I A N A S S E S S M E N T OF L A N D – B A S E D A C T I V I T I E S A S I N P U T S O F M I C R O P L A S T I C P O L L U T I O N I N S O U T H A F R I C A ’ S A Q U A T I C E N V I R O N M E N T : A C A S E S T U D Y O F D U R B A N B A Y H A R B O U R A N D T H E H E N N O P S R I V E R . Digambari Devi Sharma Latcheman A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 1 February 2023 II Candidate declaration I declare that this dissertation is my own, unaided work. It is being submitted for the Degree of Master of Science at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any other University. _______________________________________ (Signature of candidate) 1st day of February 2023 at the University of the Witwatersrand, Johannesburg. III Abstract Microplastics have been identified globally in a diverse range of environments. The extent of information on microplastic pollution in South Africa’s context is scarce and lacking. An investigation of microplastic pollution was conducted across the Durban Bay harbour in the KwaZulu Natal province and the Hennops River, Gauteng province in South Africa. Microplastics were detected in all surface water and sediment samples, with abundances up to 80.72 MP/ m3 detected in surface water samples and 1.76 MP/ g for sediment samples. Fibres and fragments were the most commonly observed morphologies in surface water and sediment samples; with fibres comprising up to 75.68 % in surface waters and 45.54 % in sediments, and fragments comprising up to 19.84 % in surface waters and 41.07 % in sediment samples. Polyethylene, polypropylene and polyester were the most dominant polymers observed in water and sediment samples, and both buoyant and non-buoyant polymers were observed of different morphologies for the study areas of Durban Bay and the Hennops River. Microplastic particles less than 1 mm in diameter were the most commonly observed size ranges relative to microplastic particles greater than 1 mm in diameter. Sites located nearby to river inflows and stormwater drains showed relatively higher microplastic abundances. A spatial analysis of the data showed the water bodies were subject to a wide array of anthropogenic activities which serve as inputs of microplastic pollution. The reporting of microplastic pollution in the selected aquatic systems substantiates real-time data which is required to implement policies to aid mitigating the risks associated with accumulating microplastic levels. IV In memory of my brother, Atul Latcheman 2001 – 2019 And darling friend who became family, Sapna Koobair 2003 - 2022 V Acknowledgements I would like to extend my sincerest gratitude to the people below, without whom this project would not have been possible: The National Research Foundation (NRF) for their financial support in carrying out this project. Having the most encouraging supervisors was the keystone in completing this dissertation. My supervisors, Dr Heidi Richards and Prof Luke Chimuka. Your support, advice and guidance has helped me in ways I cannot begin to describe in words. I will always be deeply grateful for having such incredible supervisors. Kuria Ndungu and Steven Brooks for their help and amazing hospitality during my time in Oslo. Rachel Hurley, Amy Lusher and Nina Buenaventure from the Mikroplast Research Group at the Norwegian Institute of Water research, NIVA for their willingness to help and give advice. It was a phenomenal experience working with such an exceptional group of women in STEM! Brent Newman and the CSIR in their crucial assistance with collecting samples from Durban Bay during lockdown periods. Thank you for the practical advice and suggestions you provided! Laura Bronzo who started out as a fellow intern at NIVA and has now become a lifelong friend. To my parents who have been a pillar of strength, to my family for their words of positivity , and to dear my friends, Vizelle Naidoo and Salena Pillay, who sat with me through endless hours of microscope work via WhatsApp calls – I could not have done this without you. VI Contents Table of Contents CANDIDATE DECLARATION --------------------------------------------------------------------------------------------------------------- II ABSTRACT ------------------------------------------------------------------------------------------------------------------------------------ III ACKNOWLEDGEMENTS -------------------------------------------------------------------------------------------------------------------- V CONTENTS ------------------------------------------------------------------------------------------------------------------------------------ VI LIST OF FIGURES -------------------------------------------------------------------------------------------------------------------------- VIII LIST OF TABLES------------------------------------------------------------------------------------------------------------------------------ XI LIST OF ABBREVIATIONS ---------------------------------------------------------------------------------------------------------------- XII 1. INTRODUCTION ---------------------------------------------------------------------------------------------------------------------- 1 1.1 GENERAL INTRODUCTION TO PLASTIC AND MICROPLASTIC POLLUTION -------------------------------------------------------------- 1 1.2 MICROPLASTIC POLLUTION IN THE CONTEXT OF SOUTH AFRICA’S AQUATIC SYSTEMS ---------------------------------------------- 3 1.3 PROBLEM STATEMENT AND JUSTIFICATION FOR STUDY ------------------------------------------------------------------------------ 4 1.4 AIMS AND DELIVERABLES -------------------------------------------------------------------------------------------------------------- 4 2. LITERATURE REVIEW --------------------------------------------------------------------------------------------------------------- 6 2.1 PLASTIC AND MICROPLASTIC LITTER GLOBALLY ---------------------------------------------------------------------------------------- 6 2.2 THE DEFINITION OF MICROPLASTICS --------------------------------------------------------------------------------------------------- 7 2.3 SOURCES, PATHWAYS AND FATE OF MICROPLASTIC POLLUTION --------------------------------------------------------------------- 8 2.3.1 Primary and Secondary sources of microplastics -------------------------------------------------------------------- 8 2.3.2 Sources and pathways contributing to microplastic pollution in the marine environment ------------ 10 2.4 WHAT ARE THE IMPLICATIONS OF MICROPLASTIC POLLUTION? -------------------------------------------------------------------- 14 2.5 THE PLASTIC INDUSTRY IN SOUTH AFRICA ------------------------------------------------------------------------------------------ 16 3. METHODS --------------------------------------------------------------------------------------------------------------------------- 19 3.1 STUDY AREA -------------------------------------------------------------------------------------------------------------------------- 19 3.1.1 Durban Bay ------------------------------------------------------------------------------------------------------------------ 20 3.1.2 Hennops River --------------------------------------------------------------------------------------------------------------- 22 3.2 SAMPLE COLLECTION ----------------------------------------------------------------------------------------------------------------- 24 3.2.1 Durban Bay Sampling ----------------------------------------------------------------------------------------------------- 24 3.2.2 Hennops River Sampling ------------------------------------------------------------------------------------------------- 27 3.3 SAMPLE PRE-TREATMENT ------------------------------------------------------------------------------------------------------------ 30 3.4 GRAIN SIZE ---------------------------------------------------------------------------------------------------------------------------- 30 3.5 SAMPLE PROCESSING ----------------------------------------------------------------------------------------------------------------- 31 3.5.1 Sediment sample processing -------------------------------------------------------------------------------------------- 31 3.5.2 Water sample processing ------------------------------------------------------------------------------------------------ 32 3.5.3 Organic Matter Removal ------------------------------------------------------------------------------------------------- 32 3.6 MICROPLASTIC VISUAL IDENTIFICATION --------------------------------------------------------------------------------------------- 33 3.7 POLYMER IDENTIFICATION ----------------------------------------------------------------------------------------------------------- 34 3.8 CONTAMINATION MITIGATION MEASURES ------------------------------------------------------------------------------------------ 34 3.9 STATISTICAL ANALYSIS ---------------------------------------------------------------------------------------------------------------- 35 4. RESULTS AND DISCUSSION – DURBAN BAY ------------------------------------------------------------------------------- 36 4.1 ABUNDANCE AND SPATIAL VARIATIONS OF MICROPLASTICS IN DURBAN BAY SURFACE WATER AND SEDIMENT SAMPLES ------ 36 4.1.1. Surface water samples --------------------------------------------------------------------------------------------------- 