) - - - - 7 MILD - - - - 11 MILD

Secondary lamellae branching (RC) 8 MILD 7 MILD 7 MOD. 8 MILD - - 22 MILD

Epithelial vacuolation (RC) ------11 MILD

Rupture of Pillar Cells (RC) 15 MILD 21 MILD 7 MILD 46 MILD 57 MILD 56 MILD

Epithelial lifting (RC) 8 MILD 14 MILD 13 MILD - - 7 MILD - -

Intracellular deposits (RC) - - - - 7 MILD ------

Hyperplasia - epithelium (PC) 77 MOD. 36 MILD 93 MILD 62 MILD 50 MILD 56 MOD.

Hyperplasia - mucous cells (PC) - - - - 7 MOD. - - - - 22 MILD CD – Circulatory Disturbances, RC – Regressive Changes, PC – Progressive Changes, I – Inflammation, T – Tumour, FCA – Focal Cellular Alteration * Mod. - moderate.

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Figure 4 - 7: Histological alterations in the gills A – B: Micrographs showing observed normal structure: A. Clarias gariepinus (C. g.) B. Cyprinus carpio (C. c.) C – H: Micrographs representing observed histological alterations: C. Congestion (*) of secondary lamellae (C. g.) D. Secondary lamellae branching (arrows) (C. g.) E. Epithelial hyperplasia (arrows) (C. g.) F. Severe telangiectasia/aneurysms (*) of secondary lamellae (C. c.) G. Fused secondary lamellae (fusion) from severe epithelial hyperplasia (C. g.) H. Epithelial lifting of secondary lamellae (C. g.) I – J: Micrographs showing parasites observed: I. Parasite located between the secondary lamellae (C. g.) J. Parasite, possible trematode (Class Trematoda) located between secondary lamellae (C. g.)

A B

Secondary lamella

Primary lamella

200 µm 100 µm

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C D

A *

*

*

50 µm 50 µm * E F A

* *

50 µm 100 µm

G H A

*

* 100 µm 200 µm

I J A

50 µm 50 µm

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Table 4 - 6: Mean qualitative assessment of the liver alterations in both species at all sites. PREVALENCE (%) AND DESCRIPTION OF SEVERITY (Des.) LIVER Clarias gariepinus Cyprinus carpio RD (n = 13) VD (n = 15) MBD (n = 15) RD (n = 15) VD (n = 14) MBD (n = 9)

ALTERATION % Des. * % Des. * % Des. * % Des. * % Des. * % Des. *

Intracellular Deposits (RC) 31 MOD. 20 MOD. 27 SEVERE ------

Necrosis (RC) - - 7 MILD ------

Nuclear alterations (RC) 23 MILD 7 SEVERE - - 13 MILD - - 33 MOD.

Steatosis (RC) 15 MILD 20 MILD 7 MILD 13 SEVERE - - - -

Vacuolation (Not Steatosis) (RC) 23 MOD. 13 MILD 20 MOD. 7 MILD 50 SEVERE 100 SEVERE

Melano-macrophage Center (RC) 92 MILD 33 MOD. 73 MOD. 13 MILD 7 SEVERE - -

Infiltration (I) 54 MILD 20 MILD 7 MOD. 27 MILD - - - -

Clear cell foci (FCA) - - - - 7 MILD ------

Steatosis foci (FCA) - - - - 7 MILD ------

Eosinophilic foci (FCA) - - 7 SEVERE 7 MILD ------

Hypertrophic foci (FCA) - - 7 SEVERE 13 MILD ------CD – Circulatory Disturbances, RC – Regressive Changes, PC – Progressive Changes, I – Inflammation, T – Tumour, FCA – Focal Cellular Alteration * Mod. - moderate.

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Figure 4 - 8: Histological alterations in the liver A – B: Micrographs showing observed normal structure: A. Clarias gariepinus (C. g.) with vascular system B. Cyprinus carpio (C. c.) with intra-hepatic pancreatic tissue C – H: Micrographs representing observed histological alterations: C. Steatosis or fatty vacuolation (*) (C. c.) D. Intracellular deposits (*) within hepatocytes (C. g.) E. Infiltration (*) with melano-macrophage centres (MMC’s; arrows) (C. g.) F. Focal necrosis (^) of hepatocytes, with MMC’s (arrows) (C. g.) G. Three focal cellular alterations (FCA’s; dashed circles) with MMC’s (arrows) (C. g.) H. Eosinophilic hypertrophic foci (*) (640X magnification) (C. g.)

A B

Hepatocytes

Nucleus

Vein

Intra-hepatic

Pancreatic Tissue

50 µm 50 µm

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C D

* *

* * 50 µm 50 µm * * E F

* ^ ^ * ^

50 µm 50 µm

G

200 µm H

*

50 µm

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Table 4 - 7: Mean qualitative assessment of the kidney alterations in both species at all sites. PREVALENCE (%) AND DESCRIPTION OF SEVERITY (Des.) KIDNEY Clarias gariepinus Cyprinus carpio RD (n = 13) VD (n = 12) MBD (n = 15) RD (n = 12) VD (n = 12) MBD (n = 9)

ALTERATION % Des. * % Des. * % Des. * % Des. * % Des. * % Des. * Aneurysm (CD) - - 8 MILD ------Intracellular oedema (CD) - - - - 7 MOD. - - - - 11 MILD Vacuolation (RC) - - 25 MILD 13 MILD 42 MILD - - 33 MILD Hyaline droplet degeneration (RC) 15 MILD - - 7 MILD 8 MOD. - - - - Eosinophilic cytoplasm (RC) 54 MILD 17 MILD 33 MILD 17 MILD 21 MOD. - - Granular degeneration (RC) ------7 MOD. - - Intercellular deposits (RC) ------7 MOD. 11 MILD Nuclear alterations (RC) 23 MILD ------7 MILD - - Atrophy - tubules (RC) - - 8 MILD - - - - 14 MILD - - Atrophy - glomerulus(RC) 38 MILD - - 13 MILD 17 MILD - - - - Necrosis (RC) 23 MILD 8 MILD - - 8 MOD. - - - - Melano-macrophage centers (RC) 69 MILD 17 MILD 67 MILD 8 MILD 36 MILD 11 MILD Hypertrophy (PC) 8 MILD ------Infiltration (I) 8 MILD ------CD – Circulatory Disturbances, RC – Regressive Changes, PC – Progressive Changes, I – Inflammation, T – Tumour, FCA – Focal Cellular Alteration * Mod. - moderate.

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Figure 4 - 9: Histological alterations in the kidney A – C: Micrographs showing observed normal structure: A. Clarias gariepinus (C. g.) B. Cyprinus carpio (C. c.) showing thyroid follicles C. Cyprinus carpio (C. c.) showing Corpuscle of Stannius (*) D – H: Micrographs representing observed histological alterations: D. Intercellular oedema (*) and red blood cells located in hematopoietic tissue (C. g.) E. Hyaline droplet degeneration (^) within epithelium of tubules (C. g.) F. Infiltration (*) of lymphocytes (white blood cells) (C. g.) G. Eosinophilic cytoplasm (^), tubular necrosis (arrows) and melano-macrophage centres (MMC’s; *) (C. g.) H. Glomerular atrophy (*) and MMC’s (arrows) (C. c.)

A B

Tubules

Haematopoietic tissue

Glomerulus

Thyroid follicle

50 µm 50 µm

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C D

* * 100 µm 100 µm

E F

^ * ^ * ^ *

50 µm ^ 50 µm

G H

* ^ ^ * ^ * 50 µm 50 µm

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Table 4 - 8: Mean qualitative assessment of the heart alterations in both species at all sites. PREVALENCE (%) AND DESCRIPTION OF SEVERITY (Des.) HEART Clarias gariepinus Cyprinus carpio RD (n = 15) VD (n = 15) MBD (n = 15) RD (n = 14) VD (n = 12) MBD (n = 9) ALTERATION % Des. * % Des. * % Des. * % Des. * % Des. * % Des. * MMC - Atrium (RC) - - - - 7 MILD - - 8 MILD - - MMC - Ventricle (RC) - - - - 7 MILD - - 8 MILD - - Vacuolation - Atrium(RC) - - 20 MILD - - - - 8 MILD - - Vacuolation - Ventricle (RC) 20 MOD. 27 MILD - - 7 MILD 58 MILD - - Vacuolation - BA (RC) 20 MILD 33 MILD - - - - 25 MILD - - Nuclear alterations - Atrium (RC) ------8 MILD - - Atrophy - Atrium (RC) - - 7 MOD. ------Necrosis - Atrium (RC) 13 MILD 13 MILD 20 MILD 7 MILD 17 MILD 22 MILD Necrosis - Ventricle (RC) - - 13 MILD 7 MILD - - - - 11 MILD Necrosis – BA (RC) - - - - 13 MILD ------Myocarditis - Atrium (I) 7 MILD - - - - 7 MILD - - - - Myocarditis - Ventricle (I) 13 MILD - - 13 MILD 7 MILD 8 MILD - - Myocarditis - BA (I) 7 MILD 7 MILD - - 7 MILD - - 11 MILD Epicarditis – Atrium (I) ------7 MILD - - - - Epicarditis - Ventricle (I) - - 7 MILD ------Epicarditis - BA (I) - - 27 MILD ------CD – Circulatory Disturbances, RC – Regressive Changes, PC – Progressive Changes, I – Inflammation, T – Tumour, FCA – Focal Cellular Alteration * Mod. - moderate.

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Figure 4 - 10: Histological alterations in the heart A – C: Micrographs showing observed normal structure: A. Atrio-ventricular valve located between atrium and ventricle in C. gariepinus (C. g.) B. Atrium, showing the epicardium layer (*) and myocardium layer (^) (C. g.) C. Ventricle (cardiac muscle; arrows) with red blood cells (*) (C. g.) D. Bulbus arteriosus, showing elastic fibers (arrows) (C. g.) E. Adipose tissue (^) in the epicardium of the ventricle (C. g.) F: Micrograph representing observed histological alterations: F. Epicarditis (*) of the bulbus arteriosus (C. g.)

A Blood

Atrial myocardium Atrium Atrial endocardium

Atrio-ventricular Valve

Ventricular epicardium

Ventricle

2000 µm

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B C

* * ^

100 µm 50 µm

D E

*

* *

50 µm 200 µm

F

*

50 µm

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The sexual maturity and developmental stages were determined whilst assessing the gonads for pathology (Table 4 – 9). It should be noted that numerous stages may be present within gonads simultaneously, but the developmental stage is often determined by the stage which is most prevalent at the time of sampling. The vast majority of both male fish species were fully mature (Stage 3) at the time of the assessment, whilst the majority of female specimens were mid-developed (Stage 2) and late-developed (Stage 3) at RD and VD for C. gariepinus. Figure 4 – 11 shows no pathology for the testes (A) or the ovaries (D). However, various stages of spermatogenesis (spermatogonia (B) > spermatocyte > spermatids (B) > spermatozoa (C)) and oogenesis (oogonia (E) > oocyte (E) > ovum) were present. Atretic or dissolving follicles (F) were also observed in some of the female specimens.

Table 4 - 9: Percentage prevalence of each developmental stage present within the sample group.

STAGE PREVALENCE IN MALES (%)

Clarias gariepinus Cyprinus carpio TESTES RD (n = 9) VD (n = 8) MBD (n = 11) RD (n = 7) VD (n = 1) MBD (n = 6)

Stage 0 ------

Stage 1 - 37.5 9.1 - - -

Stage 2 66.7 25.0 36.4 - - -

Stage 3 33.3 37.5 54.5 100.0 100.0 100.0

STAGE PREVALENCE IN FEMALES (%)

Clarias gariepinus Cyprinus carpio OVARIES RD (n = 6) VD (n = 5) MBD (n = 4) RD (n = 8) VD (n = 13) MBD (n = 3)

Stage 0 ------

Stage 1 - 20.0 - - - -

Stage 2 16.7 20.0 100.0 87.5 69.2 100.0

Stage 3 66.7 40.0 - 12.5 23.1 -

Stage 4 16.7 20.0 - - 7.7 -

Stage 5 ------

- denotes an absence of this stage within the sample group

Normal structure of the skin (A-C) was clearly observed (Figure 4 – 12), showing taste buds, goblet cells, and possible pigmentation. With the exception of a few parasite-induced granulomas (D) in the epidermis of the skin, no other pathology was observed.

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Figure 4 - 11: Histological alterations in the gonads (testes & ovaries) A. Testicular tissue showing various stages of spermatogenesis in C. gariepinus (C. g.) B. Spermatogonia (arrows) and early spermatids (^) (C. g.) C. Spermatozoa within seminiferous lobule (*) surrounded by lobule wall (^) (C. g.) D. Ovarian tissue showing various stages of oogenesis in C. carpio (C. c.) E. Primary oocytes (*) & mature oocytes (^) (C. c.) F. Atretic follicles (arrows) within in ovarian tissue (C. c.)

A B

^

^ 2000 µm 50 µm

C D F

*

^

50 µm 2000 µm

E F *

^ * * 100 µm ^ 200 µm

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Figure 4 - 12: Histological alterations in the skin A – C: Micrographs showing observed normal structure: A. Clarias gariepinus (C. g.), showing alarm or club cells (*), goblet cells (^) and melanin pigmentation (arrows) B. Cyprinus carpio (C. c.), showing scale (^) enveloped by epidermis C. Epidermal (*), dermal (arrows) and hypodermal (^) layers of skin (C. g.) D: Micrographs showing parasites observed: D. Epidermal parasite-induced granulomas (*) (C. c.)

A D F

* ^ ^ * *

50 µm

B

*

^

* 200 µm

C * *

*

^ 200 µm 100 µm

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4.6.2. QUANTITATIVE HISTOLOGICAL ASSESSMENT

The mean organ (Iorg) and total reaction pattern (ITot rp) indices were calculated per species per impoundment (Appendix 5) and they are representative of the mean histological response in each respective target organ assessed or reaction pattern observed.

4.6.2.1. Organ Indices (Iorg)

Figure 4 - 13 illustrates the difference of means in the assessed organ indices (Iorg). Since no histological alterations were observed within the gonads (testes & ovaries) and skin, these indices will not be illustrated or discussed.

A B

C D F

Figure 4 - 13: Graph to show differences in mean organ indices (Iorg) from quantitative assessment per species per impoundment: (A) gill index (Igill), (B) liver index (Iliver), (C) kidney index (Ikidney) & (D) heart index (Iheart). Italic letters denote statistical differences: same letter = no significant difference. Orange line denotes class category according to Van Dyk et al. (2009b).

The mean gill indices (Igill) showed that all alterations observed for all the impoundments and for both species fell into Class 1 (few histological alterations) except the C. carpio at MBD which fell into Class 2 and showed moderate histological alterations. Furthermore, there was no significant differences observed within the mean gill indices (Igill) between sites for C. gariepinus (H=2.965, d.f.=2, p=0.227) or C. carpio (H=4.344, d.f.=2, p=0.114).

With regards to the mean liver indices (Iliver), all observed alterations from all sites and between species classed each sample group into Class 1 (Few histological alterations), except C. gariepinus group at RD (Class 2). These mean index values showed no significant differences between impoundments for C. gariepinus (H=1.820, d.f.=2, p=0.403). However, the mean liver indices (Iliver) for C. carpio for both RD (U=21.0, z=-2.889, p<0.05) and VD (U=21.0, z=-2.954, p<0.05) were observed to be significantly lower than the reference site.

As for the mean kidney indices (Ikidney), moderate histological alterations (Class 2) were

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CHAPTER 4 – RESULTS again observed at RD for C. gariepinus, whilst the remaining observations showed few histological alterations (Class 1) at VD and MBD for C. gariepinus and all sites for C. carpio. The values calculated for C. gariepinus at RD were significantly greater than those values at VD (U=38.5, z=-3.164, P<0.01), but not significantly different to those at MBD (U=57.0, z=-2.359, P=0.018). No significant differences were observed within the C. carpio between all sites (H=1.289, d.f.=2, p=0.525).

All heart index (Iheart) values for both species and all sites were shown to have few histological alterations (Class 1). No significant differences were observed between sites for C. gariepinus (H=2.999, d.f.=2, p=0.223) and C. carpio (H=2.738, d.f.=2, p=0.254).

4.6.2.2. Total Reaction Pattern Indices (ITot rp) Figure 4 - 14 illustrates the difference of means in the assessed total reaction pattern indices (ITot rp). This comparison indicates the types of alterations most prevalent within assessed target organs of the fish from the assessed sites. Since there was no evidence of tumours or intersex in any of the assessed specimens, the graphs showing these total reaction pattern indices will be omitted.

A B

C D F

E F

Figure 4 - 14: Graph to show differences in mean total reaction pattern indices (ITot rp) from quantitative assessment per species per impoundment: (A) circulatory disturbance index (Icd), (B) regressive changes index (Irc), (C) progressive changes index (Ipc), (D) inflammation index (Ii) & (E) focal cellular alterations index (Ifca). Italic letters denote statistical differences: same letter = no significant difference.

Although the mean values for the circulatory disturbance index (Icd) were low for both species at all three sites, a significant difference was observed in both species. The values

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CHAPTER 4 – RESULTS from the C. gariepinus at VD were significantly different from those at the reference site (U=56.5, z=-2.669, p=<0.05), but not significantly different from the values at RD (U=88.0, z=-1.124, p=0.261). Conversely, the C. carpio showed that the values from MBD were significantly higher than RD (U=27.0, z=-2.527, p<0.05) and no significant difference was evident at VD (U=40.0, z=-1.535, p=0.125).

Comparatively-speaking, the regressive changes recorded the most number of change within the target organs of the collected fish at the selected sites. No significant differences existed between sites for the mean regressive changes index (Irc) for C. gariepinus (H=3.011, d.f.=2, p=0.222). However, within the mean values obtained for C. carpio, the MBD was found to be significantly higher than RD (U=25.5, z=-2.516, p<0.05), whilst no significant difference was evident with VD (U=50.0, z=0.824, p=0.410).

All mean values for the progressive changes index (Ipc) were found to show few histological alterations. The mean values calculated for C. gariepinus at the MBD was significantly higher than the values calculated for those specimens at VD (U=34.0, z=-3.452, p<0.01), but not significantly different from the RD (U=95.5, z=-0.737, p=0.461). No significant differences were observed for the mean progressive index (Ipc) values for C. carpio (H=0.822, d.f.=2, p=0.663).

As for the mean inflammation index (Ii) values, only a few histological alterations were observed in both species at all impoundments. The mean index (II) values for C. gariepinus at RD were significantly different to the mean values at MBD (U=59.0, z=-2.584, p<0.05), but no significant difference was evident between VD and the reference site (U=89.5, z=-

1.289, p=0.197). With regards to C. carpio, the mean values for the inflammation index (Ii) showed no significant difference between sites (H=5.592, d.f.=2, p=0.061).

With regards to the FCA’s, not many alterations were observed between the mean values for the focal cellular alterations index (Ifca). Within the C. gariepinus group, no alterations were observed within the specimens from the RD, but FCAs were observed during the assessment at the VD and MBD, which showed no significant differences from one another or RD (H=2.003, d.f.=2, p=0.367). There was no evidence of FCAs observed within the C. carpio specimens from all three sites.

4.6.2.3. Fish Index (Ifish)

Lastly, the mean fish index (Ifish) is representative of the overall condition of the sample group of fish from a histological response perspective. The mean values per species per impoundment were calculated from the sum of the means of the organ indices (Iorg) per species per impoundment (Figure 4 – 15).

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Figure 4 - 15: Mean fish indices (Ifish) per species per impoundment. Italic letters denote statistical differences: same letter = no significant difference.

The sum of the mean organ indices from the six main target organs (gills, liver, kidney, heart, gonads (testes & ovaries) and skin) yielded the overall fish health indices (Ifish), which for the C. gariepinus ranged from 19.69 to 30.32 at VD and RD, respectively. There were no significant differences between sites for the mean fish indices (Ifish) for C. gariepinus (H=2.351, d.f.=2, p=0.309). Conversely, the mean values for the overall fish index

(Ifish) for the C. carpio ranged between 16.00 at RD and 23.78 at MBD with significant differences between MBD and RD (U=26.5, z=-2.454, p<0.05), but no significant difference relative to VD (U=37.5, z=-1.617, p=0.106).

4.7. AGE ESTIMATION 4.7.1. OTOLITHS

All C. gariepinus were aged using otoliths, all counts are recorded in Appendix 6 and the mode of each specimen is shown Table 4 - 10. The group of C. gariepinus at RD age estimation ranged from 3 to 8 years with a mean age of 5.00 years and was the youngest group between sites. The VD showed a similar age group with an additional 12 year old specimen. The oldest group of catfish was from the reference site (MBD), whereby four individual specimens were estimated to be over 10 years of age. Numerous values were absent and could not be estimated because otoliths were damaged during the sectioning process. Unfortunately, the process often damaged both pairs of otoliths collected and no alternative was available.

Table 4 - 10: Estimated ages (years) of each C. gariepinus, estimated using otoliths. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN Roodekopjes Dam 8 5 6 - - - 7 5 4 3 4 5 - 4 4 5.00 Vaalkop Dam 12 3 - - - 5 - - 5 - 3 6 6 4 8 5.78 Marico-Bosveld Dam 10 16 5 - 6 5 13 - 8 4 6 - 4 12 9 8.17 - Absent

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4.7.2. SCALES

All C. carpio were aged using scales, all counts are recorded in Appendix 6, and the mode of each specimen is shown Table 4 - 11. Not much variation in age was evident within sites and between sites, whereby all specimens collected ranged between 3 and 6 years old. The mean age estimation ranged between 4.33 at MBD to 4.71 at VD. Absent values for the C. carpio indicate the specimens that were not collected during the field surveys, as the full target sample was not attained at VD and MBD.

Table 4 - 11: Estimated ages (years) of each C. carpio, estimated using scales. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN Roodekopjes Dam 5 4 5 4 3 4 6 3 3 4 6 5 6 6 5 4.60 Vaalkop Dam 4 5 4 6 4 4 4 5 6 5 5 4 5 5 - 4.71 Marico-Bosveld Dam 6 4 5 5 3 4 4 4 4 ------4.33 - Absent

4.8. EDIBILITY 4.8.1. MUSCLE ANALYSIS

The mean values (Table 4 – 12) of the organic and inorganic toxicants detected in the muscle tissue, as well as the highest concentrations detected within each impoundment per species (Table 4 – 13) were used in the human health risk assessment (HHRA) model (Appendix 7). Using the mean values in the HHRA would give a realistic probability of risk for consuming either C. gariepinus and/or C. carpio as they are caught, whereas using the highest values detected will give a risk for the worst-case scenario.

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Table 4 - 12: Mean concentration of selected toxicants detected in pooled muscle samples across both species. Highest values between impoundments are indicated by Red. TOXICANT CONCENTRATION (µg/kg or ppb) RD VD MBD Organic Compounds Chlorpyriphos - 0.419 - pp’-DDE 4.943 1.782 6.008 β-HCH 7.500 2.450 - p-NP 73883.000 91800.000 10500.000 Terbuthylazine - 0.820 - Inorganic Compounds Ag 3.958 - 11.093 Al 649.776 920.305 1320.811 As 107.921 116.708 161.016 B 4088.314 3378.022 5178.885 Ba 4880.969 4896.907 7490.591 Be 1.989 2.317 4.338 Cd 2.653 0.664 2.776 Co - 2.656 - Cr 7.287 5.303 14.261 Cu 132.479 82.891 113.794 Mn 47.426 21.551 26.176 Mo - - - Ni - - - Pb 70.190 61.682 78.144 Sb 0.330 - - Se 271.436 328.265 347.732 Sn 121.449 95.794 107.710 Sr 44.041 41.461 47.572 V 1.989 0.662 1.189 Zn 3846.159 3520.602 5992.587 - Toxicant absent or below detection limits

4.8.2. HUMAN HEALTH RISK ASSESSMENT (HHRA)

A total of nine (9) risk assessments were performed, using the mean values of detected toxicants within the muscle of both species together per impoundment (Table 4 – 12; 3 assessments) and the highest values of detected toxicants within each species per impoundment (Table 4 – 13; 6 assessments). The process described in the Background Information chapter (Chapter 2) was followed as per assessment and resulted in a defined toxic and cancer risk per assessment.

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Table 4 - 13: Highest concentrations of selected toxicants detected in pooled muscle samples per species. Highest values between impoundments are indicated by Red. TOXICANT CONCENTRATION (µg/kg or ppb) Species Clarias gariepinus Cyprinus carpio Impoundment RD VD MBD RD VD MBD Organic Compounds Chlorpyriphos - 2.514 - - - - pp’-DDE 9.350 3.130 14.310 6.660 2.870 3.610 β-HCH 11.390 8.190 - 16.230 1.540 - p-NP - 132300.000 26400.000 276000.000 257700.000 - Terbuthylazine - 2.290 - - 0.770 - Inorganic Compounds Ag 23.748 - 25.722 - - 29.744 Al 1025.134 1050.337 1499.802 965.627 986.672 136.823 As 140.594 160.874 203.799 167.331 143.227 136.823 B 4729.863 3501.784 5878.512 4728.790 4717.131 8128.098 Ba 6572.333 5051.526 8862.287 7278.884 7039.841 12482.649 Be - 13.903 21.688 9.956 - - Cd 1.987 - 3.957 7.968 1.995 5.949 Co - - - - 15.936 - Cr 3.960 5.977 37.594 19.869 7.957 9.930 Cu 108.911 93.143 92.666 227.092 99.602 249.851 Mn 17.811 21.799 27.701 201.195 31.873 41.642 Mo ------Ni ------Pb 85.149 91.360 71.899 109.562 71.828 116.994 Sb 1.980 - - - - - Se 308.727 340.864 395.726 342.629 367.119 329.169 Sn 231.683 105.263 177.445 280.151 229.084 250.248 Sr 41.559 29.727 49.466 61.753 87.649 89.233 V 5.962 3.972 1.979 3.984 - 3.966 Zn 4977.241 3686.088 6363.277 5501.992 5051.793 9367.440 - Toxicant absent or below detection limits

4.8.2.1. Hazard Identification Since the presence of many of the selected toxicants that were screened for during the laboratory analysis have the potential to cause adverse health effects, all toxicants detected within the muscle of the fish with an available oral reference dose (RfD) were included in the toxic risk assessment. Similarly, and in addition to an available oral RfD, all detected toxicants for which a slope factor (β) was available were also assessed for potential carcinogenicity in the cancer risk assessment.

The organic toxicants included in the assessment were terbuthylazine, pp’-DDE, β-HCH, Chlorpyriphos and p-NP, whilst the inorganic toxicants included were Ag, Al, As, B, Ba, Be, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Sn, Sr, V and Zn.

4.8.2.2. Dose-Response Assessment RfDs and slope factors (β) were sourced from various databases (Chapter 2 – Literature PAGE | 82

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Review) for each of the five (5) organic and twenty (20) inorganic toxicants identified in the above section. Table 4 – 14 shows the relevant values and sources for each of the respective toxicants included in the assessment.

Table 4 - 14: Oral reference doses (RfD) and slope factors (β) sourced for the human health risk assessment. Refer to Chapter 2 – Literature Review for source. ORAL RFD TOXICANT β (mg/kg/day) SOURCE (mg/kg/day) Ag 0.005 - IRIS Al 1.000 - IRIS, RAIS As 0.0003 1.5 IRIS B 0.200 - IRIS Ba 0.200 - IRIS Be 0.002 4.3 IRIS, RAIS Cd 0.001 - IRIS Co 0.0003 - CDEP, 2008 ATSDR, Chlorpyriphos 0.0003 - Christensen et al. (2009) Cr (III) 1.500 - IRIS Cr (VI) 0.003 - IRIS Cu 0.010 - CDEP (2008) pp’-DDE 0.00022 0.34 IRIS β-HCH 0.017 1.8 ATSDR Mn 0.140 - IRIS Mo 0.005 - IRIS Ni 0.020 - IRIS p-NP 0.100 - Bakke (2003) Pb 0.00071 - Watanabe et al. (2003) Sb 0.0004 - IRIS Se 0.005 - IRIS Sn 0.003 - CDEP (2008) Sr 0.600 - IRIS Terbuthylazine 0.035 - USEPA (1995) V 0.007 - RAIS Zn 0.300 - IRIS - No available data at present

4.8.2.3. Exposure Assessment Since the assessment is based solely upon the potential risk for consumption of contaminated fish, other exposure pathways such as inhalation or dermal contact were not considered. Also, due to a lack of information with regards to the socio-demographic information of the subsistence fishermen (age, sex, body weight) and fish consumption patterns (number of species, preparation methods, meal size, etc.), a number of worst- case scenario assumptions were applied in order to eliminate lesser acute risks: . Chronic exposure of an adult, with an average weight of 70kg and an expected lifetime of 70 years. . Consumption of a single 150g portion per day for an exposure period of 30 years.

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4.8.2.4. Risk Characterisation A significant toxic risk was defined to be present when the average daily dose (ADD) was greater than the reference dose (HQ≥1), whilst a significant cancer risk was present when probability of acquiring cancer was calculated to be lower than 1 in 100 000 people (≤ 1 in 100 000) (WHO, 2001). Table 4 – 15 shows the highest potential health risks from the consumption of contaminated C. gariepinus or C. carpio from any of the selected impoundments, as well as the mean potential risk from consuming either of the species at each of the selected impoundments (Appendix 7).

Table 4 - 15: Highest and mean potential health risk through consumption of contaminated fish per species and between species. Significant toxic/cancer risks are indicated by Red. TOXIC RISK (HQ) & CANCER RISK (per number of ppl.) TOXICANT Clarias gariepinus Cyprinus carpio Average/Both TR* CR* TR* CR* TR* CR* Roodekopjes Dam As 1.00 1 in 5 000 1.20 1 in 5 000 0.77 1 in 10 000 Be 0.00 1 in 125 000 0.01 1 in 25 000 0.00 1 in 125 000 pp’-DDE 0.09 1 in 333 333 0.06 1 in 500 000 0.05 1 in 500 000 β-HCH 0.00 1 in 50 000 0.00 1 in 33 333 0.00 1 in 100 000 Vaalkop Dam As 1.15 1 in 5 000 1.02 1 in 5 000 0.83 1 in 5 000 Be 0.01 1 in 20 000 No Risk 0.00 1 in 111 111 pp’-DDE 0.02 1 in 1 250 000 0.03 1 in 1 111 111 0.02 1 in 1 666 667 β-HCH 0.00 1 in 100 000 0.00 1 in 333 333 0.00 1 in 250 000 Marico-Bosveld Dam As 1.46 1 in 3 333 0.98 1 in 5 000 1.15 1 in 5 000 Be 0.02 1 in 11 111 No Risk 0.00 1 in 50 000 pp’-DDE 0.14 1 in 250 000 0.04 1 in 1 000 000 0.06 1 in 5 00 000 β-HCH No Risk No Risk No Risk *TR – Toxic Risk, CR – Cancer Risk

There were no significant toxic risks posed by individual chemicals in any of the fish muscle tissue sampled, except for those posed by As, whilst in addition to As, Be and β-HCH were the only toxicants to pose a significant carcinogenic risk (Table 4 – 15). The health effects of As, Be and β-HCH are described and characterised by Table 4 – 16.

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Table 4 - 16: Characterisation of toxicants with a significant risk. TOXICANT HEALTH EFFECTS Acute Exposure:  Small oral dosages can be associated with an irritation of the stomach and intestines, including stomach ache, nausea, vomiting and diarrhea.  Large oral dosages of this recognised human poison can result in death. Chronic Exposure: As1  Symptoms included a pattern of skin changes including darkened areas and/or the development of ‘corns’ or ‘warts’ of palms, soles and torso.  Skin cancer may also develop and the risk of developing cancer in the liver, bladder and lungs is increased. Acute Exposure:  Potential ulcers and lesions in the stomach and intestines. Be2  Weight loss, anorexia and lassitude/fatigue. Chronic Exposure:  Risk of developing some form of cancer. Acute Exposure:  Production of liver and kidney effects. β-HCH3  Reduced immunity and injury to the testes and ovaries. Chronic Exposure:  Liver cancer has been known to occur. 1 – (ATSDR, 2007a), 2 – (ATSDR, 2002b; USEPA, 1998), 3 - (ATSDR, 2005a)

Furthermore, Table 4 – 17 shows the highest overall toxic risk per species and the mean overall toxic risk for both species, at each of the selected impoundments (Appendix 7). This value is representative of an accumulative effect from a number of toxicants rather than a single isolated toxicant within the fish muscle.

Table 4 - 17: Highest and mean overall toxic risk through consumption of contaminated fish per species and between species. Highest values between impoundments are indicated by Red. IMPOUNDMENT OVERALL TOXIC RISK (SUM OF HQ VALUES) Clarias gariepinus Cyprinus carpio Average/Both Roodekopjes Dam 1.86 2.20 1.40 Vaalkop Dam 1.85 1.90 1.41 Marico-Bosveld Dam 2.39 2.06 1.90

The MBD showed the highest accumulated HQ for the highest potential toxic risk in the muscle of C. gariepinus and in the mean potential risk assessment for both species. These values were a result of the elevated HQs calculated, in addition to arsenic (As), for lead (Pb), selenium (Se) and tin (Sn). The effects of these toxicants, in addition to As can lead to a combination of health effects occurring, some of these health effects are characterised as per toxicant (Table 4 – 18).

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Table 4 - 18: Characterisation of other toxicants contributing to a high overall toxic risk. TOXICANT HEALTH EFFECTS The main target of lead toxicity is the nervous system (ATSDR, 2007b). Acute Exposure:  High levels of exposure can severely damage the brain and kidneys, and in turn cause death. Chronic Exposure: Pb  Decrease performance in tests that measure function of the nervous system.  Weakness in fingers, wrists and ankles.  Increased blood pressure.  Cause anaemia.  Potentially carcinogenic to humans. Selenium is an essential element required by the body in antioxidant enzymes and in enzymes that affect growth and metabolism (ATSDR, 2003). Acute Exposure:  Large doses can cause dizziness, fatigue and irritation of the mucous Se membranes. Chronic Exposure:  Not enough can lead to heart problems and muscle pain.  Brittle hair and deformed nails can develop.  In extreme cases, people may lose feeling in their arms and legs. Tin and its compounds can show a wide variety of health effects, depending on the specific organo-tin exposed to. Certain organo-tins mainly affect the immune system, while others affect the nervous system (ATSDR, 2005b). Acute Exposure:  Large amounts to toxic organo-tins have been known to cause stomach aches, anaemia, and liver and kidney problems. Sn  Reports of skin and eye irritation, respiratory irritation, gastrointestinal effects, and neurological problems have been shown during a short exposure to high doses. Chronic Exposure:  Has been shown to reduce fertility and cause stillbirth in pregnant rats and mice.

In addition, the overall toxic risk was observed to be higher in C. carpio, relative to the values for C. gariepinus in both of the assessments sites, but not in the reference site (MBD).

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5. DISCUSSION & CONCLUSION

CHAPTER 1: IN THIS CHAPTER PROJECT 5.1. SUBSTRATE ANALYSIS ...... 88 OVERVIEW 5.1.1. Physico-chemical Parameters ...... 88 5.1.2. Chemical Analysis ...... 89 CHAPTER 2: 5.2. NECROPSY-BASED HEALTH ASSESSMENT ...... 92 BACKGROUND 5.3. BIOMETRIC INDICES ...... 93 INFORMATION 5.4. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) ...... 94 5.4.1. Gill Histology ...... 94 5.4.2. Liver Histology ...... 95 CHAPTER 3: 5.4.3. Kidney Histology ...... 97 MATERIALS & 5.4.4. Heart Histology ...... 97 METHODS 5.4.5. Gonad Histology ...... 98 5.4.6. Skin Histology ...... 100 CHAPTER 4: 5.4.7. Combined Histological Effects ...... 100 5.5. EDIBILITY ...... 102 RESULTS 5.5.1. Muscle Analysis ...... 102 5.5.2. Human Health Risk Assessment (HHRA) ...... 103 5.6. CONCLUSION ...... 106 CHAPTER 5: 5.7. RECOMMENDATIONS ...... 108 DISCUSSION & REFERENCES ...... 109 CONCLUSION

APPENDICES

1 John 2: 16 – For all that is in the world, the lust of the flesh, and the lust of the eyes, and the pride of life, is not of the Father, but is of the world.. CHAPTER 5 – DISCUSSION & CONCLUSION

5.1. SUBSTRATE ANALYSIS

In order to assess the environment, in which the fauna and flora live and survive, the two main physical substrates associated with the impoundments were assessed. Assessing the physico-chemical parameters of the water quality resulted in a snapshot of the current and recent condition within each impoundment, whilst an analysis of the sediment inferred more of a historical condition, whereby toxicants often sequester in these ‘sinks’ of the aquatic ecosystem (Chapman & Mann, 1999).

5.1.1. PHYSICO-CHEMICAL PARAMETERS

Of all the physico-chemical parameters measured during this assessment, the pH levels and the dissolved oxygen concentrations within the assessments site (RD & VD) were a cause for concern. There were indications that the impoundments were highly enriched with nutrients and in fact eutrophic, which would already indicate that the system is stressed.

Firstly, the pH levels measured at the assessment sites were basic/alkaline ranging from 8.90 at VD to 9.43 at RD and the mean pH values exceeded the generic RWQOs for the catchment. Elevated pH values can be caused by increased biological activity in eutrophic systems and have been known to fluctuate widely from below 6 to above 10 over a 24- hour period as a result of changing rates of photosynthesis and respiration (DWAF, 1996). The relatively high prevalence of aquatic vegetation, especially Water Hyacinth (Eichhornia crassipes) (Figure 4 – 1A) and algal blooms (Figure 4 – 1B), at the assessment sites also indicates that there is an excess of nutrients supporting these large populations, which is characteristic of eutrophic systems (Dallas & Day, 2004). Conversely, the mean pH level measured at the reference site (MBD) was a neutral 7.81 and within the set generic RWQOs. The aesthetic value of the MBD, with regards to the absence of significant green algal blooms, was also noted.

Secondly, dissolved oxygen concentrations of 80-120% saturation are considered to protect all life stages of most South African aquatic organisms that are endemic or adapted to the aerobic warm water habitats of South Africa (DWAF, 1996). Furthermore, the standing (lentic) nature of impoundments discourages the loss of oxygen to the atmosphere and maintains the oxygen production during photosynthesis from aquatic plants at a high saturation level during the day (Dallas & Day, 2004). This was evident when dissolved oxygen content at RD and VD were observed to be super-saturated (>120%) throughout the day, which does not necessarily negatively affect the aquatic biota during the day. However, such elevated levels can indicate eutrophication in a water body and

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CHAPTER 5 – DISCUSSION & CONCLUSION implicates significant diurnal fluctuations that would most definitely affect the aquatic biota especially at night when oxygen saturation is expected to drop (DWAF, 1996; NWDACE, 2002).

According to the National Eutrophication Monitoring Programme (NEMP), mean annual chlorophyll a and mean annual total phosphorus values at the time of the survey showed RD to be hyper-eutrophic (122.1µg/L; 0.154mg/L), whilst VD was defined as hyper-eutrophic for the chlorophyll a (50.0µg/L) in the system and eutrophic for the total phosphorus (0.061mg/L) in the system (DWA - RQS, 2013). Lastly, the reference site was confirmed to be oligotrophic according to chlorophyll a levels (1.2 µg/L) and mesotrophic according to total phosphorus levels (0.024mg/L) (DWA - RQS, 2013).

In summary, these systems (RD & VD) showed elevated pH levels, super-saturated dissolved oxygen content, high chlorophyll a levels and excessive phosphorus levels similar to other hyper-eutrophic ecosystems, such as the Roodeplaat Dam (Marchand et al., 2012) and the Hartbeespoort Dam (Wagenaar et al., 2012). Therefore, there are numerous indirect factors that may contribute to the stress of the fish populations in each system and render them more susceptible to infection and disease.

5.1.2. CHEMICAL ANALYSIS

5.1.2.1. Organic Only five organic toxicants were detected within the water column across all three impoundments. All showed low concentrations relative to the appropriate target range or screening value, except for DDT and its metabolites.

5.1.2.1.a. DDT & metabolites (DDE & DDD) Despite the international ban on the use of DDT, the Stockholm Convention grants a number of countries – one of which being South Africa, restricted use to spray DDT on the interior surfaces of homes in high-risk malaria areas (Bornman et al., 2010). Since none of the study sites are recognised to occur within high-risk malaria areas (Bouwman, 2004), there was major cause for concern when this broad-spectrum insecticide and its metabolites were detected in the water at each of the assessed impoundments. The concentrations for DDT at VD and MBD were above the target range as set by the NOAA SQuiRTs (Buchman, 2008) and were expected to present potential human health risk implications to consumers of these fish, as the nature of the compound is easily absorbed into aquatic biota and leaves little amounts within the water column (Bornman et al., 2009). However, this was not the case, as only pp’-DDE was detected within the muscle of the fish.

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It should be noted that the op’-DDT concentration measured in the MBD was substantially higher than other op’-DDT, op’-DDE and op’-DDD values present in any of the other systems. This may be a direct result of the runoff or spray drift from upstream farming activities, one off which is, located at the mouth of the impoundment and potentially recent spraying of crops. However, this cannot be confirmed.

Technical-grade DDT is composed mostly of isomers of DDT with small amounts of DDE isomers and it is quickly broken down into its persistent metabolites (DDE & DDD) by microorganisms (ATSDR, 2002c; Bornman et al., 2010). Consequently, the types of components present within a system can indicate how long ago DDT was used. Therefore, the high concentrations of DDT and DDD present in the water at the MBD may indicate recent use of the insecticide, whilst DDE level at the assessment sites may indicate historical usage.

DDT has been described as a possible cause to a number of abnormalities in the reproductive system of fish, where sperm counts are reduced, sex ratios are skewed toward female dominance and delayed or abnormal development of ovarian tissue (Barnhoorn, et al., 2010; Bornman et al., 2010; Marchand et al., 2010).

5.1.2.1.b. Nonylphenol (p-NP) Although the p-NP (and its ethoxylates) concentrations in the sediment at all the assessed impoundments did not exceed the target objectives, as determined by NOAA SQuiRTs (USEPA, 2005), there remained a potential cause for concern. p-Nonylphenol is capable of persisting in soil/sediment for as long as a year, which indicates that the p-NP may have contaminated these impoundments within the last year. This may be a direct result from agricultural runoff within each respective catchment as p-NP and its ethoxylates form the inert base to numerous pesticides (Cox, 1996). MBD is potentially affected by farming activity that is located at the mouth of the impoundment which might explain the highest concentration detected.

Although, these concentrations (64.80 - 75.50 ppm) confirm the presence of p-NP within the sediment at all three of the assessed impoundments, two similar studies at Rietvlei Dam in Pretoria reported values much higher. According to Van Dyk et al. (2009a), the p- NP values detected in the water (1.87 µg.L-1 or 1 870 ppm) and sediment (100 µg.kg-1 or 100 000 ppm) at the time of the survey may have had potential effects on gill structure and compromised the respiratory system. In the second study by Marchand et al. (2008b), the highest recorded p-NP concentration detected in the sediment was 0.64 mg.kg-1 (or 0. 64 ppm), but an alarming 50µg.L-1 (or 50 000 ppm) was detected within the water column.

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As a result, p-NP was the primary EDC present in the system, and it was suspected that these high concentrations within the water column were responsible for disrupting sexual development in fish. Therefore, this compound can potentially exhibit adverse health effect within the ecosystem, but if the p-NP remains trapped within the sediment, as in this assessment, any p-NP-related adverse effects may be avoided.

5.1.2.2. Inorganic Relative to the set target ranges, only aluminium (Al) and silver (Ag) exhibited an excessive presence in the water of the RD and the sediment of the MBD, respectively. However, it is clear that the RD contained relatively larger quantities of each metal within the water, which may be attributed to increased mining activities within its catchment. Relatively high levels of potassium (K), sodium (Na), magnesium (Mg) and calcium (Ca) in the water at the assessment sites (RD & VD) relative to the reference site (MBD) can explain the exceedances of the RWQOs for electrical conductivity and TDS (Table 4 – 1) as all these ions carry a charge and were dissolved within the water column.

Potentially toxic metals often accumulate in the sediments, as the sinks of aquatic ecosystems. Within this partition, they are not as easily biodegraded by natural processes relative to organic toxicants, and therefore persist in the sediment for an extended period after their introduction (Tica et al., 2011). This was largely evident at all sites, whereby sediment concentration far exceed the concentrations detected in the water of each impoundment. Various mechanisms performed by the sediment i.e. redox reactions are suspected to have a natural capacity to attenuate the bioavailability and mobility of these metals (Chapman & Mann, 1999; Tica et al., 2011). When potentially toxic substances are trapped within the sediment of an impoundment and unavailable for absorption by aquatic biota, the ecosystem may function as normal. However, if these toxicants become bioavailable as a result of various in-sediment mechanisms or feeding habits of particular fish, the system’s ecology and stability is likely to change with no prior warning.

5.1.2.2.a. Aluminium (Al) Aluminium is the most abundant metal in the earth’s crust, yet elevated levels of bioavailable Al in water are toxic to a wide variety of organisms. However, there is uncertainty to the forms that are bioavailable to different organisms, as well as the mode of toxicity as the state of Al in water is governed by numerous complex interactions with other water quality parameters (DWAF, 1996; ATSDR, 2008). This value was measured and noted to exceed the tentative TWQR for water with a pH greater than 6.5. The mechanism of toxicity towards fish is related to the interference with ionic and osmotic balance as well as respiratory complications resulting in coagulation of mucous on the gills (DWAF, 1996).

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5.1.2.2.b. Silver (Ag) This naturally-occurring chemical is used as pesticide in disinfectants, sanitizers and fungicides and it is highly acutely toxic toward fish, aquatic macroinvertebrates and estuarine organisms (USEPA, 1992). The detected value trapped within the sediment sink of the MBD most likely accumulated over a number years and presents no possible risk to fish or humans unless it becomes mobilised or released. However, this is not to say that there is no risk, there is likely to be a direct toxicity toward immediate surroundings.

5.2. NECROPSY-BASED HEALTH ASSESSMENT

With the exception of a single skin lesion found on a C. carpio specimen, no other external abnormalities were observed on either fish species at any of the impoundments. A skin ulcer or lesion, such as the one found in this study, may be a result of a number of different causes, namely bacterial, fungal, dermatotoxins released from cyanobacteria, or even a eukaryotic protozoan (Vogelbein et al., 2001; Zerihun et al., 2011). To be sure, further investigations of such lesions should be performed and assessed.

The internal macroscopic examination yielded largely normal observations with potential causes for concern with regards to the cyst located in the gill of a C. gariepinus specimen from the RD and fatty or discoloured livers within various specimens of both species at each impoundment. Firstly, the gills are also vulnerable to a number of infectious diseases from a number of possible pathogens, namely amoebae, other parasites, bacteria and viruses, as they are permanently and directly exposed to the medium and its dissolved solutes (Abalaka et al., 2010; Mitchell & Rodger, 2011). No other major histological alterations were macroscopically observed, but a number of parasites were found during histological assessment (Photo Plate 4 – 1J), and this was suspected to be the source of the observed cyst.