36 4.1.2 Sediment samples ---------------------------------------------------------------------------------------------------------- 39 4.2 SIZE DISTRIBUTION -------------------------------------------------------------------------------------------------------------------- 43 4.3 MORPHOLOGY ------------------------------------------------------------------------------------------------------------------------ 44 VII 4.4 COLOUR COMPOSITION--------------------------------------------------------------------------------------------------------------- 48 4.5 POLYMER COMPOSITION ------------------------------------------------------------------------------------------------------------- 49 4.6 EXPERIMENT CONTAMINATION CONTROL ------------------------------------------------------------------------------------------- 50 4.7 COMPARISONS TO OTHER STUDIES -------------------------------------------------------------------------------------------------- 51 4.8 APPLICATION -------------------------------------------------------------------------------------------------------------------------- 53 CONCLUSION – DURBAN BAY --------------------------------------------------------------------------------------------------------- 54 5. RESULTS AND DISCUSSION – THE HENNOPS RIVER --------------------------------------------------------------------- 55 5.1 ABUNDANCE AND DISTRIBUTION OF MPS IN THE HENNOPS RIVER ---------------------------------------------------------------- 55 5.2 GRAIN SIZE PROFILE ------------------------------------------------------------------------------------------------------------------ 56 5.3 SIZE DISTRIBUTION OF MICROPLASTICS COLLECTED FROM THE HENNOPS RIVER ------------------------------------------------- 57 5.4 MORPHOLOGY OF MICROPLASTICS -------------------------------------------------------------------------------------------------- 58 5.5 COLOUR CHARACTERIZATION OF MICROPLASTICS ----------------------------------------------------------------------------------- 59 5.6 POLYMER COMPOSITION OF MICROPLASTICS --------------------------------------------------------------------------------------- 60 5.7 SOURCES OF MICROPLASTICS --------------------------------------------------------------------------------------------------------- 62 5.8 EXPERIMENT CONTAMINATION CONTROL ------------------------------------------------------------------------------------------- 64 5.9 LIMITATIONS -------------------------------------------------------------------------------------------------------------------------- 64 CONCLUSION – THE HENNOPS RIVER ----------------------------------------------------------------------------------------------- 65 6. GENERAL DISCUSSION ----------------------------------------------------------------------------------------------------------- 65 6.1 SIZE ------------------------------------------------------------------------------------------------------------------------------------ 67 6.2 MORPHOLOGY ------------------------------------------------------------------------------------------------------------------------ 68 6.3 COLOUR ------------------------------------------------------------------------------------------------------------------------------- 69 6.4 POLYMER COMPOSITION ------------------------------------------------------------------------------------------------------------- 69 6.5 LIMITATIONS -------------------------------------------------------------------------------------------------------------------------- 70 6.6 RECOMMENDATIONS FOR FUTURE WORK ------------------------------------------------------------------------------------------- 71 1. Implementation of monitoring programmes ----------------------------------------------------------------------- 71 2. Sample collection taking seasonality into consideration-------------------------------------------------------- 72 3. Microplastic risk assessment on biota and the ecosystem ----------------------------------------------------- 72 6.7 APPLICATION -------------------------------------------------------------------------------------------------------------------------- 72 7. CONCLUSION ----------------------------------------------------------------------------------------------------------------------- 76 8. REFERENCES ------------------------------------------------------------------------------------------------------------------------ 77 VIII List of Figures Figure 1. Schematic diagram depicting a selection of sources and pathways of that contribute to microplastic pollution in the marine environment. ---------------------------------------------- 11 Figure 2. Map depicting the sampling areas selected for this study, namely the Hennops River in the Centurion area, Gauteng and Durban Bay located in Durban, KwaZulu-Natal. -------- 19 Figure 3. Map depicting Durban Bay with a) rivers and tributaries, b) drainage areas (coloured blocks) and the corresponding land-use activities inputs, stormwater channels and inflows, and river inflows into the harbour. (Cited from Preston-Whyte, F., Silburn, B., Meakins, B., Bakir, A., Pillay, K., Worship, M., Paruk, S., Mdazuka, Y., Mooi, G., Harmer, R. and Doran, D., 2021. Meso-and microplastics monitoring in harbour environments: A case study for the Port of Durban, South Africa. Marine Pollution Bulletin, 163, p.111948.) ----- 21 Figure 4. Sampling at Durban Bay A) Deployment of the plankton net used for microplastic surface-water sampling in Durban Bay. B) Photograph of sampling site 7 nearby to a cruise passenger terminal within Durban Bay. ------------------------------------------------------------- 26 Figure 5. Map showing the sample collection locations, indicated by red markers, along the Hennops River located in Centurion, Gauteng. The red pins indicate where the plankton nets were deployed in the river, sediment samples were collected in the surrounding areas. Samples were collected in July 2020. The base map is produced in ArcGIS Pro. ------------- 27 Figure 6. Photographs depicting Hennops sampling site 1. The site in the river has sewage pipes discharging directly into the river with many solid waste materials and discarded debris (Figure A and B). Sed 1A, 1B and 1C depict the points where sediment samples were collected. ------------------------------------------------------------------------------------------------- 28 Figure 7. Photographs depicting Hennops sampling site 1. A) Plastic and organic debris along the river bank. B) Bridge over the river where net was suspended from C) Plankton net suspended in the centre of the river with normal river flow conditions. ------------------------ 29 Figure 8. Schematic outline of the sample processing steps taken for sediment and surface water samples to extract and analyse microplastics. ----------------------------------------------- 31 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074460 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074460 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074460 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074460 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074460 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074460 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074461 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074461 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074461 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074462 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074462 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074462 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074462 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074463 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074463 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074463 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074463 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074464 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074464 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074464 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074465 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074465 IX Figure 9. Map depicting microplastic abundance (MP/ m3) present in samples collected from surface water at Durban Bay. Each sampling site is labelled numerically from 1 to 15. ------ 37 Figure 10. Map depicting sediment abundance at Durban Bay collected using a grab sampler (n = 15). -------------------------------------------------------------------------------------------------- 40 Figure 11. Map depicting particle size distribution of sediments at each site sampled in Durban Bay (n = 15). ----------------------------------------------------------------------------------- 41 Figure 12. Box plot depicting the spread of microplastic abundances (MP/g) recovered from triplicate samples across sites at Durban Bay. ------------------------------------------------------ 42 Figure 13. Size distribution of microplastics Durban Bay surface water samples and sediment samples. -------------------------------------------------------------------------------------------------- 44 Figure 14. Photograph images of the morphologies of the anthropogenic particles extracted from sediment and surface water samples from Durban Bay identified during visual analysis using a stereomicroscope. A) multiple