A study on the livers of C. gariepinus from Roodeplaat Dam found large fatty nodules and irregular discoloured tissue regions ranging from yellow, green, black and/or red. These abnormalities were as high as 45% prevalent, whilst other NWP impoundments (Hartbeespoort-, Bospoort- and Klipvoor Dams) ranged between 5 - 9% prevalence (Van Dyk et al., 2012). By comparison, the current study showed a prevalence of 27% at RD and 46% at VD for the C. gariepinus, whilst the C. carpio specimens yielded a lesser prevalence not exceeding 7% of the observed macroscopic alterations. Therefore, there is a concern that the comparatively high prevalence’s of these observations may support hepatocellular vacuolation, of which is characteristic of steatosis (Van Dyk et al., 2012). Although, an excessive lipid accumulation or steatosis of hepatocytes can often be defined as a pre-neoplastic toxicopathic change in mammals, in fish, an increased prevalence and

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CHAPTER 5 – DISCUSSION & CONCLUSION severity of these observation, such as observed in this study, suggests possible toxicant exposure (Feist et al., 2004; Lang et al., 2006). The comparative lack of fatty nodules in C. carpio specimens may be a result of the additional visceral adipose tissue observed within the species, thus providing additional inert tissue for sequestration. The histological assessment of the liver confirmed that less alterations were observed in the C. carpio specimens relative to the livers in C. gariepinus specimens.

5.3. BIOMETRIC INDICES

With respect to C. gariepinus, the mean CF recorded at the MBD (0.68±0.09) was significantly lower than the RD (U=37.0, z=-3.132, P<0.01) and the VD (U=52.5, z=-2.489, P<0.05), and even below the mean range of (0.72±0.12 - o.80±0.3) recorded for the same species in the close-to-pristine Okavango Delta panhandle (Van Dyk et al., 2009b). In general, the CF varies directly with nutrition and it shows a negative correlation to the presence of disease/toxicants in the system (Schmitt & Dethloff, 2000). However, given the hyper-eutrophic state of RD and VD at the time of the survey, it is likely that these specimens were heavier relative to their length because of the increased biomass, algae and/or sediment that was indirectly ingested during feeding.

On the other hand, in consideration of the estimated mean age of C. gariepinus at MBD (8.17 years old) relative to 5.00 at the RD and 5.78 at the VD, it was suspected that ‘old age’ may be a contributing factor to this slightly lowered CF. Furthermore, since there exists a reducing trend (RD > VD > MBD) in the female C. gariepinus sexual maturity levels, as observed by the histological staging, which also directly relates to the reduced mean CFs, another consideration might be that the size of the gonads fluctuates seasonally and affects the CF of each collected specimen.

The HSI values for all C. carpio specimens were consistent between sites and according to Datta-Mushi and Dutta (1996), they were defined as normal for occurring within the normal range of 1-2% for all Osteicthyes. Similarly, again relative to the values obtained in the close-to-pristine Okavango Delta (HSI<1) (Van Dyk et al., 2009b), the HSI values for the C. gariepinus were also deemed normal. However, the significant difference between the C. gariepinus at MBD and the RD (U=10.0, z=-4.338, P<0.01) and VD (U=49.0, z=-2.712, P<0.05) indicated potential atrophy of the liver. However, no substantial atrophy was observed in any of the C. gariepinus samples during the macroscopic or microscopic assessments.

According to Hadidi et al. (2008), the significantly elevated mean SSI value observed at the RD for C. carpio suggested that the immune system and resistance to pathogens was

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CHAPTER 5 – DISCUSSION & CONCLUSION potentially elevated in this group (Figure 4 – 6B). By deduction, this would further suggest that the RD is more heavily contaminated with toxicants than the other two assessment sites, which would support with the increased mining impacts at RD. However, this trend does not follow for the C. gariepinus group, which warrants further investigation to determine if this is actually the case, as suggested by Hadidi et al. (2008).

The mean GSI values compared well with the gonadal development stages or sexual maturity observed in the female specimens of both species. However, in contrast, the comparatively small size of the testes in the male specimens, especially in C. gariepinus, was suspected to skew the GSI values slightly, and did not easily reflect the developmental stages of these male specimens. Chronic exposure to toxicants has shown gonadic deterioration or a reduction in the GSI values (Louiz et al., 2009; Marchand et al., 2010), but there was no evidence of histological alterations. Relative to the male (0.221±0.053) and female (4.53±0.53) C. gariepinus GSI values taken during a Rietvlei Dam study, where intersex was confirmed to occur (Barnhoorn et al., 2004), only the male C. gariepinus GSI values recorded in the MBD during the current study indicated a potential deterioration and reduced functionality despite the absence of histological alterations. Therefore, since pathology is unlikely, changes observed in the GSI values of male C. gariepinus at the MBD are likely to be related to seasonal variation of gonadal development. This was supported by a slightly more mature developmental stage observed within this group relative to RD and VD (Refer Chapter 5, Section 5.4.5.1).

5.4. HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) 5.4.1. GILL HISTOLOGY

Although, there were no significant differences between the impoundments for the Igill observed for C. gariepinus (H=2.965, d.f.=2, P=0.227) or C. carpio (H=4.344, d.f.=2, P=0.114), there was an indication of slightly worse affected gills in the C. carpio at the MBD. This is due to the elevated prevalence (78%) of mild/focal telangiectasia in the secondary gill lamellae and moderate epithelial hyperplasia, which was prevalent in 56% of the individuals collected. The occurrence of telangiectasia in the gills, which is sometimes irreversible, has been associated with exposure to numerous toxicants, such as ammonia in Nile Tilapia (Oreochromis niloticus) (Benli et al., 2008), mercury in European Sea Bass (Dicentrarchus labrax) (Giari et al., 2008), and paraquat in the “Cachama” or “Tambaqui” (Colossoma macropomum) (Salazar-Lugo et al., 2011). Furthermore, severe epithelial hyperplasia in the secondary gill lamellae (fusion) was observed in C. gariepinus when exposed to ethanolic extracts of the Parkia biglobosa tree pods (Abalaka et al., 2010). Therefore, the gills are likely to demonstrate these inflammatory cascade mechanisms, which often begin with

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CHAPTER 5 – DISCUSSION & CONCLUSION telangiectasia as a non-toxicant specific stress response (Abalaka et al., 2010; Salazar-Lugo et al., 2011).

In a similar study on the Rietvlei and Marais dams, the observed histological alterations in the gill were mostly associated with circulatory disturbances, regressive changes and progressive changes (Van Dyk et al., 2009a). This trend was similar for the current study, except that the regressive changes dominated the reaction pattern of the alteration observed, in terms of number of alterations observed.

5.4.2. LIVER HISTOLOGY

The following non-specific histological alterations were observed in the liver from the assessed impoundments, namely moderate to severe intracellular deposits (20 - 31% prevalence) within the C. gariepinus and mild to moderate cases of melano-macrophage centres (MMCs) in C. gariepinus (33 - 92% prevalence) and C. carpio (7 - 13% prevalence). These pigment-containing cells are found in a number of organs in fish (Agius & Roberts, 2003) and their main function is to concentrate heterogeneous materials (lipofuscin, melanin, ceroid, or hemosiderin). The size, number and content of these MMCs are highly variable and dependent upon species, age and health status (Agius et al. 1985). A high prevalence of MMCs can be a response to environmental or chemical stressors (Van Dyk et al., 2009b). However, MMCs and intracellular deposits present in C. gariepinus are two distinct histological characteristics which are considered a normal histological feature and only an increase in the number of MMCs is likely to indicate a toxicant-induced response (Van Dyk et al., 2012).

Pacheco & Santos (2002) described increased hepatocellular vacuolation as a signal of degenerative process that suggests metabolic damage as a result of exposure to contaminated water. However, the presence of mainly glycogen within these vacuoles can eliminate metabolic damage as a cause for concern (Camargo & Martinez, 2007). Although, vacuolation of hepatocytes can be observed under both controlled and exposed conditions, as recorded during cadmium (Cd) and zinc (Zn) exposure studies using O. mossambicus, a drastic increase relative to normal conditions is often observed (Van Dyk et al., 2007). The most prominent alterations observed in both the indicator species’ between each of the impoundments was vacuolation, namely fatty vacuolation or steatosis and other vacuolation. The 100% prevalence of the non-fatty vacuolation observed at the MBD for the C. carpio is most probably the reason that Iliver was significantly higher than both the RD (U=21.0, z=-2.889, p<0.05) and the VD (U=21.0, z=- 2.954, p<0.05).

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In a recent study in the Kruger National Park to investigate the unexplained fish and crocodile deaths in the Olifants River, researchers found the first incidence of steatitis in wild C. gariepinus. The specimen’s livers were characteristically fatty and showed signs of fatty degeneration (steatosis) with the accumulation of both ceroid and hemosiderin in hepatocytes and melano-macrophages (Huchzermeyer et al., 2011). Mild and severe steatosis in the livers of C. gariepinus (7-20% prevalence) and C. carpio (13% prevalence at RD), respectively, was observed during this assessment. However, the presence of both ceroid and/or hemosiderin was not assessed and no other signs of steatitis was observed.

An inflammatory response, in the form of leukocyte infiltration in the liver, was also observed in the C. gariepinus. This may be potentially linked to a high parasite load, which was observed in a fish health assessment study at the Okavango Delta (Van Dyk et al., 2009b). However, the higher natural incidence for the nematode larvae (Contracaecum sp.) to infect the mesenteries of the C. gariepinus was likely to cause this response as these parasites are not usually associated with the liver (Barson, 2004). However, it should be mentioned that numerous other pathogens can also cause infiltration of leukocytes to the liver.

Lastly, focal areas of cellular alterations (FCAs) are categorised by Lang et al. (2006) as pre- neoplastic lesions and their presence is considered a relatively severe histological alteration. Whilst there were no C. carpio specimens to exhibit this reaction pattern in the liver, various FCAs were observed in the livers of three separate C. gariepinus specimens: in the VD, eosinophilic and hypertrophic FCAs were observed to dominate the histological section, whilst two specimens showed comparatively smaller affected areas at the reference site. Firstly, a potential reason for the lack of observed FCAs in C. carpio specimens may be attributed to their youth with C. gariepinus specimens observed to be more mature in age. Research on the Baltic Flounder (Platichthys flesus) has shown that the occurrence of pre-neoplastic and neoplastic lesions can be correlated with the age and length of the specimens (Vethaak & Wester, 1996; Stentiford et al., 2003; Lang et al., 2006). Similarly, the individuals observed to exhibit FCAs within the liver ranged between 8 and 12 years of age according to the age estimation, which is relatively old for C. gariepinus.

Microcystin is a highly potent hepatotoxin produced by Microcystis aeruginosa, which is often associated with cyanobacterial algal blooms within hyper-eutrophic systems, such as RD and VD. It has further been implicated in causing a loss of hepatic architecture, hypertrophic hepatic FCAs, inflammation, hepatocyte vacuolar change and necrosis – some of which have been discussed (Marchand et al., 2012). It is likely that some cumulative effects of this hepatotoxin, in addition to other toxicants, may have caused some of the

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CHAPTER 5 – DISCUSSION & CONCLUSION observed alterations, especially at the hypereutrophic VD, where the FCAs were most severe. Microcystin analysis needs to be included in future studies.

5.4.3. KIDNEY HISTOLOGY

The teleost kidney, together with the gills and liver, is usually one of the major organs to be affected by toxicants in the water (Thophon et al., 2003). The presence of metals in a system frequently causes alterations in the tubules and glomerulus, such as a swelling of the glomerulus and reduction of the Bowman’s space (Takashima & Hibiya, 1995; Thophon et al., 2003; Camargo & Martinez, 2007). However, there was mild evidence of the reverse, whereby an increase in the Bowman’s space and glomerular atrophy (13-38% prevalence) was observed in both species.

Similar alterations were observed in Brown Trout (Salmo trutta) downstream of a waste water disposal, including hyaline droplet damage and intracellular deposits within the tubules (Bernet et al., 2004). Therefore, the occurrence of these alterations may indicate potential toxicants or elevated organic loads in the water, which may also originate from agricultural runoff. Other histological alterations associated with the inflammatory response, such as mononuclear leukocyte infiltration, were observed in a study in Turkey (Ayas et al., 2007). These alterations showed a higher prevalence and severity relative to the current assessment, which correlated with the polluted state of the Sariyar Reservoir in Turkey but could only allude to a lesser extent of pollution in the RD.

MMCs were common in the kidneys of both C. gariepinus and O. mossambicus in the study performed at the highly polluted, hyper-eutrophic Roodeplaat Dam (Marchand, et al., 2012). Therefore, the presence of MMCs in the kidney may also be deemed normal, but an excessive number and severity may again be related to toxicant exposure.

Overall, the alterations observed in the kidney of the indicator species’ across the assessed impoundments were largely mild with low prevalence between impoundments for both species. However, the significantly higher mean Ikidney value for C. gariepinus at the RD compared to the VD (U=38.5, z=-3.164, P<0.01) may be related to a specific toxicant present at the RD which had a greater affinity for the kidney.

5.4.4. HEART HISTOLOGY

There was evidence of mild inflammation in the myocardium and epicardium of the atrium, ventricle and the outer wall of the bulbous arteriosus between species and at all assessed impoundments. Comparatively, in a study at the Roodeplaat Dam, only a mild and focal

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CHAPTER 5 – DISCUSSION & CONCLUSION inflammatory response and vacuolation of the ventricular myocardium was identified in C. gariepinus. There were no clinical signs that the lesions would affect the fish negatively. Further investigation was recommended in order to assess any correlation between toxicant-exposure and cardiac inflammation (Marchand, 2012). These findings were seconded by the results of the current study.

Poppe & Taksdal (2000) found an abundance of fat cells in the epicardium, as well as heavy infiltration of lymphocytes and macrophages in the epicardium and outer compact muscle layer of farmed Atlantic Salmon (Salmo salar) in Norway. However, these alterations were postulated to be related to high incubation temperature during embryonic development (Poppe & Taksdal, 2000). Although, the histological alterations observed showed some resemblance to the alterations observed in this assessment, this is not likely to be the case in this assessment. However, effects of temperature and the potential effects on the heart should be further investigated.

5.4.5. GONAD HISTOLOGY

5.4.5.1. Testis Histology The testicular architecture of the C. gariepinus, and most teleost fish, corresponds with the description by Van Dyk & Pieterse (2008), which indicates the anastomosing lobular type and numerous stages of development present at the same time (Mokae et al., 2013). The testicular developmental stages, as determined by McDonald et al. (2000), were varied between impoundments for the C. gariepinus, but the older specimens in the MBD were dominated by the most mature stages (Stage 2 & 3). Also, since the oldest C. gariepinus specimens were found at MBD, age is likely to play an expected role in sexual maturity levels, where according to this assessment a sexually mature specimen may be as young as 4 years old. Developmental stage 3 was 100% prevalent in the testes of C. carpio at all of the assessed impoundments, where individuals were as young as 3 years old. This was supported by no significant difference (H=0.103, d.f.=2, p=0.950) between the impoundments that was observed for the high GSI values for C. carpio.

The presence of testicular oocytes (intersex) have been identified in a number of fish species a variety of watercourses within South Africa, including Thinface Largemouth (Serranochromis angusticeps) in the Okavango Delta (Van Dyk et al., 2009b), O. mossambicus in the Luvhuvhu River system (Barnhoorn et al., 2010; Marchand et al., 2010), and in C. gariepinus at Marais and Rietvlei Dams in the Rietvlei Nature Reserve (Pieterse et al., 2010; Barnhoorn et al., 2004). These alterations have been observed in males in areas consistent with significant exposure to selected EDCs, such as DDT and p-NP. Furthermore, a potential species-specific sensitivity to selected EDCs was evident, as a study on O.

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CHAPTER 5 – DISCUSSION & CONCLUSION mossambicus and C. gariepinus at the Roodeplaat Dam found testicular oocytes only in O. mossambicus (Marchand et al., 2012).

In the eutrophic Hartbeespoort Dam, testes of C. gariepinus were observed to have numerous abnormal growths and an increased proliferation of connective tissue (Wagenaar et al., 2012). However, there was no evidence of such alterations in the testes of assessed specimens of either species at RD, despite RD being the downstream impoundment to the Hartbeespoort Dam.

5.4.5.2. Ovary Histology Similarly, the ovaries are composed of numerous developing follicles at various stages of maturity (Grizzle & Rogers, 1976). The majority of the female C. gariepinus specimens exhibited Stage 3 and 4 developmental maturity, whilst all four specimens from the reference site (MBD) were determined to be less mature (Stage 2) and under the age of 6 years. This supported the decrease of female GSI values observed for C. gariepinus, where the trend from RD > VD > MBD was correlated with a decreasing prevalence of fully mature stages (Stage 2 & 3). On the other hand, the majority of the C. carpio specimens from all the impoundments were less mature (Stage 2) and between the ages of 3 and 6 years old. Therefore, age is likely to determine a maturation age where fish develop sexually: female C. gariepinus specimens were observed to mature between 3 and 6 years old and female C. carpio specimens matured between the ages of 4 to 5 years old. However, it should be noted that seasonal variation in sexual maturity and gonad size was likely to vary with breeding cycle and spawning.

Again, observed alterations were compared to the study in the highly polluted and hyper- eutrophic Roodeplaat Dam, in which only an increase in MMCs was identified in the ovaries of the C. gariepinus (Marchand et al., 2012). And since no alterations were observed, it is suspected that based solely on the absence of histological alterations in the ovaries, each of the assessed impoundments is in a better condition than Roodeplaat Dam.

Although, ovarian tumours are seldom found in wild fish (Roberts, 1989), two benign ovarian neoplastic growths were found in O. mossambicus at the RD and VD during the same sampling period (Mooney, 2012). This was interesting as the assessment occurred during the same period and toxicant exposure was expected to be similar, and by inference, similar alterations were expected to present themselves. However, this was not the case with respect to the ovarian structure and health during the current study, but it would substantiate the possibility of differences occurring between trophic levels.

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5.4.6. SKIN HISTOLOGY

Hyperplasia of mucous producing cells in the skin may be recognised as a sign of stress, as the increased mucous production would attempt to thicken the mucous coat on the body surface and thereby reduce contact and absorption of the toxicant (Paul & Banerjee, 1997; Abalaka et al., 2010). However, this was not evident during the histological assessment, and furthermore, no histological alterations were found in any specimens.

With the exception of granulomas observed within the epidermis of C. carpio at VD, no other alterations were observed within the skin at any of the other assessed impoundments. These granulomas were suspected to be as a result of the larvae of a parasite, but further investigation is required to identify the species of parasite.

5.4.7. COMBINED HISTOLOGICAL EFFECTS

An important consideration in evaluating histopathological alterations in wild fish is that they experience chronic exposure to a mixture of toxicants at low concentrations, and these alterations cannot be regarded as toxicant-specific (Marchand et al., 2012). However, the resulting scores are likely to be an underestimation of real conditions as a result of the small relative amount of each organ that is assessed (Lang et al., 2006).

In review of the assessed mean Iorg, the liver was the most affected target organ of the overall histological assessment in C. gariepinus, with a high number of alterations also recorded in the kidney, heart and gills at the RD, VD and MBD, respectively. This was largely expected as the liver is essential for the metabolism and excretion of toxic substances in the body and as a result is more exposed to selected toxicants (Hinton & Laurén, 1990). However, in the C. carpio, the gills were the worst-affected target organ within each of the impoundments, which may be a result of the well-known habitat-modifying feeding behaviour of the C. carpio that would likely increase gill exposure to toxicants trapped within the sediment (Chapman & Mann, 1999). Organisms living in the benthos are more exposed to the toxicants with a high affinity for undissolved organic matter in the benthic zone (Larsson et al., 1992; Ruelas-Izunza et al., 2011). Since, C. carpio is most likely associated with the sediment, the elevated Igill scores at MBD for the C. carpio might indicate that toxicants within sediment of the reference site are more prevalent relative to the RD and VD.

Furthermore, all the Iorg only exhibited few histological alterations (Class 1) except for the

Iliver and Ikidney in the C. gariepinus at the RD and the Igills for C. carpio at the MBD, which fell into a Class 2 range with moderate histological alteration observed. Comparatively

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CHAPTER 5 – DISCUSSION & CONCLUSION speaking, these values were very similar to the respective gill (Igills=8.4) and liver indices

(Iliver=12.0) calculated for the same species in the Okavango Delta panhandle (Van Dyk et al., 2009b). Furthermore, relative to a study done on the C. gariepinus at the hyper- eutrophic Roodeplaat Dam, the Iorg values in the current study were well below these observed levels (Marchand et al, 2012). Therefore, the histological response from the C. gariepinus implies a fish condition and associated ecosystem health between the largely unpolluted Okavango Delta panhandle and the hyper-eutrophic Roodeplaat Dam. In consideration of the higher accumulation of organochlorine pesticides observed in the piscivorous species (Ayas et al., 2007; Marchand, 2012), C. gariepinus may be more affected by the biomagnification of toxicants in prey fish and invertebrates. These toxicants then accumulate in the liver and kidney of these specimens and cause structural alterations that may affect their function.

Regressive changes were observed to be the dominate reaction pattern for the histological alterations observed in each of the target organs. These alterations can be associated with a functional reduction or loss of an organ. However, these alterations are still reversible in many cases should the toxicant be neutralised (Bernet et al., 1999).

Interestingly, the inflammatory response, as measured by the Ii, was significantly higher in RD (U=59.0, z=-2.584, p<0.05) for C. gariepinus and substantially higher in RD for C. carpio. This indicates a more active immune system within the fish collected at the RD. This is supported by the elevated SSI value for the C. carpio at RD mentioned earlier, which alluded to an increased immunity (Hadidi et al., 2008). Similarly, the Ikidney was also the highest at the RD in both indicator species. Since there is haematopoietic tissue scattered within the kidney, it is expected that this would affect the production of leukocytes and immunity may be potentially reduced.

In summary, the Ifish calculated for each of the two indicator species’ reflected somewhat contradictory results, whereby C. gariepinus at the RD and C. carpio at the reference site (MBD) were observed with the highest index score. Therefore, the overall health of the C. gariepinus population was determined to be lowest at the RD, whilst the C. carpio population was worst-affected at the MBD. These differences were most probably due to the differences in diet and behaviour of the indicator species: (i) C. gariepinus is an opportunist for organic matter that accumulates toxicants through biomagnification. This was especially possible at the RD, where the group’s mean size and weight was greater, relative to other assessed impoundments and allowed for more piscivorous feeding and thereby increased bioaccumulation of toxicants. On the other hand, the (ii) C. carpio is a herbivore likely to express toxicant-induced alterations from direct exposure to its habitat, which is often associated with foraging in the sediment. Therefore, according to the

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CHAPTER 5 – DISCUSSION & CONCLUSION significant difference determined between Ifish for C. carpio, the fish may have been significantly worse-affected by the toxicants trapped in the sediment relative to RD & VD. In contrast, the observed differences between impoundments shown for C. gariepinus were suspected only to indicate different levels of pollution, which indicated that RD was worse polluted of the impoundments assessed.

Although no relation between the mean age and mean Ifish for each group was evident, there was some indication that the younger specimens showed lower scores and better health. It would be expected that older fish present more histological alterations as a result of the extended exposure period, but the relative age should rather be considered where fish closer to death will be more vulnerable to surrounding toxicants and cumulative effects as a result of extended exposure. However, this should be further investigated.