fibres (multiple colours) B) red plastic pellet C) green plastic fragment. ---------------------------------------------------------------------------------------- 45 Figure 15. Proportion of microplastic morphologies recovered from samples collected from Durban Bay stations from A) surface water samples and B) sediment samples. --------------- 45 Figure 16. Morphology composition (pellets, fragments, films and fibres) percentages of microplastics observed in surface water samples collected from sites across Durban Bay harbour. -------------------------------------------------------------------------------------------------- 46 Figure 17. Morphology composition percentages of microplastics observed in sediment samples collected from sites across Durban Bay harbour. ---------------------------------------- 47 Figure 18. Pie charts indicating the colour assignments for microplastics found in Durban Bay’s A) surface water samples and B) sediment samples during visual analysis on a stereomicroscope. --------------------------------------------------------------------------------------- 49 Figure 19. Abundance proportions of selected microplastic polymer composition detected in selected samples for a) surface water samples and b) sediment samples. ----------------------- 50 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074466 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074466 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074467 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074467 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074468 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074468 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074469 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074469 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074470 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074470 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074471 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074471 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074471 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074471 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074472 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074472 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074473 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074473 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074473 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074474 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074474 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074475 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074475 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074475 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074476 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074476 X Figure 20. Box plot depicting microplastic abundance (MP/ g) in sediment samples collected from the Hennops River. ------------------------------------------------------------------------------- 55 Figure 21. Grain size distribution of sediments collected from sampling sites located along the Hennops River. ------------------------------------------------------------------------------------- 57 Figure 22. Histogram depicting the frequency of microplastics according to their morphology (fibre, film, fragment, pellet) and classed according to the lengthwise size distribution of microplastics in A) surface water samples and B) sediment samples. --------------------------- 58 Figure 23. Proportion of morphologies of microplastics recovered from samples collected from the Hennops River stations in 2019. Stereomicroscope microscope analysis revealed that microplastics recovered from A) water samples surface and B) sediment samples. ----- 58 Figure 24. Percentage of microplastic morphologies detected across the Hennops River in A) surface water samples and B) sediment samples. -------------------------------------------------- 59 Figure 25. Pie charts indicating the colour assignments for microplastics from the Hennops River found in A) surface water samples and B) sediment samples during visual analysis on a stereomicroscope. --------------------------------------------------------------------------------------- 60 Figure 26. Abundance proportions of selected microplastic polymer composition detected in selected samples for a) surface water samples and b) sediment samples. ----------------------- 61 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074478 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074478 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074479 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074479 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074479 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074480 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074480 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074480 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074481 https://witscloud-my.sharepoint.com/personal/917172_students_wits_ac_za/Documents/Dissertation.docx#_Toc102074481 XI List of Tables Table 1. Sampling site coordinates and depths with their locations in Durban Bay and associated anthropogenic activities nearby. ............................................................................. 25 Table 2. Sampling site coordinates and depths with their locations in Hennops River and associated anthropogenic activities nearby. ............................................................................. 27 XII List of Abbreviations ANOVA Analysis of Variance BOD Biological Oxygen Demand CBD Central Business District CSIR Council for Scientific and Industrial Research FTIR Fourier Transform Infrared g gram GC Gas Chromatography GDP Gross Domestic Product GESAMP Group of Experts on the Scientific Aspects of Marine Environmental Protection GF Glass Fibre GHG Green House Gases GIS Geographic Information System m meter MP Microplastic NOAA National Oceanic and Atmospheric Administration NP Nanoplastic PA Polyamide PAH Polycyclic Aromatic Hydrocarbons PCB Polychlorinated Biphenyl PE Polyethylene PET Polyethylene Terephthalate PP Polypropylene PPE Personal Protective Equipment PS Polystyrene PTFE Polytetrafluoroethylene PVC Polyvinyl Chloride SA South Africa SAHRC South African Human Rights Commission UN United Nations UNEP United Nations Environment Programme USA United States of America UV Ultraviolet WWTP Wastewater Treatment Plants WWTW Wastewater Treatment Works 1 1. INTRODUCTION 1.1 General introduction to plastic and microplastic pollution The durability, wide application and cheap production of plastics have allowed for its incorporation into a variety of products, however these very same design characteristics present as threats to the health of ecosystems (Miller et al., 2021). The increased production and exponential usage of plastic since the beginning of its industrial production in the 1950s, have led to an abundance of synthetic polymers being found across terrestrial, freshwater and marine ecosystems. (Borrelle et al., 2020; Farmen et al., 2021; Kühn et al., 2015; Wang and Wang, 2018). The large volumes of plastics waste accumulation in the environment has developed into one of the most pervasive problems of the anthropogenic era, with as much as 80% of all litter in the oceans being made up of plastic (Hartmann et al., 2019). Plastic has been detected in remote regions, originating from various sources, such as landfills, shipping, tourism, and fisheries; and are transported via ocean currents, wind, sea ice, or biota (Lusher et al., 2015). The ocean has long been looked at as a disposal sites for waste with many estimates suggesting that mismanaged waste on land is mostly responsible (Vanapalli et al., 2021a). The detection of plastic pollution in the marine environment was first reported in the 1970s and its increase in abundance since then has highlighted the adverse anthropogenic impact on the environment (Kühn et al., 2015; Wagner and Lambert, 2018). The persistence of plastic debris adversely affects wildlife by presenting an environmental hazard, which has consequential effects on global economies, biodiversity loss, ecosystem function and human health (Hartmann et al., 2019; Hidalgo-Ruz et al., 2012). Mismanaged plastic waste leaked into the environment from landfills, littering, fishing and shipping activities has led to marine ecosystems acting as a sink for plastic debris, such as the oceanic gyres of the Great Pacific Garbage Patch (Petersen and Hubbart, 2021). Rivers and estuaries in urban settings are major conduits and collectively account for a large proportion of plastics entering the oceans annually (Naidoo and Glassom, 2019). Plastics can persist in marine and coastal environments due to their long degradation times (Sparks and Immelman, 2020). Over time, the large volume of plastic accumulating in the environment breaks down into smaller polymer fragments known as microplastics (Zhu et al., 2021). Microplastics are items of synthetic or semi-synthetic polymer with average sizes between 1 μm and 5 mm. They are present in a variety of colours, chemical compositions and morphologies such as beads, 2 films, fragments and fibres, each with its own toxicological impact (Booth et al., 2017; Miller et al., 2021). Microplastics are used in industrial processes, personal care and domestic cleaning products and have been found to be present in seafood, sea salt, the air we breathe and drinking water (Kühn et al., 2015). The contamination of global environments by microplastics has gained widespread attention and there have been increased efforts to understand the fate and risk of microplastic in different environments (Pakhomova et al., 2022). Microplastics are ubiquitous and remain as highly persistent contaminants of the aquatic ecosystem long after they have been introduced into the environment (Miller et al., 2021). The half-life of microplastics is not known with certainty due to the broad variety of polymers used and the variation in environmental conditions (Miller et al., 2021). Microplastics make up the highest count of plastics in the aquatic environment and have been detected across the globe in inland lakes, estuaries, seawater, sediments and remote regions such as the Arctic and deep ocean basins (Guo and Wang, 2019; Miller et al., 2021; Zhu et al., 2021). Studies indicate that microplastics are more abundant in surface water relative to the water column (Guo and Wang, 2019). Contamination of freshwater and marine habitats with microplastics is ubiquitous throughout the world, but high pollution levels generally coincide with heavily industrialised and densely populated areas (Naidoo and Glassom, 2019). The concentration of microplastic pollution is predicted to heighten in the coming years due to the projected increase of synthetic polymer products (Petersen and Hubbart, 2021). The increased abundance of microplastics may lead to an increase in the risk of detrimental effects caused by microplastic pollution (Petersen and Hubbart, 2021). The small size and high abundance of these small plastic particles make them difficult to remove from the environment allowing for easy infiltration into terrestrial, marine and freshwater systems and as a result are introduced into the food web (Klasios et al., 2021). Microplastics have been found to be readily ingested by freshwater and marine fauna and have wide ranging effects on the health of aquatic biota ( Athey et al., 2020; Vanapalli et al., 2021a). There is also a rising potential threat of microplastic particles as a pollutant and as a vector for adsorbed toxic pollutants from the surrounding environment. The issue of microplastics has been recognized in the United Nation Sustainable Development Goals under Goal 14: Life Below Water, which aims to substantially reduce the amount of pollution in the marine environment by 2025 (Pagter et al., 2018). 3 1.2 Microplastic pollution in the context of South Africa’s aquatic systems South Africa has a coastline length of 3 400 km, with four coastal provinces out of the country’s total of nine provinces. The marine environment hosts substantial biodiversity and contributes to the economic sector through fishing, shipping, tourism and recreational activities. South Africa is a water stressed nation with approximately 300 river outlets which vary in their level of urbanisation and sources of pollution. Many communities, such as those dwelling in informal settlements, are directly dependent on freshwater resources, however raw sewage entering freshwater resources is a major concern. The high density population with high consumption of plastic, pose as threats to the network of rivers and tributaries, and ultimately the marine environment. The lack of infrastructure and waste management systems in many areas in South Africa, especially in areas of informal settlements, have resulted in large proportions of unmanaged waste entering the environment (Verster et al., 2017). Despite South Africa being a large contributor to plastic waste across the globe, the quantitative data available for environmental microplastic pollution in South Africa is relatively limited (Nel and Froneman, 2015). Few studies have been done to investigate microplastic abundances and polymer types in South Africa’s aquatic ecosystems. However such studies have been limited as more attention has been paid to the marine environment, outlining the need to identify gaps on microplastic pollution in the context of South Africa’s freshwater systems (Pereao et al., 2020). South Africa is a developing country and microplastic research is at its inception, highlighting the increasing importance of understanding the current abundances and potential sources of microplastic pollution in order to implement measures to prevent anthropogenic litter entering the aquatic ecosystems (Verster et al., 2017). Microplastics have been reported to enter the environment via various pathways, however, highly populated and urbanised areas have been found to have aquatic systems with poorer water quality and higher microplastic abundances. Globally, microplastic pollution has garnered a lot of attention in the last decade. However, there remains a dearth of data describing microplastic pollution in freshwater systems, as well as limited data on discharge from urban stormwater drains and treated wastewater outflows (Sutton et al., 2016). The sources of microplastics have been found to enter via land-based or sea-based sources. A substantive fraction of plastic present in the marine environment originates from terrestrial sources and enter the oceanic environment through freshwater systems such as estuaries and rivers, 4 effluents and run-off from surrounding urban and industrial areas (Sparks and Immelman, 2020). Industrial port environments contain potentially high levels of pollution resulting in the threat of spreading contaminants to the marine and coastal environment (Naidoo and Glassom, 2019). Special attention should be given to the sources of plastic in catchments exposed to a wide array of anthropogenic activities, especially urban areas, to understand key areas for mitigation strategies and for developing applicable management tools and policies. 1.3 Justification for study Durban Bay and the Hennops River were selected for this study given the dense populations surrounding these sites and the amount of supporting infrastructure such as roads, railways, wastewater treatment plants, stormwater drains and diverse industrial activities. A combination of all these factors is likely to lead to multiple sources of microplastic pollution for these areas making them interesting ecosystems to investigate. The aim of this study is to take a holistic approach to the investigation by looking at surface water and sediment microplastic abundances. 1.4 Aims and Deliverables Given the increasing concern on microplastic pollution, and its effects, the overarching aim of this study is to increase the cognizance of microplastic pollution. This study aims to provide a baseline description for the potential inputs, abundance and spatial distributions of microplastics in the Hennops River and Durban Bay harbour. There are currently no studies on the extent of microplastic contamination present in the highly polluted Hennops River. On the other hand, Durban Bay, located within the Durban metropolitan area, is subject to expanding anthropogenic activities and industrial activities (Naidoo et al., 2015). The area is also subject to multiple sources of pollution from the numerous stormwater outfalls and river inflows that drain into various areas of the port, making it an ideal site to assess the level of land-based inputs into the oceanic environment (Naidoo et al., 2015). A baseline description of the abundance of microplastics will help to fill the knowledge gaps, and comparisons of spatial patterns with findings from other studies will help to determine whether microplastic pollution in the study areas is a significant problem that requires focused action. This study uses the estuarine environment of Durban Bay, located in KwaZulu Natal, and the freshwater environment of the Hennops River situated in Gauteng, as case studies. The 5 deliverables from this study aims to detail an investigation into the anthropogenic activities in urban areas, and their impacts on the abundance and fates of microplastics found in surface water and sediment samples. Microplastic particles considered for the purpose of this study were within the size boundaries of 1 m to 5 mm, as per the recommendations made by the National Oceanic and Atmospheric Administration (NOAA). The upper size limit of 5 mm was chosen from a pragmatic approach by the NOAA due to particles of this size having a greater probability of being ingested by organisms (Hartmann et al., 2019). 1. Mapping the spatial distribution and abundance of microplastics in surface water and sediment samples at different sites in Durban Bay, KwaZulu Natal and the Hennops River, Gauteng. 2. Characterisation of the microplastics extracted from surface water and sediment samples (categorized according to their morphology, size, colour and polymer compositions) found at the sites potentially linked to various anthropogenic activities. 3. Reporting potential accumulation hotspots of microplastics within sediment samples across the harbour in Durban Bay and the Hennops River. 4. Identifying potential land-based anthropogenic activities contributing to the input of microplastic in the aquatic environment. 