5.5. EDIBILITY

Edibility is defined by the Oxford Dictionary as any food fit to be eaten, further elaboration would imply that the food is safe for human consumption (Barnhoorn et al., 2013). Therefore, this would exclude the presence of any particular toxicant that may cause adverse effects to human health, or potential carcinogenicity from food. With the exception of the liver, the muscle and fatty tissue in fish are target areas for sequestration of toxicants. As such, a chemical analysis of the muscle tissue would likely define the composition and the concentration of toxicants that have bioaccumulated. In addition, since the bulk of the protein is located in the muscle of the fish, subsistence fishermen and other consumers are likely to consume the muscle and its associated toxicants.

5.5.1. MUSCLE ANALYSIS

The following metals are essential in trace amounts for many aquatic organisms, as they form an integral part of one or more enzymes involved in metabolic or biochemical processes: Cr, Cu, Fe, Mn, Mo, Se, and Zn (Connell, 1999). Although some of these metals do have toxic or carcinogenic effects at levels that are either too high or too low (such essential metals), it was expected that they be detected. On the other hand, many other organic and inorganic components found within the muscle of the fish in this assessment are probably a result of natural processes i.e. leaching from geology.

It should be noted, that for all the organic and inorganic toxicants (except vanadium (V) and p-NP) present in the C. gariepinus muscle at the reference site (MBD), the mean concentrations were recorded as the highest values relative to the other assessed

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CHAPTER 5 – DISCUSSION & CONCLUSION impoundments. This may be a result of the geology in the area, but the agricultural runoff within the catchment is also likely to play a major role.

The presence of organic toxicants, such as pp’-DDE and p-NP, in the muscle of both species at each of the impoundments was a cause for concern given their high affinity for muscular and adipose tissue in fish (ATSDR, 2002; Dai, et al., 2011). Although, it has been shown that p-NP can be metabolised by hepatic cytochrome P450 enzymes in the Rainbow Trout (Oncorhynchus mykiss) and excreted within bile with a half-life of 24-48 hours following exposure (USEPA, 2005), it remained a cause for concern because of its estrogen-mimicing properties. Furthermore, in addition to the potential effects on the endocrine system within aquatic biota, recent studies have demonstrated that exposure to EDCs can modulate expression of the genes critical in tumour development and regulations, and as a result, cancer may be a further distress within such systems (Raisuddin & Lee, 2008; Bornman et al., 2009).

Relative to the levels of β-HCH (512 ppb) and p, p’-DDE (307 ppb) found in the muscle of C. carpio in the Sariyar Reservoir in Turkey (Ayas et al., 2007), the respective levels were substantially lower in this study. However, there was still a potential cause for concern regarding the cumulative carcinogenic effect of these compounds.

Arsenic levels were a potential cause for concern relative to the low reference dose of 0.1 µg.kg-1.day-1 used for the HHRA process. The detected mean levels of As in both fish species were similar to studies on various fish species obtained from local markets in Spain by Falco et al. (2006) and in C. carpio in Croatia by Has-Schon et al. (2006), which were considered low relative to allowable consumption limits (Castro-Gonzalez & Mendez-Armenta, 2008). However, As levels were also substantially lower than those found in coastal fish species on the coast of Spain (Usero et al., 2003).

Therefore, despite there being no significant human health risk posed by these toxicants, their presence remains a concern from a fish health aspect of the assessment and also for future human health related risks.

5.5.2. HUMAN HEALTH RISK ASSESSMENT (HHRA)

The health benefits of a diet rich in fish has been extensively recognised in recent years, however, fish can often also be a source of pernicious toxicants, especially within systems known to be polluted and affected by anthropogenic activities (Cardoso et al., 2010a).

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Numerous studies have been done on the presence of elevated levels of mercury in human cohorts or communities, as a result of eating wild fish and/or canned fish and associated products (Harnly, et al., 1997; Xue et al., 2007). The alternative approach to determine health risks related to the consumption of fish has also been frequently applied by inputting concentrations from selected toxicants found within aquatic biota into HHRA models and determining a probability of contracting a disease and/or cancer (MacIntosh et al., 1996; Burger et al., 2005; Ginsberg & Toal, 2009; Cardoso et al., 2010b; Ruelas-Izunza et al., 2011; Dai et al., 2011, Barnhoorn et al., 2013).

Three toxicants were calculated and defined to pose some form of individual potential health risk to the consumers of the fish from these impoundments: (i) Arsenic was the only metal present in the muscle of the fish at high enough concentrations to present both a toxic and carcinogenic risk. The high mean concentrations were detected in the muscle of both species at the reference site, which implicated the upstream agricultural activities as As is often used in pesticides. The hazard quotient (HQ) values ranged between 1.00 and 1.46 times that of the reference dose, and therefore were not severely dangerous as single entities. Since, (ii) Be is not easily accumulated into the bodies of fish (ATSDR, 2002b) and no Be was detected in the water and sediment samples, it was surprising to find a carcinogenic risk posed by Be. Upstream coal mining is the most likely of impacts associated with a Be concentration in the muscle high enough to pose a carcinogenic risk as high as 1 in 11 111 people. However, coal mining is not known to occur within each respective catchment area and therefore, the natural process of erosion and leeching may be the cause for such elevated Be concentrations. Similarly, (iii) β-HCH was not measured within either of the substrates, but occurred in low concentrations of the fish muscle at RD and VD. β-HCH is highly lipophilic and persistent substance and it tends to accumulate in the fatty tissue of the fish (Dai et al., 2011). The presence of β-HCH is also most likely to implicate the agricultural activities upstream of RD and VD as contributing impacts.

Marchand (2009) found elevated levels of polychlorinated biphenols in the muscle of the C. gariepinus and O. mossambicus at the Roodeplaat Dam and the scenario-based health risk assessment determined that the long-term consumption (chronic exposure) of these fish would present adverse health effects. However, under realistic conditions in this assessment, there would be no significant toxic risks posed, except for at the reference site, where the relatively low risk may be deemed acceptable with regards to consumption.

Since the fish are exposed to a cocktail of toxicants and solutes within each system the overall toxic risk can gauge the worst contaminated impoundments i.e. most toxicants are bioavailable to fish. Accordingly, the MBD was specified as the impoundment with the

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CHAPTER 5 – DISCUSSION & CONCLUSION most intoxicated fish, as demonstrated by the highest overall toxic risk for the community of C. gariepinus.

Lastly, the carcinogenic potential from consuming the two species of fish in these impoundments, in realistic terms, was quite sobering, as As alone would cause 1 person in every 5000 consumers or subsistence fishermen to contract some form of cancer. The cumulative effects of Be and β-HCH may reduce this number even further i.e. increase the risk.

5.5.2.1. Risk Communication Under worst-case scenario conditions, it has been shown that there is a slight toxic risk posed through the consumption of either C. gariepinus from RD, VD and MBD or C. carpio from RD and VD. Furthermore, there are accumulative toxic risks posed from a combination of toxicants present in the fish muscle in both species from each impoundment. However, it is expected that any adverse effects from this risk can be controlled through eating less than 150g of fish muscle per day for 350 days a year for 30 years. There was also a significant cancer risk posed by the consumption of either species and even between species, as a result of the high concentrations of As present in the fish muscle. However, since these calculations represent worst-case scenario conditions, the recommendation made to avoid any adverse effects from toxic effect should also be adhered to, so as to avoid any potential carcinogenic effects.

5.5.2.2. Uncertainty Analysis There are a number of assumptions and uncertainties that should be identified in this HHRA process, so as to better understand the limitations of these assessments. This uncertainty analysis is notes that: . The calculated risks are specific to the assumed socio-demographic information i.e. the calculated risks are correct and effective for adults of average weight (70kg) with a life expectancy 70 years and eat a single 150g portion of fish muscle per day for 30 years. . These calculated risks do not account for alternate and/or accumulative exposure pathways, whereby the risks are calculated in assessment of consumption of the fish muscle in isolation. . Only risks for the toxicants included in the assessment are represented, and if additional data becomes available in the form of new or refined RfDs and slope factors, the assessment should be adjusted and corrected. . These risks are based solely upon the accumulation of toxicants within the muscle, and they should be adjusted to account for preparation methods, especially when fish are prepared whole. This allows for toxicants that have sequestered in other

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areas of the fish to enter the muscle and further contaminant or dilute the edible part of the fish.

RfD and slope factors are extrapolations of dose-response curves developed from animal exposure studies, which incorporate an uncertainty factor and may skew values slightly. 5.6. CONCLUSION

The substrate analysis of each impoundment showed that the physico-chemical water quality was largely favourable to all life stages of aquatic organisms at all impoundments. However, given the hyper-eutrophic state of the assessment sites (RD & VD), the aquatic biota were expected to be under some stress from substantial diurnal fluctuations in pH and dissolved oxygen, as observed in other hyper-eutrophic systems. On the other hand, the chemical analysis documented the presence of numerous organic and inorganic toxicants in both the water and more so in the sediments of these systems. The high affinity of many of these toxicants to the ‘sinks’ of the aquatic ecosystem was a result of their molecular nature, as many of these toxicants eventually settle out of suspension and accumulate in the sediment of the impoundment. This could mean that all previous contaminants to the impoundment may be found trapped in some layer of the sediment. Hence, the potential danger to fish and other aquatic biota that feed within the sediments. They are likely to be directly exposed to these historic contaminants.

An assessment of the health of fish populations in any system would render a snapshot of their overall condition after chronic exposure to the ‘cocktail’ of toxicants and stressors in a system. Whilst, any deterioration in health and especially immunity of these populations may not make fish more vulnerable to the accumulation of potentially dangerous toxicants, it is likely to make them more vulnerable to the adverse effects from these bioaccumulated toxicants. Therefore, a slight positive correlation is expected, where a fish population in good health in a contaminated impoundment is expected to tolerate and/or regulate the accumulated toxicants. Thus, the consumption of a highly tolerable fish may render a greater human health risk to the consumer, as the exposure to the toxicants accumulated within the fish may be greater.

With regards to the health of C. gariepinus and C. carpio sample populations from each of the selected impoundments in this study, the necropsy-based macroscopic assessment indicated a naturally higher incidence of Contracaecum sp. larvae within the mesenteries of C. gariepinus compared to C. carpio. The gross body indices, including condition factor (CF) and selected organosomatic indices, helped to indicate the health condition and sexual maturity of collected specimens and potentially, their immunity levels with regards to their splenosomatic index (SSI). However, exposure studies that monitor changes in the

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SSI of fish were required to properly investigate the potential indicator strength of the SSI as an indicator of immunity. In conjunction with the age estimation and the histological staging of the gonads, it was further suggested that the male fish from this study become sexually mature at an earlier age than their female counterparts.

Furthermore, the microscopic HBFHA showed few to moderate structural alterations in four of the six target organs of both species at all sites, with regressive changes dominating the reaction pattern type. These types of alterations can be associated with a functional reduction, which is often a reversible alteration once the toxicant is neutralised, or loss of an organ in the cases of severe necrosis, which is largely irreversible. The overall health, as indicated by the fish index (Ifish), of the C. gariepinus population was determined to be lowest at the RD, whilst the C. carpio population was worst-affected at the MBD. These species differences were likely due to the increased biomagnification of toxicants in C. gariepinus through its piscivorous feeding habits, relative to the more herbivorous nature of C. carpio. Moreover, the increased biomagnification in C. gariepinus was likely to worse-affect the liver, kidney and heart, as shown by their respective organ indices, (Iliver,

Ikidney & Iheart). Conversely, the gill index (Igills) was observed to be worst-affected in the sample population of C. carpio at the reference site (MBD). This was potentially related to its sediment-modifying behaviour and the increased exposure to sediment-trapped toxicants.

A significant toxic risk only exists when the average daily dose (ADD) for a specific toxicant exceeds the reference dose (RfD) for that specific toxicant, which can alternatively be expressed as a hazard quotient (HQ) greater than or equal to 1.0. It should be noted that RfDs have been conservatively estimated by applying numerous uncertainty factors and a number of safety buffer factors, and as a result the human health risk assessment (HHRA) process is perceived as a conservative estimate. Consequently, it was concluded that under worst-case scenario conditions, a slight toxic risk was posed through the consumption of either C. gariepinus from RD, VD and MBD or C. carpio from RD and VD as a result of the highest arsenic levels detected within each fish species. However, under realistic conditions, no significant toxic risk through arsenic exists at any of the sites through the consumption of either of the selected species, except at the MBD which marginally exceeded an HQ of 1.0. Alternatively, there were cumulative toxic risks posed from a combination of the detected toxicants present in the fish muscle in both species from each impoundment. However, synergistic and antagonistic interactions between the toxicants present were not determined and it is expected that any potential adverse effects from this ‘cocktail’ of toxicants can be controlled by eating less than 150 g of fish muscle per day for 350 days a year for 30 years.

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Finally, with regards to the carcinogenic potential of the toxicants present in the fish, a cancer risk was evident due to the arsenic levels detected, as well as cumulatively with the carcinogenic effects of beryllium (Be) and β-HCH. The risk was defined that 1 in 5 000 fish consumers consuming only 150 g of fish per day over an extended period have a high probability of acquiring some form of cancer. Therefore, in order to avoid any carcinogenic effects, it is advised that consumers eat less than 150 g per day or eat the collected fish less frequently.

5.7. RECOMMENDATIONS

A number of recommendations can be made for similar future studies on fish health and HHRA:

 The algal species present in the assessment site (RD & VD) should be identified in order to determine whether the toxic Microcystis sp. occurs and to measure the concentration present in the fish.  Exposure studies for Be and p-NP should be conducted on fish and on a terrestrial species to assess both the accumulative potential and the potential effects from consumption of Be.  PAS stains, or more preferably chemical analysis, should be used in organs that contain vacuolation to determine the contents thereof, which may be glycogen or any other heterogeneous substance, which would allow for further insight of the types of alterations present.  During the histological assessment, the prevalence of MMC and intercellular deposits are relatively normal in some species. Therefore, alterations should be assessed against a species-specific reference group or research.  There appears to be a potential correlation between the age of the fish collected and the state of it histopathology. Age-related exposure studies should be performed to confirm these findings.  A fat (or adipose tissue) analysis should be included during the HHRA as many toxicants are lipophilic and accumulate in this tissue. Also, this affects the concentrations of the toxicants during various different cooking methods, which can concentrate or dilute the toxicants.

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APPENDICES

CHAPTER 1: IN THIS CHAPTER PROJECT Appendix 1 – Record & Assessment Sheets ...... 126 OVERVIEW Appendix 2 – Substrate Analysis Data ...... 137 Appendix 3 – Haematological Assessment Data...... 142 CHAPTER 2: Appendix 4 - Biometric Indices Data ...... 144 BACKGROUND Appendix 5 – Histology-Based Fish Health Assessment (HBFHA) Data ...... 154 INFORMATION Appendix 6 - Age Estimation Data ...... 158 Appendix 7 – Edibility Assessment Data ...... 159 Appendix 8 – List of Histopathological Alterations ...... 172 CHAPTER 3: Appendix 9 – List of Organic Toxicants ...... 176 MATERIALS & METHODS

CHAPTER 4:

RESULTS

CHAPTER 5: DISCUSSION & CONCLUSION

APPENDICES

1 John 2: 16 – For all that is in the world, the lust of the flesh, and the lust of the eyes, and the pride of life, is not of the Father, but is of the world.. APPENDIX 1 – RECORD & ASSESSMENT SHEETS

APPENDIX 1 – RECORD & ASSESSMENT SHEETS

SITE: SPECIES: GPS:

BODY MASS (g) BODY LENGTH LIVER MASS SPLEEN MASS GONAD MASS TESTIS LENGTH DATE FISH # SEX (including viscera) (cm) (g) (g) Left Right Left Right

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

NECROPSY-BASED HEALTH ASSESSMENT DATA SHEET

Fish # Fish Gender Eyes Skin Fins Opercula Gills Bile Mesenteric Fat Liver Spleen Hindgut Kidney Parasites Comments

Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal None Additional samples collected: Hemorr. Aberrations : Erosion: Shortening Frayed D Straw <50% Fatty Granular Inflamed: Swollen Few

Missing Mild Mild Clubbed L Green 50% Nodules/Cysts Nodular Mild Mottled Moderate Macroscopic pathology: Other Moderate Moderate Discolour. D Green >50% Focal Discolour. Enlarged Moderate Granular Severe

Severe Severe Pale 100% Discolouration Other Severe Urolithiasis Developing stage: Other Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal None Additional samples collected: Hemorr. Aberrations : Erosion: Shortening Frayed D Straw <50% Fatty Granular Inflamed: Swollen Few

Missing Mild Mild Clubbed L Green 50% Nodules/Cysts Nodular Mild Mottled Moderate Macroscopic pathology: Other Moderate Moderate Discolour. D Green >50% Focal Discolour. Enlarged Moderate Granular Severe

Severe Severe Pale 100% Discolouration Other Severe Urolithiasis Developing stage: Other Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal None Additional samples collected: Hemorr. Aberrations : Erosion: Shortening Frayed D Straw <50% Fatty Granular Inflamed: Swollen Few

Missing Mild Mild Clubbed L Green 50% Nodules/Cysts Nodular Mild Mottled Moderate Macroscopic pathology: Other Moderate Moderate Discolour. D Green >50% Focal Discolour. Enlarged Moderate Granular Severe

Severe Severe Pale 100% Discolouration Other Severe Urolithiasis Developing stage: Other Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal None Additional samples collected: Hemorr. Aberrations : Erosion: Shortening Frayed D Straw <50% Fatty Granular Inflamed: Swollen Few

Missing Mild Mild Clubbed L Green 50% Nodules/Cysts Nodular Mild Mottled Moderate Macroscopic pathology: Other Moderate Moderate Discolour. D Green >50% Focal Discolour. Enlarged Moderate Granular Severe

Severe Severe Pale 100% Discolouration Other Severe Urolithiasis Developing stage: Other Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal None Additional samples collected: Hemorr. Aberrations : Erosion: Shortening Frayed D Straw <50% Fatty Granular Inflamed: Swollen Few

Missing Mild Mild Clubbed L Green 50% Nodules/Cysts Nodular Mild Mottled Moderate Macroscopic pathology: Other Moderate Moderate Discolour. D Green >50% Focal Discolour. Enlarged Moderate Granular Severe

Severe Severe Pale 100% Discolouration Other Severe Urolithiasis Developing stage: Other Other Other Normal Normal Normal Normal Normal L Straw None Normal Normal Normal Normal None Additional samples collected: Hemorr. Aberrations : Erosion: Shortening Frayed D Straw <50% Fatty Granular Inflamed: Swollen Few

Missing Mild Mild Clubbed L Green 50% Nodules/Cysts Nodular Mild Mottled Moderate Macroscopic pathology: Other Moderate Moderate Discolour. D Green >50% Focal Discolour. Enlarged Moderate Granular Severe

Severe Severe Pale 100% Discolouration Other Severe Urolithiasis Developing stage: Other Other Other *Adapted from Adams et al. (1993)

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

HISTOPATHOLOGY ASSESSMENT SHEETS

Gill Quantitative histological assessment (Adapted from Bernet et al., 1999 by Van Dyk, JC & Marchand, MJ) Species: Assessors: Site: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Notes Date: 1 RP Functional Unit Alterations CD Aneurysm/Haemor. e.g. telangiectasia 2 e.g. congestion Intercellular oedema 3 RC Epithelium Structural alterations e.g. sec lam branching Plasma alterations e.g. vacuolation 4 Inter cellular deposits Nuclear alterations 5 Atrophy Necrosis 6 Rupture of pilar cells Supporting tissue Structural alterations e.g. prim lam branching 7 Plasma alterations Intercellular deposits 8 Nuclear alterations Atrophy 9 Necrosis PC Epithelium Hypertrophy 10 Hyperplasia Mucous cells Hypertrophy 11 Hyperplasia I Exudate 12 Activation of RES Infiltration Lymphocytes 13 Infiltration Granulocytes T Benign 14 Malignant

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

Liver Quantitative histological assessment (Adapted from Bernet et al., 1999 by Van Dyk, JC & Marchand, MJ) Scoring dispersed lesions: 2 = Multiple single cells / blood Site: vessels Scoring MMCs: 2 = Few (< 5) small or large Scoring FCAs: 2 = One Species: 4 = 50% of cells / blood vessels 4 = Multiple small 4 = Multiple small Date assessed: 6 = All cells / blood vessels 6 = Multiple small and large 6 = Multiple large Assessors: 1 1 1 1 1 1 Note 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 s A. Cytoplasmic characteristics Clear cell hepatocytes (Majority) 1 Granular cell hepatocytes (Majority) - Basophilic Granular cell hepatocytes (Majority) - Eosinophilic 2 Dense cytoplasmic hepatocytes (Majority) - Basophilic Dense cytoplasmic hepatocytes (Majority) - Eosinophilic 3 Clumped cytoplasmic hepatocytes (Majority) B. Diffused or dispersed lesions 4 Circulatory 1. Peliosis 2. Intercellular haemorrhage / Vascular congestion 5 Regressive 1. Intracellular deposits 2. Atrophy 6 3. Frank necrosis 4. Hepatocellular nuclear pleomorphism / chromatin clearing 7 5. Hepatocellular pleomorphism 6. Steatosis 8 7. Vacuolation other than steatosis 8. Melano-macrophage centres (Light gold granular) 9 (Light gold vacuolated) 1 (Dark brown solid large) 0 Progressive 1. Hypertrophy: Hydropic change 1 2. Hypertrophy: Steatosis 1 3. Wall proliferation / Fibrosis 1 Inflammatory 1. Infiltration 2 2. Granulomatosis 1 D. Foci of cellular alteration (FCA) or liver nodules 1. Clear cell foci (central nuclei, glass appearance of cytoplasm) 3 Criteria: 2. Vacuolated foci (Displaced nuclei, fatty change) 3. Eosinophilic foci (pale to dark pink) Proliferation of smooth 1 a. Ten or more cells ER 4 b. No compression of the surrounding tissue 4. Basophilic Foci - Proliferation of rough ER 1 c. Continuity of the trabecular structure 5. Mixed foci 5 d. Mitotic figures are rare or absent 6. Necrotic foci e. Relative absence of MMCs, hepatopancreas, bile ducts 7. Hydropic foci f. Differ in morphology and staining from surrounding cells 8. Hypertrophic foci E. Benign tumours 1. Hepatocellular adenoma Criteria: 2. Cholangioma a. Clear separation of tubules in tumour from surrounding tissue 3. Hemangioma b. Compression of the surrounding tissue 4. Pancreatic acinar cell adenoma

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS c. Tubular thickening (more than three cells) & disorganization d. Relative absence of MMCs, Bile ducts and Hepatopancreas e. Increased cellular density f. Cellular and nuclear atypia will be absent or insignificant F. Malignant neoplasms 1. Hepatocellular carcinoma Criteria 2. Cholangiocarcinoma a. Cellular atypia including cellular and nuclear pleomorphism 3. Pancreatis acinar cell carcinoma b. Invasion of surrounding tissue, irregular borders 4. Mixed hepatobiliary cell carcinoma c. Satellite lesions 5. Mixed angiosarcoma/hepatocellular carcinoma d. Cystic structures, enlarged blood vessels and necrosis 6. Hemangiosarcoma e. Normal tissue architecture is lost 7. Hemangiopericytic sarcoma f. Increase in mitotic structures g. Relative absence of MMCs, bile ducts and hepatopancreas

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

Kidney Quantitative histological assessment (Adapted from Bernet et al., 1999 by Van Dyk, JC & Marchand, MJ) Species: Assessors: Site: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Notes Date: 1 RP Functional Unit Alterations CD Aneurysm/Haemor. e.g. induce congestion 2 Intercellular oedema Dilation of glomerulus capillaries 3