6 2. LITERATURE REVIEW 2.1 Plastic and microplastic litter globally The global production of plastics has increased exponentially over recent decades due to the broad array of applications, versatility and low production costs of plastics and the demand is predicted to continually grow in the coming decades (Bronzo et al., 2021; Duis and Coors, 2016). The production of plastics began in the 1950s and up to 2016, an estimated 8.3 billion metric tonnes of plastic have been produced (Rose and Webber, 2019). The increased plastic production is outpacing the capacity for disposal, recycle and reuse and has raised concern with plastic waste management systems whereby a large portion of plastics is being discarded into the environment where it serves no intended function ( Bronzo et al., 2021; Hidalgo-Ruz et al., 2012). The durability of plastics which makes for a persistent and indispensable material is also responsible for the environmental strain from the amount of plastic pollution present in the environment. Plastics can travel between terrestrial and aquatic environments with marine environments functioning as recipients for plastic from land and sea-based sources (Bronzo et al., 2021). Plastic pollution was first reported in coastal environments in the 1970s and studies have shown that oceans act as sinks for plastic debris due to the large volume that travels through rivers that eventually enter the ocean. (Pagter et al., 2018). Marine debris has been defined by the Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP) as: ‘any persistent, manufactured or processed solid material discarded, disposed of or abandoned in the marine and coastal environment’ (Agamuthu et al., 2019). Many countries lack proper infrastructure, waste facilities and waste management practices and as a result struggle with discarding the large volumes of plastic waste (Borrelle et al., 2020). The entry of plastics into the environment is a result of multiple deficiencies across the plastic life cycle, commencing at the extraction of raw materials, followed by polymer design and production to the disposal of plastic materials (Lange, 2021). The after effects of plastic disposal is easily observable due to the large amounts of plastic polluting the environment, however, the true lifecycle toll of plastic materials is not holistically taken into consideration. The production of plastic is largely fossil fuel derived, therefore leading to non-biodegradable materials, and the emission of chemical pollutants and greenhouse gases (GHGs) exacerbates the profound effects of climate change on human and ecosystem health (Adeleke et al., 2020). 7 The pressures from increased production and consumption of single-use plastics coupled with inadequate waste management in high population density cities have severe ecological and socio-economic consequences. The improper waste disposal of plastic and introduction of microplastic pollution in a multitude of shapes, colours, and polymers to aquatic and terrestrial environments have resulted in environmental protection concerns in many countries throughout the world (Petersen and Hubbart, 2021). The degradation of plastics can take several years resulting in lengthened exposure times of plastics. The breakdown of plastics in the environment into smaller microplastics (MPs) makes these particles more accessible to terrestrial and aquatic organisms. Microplastics are the most commonly found anthropogenic debris found in marine and freshwater environments (Athey et al., 2020). The United Nations Environment Program (UNEP) have established MPs and nanoplastics (NPs) as worldwide contaminants of concern and are amongst the most important environmental issues to be addressed (Petersen and Hubbart, 2021). The particle dimensions of nanoplastics allow for potential penetration across cell membranes. Microplastics have become a pollutant of emerging concern due to their resistance to degradation, ability to adsorb toxicants and their increasing widespread pollution in the environment in conjunction with their small dimensions and risk for ingestion which is a growing concern for marine organisms (Lutz et al., 2021). Studies conducted across terrestrial, aquatic and atmospheric environments have reported the presence of microplastics in various environmental matrices such as water, air, sand and biota (Huntington et al., 2020). Microplastics have been detected in beaches, snow, in surface-, subsurface- and seafloor water samples and have a tendency to accumulate in coastal zones, estuaries and soils further increasing the complexities of water pollution (Booth et al., 2017). Previous studies have concluded that marine sediments act as a sink and accumulation zone for microplastics with shoreline sediments having higher concentrations than deep sea sediments (Booth et al., 2017). In the years to come, microplastic pollution is expected to increase with the production of plastics on a global scale and the further degradation of these plastics. 2.2 The definition of microplastics There has been an increase in the research of microplastic pollution in recent years, however, there is a lack of international agreements of size standardisation (Su et al., 2020). Plastic debris are typically categorized into items and particles of macroplastics (> 25 mm in dimension), mesoplastics (between 5 to 25 mm), microplastics (between 5 mm and 1 m), and nanoplastics (< 1000 nm in their largest dimension) (Cai et al., 2021; McWilliams et al., 2018). The label 8 of ‘microplastics’ was first used in 2004 and refers to all plastic particles with diameters of 5 mm or less, however, there has been difficulty reaching consensus for the size boundaries of microplastics (Frias and Nash, 2019; Pagter et al., 2018). The National Oceanic and Atmospheric Administration (NOAA) and European Marine Strategy Framework Directive (EMSFD) have proposed that the term of “microplastics” be employed for all plastic particles less than 5 mm, whereas the United Nation Environment Programme (UNEP) and Global Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP) frameworks define microplastic dimensions as between 1 m and 1 mm, with particles smaller than 1 m being classified as nanoplastics (Besley et al., 2017; Pagter et al., 2018). The definition of microplastics in this study, in accordance with the NOAA report, includes synthetic polymeric material (produced from hydrocarbon or raw materials), material fibres, monofilament lines and coatings, with size dimensions lower than 5 mm as these have been found to be ingested by biota. Microplastics have been defined as “any synthetic solid particle or polymeric matrix, with regular or irregular shape and with size ranging from 1 μm to 5 mm, of either primary or secondary manufacturing origin, which are insoluble in water” (Frias and Nash, 2019). Plastic covers a wide range of polymeric compositions and can consist of blends of different synthetic blend types and properties (Weithmann et al., 2018). Identifying the characteristics of microplastics are important for identifying the sources of microplastic pollution. The size, colour, polymer type and morphology (fragments, fibres, pellets and films) of plastic particles may influence their fate and impacts (Weithmann et al., 2018). Characteristic polymers are used to make different products which can aid with identifying the source (Zhu et al., 2021). The most commonly produced polymers include polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) (Weithmann et al., 2018). 2.3 Sources, pathways and fate of microplastic pollution 2.3.1 Primary and Secondary sources of microplastics Microplastic sources are largely classified according to primary and secondary sources (Pagter et al., 2018). The distinction between the two classes can help identify the main sources of 9 microplastic pollution. The identification of microplastic sources is critical for implementing regulations to prevent pollution. Primary microplastics are manufactured as microscopic dimensions for application as industrial pellets, abrasives, and microbeads (Guo and Wang, 2019; Pagter et al., 2018; Petersen and Hubbart, 2021). Primary microplastics are employed as industrial materials, scrubbers and exfoliants in personal care products (such as facial and body scrubs, toothpastes, shaving cream and make-up), and domestic cleaning product additives (Pagter et al., 2018; Petersen and Hubbart, 2021). Industrial pellets constitute the main forms of transporting plastic between producers and manufacturers. The pellets can enter the environment through accidental spillages and poor handling during transportation, which is especially common near ports and in industrialised areas (Zhu et al., 2021). Primary microplastics may also directly enter the environment through streams, surface runoff and discharge from wastewater treatment plants (WWTPs). Secondary microplastics are fragments derived from larger plastics and are formed through the disintegration and degradation of larger synthetic polymers (Naidoo and Glassom, 2019; Pagter et al., 2018; Petersen and Hubbart, 2021). The breakdown of fishing nets disposed of from fishing activities or fibres from synthetic textiles results in secondary microplastics. Plastics are durable and can take a long time to breakdown from weathering and aging depending on the types of plastic and the conditions they are exposed to (Guo and Wang, 2019). The degradation of plastics can occur via mechanical abrasion (wind, wave and water current