RC Tubule Structural alterations 4 Plasma alterations Vacuolation Hyaline droplet degeneration 5 Eosinophilic cytoplasm Granular degeneration 6 Inter cellular deposits Nuclear alterations 7 Atrophy Necrosis 8 Glomerulus Structural alterations e.g. granular degeneration / Plasma alterations intra cellular deposits 9 Intercellular deposits Nuclear alterations 10 Atrophy Necrosis 11 Interstitial tissue Structural alterations e.g. granular degeneration / Plasma alterations intra cellular deposits / vacuolation 12 Intercellular deposits eg. MMC Nuclear alterations 13 Atrophy Necrosis 14

e.g. albuminous degeneration PC Tubule Hypertrophy (reversible) (cloudy swelling) 15 Hyperplasia Glomerulus Thickening of BC membrane Hypertrophy Hyperplasia Interstitial tissue Hypertrophy Hyperplasia e.g. cirrhosis

I Exudate Activation of RES Infiltration Leucocytes (MNL) - lymphocytes Granulocytes T Benign Malignant

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

Heart Quantitative histological assessment (Adapted from Bernet et al., 1999 by Van Dyk, JC & Marchand, MJ) Species: Assessors: Site: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Notes Date: 1 RP Functional Unit Alterations CD Aneurysm/H/Haemor. e.g. induce congestion 2 Intercellular oedema RC Atrium MMC Myocardium 3 Epicardium Vacuolation Myocardium 4 Epicardium Nuclear alterations Myocardium 5 Atrophy e.g. muscle fibres Necrosis e.g. muscular necrosis 6 Ventricle MMC Myocardium Epicardium 7 Vacuolation Myocardium Epicardium 8 Nuclear alterations Atrophy e.g. muscle fibres 9 Necrosis BA MMC Elastic fibres 10 Epicardium Vacuolation Elastic fibres 11 Epicardium Nuclear alterations 12 Atrophy e.g. elastic fibres Necrosis 13 PC Atrium Hypertrophy Hyperplasia e.g. proliferation of collagen fibres 14 Ventricle Hypertrophy Hyperplasia e.g. proliferation of collagen fibres 15 I Endocardium Endocarditis Sinoatrial valve Atrio-ventricular valve Semi-lunar valve Atrium Ventricle Myocardium Myocarditis Atrium Ventricle BA Epicardium Epicarditis Atrium Ventricle BA T Benign Malignant

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

Testis Quantitative histological assessment (Adapted from Bernet et al., 1999 by Van Dyk, JC & Marchand, MJ) Species: Assessors: Site: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Notes Date: 1 RP Functional Unit Alterations Developmental stage: CD Aneurysm//Haemor. e.g. induce congestion 2 Intercellular oedema RC Lobule cysts Disorganization of lobules 3 Detachment of basal membrane Inhibition of spermatogenesis 4 Degeneration of Sertoli cells Interstitial tissue Structural alterations 5 Plasma alteration in Leydig cells Deposits MMC 6 Vacuolation Nuclear alterations in Leydig cells 7 Atrophy Necrosis 8 Developmental stages (1) Spermatogonia Structural alterations 9 Plasma alterations e.g. intra cellular deposits Vacuolation 10 Inter cellular deposits Nuclear alterations 11 Atrophy Necrosis 12 (2) Spermatocytes Structural alterations Plasma alterations e.g. intra cellular deposits 13 Vacuolation Intercellular deposits 14 Nuclear alterations Atrophy 15 Necrosis (3) Spermatids Structural alterations Plasma alterations e.g. intra cellular deposits Vacuolation Intercellular deposits Nuclear alterations Atrophy Necrosis (4) Spermatozoa Structural alterations Plasma alterations e.g. intra cellular deposits Vacuolation Intercellular deposits Nuclear alterations Atrophy Necrosis PC Lobule cysts Wall proliferation e.g. basal membrane / Tunica A Wall proliferation eg. blood vessels Interstitial tissue Hypertrophy

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

Hyperplasia Developmental stages (1) Spermatogonia Hypertrophy Hyperplasia (2) Spermatocytes Hypertrophy Hyperplasia (3) Spermatids Hypertrophy Hyperplasia (4) Spermatozoa Hypertrophy Hyperplasia I Exudate Activation of RES Infiltration Leucocytes (MNL) T Benign Malignant IS Intersex

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

Ovary Quantitative histological assessment (Adapted from Bernet et al., 1999 by Van Dyk, JC & Marchand, MJ) Species: Assessors: Site: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Notes Date: 1 RP Functional Unit Alterations Developmental stage: CD Aneurysm//Haem. e.g. induce congestion 2 Intercellular oedema RC Ovary Inhibition of oogenesis 3 Develop. stages Oogonia Structural alterations 4 Plasma alterations Inter cellular deposits 5 Nuclear alterations Atrophy 6 Necrosis Oocytes Structural alterations 7 Plasma alterations Intercellular deposits 8 Nuclear alterations e.g. germinal vesicle Atrophy 9 Necrosis Interstitial tissue Structural alterations 10 Plasma alterations e.g. vacuolation Intercellular deposits MMC 11 Nuclear alterations Atrophy 12 Necrosis PC Develop. stages 13 Oogonia Hypertrophy Hyperplasia 14 Oocytes Hypertrophy Hyperplasia 15 Interstitial tissue Hypertrophy Hyperplasia Tunica albuginea Thickening I Exudate Activation of RES Infiltration leucocytes (MNL) - lymphocytes granulocytes T Benign Malignant IS Intersex

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APPENDIX 1 – RECORD & ASSESSMENT SHEETS

Skin Quantitative histological assessment (Adapted from Bernet et al., 1999 by Van Dyk, JC & Marchand, MJ) Species: Assessors: Site: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Notes Date: 1 RP Functional Unit Alterations CD Aneurysm/Haem e.g. induce congestion 2 Intercellular oedema RC Epidermis Structural alterations 3 Plasma alterations granular degeneration / intra cellular deposits fatty degeneration (e.g. fatty change) 4 glycogen vacuoles vacuolation (content unknown) 5 Nuclear alterations pleomorphism / chromatin clearing pyknosis 6 Atrophy Necrosis 7 Basement Membrane Defect Dermis Structural alterations 8 Plasma alterations granular degeneration / intra cellular deposits fatty degeneration (e.g. fatty change) 9 glycogen vacuoles vacuolation (content unknown) 10 Nuclear alterations pleomorphism / chromatin clearing pyknosis 11 Atrophy Necrosis 12 PC Epidermis Hypertrophy Hyperplasia eg. mucous cells 13 Dermis Hypertrophy Hyperplasia 14 I Exudate Activation of RES 15 Infiltration e.g. leucocytes (MNL) - Lymphocytes granulocytes T Benign Malignant

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APPENDIX 2– SUBSTRATE ANALYSIS DATA

APPENDIX 2 – SUBSTRATE ANALYSIS DATA PHYSICO-CHEMICAL PARAMETERS Roodekopjes Dam

Table A - 1: Physico-chemical water parameters recorded on site at Roodekopjes Dam. Temperature Conductivity TDS Dissolved Oxygen Time pH (oC) (mS/m) (ppm) (mg/L) (% saturation) Site 1 – East of Dam Wall 09h00 9.20 25.0 46.3 231.0 12.90 169.80 12h00 9.43 28.1 46.4 233.0 19.64 251.10 17h00 9.60 27.3 46.8 234.0 23.47 291.50 Site 2 – Outlet 09h00 9.31 25.6 46.7 233.0 16.80 203.50 12h00 9.34 27.6 46.3 231.0 19.75 254.10 17h00 9.23 27.7 47.1 235.0 14.65 103.60 Site 3 – Middle of Impoundment 09h00 9.40 26.5 47.3 237.0 18.40 228.00 12h00 9.49 28.4 47.6 238.0 21.22 277.10 17h00 9.27 26.6 46.9 234.0 16.49 209.60 Site 4 – South of north-west Bay 09h00 9.40 26.7 42.4 212.0 17.40 213.20 12h00 9.60 28.7 42.7 214.0 20.16 264.80 17h00 9.42 27.2 47.2 235.0 20.90 255.10 Site 5 – North of north-west Bay 09h00 9.44 25.7 42.9 213.0 20.07 245.70 12h00 9.64 28.6 41.5 207.0 20.73 266.40 17h00 9.62 29.7 42.4 211.0 23.48 309.60 Site 6 – Crocodile River Inlet 09h00 No measurements taken 12h00 8.84 30.2 59.8 300.0 13.46 163.30 17h00 No measurements taken Site 7 – Sterkstroom River Inlet 09h00 No measurements taken 12h00 7.92 26.5 33.1 165.0 8.65 107.10 17h00 No measurements taken

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APPENDIX 2– SUBSTRATE ANALYSIS DATA

Vaalkop Dam

Table A - 2: Physico-chemical water parameters recorded on site at Vaalkop Dam. Temperature Conductivity TDS Dissolved Oxygen Time pH (oC) (mS/m) (ppm) (mg/L) (% saturation) Site 1 – Eastern Shore 09h00 9.34 26.4 41.4 207.0 17.68 219.30 12h00 9.30 31.1 42.5 211.0 17.23 231.90 17h00 9.34 32.6 41.5 209.0 15.99 221.00 Site 2 – East of Dam Wall 09h00 9.25 26.8 41.6 206.0 17.04 212.00 12h00 9.19 30.4 41.5 208.0 17.39 231.60 17h00 9.29 30.9 42.0 210.0 17.06 229.50 Site 3 – Middle of Eastern Portion of Impoundment 09h00 9.25 26.7 42.0 210.0 17.65 220.10 12h00 9.19 30.1 43.0 215.0 16.90 224.40 17h00 9.35 30.5 42.3 211.0 18.96 255.40 Site 4 – Outlet 09h00 9.34 27.0 21.0 204.0 17.65 221.70 12h00 9.31 29.5 42.2 210.0 17.73 231.40 17h00 9.34 30.7 42.1 211.0 19.99 268.60 Site 5 – Hex River Inlet 09h00 No measurements taken 12h00 8.36 27.6 53.4 257.0 9.67 123.50 17h00 No measurements taken Site 6 – Elands River Inlet 09h00 No measurements taken 12h00 8.06 31.9 35.3 177.0 7.83 113.90 17h00 No measurements taken

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APPENDIX 2– SUBSTRATE ANALYSIS DATA

Marico-Bosveld Dam

Table A - 3: Physico-chemical water parameters recorded on site at Marico-Bosveld Dam. Temperature Conductivity TDS Dissolved Oxygen Time pH (oC) (mS/m) (ppm) (mg/L) (% saturation) Site 1 – Eastern Shoreline 09h00 7.70 23.0 19.1 96.0 * * 12h00 7.66 23.0 18.7 93.5 * * 17h00 7.88 24.0 18.9 94.6 * * Site 2 – Middle of Impoundment 09h00 7.80 23.0 18.9 95.1 * * 12h00 7.78 24.0 18.6 92.8 * * 17h00 8.02 23.0 18.8 93.9 * * Site 3 – Outlet 09h00 7.99 23.0 18.7 93.7 * * 12h00 7.83 24.0 18.7 93.7 * * 17h00 7.81 23.0 18.7 93.5 * * Site 4 – Groot-Marico River Inlet 09h00 No measurements taken 12h00 7.55 24.0 19.1 95.6 * * 17h00 No measurements taken Site 5 – Marico Eye 09h00 No measurements taken 12h00 7.50 20.5 27.1 135.0 * * 17h00 No measurements taken *Equipment malfunction – meters were serviced and checked before the Field Surveys.

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APPENDIX 2– SUBSTRATE ANALYSIS DATA

CHEMICAL ANALYSIS ORGANIC

Table A - 4: Incidence of organic toxicants measured in pooled water & sediment samples, from the outlets and inlets of each impoundment. CONCENTRATION TOXICANT TYPE TOXICANT (ppb)* WATER SEDIMENT Roodekopjes Dam Hormone Estrone <10.00 - Hormone Estriol <10.00 - Hormone 17-Beta Estradiol <10.00 - Hormone Ethynyl Estradiol <10.00 - Organochlorine op'-DDE 0.12 - Organochlorine op’-DDD 0.11 - Pesticides All* - <66.00 Phenol Nonylphenol Technical <2000.00 64800.00 Polychlorinated Biphenols All* - <1000.00 Vaalkop Dam Herbicide Terbuthylazine <2.00 - Hormone Estrone <10.00 - Hormone Estriol <10.00 - Hormone 17-Beta Estradiol <10.00 - Hormone Ethynyl Estradiol <10.00 - Organochlorine op’-DDE 0.15 - Organochlorine op'-DDT 0.58 - Pesticides All* - <66.00 Phenol Nonylphenol Technical 3000.00 69900.00 Polychlorinated Biphenols All* - <10000.00 Marico-Bosveld Dam Hormone Estrone <10.00 - Hormone Estriol <10.00 - Hormone 17-Beta Estradiol <10.00 - Hormone Ethynyl Estradiol <10.00 - Organochlorine Aldrin 0.59 - Organochlorine op’-DDD 0.62 - Organochlorine op'-DDT 1.41 - Pesticides All* - <66.00 Phenol Nonylphenol Technical <2000.00 75500.00 Polychlorinated Biphenols All* - <10000.00 * See Appendix 9 for the list of PCB’s and pesticides screened for.

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APPENDIX 2– SUBSTRATE ANALYSIS DATA

INORGANIC

Table A - 5: Inorganic toxicants found in the pooled water samples, from the outlets and inlets of each impoundment. CONCENTRATION (ppb) METAL/ELEMENT WATER SEDIMENT RD VD MBD RD VD MBD Ag - 0.073 - 493.014 255.949 760.697 Al 19.560 5.454 1.540 29 520.958 40 511.898 44 096.672 As 0.031 0.010 - 131.737 107.978 130.745 B 1.295 0.161 0.152 3 067.864 3 331.334 3 199.287 Ba 0.105 0.086 0.044 4 900.200 5 560.888 5 225.832 Be ------Bi ------Ca 84.910 42.760 22.920 21 556.886 48 850.230 20 839.937 Cd - - - - 3.999 - Co - - - 17.964 51.990 29.715 Cr - 0.002 - 389.222 405.919 657.686 Cu 0.048 0.034 0.052 297.405 301.940 336.767 Fe 2.144 3.808 0.655 104 431.138 106 918.616 126 208.399 K 6.846 6.459 1.305 8 321.357 8 530.294 13 801.506 Li 0.015 - - 23.952 - 33.677 Mg 18.360 18.680 12.770 8 351.297 17 504.499 17 373.217 Mn 0.353 0.171 0.045 1 876.248 2 541.492 1 555.071 Mo ------Na 57.000 31.690 5.995 17 704.591 18 734.253 20 740.887 Ni - - - 119.760 183.963 190.174 P 1.361 2.009 1.055 2 497.006 - 774.564 Pb 0.033 - 0.017 109.780 63.987 87.163 Sb - - 0.001 1.996 - - Se 0.176 0.180 0.136 299.401 269.946 283.281 Si 5.240 8.064 3.645 1 299.401 1 659.668 1 372.821 Sn 0.091 0.047 - 67.864 23.995 245.642 Sr 0.496 0.257 0.059 83.832 149.970 65.372 Ti - 0.043 0.003 293.413 93.981 576.466 V 0.019 0.014 0.001 259.481 311.938 289.223 W 0.033 0.051 0.036 - 55.989 17.829 Zn 0.007 0.001 - 3 465.070 3 731.254 3 664.818 Zr 0.003 0.003 0.001 29.940 27.994 63.391

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APPENDIX 3 – HAEMATOLOGICAL ASSESSMENT DATA

APPENDIX 3 – HAEMATOLOGICAL ASSESSMENT DATA HAEMATOLOGICAL ASSESSMENT Sharptooth Catfish (CLARIAS GARIEPINUS)

Table A - 6: Raw data for the calculation of the haematocrit and leukocrit of each individual Clarias gariepinus at each assessment site. RBC* WBC* RBC* WBC* RBC* WBC* FISH # TOTAL FISH # TOTAL FISH # TOTAL (%) (%) (%) (%) (%) (%) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD4 54 44.44 (24) 3.70 (2) VD1 59 38.98 (23) 3.39 (2) MBD2 50 24.00 (12) 2.00 (1) RD5 55 47.27 (26) 3.64 (2) VD3 49 40.82 (20) 2.04 (1) MBD3 36 36.11 (13) 2.78 (1) RD6 48 31.25 (15) 0.00 (0) VD5 58 41.38 (24) 1.72 (1) MBD4 47 42.55 (20) 4.26 (2) RD10 42 38.10 (16) 2.38 (1) VD6 55 38.18 (21) 1.82 (1) MBD5 51 17.65 (9) 1.96 (1) RD11 53 39.62 (21) 1.89 (1) VD7 46 43.48 (20) 4.35 (2) MBD6 51 39.22 (20) 1.96 (1) RD12 47 29.79 (14) 2.13 (1) VD8 54 35.19 (19) 3.70 (2) MBD7 37 37.84 (14) 2.70 (1) RD17 61 32.79 (20) 1.64 (1) VD11 47 44.68 (21) 2.13 (1) MBD8 51 33.33 (17) 3.92 (2) RD18 51 33.33 (17) 1.96 (1) VD14 Laboratory Accident BMB9 57 MBD9 1.75 (1) RD19 56 39.29 (22) 1.79 (1) VD16 Laboratory Accident BMB10 56 MBD10 3.57 (2) RD20 62 32.26 (20) 1.61 (1) VD17 54 11.11 (6) 0.00 (0) MBD11 53 37.74 (20) 3.77 (2) RD21 65 41.54 (27) 1.54 (1) VD18 56 35.71 (20) 1.79 (1) MBD12 56 32.14 (18) 1.79 (1) RD22 50 28.00 (14) 2.00 (1) VD19 52 34.62 (18) 1.92 (1) MBD15 64 25.00 (16) 3.13 (2) RD23 54 38.89 (21) 1.85 (1) VD20 58 29.31 (17) 1.72 (1) MBD16 51 33.33 (17) 1.96 (1) RD24 58 46.55 (27) 0.00 (0) VD21 51 31.37 (16) 1.96 (1) MBD17 31 29.03 (9) 3.23 (1) RD25 56 41.07 (23) 1.79 (1) VD22 39 30.77 (12) 2.56 (1) MBD18 46 26.09 (12) 2.17 (1) MEAN 37.61 (20.47) 1.86 (1.00) MEAN 35.05 (18.23) 2.24 (1.15) MEAN 32.68 (16.00) 2.73 (1.33) STDEV 6.10 (4.50) 1.00 (0.53) STDEV 8.66 (4.80) 1.09 (0.55) STDEV 6.97 (4.29) 0.86 (0.49) * RBC – Red blood cell count and percentage, WBC – White blood cell count and percentage. **RBC & WBC shows physical counts in parenthesis and percentage out of total.

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APPENDIX 3 – HAEMATOLOGICAL ASSESSMENT DATA

Common Carp (Cyprinus carpio)

Table A - 7: Raw data for the calculation of the haematocrit and leukocrit of each individual Cyprinus carpio at each assessment site. RBC* WBC* RBC* WBC* RBC* WBC* FISH # TOTAL FISH # TOTAL FISH # TOTAL (%) (%) (%) (%) (%) (%) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD1 54 37.04 (20) 1.85 (1) VD2 56 41.07 (23) 1.79 (1) MBD1 62 29.03 (18) 0.00 (0) RD2 44 50.00 (22) 2.27 (1) VD4 55 34.55 (19) 1.82 (1) MBD13 49 42.86 (21) 2.04 (1) RD3 52 36.54 (19) 1.92 (1) VD9 55 29.09 (16) 1.82 (1) MBD14 46 26.09 (12) 2.17 (1) RD7 40 37.50 (15) 2.50 (1) VD10 51 25.49 (13) 1.96 (1) MBD19 60 30.00 (18) 0.00 (0) RD8 48 29.17 (14) 2.08 (1) VD12 59 20.34 (12) 1.69 (1) MBD20 76 23.68 (18) 1.32 (1) RD9 44 22.73 (10) 2.27 (1) VD13 59 28.81 (17) 1.69 (1) MBD21 57 43.86 (25) 1.75 (1) RD13 53 30.19 (16) 1.89 (1) VD15 57 19.30 (11) 1.75 (1) MBD22 30 70.00 (21) 3.33 (1) RD14 46 39.13 (18) 2.17 (1) VD23 55 43.64 (24) 1.82 (1) MBD23 66 36.36 (24) 1.52 (1) RD15 50 32.00 (16) 2.00 (1) VD24 61 40.98 (25) 1.64 (1) MBD24 63 38.10 (24) 1.59 (1) RD16 58 29.31 (17) 1.72 (1) VD25 59 33.90 (20) 1.69 (1) - - - - RD26 24 25.00 (6) 4.17 (1) VD26 56 19.64 (11) 1.79 (1) - - - - RD27 39 33.33 (13) 5.13 (2) VD27 54 38.89 (21) 1.85 (1) - - - - RD28 39 43.59 (17) 2.56 (1) VD28 58 36.21 (21) 1.72 (1) - - - - RD29 46 26.09 (12) 2.17 (1) VD29 63 28.57 (18) 1.59 (1) - - - - RD30 61 42.62 (26) 0.00 (0) ------MEAN 34.28 (16.07) 2.31 (1.00) MEAN 31.46 (17.93) 1.76 (1.00) MEAN 37.78 (20.11) 1.52 (0.78) STDEV 7.61 (4.88) 1.13 (0.38) STDEV 8.29 (4.78) 0.10 (0.00) STDEV 14.03 (4.11) 1.04 (0.44) * RBC – Red blood cell count and percentage, WBC – White blood cell count and percentage. **RBC & WBC shows physical counts in parenthesis and percentage out of total.

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APPENDIX 4 – BIOMETRIC INDICES DATA

APPENDIX 4 - BIOMETRIC INDICES DATA CONDITION FACTOR (CF) Sharptooth Catfish (CLARIAS GARIEPINUS)

Table A - 8: Body mass and total lengths are used to calculate the condition factor (CF) for each individual Clarias gariepinus, as per site.

BODY TOTAL BODY TOTAL BODY TOTAL FISH # CF FISH # CF FISH # CF MASS (KG) LENGTH (CM) MASS (KG) LENGTH (CM) MASS (KG) LENGTH (CM) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD4 4.42 86.0 0.69 VD1 3.83 73.0 0.98 MBD2 2.16 72.7 0.56 RD5 8.64 105.0 0.75 VD3 3.15 72.0 0.84 MBD3 1.26 58.5 0.63 RD6 4.62 84.5 0.77 VD5 4.12 81.0 0.78 MBD4 1.44 58.0 0.74 RD10 3.66 75.0 0.87 VD6 4.64 94.0 0.56 MBD5 1.33 60.0 0.62 RD11 3.70 66.0 1.29 VD7 3.58 78.2 0.75 MBD6 1.29 57.5 0.68 RD12 3.80 76.0 0.87 VD8 2.95 71.6 0.80 MBD7 1.50 61.5 0.64 RD17 5.70 93.5 0.70 VD11 3.08 75.0 0.73 MBD8 1.31 56.5 0.73 RD18 6.17 97.8 0.66 VD14 1.55 58.0 0.79 MBD9 2.88 73.0 0.74 RD19 6.30 94.0 0.76 VD16 3.37 75.5 0.78 MBD10 8.69 109.0 0.67 RD20 1.88 65.7 0.66 VD17 2.22 64.0 0.85 MBD11 1.33 58.0 0.68 RD21 3.81 78.5 0.79 VD18 2.04 66.0 0.71 MBD12 1.50 54.0 0.95 RD22 2.75 70.0 0.80 VD19 1.58 62.0 0.66 MBD15 1.68 63.0 0.67 RD23 2.06 66.3 0.71 VD20 1.85 63.0 0.74 MBD16 1.33 60.0 0.62 RD24 3.68 73.0 0.95 VD21 1.99 63.5 0.78 MBD17 1.86 67.0 0.62 RD25 3.18 75.0 0.75 VD22 4.17 88.0 0.61 MBD18 2.15 71.6 0.59 MEAN 4.29 80.42 0.80 MEAN 2.94 72.32 0.76 MEAN 2.11 65.35 0.68 STDEV 1.78 12.48 0.16 STDEV 1.02 10.11 0.10 STDEV 1.87 13.53 0.09

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APPENDIX 4 – BIOMETRIC INDICES DATA

Common Carp (Cyprinus carpio)

Table A - 9: Body mass and total lengths are used to calculate the condition factor (CF) for each individual Cyprinus carpio, as per site.