action), chemical disintegration (UV degradation) or biodegradation (microbial breakdown) causing physical stress and brittleness (Vanapalli et al., 2021a). There is greater potential for abiotic degradation of microplastic in coastal regions and along shorelines because of the combination of environmental factors such as wave action and UV radiation, therefore it is suggested that coastal areas are the main source of marine generated microplastic (Booth et al., 2017). Secondary microplastics have been found to be prevalent in the environment in comparison to primary microplastics (Booth et al., 2017; Naidoo and Glassom, 2019; Petersen and Hubbart, 2021). A combination of environmental conditions such as water chemistry, temperature, sunlight and microorganisms determine the kinetics of polymer degradation (Booth et al., 2017). The degradation of secondary and primary microplastics may result in morphological changes such 10 as discolouration, density changes, crystallinity changes and increased surface area which affects their fate and behaviour in the environment (Guo and Wang, 2019). The degradation of plastics into microplastics are influenced by polymer type, physicochemical properties and the presence of additives (Booth et al., 2017). Microplastics can be categorized according to the morphologies of the particles, the morphologies include fragments, pellets, fibres and films ( Pagter et al., 2018; Sparks and Immelman, 2020). The sizes of microplastics is also an important factor contributing to the potential ingestion rates of marine biota and is a crucial factor for the health of organisms and subsequently ecosystems (Sparks and Immelman, 2020). Shape and size can affect toxicity, as well as associated chemicals. The introduction of plastics and microplastics into aquatic ecosystems encompasses a variety of inputs such as fishing waste, packaging litter, beauty products with microbeads, synthetic textiles shedding fibres or rubber from tyres (Zhu et al., 2021). Anthropogenic activities from terrestrial sources are largely responsible for the amount of plastic debris present in the marine environment (Verster and Bouwman, 2020). The widespread distribution of microplastics highlights the need to identify the pathways of these plastics from their sources. 2.3.2 Sources and pathways contributing to microplastic pollution in the marine environment There are several pathways and a variety of sources responsible for the origination of microplastic pollution in the marine environment (Figure 1) (Naidoo and Glassom, 2019). It is helpful to understand the fate and source of microplastic inputs to inform the implementation of regional policies to aid in reducing the amount of plastic pollution. The point of entry of various microplastic pollution sources into the marine environment can be classified as land- based or sea-based sources (Vanapalli et al., 2021a). Direct sea-based inputs of microplastics entering the marine environment include marine litter released during sea activities including recreational activities and tourism, ghost fishing gear, activities near ports and harbours, offshore industrial activities, discarded plastic equipment from aquaculture activities and accidental spillages during shipping (Sparks and Immelman, 2020; Vanapalli et al., 2021a). Fibres from textiles have also been identified as a large contributor to microplastic pollution. Microplastics can also be introduced into the environment through the utilization and disintegration of products such as tyres, paint, textiles and surfaces composed of or coated with polymers such as artificial grass pitches. These particles are persistent and lightweight allowing 11 for transportation over long distances by means of ocean currents, air currents or biota (Farmen et al., 2021; Huntington et al., 2020). Studies have shown that over 80 % of microplastics present in the marine environment originate from land-based sources, entering through various pathways such as rivers, effluents and run-off ((Preston-Whyte et al., 2021: Sparks and Immelman, 2020; Zhu et al., 2021). This outlines the need for focused attention towards strategies on reducing microplastic leakages from the identified land-based sources. Land-based sources of pollution encompass illegal dumping and littering, river inflows, sewage outlets and stormwater inputs (Preston-Whyte et al., 2021). Riverine and atmospheric inputs can introduce contaminants from landfills, commercial- and domestic sewage effluents, urban stormwater and agricultural runoff (Farmen et al., 2021; Zhu et al., 2021). Periods of heavy rainfall can result in the contamination of aquatic waterways due to leakages from municipal solid waste deposits in urban areas (Su et al., 2020). These plastics undergo fragmentation during transportation or once they enter the marine environment (Vanapalli et al., 2021a). The UN Environment Programme (UNEP) reported mismanaged solid waste and tyre abrasion as the largest contributors to macro- and microplastic pollution from land-based activities globally. Figure 1. Schematic diagram depicting a selection of sources and pathways of that contribute to microplastic pollution in the marine environment. Microplastics enter waterways through discharge from waste-water treatment plants (WWTPs) and storm water drains. Storm water drains are a major source of microplastics to aquatic 12 systems; especially when systems overflow stormwater can pick up numerous micropollutants originating from packaging material, degraded road paints and tyre wear from vehicles (Shruti et al., 2021). Traditional WWTPs are not designed to remove microplastics, such as microbeads present in personal care products, enabling them to escape through treatment systems employed at these plants and ultimately enter the marine environment as a result of their microscale particle dimensions and buoyancy (Shen et al., 2019). Microplastics are discharged into the environment via the use of sewage sludge to agricultural soil in countries with adequate wastewater treatment facilities. The microplastic particles concentrate in the biosolids during wastewater treatment, which is used in the application of sludge as fertilizer in agricultural activities (van den Berg et al., 2020). The introduction of microplastics into soil poses threats to soil life as the plastics remain in the soil much longer than soil nutrients (van den Berg et al., 2020). The overuse of sewage sludge as fertilizers for agricultural land potentially leads to anthropogenic enrichment of agricultural land. The pollutants from the soil can enter freshwater systems through run-off, subsequently ending in the marine environment run off placing the risk of microplastic and chemical toxicity and accumulation on biota (Weithmann et al., 2018). 2.3.2.1 Microplastics in rivers The high volumes of microplastics in freshwater has sparked recent interest in understanding the sources (Su et al., 2020). River inlets and coastal regions in urban areas serve as conduits of microplastic pollution into the marine environment. Rivers, streams and estuaries, more particularly those located in urban settings, are recognized as major contributing sources of microplastics into the marine environment (Naidoo and Glassom, 2019; Vanapalli et al., 2021a). These freshwater environments pose as recipients and transport pathways of microplastics. An estimated 80 % of microplastics present in the marine environment originates from land based sources with rivers being the most dominant pathway therefore investigating the role of rivers as microplastic pollution pathways is of importance (Eo et al., 2018; Mani et al., 2015). The declining water quality and deteriorating integrity of river systems are strongly associated with human-induced changes, particularly changes involving land-usage (Moodley et al., 2015). River systems, especially those situated in urban areas, have been identified as major transport pathways for plastic debris entering the ocean (Su et al., 2020). Rivers act as conduits for microplastics discharged from WWTPs and the concentration found in urban regions is especially high (Bulannga and Schmidt, 2022). Microbeads used in personal care products and domestic cleaning products often end up in rivers due to current clean-up methods employed by wastewater treatment plants that are insufficient at removing such small particles. 13 Rivers serve as crucial pathways in transporting waste generated inland and in landlocked regions to the sea (Siegfried et al., 2017). This highlights the need to improve the knowledge of point sources, such as WWTPs and stormwater outfalls, in their processes that lead to the pollution of waterbodies with plastics (Siegfried et al., 2017). Non-point sources can also lead to microplastic pollution. These are sources without a direct discharge point and inputs are diffused over a wide area. Examples of non-point sources of pollution include plastic debris entering aquatic systems via surface run-off, precipitation or atmospheric deposition (Siegfried et al., 2017). Non-point sources of pollution can occur over a wide area range and involve complications in characterising these sources due to the processes not being easily governable. There is a lack of data available on the microplastic discharge from wastewater treatment plants in South Africa, however rivers may lead to a substantial input as WWTPs in USA and Netherlands have reported microplastics discharge of 1010 particles per day (Bulannga and Schmidt, 2022). Riverine plastic pollution is expected to worsen in as a result of increased flood risk around the world’s rivers. Flooding events of rivers surrounded by poor waste management systems contributes to heightened debris mobilisation resulting in increased plastics entering the marine environment. Heavy rainfall as a result of seasonality changes during the summer seasons, and the risk of climate change, is expected to elevate the extent of land-based pollution inputs into the marine environment (Botterell et al., 2019). It is therefore essential to understand the fate and behaviour of microplastics in river ecosystems, especially rivers located in urban areas. The mitigation of microplastic pollution in the environment requires extensive research efforts in mapping the sources and distribution of microplastics from land-based activities into the marine environment (Su et al., 2020). 2.3.2.2 Microplastics in ports Estuarine ecosystems are undergoing rapid modification and threatened due to the rising threats from the impacts of urbanisation, industrial development, agriculture and mining activities (Adeleke et al., 2020; Barletta et al., 2019). Urbanised water bodies increasingly exposed to anthropogenic activities, such as harbours, have been shown to be contaminated with microplastics and act as conduits for microplastics entering the marine environment (Preston- Whyte et al., 2021). Ports and harbours have functioned as accumulation hotspots for anthropogenic contamination from surrounding urban areas and industrial activities (Preston- Whyte et al., 2021). Studies have shown that microplastics tend to increase downstream of 14 rivers and accumulates in estuaries (Su et al., 2020). There have been numerous studies that investigated microplastics found in sediments and water in the oceans, however less attention has been directed at investigating microplastic pollution in harbours (Preston-Whyte et al., 2021). It is essential to remediate contaminated sediments to protect human and environmental health (Preston-Whyte et al., 2021). Studies done in densely populated urban cities with high anthropogenic activity and poor waste management infrastructure, such as Chennai and Cochin in India, found major shipping and fishing harbours to have high MP abundances in marine biota (Vanapalli et al., 2021a). Studies conducted along the Belgian coast found microplastic concentrations to be highest in harbours where microplastic abundances of up to 390 particles/kg dry sediment were recorded, which is significantly higher than observed abundances of similar study areas (Claessens et al., 2011). 2.4 What are the implications of microplastic pollution? Many marine animals, including seabirds, fish, turtles and mammals, are harmed by plastic through ingestion, lacerations and entanglement. However, the ecological and public health effects of microplastics are not fully observable to the naked eye and are still to be fully elucidated ( de Villiers, 2018; Kühn et al., 2015). The widespread presence and distribution of microplastics in the environment suggests that these particles frequently interact with biota and the smaller size ranges are available to a broader range of organisms (Vanapalli et al., 2021a). Microplastics are potentially toxic and can adversely impact organisms through bioaccumulation in the food web due to their small size (Guo and Wang, 2019; Tsang et al., 2017). Aquatic organisms at every level are exposed to microplastics but the health risks associated therewith is largely unknown due to variations in plastic size, shape and chemical composition (Miller et al., 2021). Ingestion of microplastic particles compromises their ability to capture and digest food, and reproduce. The size of microplastics is important in terms of potential ingestion rates of marine organisms (Sparks and Immelman, 2020). Microplastics in surface water are within the similar measurement ranges as plankton (1 μm – 5 mm). This results in microplastics being bioavailable for ingestion by a number of lower trophic biota, potentially leading to bioaccumulation by predation in higher trophic levels along the food web (Bulannga and Schmidt, 2022; Guo and Wang, 2019; Sparks and Immelman, 2020). Marine organisms such as zooplankton, copepods, mussels, oysters, corals, rotifers fish, turtles and seabirds have observably ingested microplastics (Bulannga and Schmidt, 2022; Guo and 15 Wang, 2019; Tsang et al., 2017). Suspension and filter feeders, such as mussels and clams, are particularly susceptible to microplastic ingestion as these organisms sustain on suspended particulate matter and are ineffectual at differentiating between nutritional food items and indigestible non-food items in similar size ranges (Bulannga and Schmidt, 2022). Microplastics lead to mechanical damages in organisms through obstructing the digestive tract and altering the filtering capabilities of organisms (Farmen et al., 2021; Guo and Wang, 2019). The ingestion of microplastics may lead to the death of organisms as they are affected by exposure to toxic chemicals, lack nutritional food intake and subsequently reduced energy reserves (Guo and Wang, 2019). Microplastics detected in marine and freshwater dietary protein sources may cause potential health risks to humans through ingestion and bioaccumulation (Guo and Wang, 2019). The exposure to airborne microplastics via atmospheric deposition in urban areas has caused public health concerns as the inhalation of plastic dust particles triggers respiratory inflammation (Amato-Lourenço et al., 2020). A recent study found approximately 5 grams of microplastics can be consumed by human individuals based on weekly food and beverage consumption (Senathirajah et al., 2021). The intake of toxic chemicals incorporated in plastic can lead to accumulation in tissues and organs of humans however further research is required to discern the human health implications of ingesting microplastics (Senathirajah et al., 2021). Microplastic toxicity may originate within two processes. Firstly, the higher surface area to volume ratio and hydrophobicity of microplastics may result in the adsorption of marine pollutants, such as metal ions and organic pollutants. These particles may accumulate onto the microplastic particles and allow them to act as a vector for these toxic pollutants (Guo and Wang, 2019; Tsang et al., 2017). Microplastics can also serve as a vector for microbial pathogens as different conditions enable microorganisms to colonize surfaces of plastics and develop biofilms (Amato-Lourenço et al., 2020; Gopinath et al., 2020). Secondly, additives mixed with polymers during synthesis to improve physical and chemical properties of plastics are inherently toxic and the breakdown of plastic debris into smaller fragments allows for the release of these toxins (Tsang et al., 2017). Microplastics act as vectors for toxic pollutants and pathogens introduce these additives to marine organisms and the food web (Guo and Wang, 2019). Chemicals and additives are often incorporated into plastic products and packaging to improve properties such as flexibility or fire resistance. While additives may not be of focused attention in the environmental pollution perspective, the recycling of plastics with additives is 16 increasingly complex and poses challenges for adopting a circular economy. Additives comprise of pigments, flame retardants and stabilisers which are incorporated to enhance the plastic product’s functionalities and also pose risks to environmental and human health. Plasticizers are toxic substances incorporated in products and these substances accumulate onto plastics from the surrounding seawater (Kühn et al., 2015). The hydrophobic nature of microplastics allows them to adsorb additives and contaminants such as pigments and metal ions from the surrounding environment (Huang et al., 2021; Mani et al., 2015). The biomagnification of toxic chemicals through organism transfers in the food chain leads to increased morbidity, accumulation of toxic substances in the liver, interferences in endocrine functioning and neurotoxicity (Sparks and Immelman, 2020). The ingestion of microplastic particles alters physiology and impairs development of organisms which has consequences on growth, reproduction and population size (Botterell et al., 2019). The knock-on effects may result in diminishing population sizes for successive generations and subsequently reducing food availability for higher trophic levels (Botterell et al., 2019). Extreme weather events, such as tropical storms, caused by climate change leads to dispersed mismanaged waste between terrestrial and aquatic ecosystems and can increase the abundance of microplastics within seawater and sediments (Botterell et al., 2019). Fluctuations in temperature from climate change may additively impact the effects of microplastics, and place multiple stressors on marine biota such as oxidative stress, immune functioning and thermal stress affecting energy reserves (Botterell et al., 2019). Pristine and well-maintained natural resources are necessary for the consistent operation of aquaculture and fisheries. The potential for growth in the blue economy is hindered by the continuous breakdown of plastic which leads to the growing abundance of microplastics present in waterbodies. The increasing abundance of microplastics subsequently leads to ingestion by aquatic biota which has knock- on effects on global economies (da Costa, 2018). 