BODY TOTAL BODY TOTAL BODY TOTAL FISH # CF FISH # CF FISH # CF MASS (KG) LENGTH (CM) MASS (KG) LENGTH (CM) MASS (KG) LENGTH (CM) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD1 3.04 58.0 1.56 VD2 2.14 52.0 1.52 MBD1 1.03 48.0 0.93 RD2 2.12 51.0 1.60 VD4 3.18 64.0 1.21 MBD13 1.85 52.5 1.28 RD3 2.62 54.5 1.62 VD9 3.10 63.0 1.24 MBD14 1.57 50.0 1.26 RD7 3.12 61.0 1.37 VD10 4.37 70.3 1.26 MBD19 2.14 53.5 1.40 RD8 2.98 59.0 1.45 VD12 3.07 62.0 1.29 MBD20 2.35 59.5 1.12 RD9 2.66 57.0 1.44 VD13 2.59 61.5 1.11 MBD21 1.35 49.3 1.13 RD13 4.04 63.0 1.62 VD15 2.02 55.5 1.18 MBD22 1.28 47.3 1.21 RD14 2.34 54.0 1.49 VD23 3.13 64.0 1.19 MBD23 1.26 47.0 1.21 RD15 2.50 56.5 1.39 VD24 4.00 70.5 1.14 MBD24 1.32 48.2 1.18 RD16 2.62 59.0 1.28 VD25 3.44 63.5 1.34 - - - - RD26 3.56 62.0 1.49 VD26 3.48 64.0 1.33 - - - - RD27 1.38 45.0 1.51 VD27 2.70 61.5 1.16 - - - - RD28 3.66 62.0 1.54 VD28 2.80 57.0 1.51 - - - - RD29 2.35 56.5 1.30 VD29 2.89 60.0 1.34 - - - - RD30 2.35 56.0 1.34 ------MEAN 2.76 56.97 1.47 MEAN 3.07 62.06 1.27 MEAN 1.57 50.59 1.19 STDEV 0.67 4.69 0.11 STDEV 0.64 5.02 0.13 STDEV 0.45 4.03 0.13

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APPENDIX 4 – BIOMETRIC INDICES DATA

ORGANO-SOMATIC INDICES Hepatosomatic Index (HSI) Sharptooth Catfish (Clarias gariepinus)

Table A - 10: Hepatosomatic Index (HSI), calculated from body mass and liver mass, from Clarias gariepinus at each site.

LIVER BODY LIVER LIVER FISH # BODY MASS (KG) HSI FISH # HSI FISH # BODY MASS (KG) HSI MASS (G) MASS (KG) MASS (G) MASS (G)

Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD4 4.42 32.016 0.724 VD1 3.83 21.224 0.554 MBD2 2.16 10.849 0.502 RD5 8.64 65.480 0.758 VD3 3.15 16.842 0.535 MBD3 1.26 6.194 0.492 RD6 4.62 39.310 0.851 VD5 4.12 20.235 0.491 MBD4 1.44 9.331 0.648 RD10 3.66 21.101 0.577 VD6 4.64 28.193 0.608 MBD5 1.33 6.895 0.518 RD11 3.70 24.872 0.672 VD7 3.58 27.340 0.764 MBD6 1.29 5.691 0.441 RD12 3.80 23.175 0.610 VD8 2.95 25.118 0.852 MBD7 1.50 9.292 0.620 RD17 5.70 35.836 0.629 VD11 3.08 22.910 0.744 MBD8 1.31 7.239 0.553 RD18 6.17 43.982 0.713 VD14 1.55 13.192 0.851 MBD9 2.88 18.181 0.631 RD19 6.30 66.444 1.055 VD16 3.37 18.837 0.559 MBD10 8.69 45.739 0.526 RD20 1.88 16.096 0.856 VD17 2.22 18.215 0.821 MBD11 1.33 5.802 0.436 RD21 3.81 31.271 0.821 VD18 2.04 12.846 0.630 MBD12 1.50 6.114 0.408 RD22 2.75 26.685 0.970 VD19 1.58 7.870 0.498 MBD15 1.68 8.734 0.520 RD23 2.06 22.492 1.092 VD20 1.85 2.300 0.124 MBD16 1.33 5.800 0.436 RD24 3.68 20.375 0.554 VD21 1.99 18.210 0.915 MBD17 1.86 11.340 0.610 RD25 3.18 23.322 0.733 VD22 4.17 30.159 0.723 MBD18 2.15 10.110 0.470 MEAN 0.774 MEAN 0.645 MEAN 0.521 STDEV 0.166 STDEV 0.201 STDEV 0.078

PAGE | 146

APPENDIX 4 – BIOMETRIC INDICES DATA

Common Carp (Cyprinus carpio)

Table A - 11: Hepatosomatic Index (HSI), calculated from body mass and liver mass, from Cyprinus carpio at each site.

LIVER BODY LIVER LIVER FISH # BODY MASS (KG) HSI FISH # HSI FISH # BODY MASS (KG) HSI MASS (G) MASS (KG) MASS (G) MASS (G)

Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD1 3.04 59.246 1.949 VD2 2.14 32.022 1.496 MBD1 1.03 11.540 1.120 RD2 2.12 33.596 1.585 VD4 3.18 48.000 1.509 MBD13 1.85 34.070 1.842 RD3 2.62 13.477 0.514 VD9 3.10 38.260 1.234 MBD14 1.57 25.530 1.626 RD7 3.12 38.599 1.237 VD10 4.37 34.000 0.778 MBD19 2.14 41.464 1.938 RD8 2.98 41.885 1.406 VD12 3.07 40.782 1.328 MBD20 2.35 28.982 1.233 RD9 2.66 32.831 1.234 VD13 2.59 36.000 1.390 MBD21 1.35 16.870 1.250 RD13 4.04 56.542 1.400 VD15 2.02 27.520 1.362 MBD22 1.28 11.665 0.911 RD14 2.34 27.762 1.186 VD23 3.13 28.490 0.910 MBD23 1.26 18.764 1.489 RD15 2.50 30.985 1.239 VD24 4.00 37.800 0.945 MBD24 1.32 13.843 1.049 RD16 2.62 36.922 1.409 VD25 3.44 52.000 1.512 - - - - RD26 3.56 35.665 1.002 VD26 3.48 21.347 0.613 - - - - RD27 1.38 19.674 1.426 VD27 2.70 36.819 1.364 - - - - RD28 3.66 45.000 1.230 VD28 2.80 41.550 1.484 - - - - RD29 2.35 27.825 1.184 VD29 2.89 28.501 0.986 - - - - RD30 2.35 20.302 0.864 ------MEAN 1.258 MEAN 1.208 MEAN 1.384 STDEV 0.323 STDEV 0.302 STDEV 0.359

PAGE | 147

APPENDIX 4 – BIOMETRIC INDICES DATA

Splenosomatic Index (SSI) Sharptooth Catfish (Clarias gariepinus)

Table A - 12: Splenosomatic Index (SSI), calculated from body mass and spleen mass, from Clarias gariepinus at each site.

SPLEEN BODY SPLEEN SPLEEN FISH # BODY MASS (KG) SSI FISH # SSI FISH # BODY MASS (KG) SSI MASS (G) MASS (KG) MASS (G) MASS (G)

Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD4 4.42 14.055 0.318 VD1 3.83 5.065 0.132 MBD2 2.16 4.940 0.229 RD5 8.64 15.165 0.176 VD3 3.15 2.892 0.092 MBD3 1.26 5.578 0.443 RD6 4.62 9.343 0.202 VD5 4.12 2.742 0.067 MBD4 1.44 2.315 0.161 RD10 3.66 8.645 0.236 VD6 4.64 8.113 0.175 MBD5 1.33 4.069 0.306 RD11 3.70 3.966 0.107 VD7 3.58 3.082 0.086 MBD6 1.29 2.368 0.184 RD12 3.80 5.430 0.143 VD8 2.95 6.360 0.216 MBD7 1.50 1.833 0.122 RD17 5.70 5.832 0.102 VD11 3.08 3.660 0.119 MBD8 1.31 2.488 0.190 RD18 6.17 9.336 0.151 VD14 1.55 1.138 0.073 MBD9 2.88 6.584 0.229 RD19 6.30 22.972 0.365 VD16 3.37 3.924 0.116 MBD10 8.69 17.835 0.205 RD20 1.88 1.962 0.104 VD17 2.22 4.667 0.210 MBD11 1.33 1.793 0.135 RD21 3.81 16.812 0.441 VD18 2.04 3.082 0.151 MBD12 1.50 1.035 0.069 RD22 2.75 2.886 0.105 VD19 1.58 1.145 0.073 MBD15 1.68 2.333 0.139 RD23 2.06 1.650 0.080 VD20 1.85 2.226 0.120 MBD16 1.33 3.120 0.235 RD24 3.68 3.729 0.101 VD21 1.99 1.853 0.093 MBD17 1.86 3.810 0.205 RD25 3.18 3.525 0.111 VD22 4.17 6.636 0.159 MBD18 2.15 2.070 0.096 MEAN 0.183 MEAN 0.126 MEAN 0.196 STDEV 0.110 STDEV 0.048 STDEV 0.091

PAGE | 148

APPENDIX 4 – BIOMETRIC INDICES DATA

Common Carp (Cyprinus carpio)

Table A - 13: Splenosomatic Index (SSI), calculated from body mass and spleen mass, from Cyprinus carpio at each site.

LIVER BODY LIVER LIVER FISH # BODY MASS (KG) SSI FISH # SSI FISH # BODY MASS (KG) SSI MASS (G) MASS (KG) MASS (G) MASS (G)

Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD1 3.04 15.150 0.498 VD2 2.14 2.768 0.129 MBD1 1.03 0.840 0.082 RD2 2.12 14.410 0.680 VD4 3.18 2.386 0.075 MBD13 1.85 1.901 0.103 RD3 2.62 2.946 0.112 VD9 3.10 2.510 0.081 MBD14 1.57 2.298 0.146 RD7 3.12 11.160 0.358 VD10 4.37 6.418 0.147 MBD19 2.14 2.101 0.098 RD8 2.98 64.180 2.154 VD12 3.07 4.803 0.156 MBD20 2.35 2.341 0.100 RD9 2.66 46.220 1.738 VD13 2.59 3.423 0.132 MBD21 1.35 1.230 0.091 RD13 4.04 10.711 0.265 VD15 2.02 2.525 0.125 MBD22 1.28 0.880 0.069 RD14 2.34 7.070 0.302 VD23 3.13 3.335 0.107 MBD23 1.26 0.980 0.078 RD15 2.50 3.955 0.158 VD24 4.00 3.479 0.087 MBD24 1.32 1.016 0.077 RD16 2.62 6.315 0.241 VD25 3.44 3.516 0.102 - - - - RD26 3.56 5.696 0.160 VD26 3.48 0.095 0.003 - - - - RD27 1.38 5.734 0.416 VD27 2.70 4.290 0.159 - - - - RD28 3.66 6.000 0.164 VD28 2.80 3.135 0.112 - - - - RD29 2.35 8.695 0.370 VD29 2.89 3.247 0.112 - - - - RD30 2.35 1.824 0.078 ------MEAN 0.513 MEAN 0.109 MEAN 0.094 STDEV 0.608 STDEV 0.040 STDEV 0.023

PAGE | 149

APPENDIX 4 – BIOMETRIC INDICES DATA

Gonadosomatic Index (GSI) Sharptooth Catfish (Clarias gariepinus): Male (Testes)

Table A - 14: Male gonadosomatic index (GSI), calculated only from body mass and combined testes mass, from Clarias gariepinus. BODY MASS TOTAL TESTES BODY MASS TOTAL TESTES BODY MASS TOTAL TESTES FISH # GSI FISH # GSI FISH # GSI (KG) MASS (G) (KG) MASS (G) (KG) MASS (G) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD4 4.420 10.125 0.229 VD1 3.830 1.605 0.042 MBD2 2.160 1.804 0.084 RD5 8.640 18.292 0.212 VD3 3.150 0.787 0.025 MBD3 1.260 1.186 0.094 RD12 3.800 10.727 0.282 VD5 4.120 0.918 0.022 MBD4 1.440 0.339 0.024 RD18 6.170 21.568 0.350 VD6 4.640 10.670 0.230 MBD5 1.330 1.363 0.102 RD19 6.300 22.362 0.355 VD8 2.950 3.135 0.106 MBD6 1.290 0.853 0.066 RD20 1.880 0.800 0.043 VD11 3.080 2.422 0.079 MBD8 1.310 1.172 0.089 RD21 3.810 11.991 0.315 VD16 3.370 7.766 0.230 MBD10 8.690 8.878 0.102 RD24 3.680 8.010 0.218 VD17 2.220 0.253 0.011 MBD15 1.680 1.110 0.066 RD25 3.180 3.651 0.115 VD20 1.850 3.114 0.168 MBD16 1.330 1.950 0.147 VD22 4.170 10.850 0.260 MBD17 1.860 1.360 0.073 MBD18 2.150 1.380 0.064 MEAN 0.235 MEAN 0.117 MEAN 0.083 STDEV 0.105 STDEV 0.097 STDEV 0.031

PAGE | 150

APPENDIX 4 – BIOMETRIC INDICES DATA

Common Carp (Cyprinus carpio): Male (Testes)

Table A - 15: Male gonadosomatic index (GSI), calculated only from body mass and combined testes mass, from Cyprinus carpio.

BODY MASS TOTAL TESTES BODY MASS TOTAL TESTES BODY MASS TOTAL TESTES FISH # GSI FISH # GSI FISH # GSI (KG) MASS (G) (KG) MASS (G) (KG) MASS (G) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD2 8.640 158.975 1.840 VD2 3.830 139.839 3.651 MBD13 1.260 58.865 4.672 RD3 4.620 129.485 2.803 MBD14 1.440 64.042 4.447 RD8 3.700 171.325 4.630 MBD21 1.500 68.840 4.589 RD14 6.170 179.406 2.908 MBD22 1.310 53.963 4.119 RD27 2.750 80.716 2.935 MBD23 2.880 61.853 2.148 RD29 3.680 180.510 4.905 MBD24 8.690 61.223 0.705 RD30 3.180 72.150 2.269 MEAN 3.184 MEAN 3.651 MEAN 3.447 STDEV 1.153 STDEV 0.000 STDEV 1.641

PAGE | 151

APPENDIX 4 – BIOMETRIC INDICES DATA

Sharptooth Catfish (Clarias gariepinus): Female (Ovaries)

Table A - 16: Female gonadosomatic index (GSI), calculated only from body mass and ovary mass, from Clarias gariepinus. BODY MASS TOTAL OVARY BODY MASS TOTAL OVARY BODY MASS TOTAL OVARY FISH # GSI FISH # GSI FISH # GSI (KG) MASS (G) (KG) MASS (G) (KG) MASS (G) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD6 4.620 461.923 9.998 VD7 3.580 146.000 4.078 MBD7 1.500 1.833 0.122 RD10 3.660 312.517 8.539 VD14 1.550 94.000 6.065 MBD9 2.880 41.891 1.455 RD11 3.700 97.163 2.626 VD18 2.040 44.568 2.185 MBD11 1.330 16.621 1.250 RD17 5.700 397.887 6.980 VD19 1.580 4.883 0.309 MBD12 1.500 13.883 0.926 RD22 2.750 269.751 9.809 VD21 1.990 116.000 5.829 RD23 2.060 11.878 0.577 MEAN 6.422 MEAN 3.693 MEAN 0.938 STDEV 3.940 STDEV 2.453 STDEV 0.586

PAGE | 152

APPENDIX 4 – BIOMETRIC INDICES DATA

Common Carp (Cyprinus carpio): Female (Ovaries)

Table A - 17: Female gonadosomatic index (GSI), calculated only from body mass and ovary mass, from Cyprinus carpio.

BODY MASS TOTAL OVARY BODY MASS TOTAL OVARY BODY MASS TOTAL OVARY FISH # GSI FISH # GSI FISH # GSI (KG) MASS (G) (KG) MASS (G) (KG) MASS (G) Roodekopjes Dam Vaalkop Dam Marico-Bosveld Dam RD1 4.420 195.256 4.418 VD4 3.150 174.000 5.524 MBD1 2.160 18.947 0.877 RD7 3.660 139.556 3.813 VD9 4.120 236.000 5.728 MBD19 1.330 72.000 5.414 RD9 3.800 143.711 3.782 VD10 4.640 366.000 7.888 MBD20 1.290 97.657 7.570 RD13 5.700 321.228 5.636 VD12 3.580 212.000 5.922 RD15 6.300 87.295 1.386 VD13 2.950 232.000 7.864 RD16 1.880 83.275 4.430 VD15 3.080 124.000 4.026 RD26 3.810 120.021 3.150 VD23 1.550 102.000 6.581 RD28 2.060 232.000 11.262 VD24 3.370 148.000 4.392 VD25 2.220 170.000 7.658 VD26 2.040 124.000 6.078 VD27 1.580 232.000 14.684 VD28 1.850 198.000 10.703 VD29 1.990 284.000 14.271 MEAN 4.734 MEAN 7.794 MEAN 4.620 STDEV 2.906 STDEV 3.428 STDEV 3.416

PAGE | 153

APPENDIX 5 – HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) DATA

APPENDIX 5 – HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) DATA QUANTITATIVE HISTOLOGICAL ASSESSMENT -

The following tables show the calculated organ indices (Iorg) and total reaction pattern indices (ITot rp) for each individual fish species assessed per species per impoundment. The arithmetic mean has been calculated per target organ present. Each target organ sample number differs depending on the number of lost target organs during tissue processing or absence of fish due to incomplete specimen sample.

The sum of these means determines the average fish index (Ifish) per species per site. It should be noted that the method applied shows some degree of uncertainty when the full target sample is not attained within all target organs, as indicated by the difference in the sum of the total reaction pattern indices (ITot rp) and the organ indices (Iorg), respectively.

Table A - 18: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. gariepinus at Roodekopjes Dam. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN GILLS (Igills) 8 8 12 16 10 2 4 2 L L 6 0 4 8 6 6.62 LIVER (Iliver) 18 8 4 12 18 8 2 L 14 0 14 16 14 L 4 10.15 KIDNEY (Ikidney) 18 22 20 10 2 8 8 L 6 8 8 8 14 6 L 10.62 HEART (Iheart) 0 0 10 4 10 0 2 10 2 0 2 0 4 0 0 2.93 TESTIS (Itestis) 0 0 - - - 0 - 0 0 0 0 - - 0 0 0.00 OVARY (Iovary) - - 0 0 0 - 0 - - - - 0 0 - - 0.00 SKIN (Iskin) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FISH (Ifish) 44 38 46 42 40 18 16 12 22 8 30 24 36 14 10 30.32 CIRCULATORY 0 0 0 4 2 2 0 2 0 0 2 0 0 0 2 1.04 DISTURBANCES (Icd) REGRESSIVE 30 30 26 18 14 12 12 6 18 8 16 24 24 6 4 18.37 CHANGES (Irc) PROGRESSIVE 10 8 12 12 8 0 4 0 0 0 4 0 4 8 4 5.48 CHANGES (Ipc) INFLAMMATION (Ii) 4 0 8 8 16 4 0 4 4 0 8 0 8 0 0 4.74 TUMOURS (It) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FOCAL CELLULAR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 ALTERATIONS (Ifca) FISH (Ifish) 44 38 46 42 40 18 16 12 22 8 30 24 36 14 10 29.63 *L – lost during histological processing, A – absent as a result of sample size

PAGE | 154

APPENDIX 5 – HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) DATA

Table A - 19: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. gariepinus at Vaalkop Dam Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN GILLS (Igills) 6 8 6 6 6 0 2 10 10 2 4 0 0 0 L 4.29 LIVER (Iliver) 8 20 4 10 2 8 0 0 4 0 2 0 0 6 36 6.67 KIDNEY (Ikidney) L 0 0 L 4 4 6 0 0 L 0 2 0 2 10 2.33 HEART (Iheart) 14 6 0 6 0 10 2 14 6 4 0 0 4 18 12 6.40 TESTIS (Itestis) 0 0 0 0 - 0 0 - 0 0 - - 0 - 0 0.00 OVARY (Iovary) - - - - 0 - - 0 - - 0 0 - 0 - 0.00 SKIN (Iskin) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FISH (Ifish) 28 34 10 22 12 22 10 24 20 6 6 2 4 26 58 19.69 CIRCULATORY 2 4 2 2 2 0 2 2 4 2 0 2 0 0 0 1.71 DISTURBANCES (Icd) REGRESSIVE 22 30 4 16 6 10 8 10 16 4 2 0 0 10 34 12.29 CHANGES (Irc) PROGRESSIVE 4 0 4 0 4 0 0 8 0 0 4 0 0 0 0 1.71 CHANGES (Ipc) INFLAMMATION (Ii) 0 0 0 4 0 12 0 4 0 0 0 0 4 16 0 2.86 TUMOURS (It) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FOCAL CELLULAR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 24 1.60 ALTERATIONS (Ifca) FISH (Ifish) 28 34 10 22 12 22 10 24 20 6 6 2 4 26 58 20.17 *L – lost during histological processing, A – absent as a result of sample size

Table A - 20: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. gariepinus at Marico-Bosveld Dam. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN GILLS (Igills) 4 24 8 4 12 4 8 8 6 4 8 0 4 6 8 7.20 LIVER (Iliver) 6 4 0 0 6 6 8 0 22 2 8 6 4 30 12 7.60 KIDNEY (Ikidney) 6 6 0 0 6 6 2 4 4 4 6 4 2 0 2 3.47 HEART (Iheart) 0 0 12 4 0 4 0 0 0 0 0 6 8 12 8 3.60 TESTIS (Itestis) 0 0 0 0 0 - 0 - 0 - - 0 0 0 0 0.00 OVARY (Iovary) - - - - - 0 - 0 - 0 0 - - - - 0.00 SKIN (Iskin) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FISH (Ifish) 16 34 20 8 24 20 18 12 32 10 22 16 18 48 30 21.87 CIRCULATORY 0 6 0 0 0 0 0 0 2 0 0 0 0 0 0 0.53 DISTURBANCES (Icd) REGRESSIVE 12 12 12 0 16 12 10 4 18 6 14 16 14 20 22 12.53 CHANGES (Irc) PROGRESSIVE 4 16 8 4 8 4 8 8 4 4 8 0 4 4 8 6.13 CHANGES (Ipc) INFLAMMATION (Ii) 0 0 0 4 0 4 0 0 0 0 0 0 0 12 0 1.33 TUMOURS (It) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FOCAL CELLULAR 0 0 0 0 0 0 0 0 8 0 0 0 0 12 0 1.33 ALTERATIONS (Ifca) FISH (Ifish) 16 34 20 8 24 20 18 12 32 10 22 16 18 48 30 21.87 *L – lost during histological processing, A – absent as a result of sample size

PAGE | 155

APPENDIX 5 – HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) DATA

Table A - 21: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. carpio at Roodekopjes Dam. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN GILLS (Igills) 16 10 10 L 22 6 6 4 L 0 6 8 0 8 0 7.38 LIVER (Iliver) 0 6 6 0 4 4 6 8 0 0 0 0 4 0 8 3.07 KIDNEY (Ikidney) L 2 4 0 10 0 0 2 0 2 L 4 L 2 20 3.83 HEART (Iheart) 0 0 4 0 10 6 0 0 L 0 0 0 4 0 0 1.71 TESTIS (Itestis) - 0 0 - 0 - - 0 - - - 0 - 0 0 0.00 OVARY (Iovary) 0 - - 0 - 0 0 - 0 0 0 - 0 - - 0.00 SKIN (Iskin) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FISH (Ifish) 16 18 24 0 46 16 12 14 0 2 6 12 8 10 28 16.00 CIRCULATORY 4 2 2 0 6 2 2 0 0 0 2 0 0 0 0 1.48 DISTURBANCES (Icd) REGRESSIVE 8 8 10 0 28 6 10 6 0 2 0 4 4 2 28 8.59 CHANGES (Irc) PROGRESSIVE 4 4 4 0 8 0 0 4 0 0 4 8 0 8 0 3.26 CHANGES (Ipc) INFLAMMATION (Ii) 0 4 8 0 4 8 0 4 0 0 0 0 4 0 0 2.37 TUMOURS (It) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 FOCAL CELLULAR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 ALTERATIONS (Ifca) FISH (Ifish) 16 18 24 0 46 16 12 14 0 2 6 12 8 10 28 15.70 *L – lost during histological processing, A – absent as a result of sample size

Table A - 22: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. carpio at Vaalkop Dam. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN GILLS (Igills) 8 2 6 6 4 16 12 16 10 8 0 0 8 12 A 7.71 LIVER (Iliver) 6 4 0 6 0 0 6 6 0 6 0 6 6 0 A 3.29 KIDNEY (Ikidney) 8 0 0 4 6 2 4 0 4 0 2 8 0 8 A 3.29 HEART (Iheart) 8 4 0 4 2 0 6 6 12 8 0 0 L L A 4.17 TESTIS (Itestis) 0 ------A 0.00 OVARY (Iovary) - 0 0 0 0 0 0 0 0 0 0 0 0 0 A 0.00 SKIN (Iskin) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A 0.00 FISH (Ifish) 30 10 6 20 12 18 28 28 26 22 2 14 14 20 0 18.45 CIRCULATORY 4 2 2 2 0 4 4 4 2 4 0 0 0 4 A 2.37 DISTURBANCES (Icd) REGRESSIVE 26 8 4 18 8 6 12 16 20 18 2 14 6 12 A 12.59 CHANGES (Irc) PROGRESSIVE 0 0 0 0 4 8 8 8 4 0 0 0 8 4 A 3.26 CHANGES (Ipc) INFLAMMATION (Ii) 0 0 0 0 0 0 4 0 0 0 0 0 0 0 A 0.30 TUMOURS (It) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A 0.00 FOCAL CELLULAR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A 0.00 ALTERATIONS (Ifca) FISH (Ifish) 30 10 6 20 12 18 28 28 26 22 2 14 14 20 0 18.52 *L – lost during histological processing, A – absent as a result of sample size

PAGE | 156

APPENDIX 5 – HISTOLOGY-BASED FISH HEALTH ASSESSMENT (HBFHA) DATA

Table A - 23: Organ (Iorg) and total reaction pattern indices (ITot rp) for C. carpio at Marico-Bosveld Dam. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MEAN GILLS (Igills) 14 8 8 22 20 6 4 12 12 A 11.78 LIVER (Iliver) 6 6 6 6 6 10 6 14 14 A 8.22 KIDNEY (Ikidney) 2 0 2 4 0 2 2 0 0 A 1.33 HEART (Iheart) 4 6 0 0 0 12 0 0 0 A 2.44 TESTIS (Itestis) - 0 - - 0 0 0 0 0 A 0.00 OVARY (Iovary) 0 - 0 0 - - - - - 0.00 SKIN (Iskin) 0 0 0 0 0 0 0 0 0 A 0.00 FISH (Ifish) 26 20 16 32 26 30 12 26 26 0 0 0 0 0 0 23.78 Circulatory 6 0 4 4 2 2 6 4 4 A 3.56 Disturbances (Icd) Regressive 16 12 12 16 8 28 6 18 14 A 14.44 Changes (Irc) Progressive 0 8 0 12 16 0 0 4 8 A 5.33 Changes (Ipc) Inflammation (Ii) 4 0 0 0 0 0 0 0 0 A 0.44 Tumours (It) 0 0 0 0 0 0 0 0 0 A 0.00 Focal Cellular 0 0 0 0 0 0 0 0 0 A 0.00 Alterations (Ifca) FISH (Ifish) 26 20 16 32 26 30 12 26 26 0 0 0 0 0 0 23.78 *L – lost during histological processing, A – absent as a result of sample size

PAGE | 157

APPENDIX 6– AGE ESTIMATION DATA

APPENDIX 6 - AGE ESTIMATION DATA SHARPTOOTH CATFISH (Clarias gariepinus): OTOLITHS

Table A - 24: Ageing counts and calculated mode of each C. gariepinus at each impoundment. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 8 6 6 7 6 5 3 4 5 5 4 RD 2 5 5 6 D/L D/L D/L 4 5 3 2 4 3 D/L 2 2 3 8 5 5 7 5 4 3 4 5 4 4 MODE 8 5 6 - - - 7 5 4 3 4 5 - 4 4 1 10 3 5 5 3 6 5 4 8 VD 2 12 2 D/L D/L D/L 4 D/L D/L 2 D/L 2 4 4 3 4 3 12 3 5 5 3 6 8 4 8 MODE 12 3 - - - 5 - - 5 - 3 6 6 4 8 1 9 15 5 6 5 13 8 4 6 5 18 10 MBD 2 7 14 4 D/L 4 5 14 D/L 4 2 5 D/L 4 12 9 3 13 18 6 7 7 13 8 5 6 4 12 9 MODE 10 16 5 - 6 5 13 - 8 4 6 - 4 12 9 *D/L – Damaged/Lost otoliths during preparation process.

COMMON CARP (CYPRINUS CARPIO): SCALES

Table A - 25: Ageing counts and calculated mode of each C. carpio at each impoundment. Specimen No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 4 4 4 5 4 4 6 4 4 4 6 4 5 5 5 RD 2 5 4 5 4 3 4 6 3 3 4 6 5 6 6 5 3 5 4 5 4 3 4 5 3 3 4 6 5 6 6 5 MODE 5 4 5 4 3 4 6 3 3 4 6 5 6 6 5 1 3 6 4 6 4 4 4 5 6 5 5 4 4 5 VD 2 4 5 4 4 5 4 4 5 5 4 4 4 5 6 A 3 4 5 4 6 4 4 4 5 6 5 5 4 5 5 MODE 4 5 4 6 4 4 4 5 6 5 5 4 5 5 - 1 5 4 5 5 5 4 4 4 4 MBD 2 6 4 5 5 3 4 4 5 3 A A A A A A 3 6 3 4 5 3 4 4 4 4 MODE 6 4 5 5 3 4 4 4 4 ------* A – absent as a result of incomplete sample collection.

PAGE | 158

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

APPENDIX 7 – EDIBILITY ASSESSMENT DATA MUSCLE ANALYSIS: Organic

Table A - 26: Incidence of EDCs measured in grouped muscle samples, evidence of bioaccumulation. TOXICANT TYPE TOXICANT CONCENTRATION (µg/kg or ppb) Clarias gariepinus Cyprinus carpio MEAN MEAN Grp 1 Grp 2 Grp 3 Grp 1 Grp 2 Grp 3 Roodekopjes Dam Organochlorine β-HCH - 7.150 11.390 6.180 16.230 7.730 2.500 8.820 Organochlorine pp’-DDE 9.350 3.610 2.200 5.053 6.660 6.210 1.630 4.833 Phenol n-NP - - - - 276.000 116.900 50.400 147.767 Vaalkop Dam Organophosphate Chlorpyriphos 2.514 - - 0.838 - - - - Organochlorine β-HCH 8.190 2.330 2.640 4.387 1.540 - - 0.513 Herbicide Terbuthylazine 2.290 0.520 0.720 1.177 0.770 - 0.620 0.463 Organochlorine pp’-DDE 3.130 0.930 2.440 2.167 2.870 - 1.320 1.397 Phenol n-NP - 29.900 132.300 54.067 64.600 257.700 66.300 129.533 Marico-Bosveld Dam Organochlorine pp’-DDE 1.850 14.310 8.300 8.153 3.610 1.970 N/R* 2.790 Phenol n-NP 26.400 26.100 - 17.500 - - N/R* - - Absent or below detection limits. *N/R – Not enough fish caught to make up Group 3.

PAGE | 159

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Inorganic Roodekopjes Dam (RD)

Table A - 27: Incidence of metals/elements measured in grouped muscle samples from Roodekopjes Dam. METAL/ CONCENTRATION (µg/kg or ppb) ELEMENT Clarias gariepinus Cyprinus carpio Grp 1 Grp 2 Grp 3 MEAN Grp 1 Grp 2 Grp 3 MEAN Ag - 23.748 - 7.916 - - - - Al - 1025.134 1019.802 681.645 - 965.627 888.092 617.906 As - 98.951 140.594 79.848 167.331 131.135 109.518 135.994 B 4125.596 4729.863 3217.822 4024.427 4535.857 4728.790 3191.955 4152.201 Ba - 6572.333 4572.277 3714.870 7278.884 6312.339 4549.980 6047.068 Be - - 1.980 0.660 - - 9.956 3.319 Bi ------

Ca 8416.137 9802.098 8059.406 8759.213 0.000 21517.981 14643.568 12053.850 Cd 1.987 - - 0.662 7.968 5.961 0.000 4.643 Co ------Cr - 3.958 3.960 2.639 7.968 19.869 7.965 11.934 Cu 91.415 77.182 108.911 92.503 227.092 204.649 85.623 172.455 Fe 957.870 1765.288 1421.782 1381.647 1103.586 1007.351 872.162 994.367 K 100258.347 122719.177 108970.297 110649.273 102390.438 129465.528 146475.508 126110.491 Li - - 59.406 19.802 - - - - Mg 8064.388 10403.721 9005.941 9158.016 10288.845 10001.987 12134.608 10808.480 Mn 15.898 17.811 15.842 16.517 201.195 17.882 15.930 78.336 Mo ------Na 32571.542 39244.013 28811.881 33542.479 39442.231 43632.029 35882.119 39652.126 Ni ------P - 134217.297 102019.802 78745.700 139701.195 152632.625 182158.503 158164.107 Pb 55.644 55.413 85.149 65.402 109.562 53.646 61.728 74.979 Sb - - 1.980 0.660 - - - - Se 97.377 308.727 289.109 231.738 342.629 286.112 304.659 311.134 Si 1420.906 1792.994 1259.406 1491.102 1310.757 1424.598 1457.587 1397.647 Sn - 29.685 231.683 87.123 - 280.151 187.176 155.776 Sr 35.771 41.559 27.723 35.018 61.753 59.607 37.834 53.064 Ti ------V 5.962 - - 1.987 3.984 1.987 - 1.990 W 7.949 37.601 49.505 31.685 59.761 67.554 109.518 78.944 Zn - 4977.241 3419.802 2799.014 5501.992 5316.908 3861.012 4893.304 Zr 7.949 3.958 7.921 6.609 1.992 5.961 3.982 3.978

PAGE | 160

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Vaalkop Dam (VD)

Table A - 28: Incidence of metals/elements measured in grouped muscle samples from Vaalkop Dam. METAL/ CONCENTRATION (µg/kg or ppb) ELEMENT Clarias gariepinus Cyprinus carpio Grp 1 Grp 2 Grp 3 MEAN Grp 1 Grp 2 Grp 3 MEAN Ag ------Al 1050.337 901.688 858.737 936.921 808.061 986.672 916.335 903.689 As 87.198 160.874 105.599 117.890 103.751 143.227 99.602 115.526 B 3501.784 3102.284 2853.158 3152.409 2893.057 3200.716 4717.131 3603.635 Ba 5051.526 4297.915 3996.812 4448.751 4640.862 4354.486 7039.841 5345.063 Be - 13.903 - 4.634 - - - - Bi ------

Ca 7540.626 8307.845 8304.443 8050.971 11430.567 17614.880 30737.052 19927.499 Cd - - - - 1.995 1.989 - 1.328 Co ------15.936 5.312 Cr 5.945 5.958 5.977 5.960 1.995 7.957 3.984 4.645 Cu 93.143 77.458 55.788 75.463 93.775 77.581 99.602 90.319 Fe 1012.683 1642.502 1550.110 1401.765 1582.203 1778.397 930.279 1430.293 K 132065.002 113604.767 182107.990 142592.586 148383.879 123692.063 165219.124 145765.022 Li - 49.652 - 16.551 - 1.989 - 0.663 Mg 8565.200 9058.590 12944.810 10189.533 12823.224 11004.575 12850.598 12226.132 Mn 21.799 19.861 15.939 19.200 15.962 23.871 31.873 23.902 Mo ------Na 31965.914 30526.316 32775.453 31755.894 32142.857 30853.392 45478.088 36158.112 Ni ------P 84403.488 108023.833 136760.311 109729.211 138627.294 152456.734 218924.303 170002.777 Pb 37.654 91.360 57.780 62.265 71.828 61.667 49.801 61.098 Sb ------Se 340.864 327.706 278.940 315.837 367.119 300.378 354.582 340.693 Si 1619.104 1453.823 1004.184 1359.037 1193.136 1229.361 1364.542 1262.347 Sn 75.307 105.263 - 60.190 - 165.108 229.084 131.397 Sr 29.727 25.819 25.902 27.149 33.919 45.753 87.649 55.774 Ti ------V - 3.972 - 1.324 - - - - W 55.489 - 67.743 41.077 31.923 87.527 3.984 41.145 Zn 3686.088 2307.249 3189.878 3061.072 3268.156 3620.450 5051.793 3980.133 Zr 5.945 5.958 5.977 5.960 3.990 3.979 1.992 3.320

PAGE | 161

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Marico-Bosveld Dam (MBD)

Table A - 29: Incidence of metals/elements measured in grouped muscle samples from Marico-Bosveld Dam. METAL/ CONCENTRATION (µg/kg or ppb) ELEMENT Clarias gariepinus Cyprinus carpio Grp 1 Grp 2 Grp 3 MEAN Grp 1 Grp 2 Grp 3 MEAN Ag - 25.722 - 8.574 - 29.744 14.872 Al 918.770 1499.802 850.809 1089.794 846.077 2488.598 1667.338 As 167.587 203.799 169.762 180.383 127.110 136.823 131.967 B 4548.502 5878.512 3337.328 4588.114 4001.986 8128.098 6065.042 Ba 6336.751 8862.287 4583.583 6594.207 5187.686 12482.649 8835.168 Be 21.688 - - 7.229 - - - Bi ------Ca 10772.871 11778.789 9290.993 10614.217 11833.168 37477.692 24655.430 Cd - 3.957 - 1.319 3.972 5.949 4.961 Co ------Cr 9.858 37.594 5.992 17.815 9.930 7.932 8.931 Cu 92.666 81.124 61.913 78.568 83.416 249.851 166.634 Fe 849.763 1958.844 1573.797 1460.802 1223.436 1820.345 1521.890 K 197062.303 131084.290 172099.061 166748.551 141251.241 119869.126 130560.183 Li 53.233 - - 17.744 - - - Mg 14615.536 11580.926 12630.318 12942.260 11729.891 11308.745 N/R 11519.318 Mn 17.744 27.701 17.975 21.140 25.819 41.642 33.731 Mo ------Na 47949.527 44380.689 34511.684 42280.633 34597.815 59627.206 47112.511 Ni ------P 232847.003 115690.542 165328.540 171288.695 142343.595 138826.096 140584.845 Pb 69.006 65.295 71.899 68.733 67.527 116.994 92.261 Sb ------Se 388.407 395.726 309.567 364.567 315.789 329.169 322.479 Si 1666.009 1485.952 1581.786 1577.916 1465.740 1945.271 1705.505 Sn 177.445 87.060 - 88.168 250.248 23.795 137.022 Sr 39.432 49.466 25.964 38.287 33.764 89.233 61.498 Ti 19.716 - - 6.572 - - - V - 1.979 - 0.660 - 3.966 1.983 W 45.347 69.252 63.911 59.503 57.597 79.318 68.457 Zn 5161.672 6363.277 5006.990 5510.646 4063.555 9367.440 6715.498 Zr 7.886 7.915 3.994 6.598 5.958 9.915 7.937 *N/R – Not enough fish caught to make up Group 3.

PAGE | 162

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

HUMAN HEALTH RISK ASSESSMENT (HHRA): MEAN POTENTIAL RISK (AVERAGE FOR BOTH SPECIES) Roodekopjes Dam (RD)

Table A – 30: Mean potential human health risk for consumption of C. gariepinus and/or C. carpio at Roodekopjes Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag 0.004 0.00500 6.575 0.000009 0.00 0.000004 0.E+00 Al 0.650 1.00000 1 067.988 0.001392 0.00 0.000597 0.E+00 As 0.108 0.00030 177.382 0.000231 0.77 0.000099 1.5 1.E-04 B 4.088 0.20000 6 719.655 0.008761 0.04 0.003755 0.E+00 Ba 4.881 0.20000 8 022.483 0.010459 0.05 0.004483 0.E+00 Be 0.002 0.00200 3.270 0.000004 0.00 0.000002 4.3 8.E-06 Cd 0.003 0.00100 4.360 0.000006 0.01 0.000002 0.E+00 Co 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Cr 0.007 0.00300 11.977 0.000007 0.00 0.000003 0.E+00 Cu 0.132 0.01000 217.745 0.000284 0.03 0.000000 0.E+00 Mn 0.047 0.14000 77.951 0.000102 0.00 0.000044 0.E+00 Mo - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni - 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.070 0.00071 115.366 0.000150 0.21 0.000000 0.E+00 Sb - 0.00040 0.542 0.000001 0.00 0.000000 0.E+00 Se 0.271 0.00500 446.138 0.000582 0.12 0.000249 0.E+00 Sn 0.121 0.00300 199.617 0.000260 0.09 0.000000 0.E+00 Sr 0.044 0.60000 72.320 0.000094 0.00 0.000040 0.E+00 V 0.002 0.00700 3.287 0.000004 0.00 0.000002 0.E+00 Zn 3.846 0.30000 6 321.382 0.008241 0.03 0.003532 0.E+00 pp’-DDE 0.005 0.00022 8.125 0.000011 0.05 0.000005 0.34 2.E-06 β-HCH 0.008 0.01700 12.327 0.000016 0.00 0.000007 1.8 1.E-05 Chlorpyriphos - 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Terbuthylazine - 0.03500 0.000 0.000000 0.00 0.000000 0.E+00 n-NP 0.074 0.10000 121.436 0.000158 0.00 0.000068 0.E+00 Overall HQ (Sum of all HQ values) 1.40

PAGE | 163

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Vaalkop Dam (VD)

Table A - 31: Mean potential human health risk for consumption of C. gariepinus and/or C. carpio at Vaalkop Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Al 0.920 1.00000 1 512.636 0.001972 0.00 0.000845 0.E+00 As 0.117 0.00030 191.825 0.000250 0.83 0.000107 1.5 2.E-04 B 3.378 0.20000 5 552.201 0.007239 0.04 0.003102 0.E+00 Ba 4.897 0.20000 8 048.678 0.010493 0.05 0.004497 0.E+00 Be 0.002 0.00200 3.808 0.000005 0.00 0.000002 4.3 9.E-06 Cd 0.001 0.00100 1.091 0.000001 0.00 0.000001 0.E+00 Co 0.003 0.00030 4.366 0.000006 0.02 0.000000 0.E+00 Cr 0.005 0.00300 8.716 0.000005 0.00 0.000002 0.E+00 Cu 0.083 0.01000 136.242 0.000178 0.02 0.000000 0.E+00 Mn 0.022 0.14000 35.422 0.000046 0.00 0.000020 0.E+00 Mo - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni - 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.062 0.00071 101.382 0.000132 0.19 0.000000 0.E+00 Sb - 0.00040 0.000 0.000000 0.00 0.000000 0.E+00 Se 0.328 0.00500 539.544 0.000703 0.14 0.000301 0.E+00 Sn 0.096 0.00300 157.449 0.000205 0.07 0.000000 0.E+00 Sr 0.041 0.60000 67.389 0.000088 0.00 0.000038 0.E+00 V 0.001 0.00700 1.644 0.000002 0.00 0.000001 0.E+00 Zn 3.521 0.30000 5 787.204 0.007545 0.03 0.003234 0.E+00 pp’-DDE 0.002 0.00022 2.928 0.000004 0.02 0.000002 0.34 6.E-07 β-HCH 0.002 0.01700 4.027 0.000005 0.00 0.000002 1.8 4.E-06 Chlorpyriphos - 0.00030 0.689 0.000001 0.00 0.000000 0.E+00 Terbuthylazine 0.001 0.03500 1.348 0.000002 0.00 0.000001 0.E+00 n-NP 0.092 0.10000 150.885 0.000197 0.00 0.000084 0.E+00 Overall HQ (Sum of all HQ values) 1.41

PAGE | 164

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Marico-Bosveld Dam (MBD)

Table A - 32: Mean potential human health risk for consumption of C. gariepinus and/or C. carpio at Marico-Bosveld Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag 0.011 0.00500 18.080 0.000024 0.00 0.000000 0.E+00 Al 1.321 1.00000 2 170.918 0.002830 0.00 0.001213 0.E+00 As 0.161 0.00030 264.650 0.000345 1.15 0.000148 1.5 2.E-04 B 5.179 0.20000 8 512.145 0.011098 0.06 0.004756 0.E+00 Ba 7.491 0.20000 12 311.723 0.016051 0.08 0.006879 0.E+00 Be 0.004 0.00200 7.129 0.000009 0.00 0.000004 4.3 2.E-05 Cd 0.003 0.00100 4.562 0.000006 0.01 0.000003 0.E+00 Co 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Cr 0.014 0.00300 23.440 0.000013 0.00 0.000006 0.E+00 Cu 0.114 0.01000 187.035 0.000244 0.02 0.000000 0.E+00 Mn 0.026 0.14000 43.024 0.000056 0.00 0.000024 0.E+00 Mo - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni - 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.078 0.00071 128.440 0.000167 0.24 0.000000 0.E+00 Sb - 0.00040 0.000 0.000000 0.00 0.000000 0.E+00 Se 0.348 0.00500 571.540 0.000745 0.15 0.000319 0.E+00 Sn 0.108 0.00300 177.034 0.000231 0.08 0.000000 0.E+00 Sr 0.048 0.60000 78.894 0.000103 0.00 0.000000 0.E+00 V 0.001 0.00700 1.644 0.000002 0.00 0.000000 0.E+00 Zn 5.993 0.30000 9 850.245 0.012842 0.04 0.000000 0.E+00 pp’-DDE 0.006 0.00022 9.875 0.000013 0.06 0.000006 0.34 2.E-06 β-HCH - 0.01700 0.000 0.000 0.00 0.000015 1.8 3.E-05 Chlorpyriphos - 0.01000 0.000 0.000000 0.00 0.000000 0.E+00 Terbuthylazine - 0.03500 0.000 0.000000 0.00 0.000000 0.E+00 n-NP 0.013 0.10000 21.573 0.000028 0.00 0.000012 0.E+00 Overall HQ (Sum of all HQ values) 1.90

PAGE | 165

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

HIGHEST POTENTIAL RISK (PER SPECIES): SHARPTOOTH CATFISH (Clarias gariepinus) Roodekopjes Dam (RD)

Table A - 33: Highest potential human health risk for consumption of C. gariepinus at Roodekopjes Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag 0.024 0.00500 39.447 0.000051 0.01 0.000022 0.E+00 Al 1.025 1.00000 1 684.935 0.002197 0.00 0.000941 0.E+00 As 0.141 0.00030 231.084 0.000301 1.00 0.000129 1.5 2.E-04 B 4.730 0.20000 7 774.122 0.010135 0.05 0.004344 0.E+00 Ba 6.572 0.20000 10 802.451 0.014084 0.07 0.006036 0.E+00 Be 0.002 0.00200 3.255 0.000004 0.00 0.000002 4.3 8.E-06 Cd 0.002 0.00100 3.266 0.000004 0.00 0.000002 0.E+00 Co 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Cr 0.004 0.00300 6.509 0.000004 0.00 0.000002 0.E+00 Cu 0.109 0.01000 179.009 0.000233 0.02 0.000000 0.E+00 Mn 0.018 0.14000 29.275 0.000038 0.00 0.000016 0.E+00 Mo - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni - 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.085 0.00071 139.952 0.000182 0.26 0.000000 0.E+00 Sb 0.002 0.00040 3.255 0.000004 0.01 0.000002 0.E+00 Se 0.309 0.00500 507.432 0.000662 0.13 0.000284 0.E+00 Sn 0.232 0.00300 380.800 0.000496 0.17 0.000000 0.E+00 Sr 0.042 0.60000 69.032 0.000090 0.00 0.000039 0.E+00 V 0.006 0.00700 9.862 0.000013 0.00 0.000006 0.E+00 Zn 4.977 0.30000 8 180.322 0.010665 0.04 0.004571 0.E+00 pp’-DDE 0.009 0.00022 15.368 0.000020 0.09 0.000009 0.34 3.E-06 β-HCH 0.011 0.01700 18.721 0.000024 0.00 0.000010 1.8 2.E-05 Chlorpyriphos - 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Terbuthylazine - 0.03500 0.000 0.000000 0.00 0.000000 0.E+00 n-NP - 0.10000 0.000 0.000000 0.00 0.000000 0.E+00 Overall HQ (Sum of all HQ values) 1.86

PAGE | 166

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Vaalkop Dam (VD)

Table A - 34: Highest potential human health risk for consumption of C. gariepinus at Vaalkop Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Al 1.050 1.00000 1 726.360 0.002251 0.00 0.000965 0.E+00 As 0.161 0.00030 264.416 0.000345 1.15 0.000148 1.5 2.E-04 B 3.502 0.20000 5 755.619 0.007504 0.04 0.003216 0.E+00 Ba 5.052 0.20000 8 302.814 0.010825 0.05 0.004639 0.E+00 Be 0.014 0.00200 22.851 0.000030 0.01 0.000013 4.3 5.E-05 Cd - 0.00100 0.000 0.000000 0.00 0.000000 0.E+00 Co 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Cr 0.006 0.00300 9.824 0.000005 0.00 0.000002 0.E+00 Cu 0.093 0.01000 153.092 0.000200 0.02 0.000000 0.E+00 Mn 0.022 0.14000 35.830 0.000047 0.00 0.000020 0.E+00 Mo - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni - 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.091 0.00071 150.162 0.000196 0.28 0.000000 0.E+00 Sb - 0.00040 0.000 0.000000 0.00 0.000000 0.E+00 Se 0.341 0.00500 560.253 0.000730 0.15 0.000313 0.E+00 Sn 0.105 0.00300 173.013 0.000226 0.08 0.000000 0.E+00 Sr 0.030 0.60000 49.309 0.000064 0.00 0.000028 0.E+00 V 0.004 0.00700 6.575 0.000009 0.00 0.000004 0.E+00 Zn 3.686 0.30000 6 058.402 0.007899 0.03 0.003385 0.E+00 pp’-DDE 0.002 0.00022 4.010 0.000005 0.02 0.000002 0.34 8.E-07 β-HCH 0.008 0.01700 13.461 0.000018 0.00 0.000008 1.8 1.E-05 Chlorpyriphos 0.003 0.00030 4.132 0.000005 0.02 0.000002 0.E+00 Terbuthylazine 0.002 0.03500 3.764 0.000005 0.00 0.000002 0.E+00 n-NP 0.132 0.10000 217.452 0.000284 0.00 0.000122 0.E+00 Overall HQ (Sum of all HQ values) 1.85

PAGE | 167

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Marico-Bosveld Dam (MBD)

Table A - 35: Highest potential human health risk for consumption of C. gariepinus at Marico-Bosveld Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag 0.026 0.00500 42.734 0.000056 0.01 0.000024 0.E+00 Al 1.500 1.00000 2 465.112 0.003214 0.00 0.001377 0.E+00 As 0.204 0.00030 334.969 0.000437 1.46 0.000187 1.5 3.E-04 B 5.879 0.20000 9 662.069 0.012597 0.06 0.005399 0.E+00 Ba 8.862 0.20000 14 566.277 0.018991 0.09 0.008139 0.E+00 Be 0.022 0.00200 35.646 0.000046 0.02 0.000020 4.3 9.E-05 Cd 0.004 0.00100 6.504 0.000008 0.01 0.000004 0.E+00 Co 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Cr 0.038 0.00300 61.790 0.000035 0.01 0.000015 0.E+00 Cu 0.093 0.01000 152.308 0.000199 0.02 0.000000 0.E+00 Mn 0.028 0.14000 45.530 0.000059 0.00 0.000025 0.E+00 Mo - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni - 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.072 0.00071 118.176 0.000154 0.22 0.000000 0.E+00 Sb - 0.00040 0.000 0.000000 0.00 0.000000 0.E+00 Se 0.396 0.00500 650.425 0.000848 0.17 0.000363 0.E+00 Sn 0.177 0.00300 291.653 0.000380 0.13 0.000000 0.E+00 Sr 0.049 0.60000 80.538 0.000105 0.00 0.000045 0.E+00 V 0.002 0.00700 3.287 0.000004 0.00 0.000002 0.E+00 Zn 6.363 0.30000 10 458.386 0.013635 0.05 0.005844 0.E+00 pp’-DDE 0.006 0.00022 9.87 0.000031 0.14 0.000013 0.34 4.E-06 β-HCH - 0.01700 0.000 0.000000 0.00 0.000000 1.8 0.E+00 Chlorpyriphos - 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Terbuthylazine - 0.03500 0.000 0.000000 0.00 0.000000 0.E+00 n-NP 0.026 0.10000 43.392 0.000057 0.00 0.000024 0.E+00 Overall HQ (Sum of all HQ values) 2.39

PAGE | 168

APPENDIX 7 – EDIBILITY ASSESSMENT DATA

COMMON CARP (CYPRINUS CARPIO) Roodekopjes Dam (RD)

Table A - 36: Highest potential human health risk for consumption of C. carpio at Roodekopjes Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Al 0.966 1.00000 1 587.128 0.002069 0.00 0.000887 0.E+00 As 0.167 0.00030 275.029 0.000359 1.20 0.000154 1.5 2.E-04 B 4.729 0.20000 7 772.357 0.010133 0.05 0.004343 0.E+00 Ba 7.279 0.20000 11 963.756 0.015598 0.08 0.006685 0.E+00 Be 0.010 0.00200 16.364 0.000021 0.01 0.000009 4.3 4.E-05 Cd 0.008 0.00100 13.097 0.000017 0.02 0.000007 0.E+00 Co 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Cr 0.020 0.00300 32.657 0.000018 0.01 0.000008 0.E+00 Cu 0.227 0.01000 373.253 0.000487 0.05 0.000000 0.E+00 Mn 0.201 0.14000 330.689 0.000431 0.00 0.000185 0.E+00 Mo - 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni - 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.110 0.00071 180.078 0.000235 0.33 0.000000 0.E+00 Sb - 0.00040 0.000 0.000000 0.00 0.000000 0.E+00 Se 0.343 0.00500 563.154 0.000734 0.15 0.000315 0.E+00 Sn 0.280 0.00300 460.463 0.000600 0.20 0.000000 0.E+00 Sr 0.062 0.60000 101.905 0.000133 0.00 0.000057 0.E+00 V 0.004 0.00700 6.575 0.000009 0.00 0.000004 0.E+00 Zn 5.502 0.30000 9 043.225 0.011790 0.04 0.005053 0.E+00 pp’-DDE 0.007 0.00022 10.947 0.000014 0.06 0.000006 0.34 2.E-06 β-HCH 0.016 0.01700 26.676 0.000035 0.00 0.000015 1.8 3.E-05 Chlorpyriphos - 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Terbuthylazine - 0.03500 0.000 0.000000 0.00 0.000000 0.E+00 n-NP 0.276 0.10000 453.641 0.000591 0.01 0.000253 0.E+00 Overall HQ (Sum of all HQ values) 2.20

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APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Vaalkop Dam (VD)

Table A - 37: Highest potential human health risk for consumption of C. carpio at Vaalkop Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day) -1

Ag 0.000 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Al 0.987 1.00000 1 621.719 0.002114 0.00 0.000906 0.E+00 As 0.143 0.00030 235.411 0.000307 1.02 0.000132 1.5 2.E-04 B 4.717 0.20000 7 753.195 0.010108 0.05 0.004332 0.E+00 Ba 7.040 0.20000 11 570.858 0.015085 0.08 0.006465 0.E+00 Be 0.000 0.00200 0.000 0.000000 0.00 0.000000 4.3 0.E+00 Cd 0.002 0.00100 3.279 0.000004 0.00 0.000002 0.E+00 Co 0.016 0.00030 26.193 0.000034 0.11 0.000000 0.E+00 Cr 0.008 0.00300 13.078 0.000007 0.00 0.000003 0.E+00 Cu 0.100 0.01000 163.708 0.000213 0.02 0.000000 0.E+00 Mn 0.032 0.14000 52.386 0.000068 0.00 0.000029 0.E+00 Mo 0.000 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni 0.000 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.072 0.00071 118.058 0.000154 0.22 0.000000 0.E+00 Sb 0.000 0.00040 0.000 0.000000 0.00 0.000000 0.E+00 Se 0.367 0.00500 603.406 0.000787 0.16 0.000337 0.E+00 Sn 0.229 0.00300 376.528 0.000491 0.16 0.000000 0.E+00 Sr 0.088 0.60000 144.639 0.000189 0.00 0.000081 0.E+00 V 0.000 0.00700 0.000 0.000000 0.00 0.000000 0.E+00 Zn 5.052 0.30000 8 303.594 0.010826 0.04 0.004640 0.E+00 pp’-DDE 0.003 0.00022 4.717 0.000006 0.03 0.000003 0.34 9.E-07 β-HCH 0.002 0.01700 2.531 0.000003 0.00 0.000001 1.8 3.E-06 Chlorpyriphos 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Terbuthylazine 0.001 0.03500 1.266 0.000002 0.00 0.000001 0.E+00 n-NP 0.258 0.10000 423.562 0.000552 0.01 0.000237 0.E+00 Overall HQ (Sum of all HQ values) 1.90

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APPENDIX 7 – EDIBILITY ASSESSMENT DATA

Marico-Bosveld Dam (MBD)

Table A - 38: Highest potential human health risk for consumption of C. carpio at Marico-Bosveld Dam.

MUSCLE RFD TOTAL DOSE ADD TOXIC RISK LADD SLOPE FACTOR TOXICANT CANCER RISK CONC. (mg/kg) (mg/kg/day) (mg) (mg/kg/day) (HQ) (mg/kg/day) (mg/kg/day)-1

Ag 0.030 0.00500 49.309 0.000064 0.01 0.000028 0.E+00 Al 2.489 1.00000 4 090.322 0.005333 0.01 0.002285 0.E+00 As 0.137 0.00030 224.886 0.000293 0.98 0.000126 1.5 2.E-04 B 8.128 0.20000 13 359.546 0.017417 0.09 0.007465 0.E+00 Ba 12.483 0.20000 20 516.794 0.026749 0.13 0.011464 0.E+00 Be 0.000 0.00200 0.000 0.000000 0.00 0.000000 4.3 0.E+00 Cd 0.006 0.00100 9.778 0.000013 0.01 0.000005 0.E+00 Co 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Cr 0.010 0.00300 16.322 0.000009 0.00 0.000004 0.E+00 Cu 0.250 0.01000 410.662 0.000535 0.05 0.000000 0.E+00 Mn 0.042 0.14000 68.444 0.000089 0.00 0.000038 0.E+00 Mo 0.000 0.00500 0.000 0.000000 0.00 0.000000 0.E+00 Ni 0.000 0.02000 0.000 0.000000 0.00 0.000000 0.E+00 Pb 0.117 0.00071 192.294 0.000251 0.35 0.000000 0.E+00 Sb 0.000 0.00040 0.000 0.000000 0.00 0.000000 0.E+00 Se 0.329 0.00500 541.031 0.000705 0.14 0.000302 0.E+00 Sn 0.250 0.00300 411.314 0.000536 0.18 0.000000 0.E+00 Sr 0.089 0.60000 146.283 0.000191 0.00 0.000082 0.E+00 V 0.004 0.00700 6.575 0.000009 0.00 0.000004 0.E+00 Zn 9.367 0.30000 15 395.835 0.020072 0.07 0.008602 0.E+00 pp’-DDE 0.004 0.00022 5.933 0.000008 0.04 0.000003 0.34 1.E-06 β-HCH 0.000 0.01700 0.000 0.000000 0.00 0.000000 1.8 0.E+00 Chlorpyriphos 0.000 0.00030 0.000 0.000000 0.00 0.000000 0.E+00 Terbuthylazine 0.000 0.03500 0.000 0.000000 0.00 0.000000 0.E+00 n-NP 0.000 0.10000 0.000 0.000000 0.00 0.000000 0.E+00 Overall HQ (Sum of all HQ values) 2.06

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APPENDIX 8 – LIST OF HISTOPATHOLOGICAL ALTERATIONS

APPENDIX 8 – LIST OF HISTOPATHOLOGICAL ALTERATIONS

Table A - 39: Breakdown of alterations and associated importance factors (IFs) for each of the six assessed organs, as per reaction pattern. Reaction Functional Unit Importance Alteration Pattern of Tissue Factor (IF) Target Organ: GILLS CD Aneurysm/Haemor. 1 Intercellular oedema 1 RC Epithelium Structural alterations 1 Plasma alterations 1 Inter cellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 Rupture of pilar cells 2 Supporting tissue Structural alterations 1 Plasma alterations 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 PC Epithelium Hypertrophy 1 Hyperplasia 2 Mucous cells Hypertrophy 1 Hyperplasia 2 I Exudate 1 Activation of RES 1 Infiltration 2 T Benign 2 Malignant 3 Target Organ: LIVER CD Peliosis 1 Intercellular haemorrhage / Vascular congestion 1 RC Intracellular deposits 1 Atrophy 2 Frank necrosis 2 Hepatocellular nuclear pleomorphism / chromatin clearing 2 Hepatocellular pleomorphism 2 Steatosis 1 Vacuolation other than steatosis 1 Melano-macrophage centres 1 PC Hypertrophy: Hydropic change 1 Hypertrophy: Steatosis 1 Wall proliferation / Fibrosis 3 I Infiltration 2 Granulomatosis 2 T Benign 2 Malignant 3 FCA Clear cell foci 2 Vacuolated foci 2 Eosinophilic foci 2 Basophilic Foci 2 Mixed foci 2 Necrotic foci 3 Hydropic foci 2 Hypertrophic foci 2 Target Organ: KIDNEY CD Aneurysm/Haemor. 1 Intercellular oedema 1 Dilation of glomerulus capillaries 1 RC Tubule Structural alterations 1 Plasma alterations 1 Inter cellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3

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APPENDIX 8 – LIST OF HISTOPATHOLOGICAL ALTERATIONS

Glomerulus Structural alterations 1 Plasma alterations 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 Interstitial tissue Structural alterations 1 Plasma alterations 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 PC Tubule Hypertrophy 1 Hyperplasia 2 Glomerulus Thickening of BC membrane 1 Hypertrophy 1 Hyperplasia 2 Interstitial tissue Hypertrophy 1 Hyperplasia 2 I Exudate 1 Activation of RES 1 Infiltration 2 T Benign 2 Malignant 3 Target Organ: HEART CD Aneurysm/H/Haemor. 1 Intercellular oedema 1 RC Atrium MMC 1 Vacuolation 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 Ventricle MMC 1 Vacuolation 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 BA MMC 1 Vacuolation 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 PC Atrium Hypertrophy 1 Hyperplasia 2 Ventricle Hypertrophy 1 Hyperplasia 2 I Endocardium Endocarditis 2 Myocardium Myocarditis 2 Epicardium Epicarditis 2 T Benign 2 Malignant 3 Target Organ: GONADS (TESTIS) CD Aneurysm//Haemor. 1 Intercellular oedema 1 RC Lobule cysts Disorganization of lobules 1 Detachment of basal membrane 1 Inhibition of spermatogenesis 3 Degeneration of Sertoli cells 2 Interstitial tissue Structural alterations 1 Plasma alteration 1 Deposits 1 Vacuolation 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 (1) Spermatogonia Structural alterations 1 Plasma alterations 1 Vacuolation 1 Inter cellular deposits 1 Nuclear alterations 2

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APPENDIX 8 – LIST OF HISTOPATHOLOGICAL ALTERATIONS

Atrophy 2 Necrosis 3 (2) Spermatocytes Structural alterations 1 Plasma alterations 1 Vacuolation 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 (3) Spermatids Structural alterations 1 Plasma alterations 1 Vacuolation 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 (4) Spermatozoa Structural alterations 1 Plasma alterations 1 Vacuolation 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 PC Lobule cysts Wall proliferation (Basal membrane/Tunica) 1 Wall proliferation (Blood vessels) 1 Interstitial tissue Hypertrophy 1 Hyperplasia 2 (1) Spermatogonia Hypertrophy 1 Hyperplasia 2 (2) Spermatocytes Hypertrophy 1 Hyperplasia 2 (3) Spermatids Hypertrophy 1 Hyperplasia 2 (4) Spermatozoa Hypertrophy 1 Hyperplasia 2 I Exudate 1 Activation of RES 1 Infiltration 2 T Benign 2 Malignant 3 IS Intersex 3 Target Organ: GONADS (OVARIES) CD Aneurysm//Haem. 1 Intercellular oedema 1 RC Ovary Inhibition of oogenesis 3 (1) Oogonia Structural alterations 1 Plasma alterations 1 Inter cellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 (2) Oocytes Structural alterations 1 Plasma alterations 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 Interstitial tissue Structural alterations 1 Plasma alterations 1 Intercellular deposits 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 PC (1) Oogonia Hypertrophy 1 Hyperplasia 2 (2) Oocytes Hypertrophy 1 Hyperplasia 2 Interstitial tissue Hypertrophy 1 Hyperplasia 2 Tunica albuginea Thickening 2

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APPENDIX 8 – LIST OF HISTOPATHOLOGICAL ALTERATIONS

I Exudate 1 Activation of RES 1 Infiltration 2 T Benign 2 Malignant 3 IS Intersex 3 Target Organ: SKIN CD Aneurysm/Haem 1 Intercellular oedema 1 RC Epidermis Structural alterations 1 Plasma alterations 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 Basement Membrane Defect 2 Dermis Structural alterations 1 Plasma alterations 1 Nuclear alterations 2 Atrophy 2 Necrosis 3 PC Epidermis Hypertrophy 1 Hyperplasia 2 Dermis Hypertrophy 1 Hyperplasia 2 I Exudate 1 Activation of RES 1 Infiltration 2 T Benign 2 Malignant 3

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APPENDIX 9 – LIST OF ORGANIC TOXICANTS

APPENDIX 9 – LIST OF ORGANIC TOXICANTS

Table A - 40: List of PCB’s and pesticides screened for in the muscle samples, at FDA Labs. Polychlorinated Biphenols (PCB’s) and Pesticide List 2,3,4,5,6-Pentachloroaniline Cyanophos Fluchloralin Phenothrin op’-DDE Cycloate Fluvalinate Phenthoate op’-DDT Cyfluthrin Fonofos Phorate pp’-DDD Cypermethrin Furalaxyl Phosalone pp’-DDE Cyprofuram Gamma-Lindane Phosmet pp’-DDT Decachlorobiphenyl Heptachlor Piperonyl butoxide pp’-Dichlorobenzophenone Delta- Lindane Heptachlor epoxide Pirimicarb Alachlor Demeton-o Heptenophos Pirimiphos ethyl Aldrin Demeton-s Hexazinone Pirimiphos methyl Alpha-Lindane Desmetryn Iodofenphos Procymidone Ametryn Di-Allate Isobenzan Propachlor Anisole,2,3,4,5,6-pentachloro Diazinone Isodrin Propargite Atrazine Dichlobutrazol Isofenphos Propazine Azinphos ethyl PCB 138 Kepone Propiconazole Aziprotryne PCB 153 Leptophos Propyzamide Benalaxyl Dichlofenthion m-Chloroaniline Prothiophos Bendiocarb Dieldrin Mecarbam Pyrazophos Benzenamine,2,6-dimethyl Diethatyl-ethyl Metalaxyl Pyridaben Benzene,hexachloro Difenoconazole Metazachlor Pyridaphenthion Benzene,pentachloro Diflufenican Methacrifos Quinalphos Benzene,pentachloronitro Dimefox Methidathion Quizalofop ethyl Beta-Lindane Dimethachlor Methoxychlor Resmethrin Bifenox Dimethipin Methyl Parathion Sulfotepp Binapacryl Dioxathion Metolachlor Sulprofos Biphenthrin Dioxathion Myclobutanil Tebuconazole Bromophos-ethyl Diphenylamine Nitrofen Terbufos Bupirimate Disulfoton Nitrothal-isopropyl Terbuthylazine Buprofezin Dodemorph Nuarimol Tetrachlorvinphos Carbofenotion Endosulfan I o,p'-Methoxychlor Tetradifon Carbofuran Endosulfan II Ovex Tetramethrin Chlorbenside Endosulfan sulfate Oxychlordan Tetrasul Chlorbromuron Endrin Paclobutrazol Thiobencarb Chlordimeform Eptc Paraoxon Thiometon Chlorfenprop-methyl Ethion Parathion Thionazin Chlormephos Ethofumesate PCB 28 Tolclofos methyl Chloropropham Ethoprophos PCB 52 Trans-Chlordane Chloropyriphos Ethoxyquin PCB 101 Triadimenol Chloropyriphos-methyl Etridazole PCB 118 Triallate Chlorthiophos i Fenarimol PCB 180 Triazophos Chlorthiophos II Fenchlorphos PCB 194 Trietazine Chlorthiophos III Fenpropathrin Penconazole Trifenmorph Clofenvinfos Fenson Pentanochlor Triflumenol Crimidine Fenthion Permethrin Trifluralin Cyanazine Fenvalerate Permethrin cis Vernolate Cyanophenphos Flamprop isopropyl Phenitrothion Vinclozolin

PAGE | 176