2.5 The Plastic Industry in South Africa South Africa has a well-developed plastics market catering to local and global markets with approximately 60 000 employees in the plastics sector (Tsotsi and Jenkins, 2019). The plastic industry is fundamental for South Africa’s economy, contributing 2.1 % to the GDP and 21.8 % to the manufacturing sector in 2018 (“Plastics – The Department of Trade Industry and Competition,” 2018). The plastics packaging sector is responsible for 52 % of South Africa’s plastic market and large proportions of domestic consumption comprises of ethylene from 17 domestic production whereas polypropylene is largely exported due to production exceeding domestic demand (“Plastics – The Department of Trade Industry and Competition,” 2018). South Africa has been ranked as the 11th most contributing country of plastic waste throughout the globe with an estimated 630 000 metric tonnes of trash leakage polluting the marine environment (Bulannga and Schmidt, 2022). The poor waste management system is a result of inadequate waste disposal protocols and the lack of proper infrastructure which results in the leakage of plastic into the environment (Verster et al., 2017). South Africa, similar to many countries across the global, is a water stressed country facing deteriorating water quality at varying extents across its freshwater sources (Morole, 2020). Aquatic systems situated nearby densely populated informal settlements are subjected to escalating levels of pollution from inadequate waste disposal of single-use plastic packaging and other plastic products. The plastic recycling sector creates approximately 7 800 jobs in South Africa (Plastics SA, 2019). Informal waste collectors account for the self-created livelihoods in the waste sector. The pickers earn their livelihoods from collecting waste found in streets and landfills, or sourced directly from households. The collected items are sold as recyclable waste to formal recyclers (Godfrey and Oelofse, 2017). The collection and recycling of plastic and other wastes are important activities in a circular economy, as they help to divert plastic waste from landfill, thus reducing plastic leakage into the environment. The services of waste collection and recycling are largely the responsibility of municipalities however the informal waste sector has been fulfilling the role. The informal workers are exposed to hostile living and working conditions, with minimal to no financial compensation whilst saving local municipalities costs for landfill space (Godfrey and Oelofse, 2017). Economic activities, such as the tourism and fishing industries, are heavily dependent on the marine environment (Jain, Raes, et al., 2021; Rose and Webber, 2019). According to a 2021 WWF report, approximately $ 60.72 billion (±28 %) damages in cost was inflicted on South Africa from the lifecycle of plastics that were produced in 2019 (WWF, 2021). The aforementioned damages are inclusive of income, population health, economic sectors such as tourism and fisheries, and costs incurred by the government for clean-up initiatives (WWF, 2021). Plastic pollution and the associated loss of aesthetic appeal of tourism hotspots such as beaches has critical impacts on the economy, with an investigation into the City of Cape Town reporting tourism revenue and employment decreasing by as much as 91 % due to plastic 18 pollution of its beaches (Jain et al., 2021). The City of Cape Town spends R13 million per year on regular beach clean-ups in order to avoid estimated damage R8.5 billion to the tourism sector (Jain et al., 2021). The extent and type of microplastic pollution in South Africa is largely unknown (Sparks and Immelman, 2020). The predicted growth of microplastic pollution in the forthcoming decades highlights the importance of understanding and identifying main pollution sources (Verster et al., 2017). Rapid population growth coupled with increased urbanisation and poor waste management have led to freshwater systems being threatened. Studies on microplastics in South Africa have reported their detection in freshwater, beach sediments, coastal zones, species of marine commercial fish and other biota (Naidoo and Glassom, 2019; Preston-Whyte et al., 2021; Verster et al., 2017). Microplastics have been detected in seven species of commercially targeted fish from the Agulhas Bank representative of South African fishing grounds along the western coast. Microplastic abundances ranged from 2.8 to 4.6 items/fish with fibres being predominantly reported of sizes from 500 to 1000 μm (Sparks and Immelman, 2020). Sewage sludge and wastewater from residential areas have been associated with higher abundances of microplastic fibres (de Villiers, 2018). Studies in South Africa have shown microfibres to be the most abundant microplastic type in coastal water and beach sediment sites linked to wastewater outfalls (de Villiers, 2018). 19 3. METHODS 3.1 Study Area An assessment of microplastic abundance, its key potential sources, and polymer types was carried out by analysing microplastics in sediment and surface water samples. Various sites were sampled along the selected study areas of Durban Bay and the Hennops River to understand the distribution of microplastics in surface water and sediments, and how nearby anthropogenic activities influence the abundance and distribution of microplastics present. The abundance, spatial distribution and characteristics of microplastics present in surface water and sediment was investigated in Durban Bay and the Hennops River (Figure 2), areas which are heavily subjected to anthropogenic activity. The results from this study will identify potential anthropogenic sources of microplastic input into Durban Bay and the Hennops River, and potentially be used to identify key areas to target for mitigating microplastic pollution. Figure 2. Map depicting the sampling areas selected for this study, namely the Hennops River in the Centurion area, Gauteng and Durban Bay located in Durban, KwaZulu- Natal. 20 3.1.1 Durban Bay Durban Bay (29° 52′ S; 31° 04′ E) is an estuarine embayment comprising of the largest port in sub-Saharan Africa, and one of the most economically active international shipping harbours and container handling ports in Africa (Adeleke et al., 2020; Preston-Whyte et al., 2021; Vogt et al., 2019). The estuarine harbour is located on the eastern coast of South Africa in the country’s third largest metropolitan of Durban, and drains into the Indian Ocean. Durban is characterised by humid subtropical climate with annual rainfall with an average annual rainfall of 1 054 mm in the hot summer season (between the months of December and February) (Moodley et al., 2015). Durban is a highly urbanised, major industrial and economic hub administered by the eThekwini Municipality in the province of KwaZulu-Natal (Adeleke et al., 2020; Naidoo and Glassom, 2019). The municipality supports a high population density of approximately 3.5 million people, and many inhabitants occupy informal settlements that are densely populated and lacking basic necessities such as piped water, basic health care and sanitation services (Vogt et al., 2018) . Numerous river inflows and estuaries are subject to a wide array of urbanised and industrialised activities surrounding the harbour, which have led to water rich in contaminants entering the marine environment (Vogt et al., 2018). A number of rivers and canals that flow through and drain the industrial and urban catchment areas of the Durban metropolitan, drain surface run off into the Durban Bay. The major catchments that contribute to perennial freshwater inputs into the Bayhead Canal of Durban Bay are the Umhlatuzana, Umbilo and aManzimnyama rivers (Figure 3.a & b) (Moodley et al., 2015; Preston-Whyte et al., 2021). Debris from waste gets washed downstream into the rivers during heavy rainfall after the build-up of pollution during the winter dry season, resulting in polluted waters of poor quality (Vogt et al., 2018). There has also been an increase in pollution from sewage, storm water, industrial runoff and wastewater outfalls into the harbour. The numerous stormwater outfalls and rivers that drain into the harbour are expected to carry the highest plastic abundances. The stormwater drains from the industries surrounding the harbour and Durban’s Central Business District (CBD) are indicated numerically (Figure 3.b) (Preston-Whyte et al., 2021). 21 The Gandhi Sewage Pump Station located at Durban Point is utilised by the Durban CBD, Berea and neighbouring areas (Preston-Whyte et al., 2021) The stormwater outfall located nearby the Yacht Club (Figure 3, point 4) is an overflow site for the sewage pump station. The estuarine harbour is highly degraded due to its exposure to anthropogenic activities and surrounding land-usage involving boardwalk restaurants, agricultural activities, dry docks, Figure 3. Map depicting Durban Bay with a) rivers and tributaries, b) drainage areas (coloured blocks) and the corresponding land-use activities inputs, stormwater channels and inflows, and river inflows into the harbour. Letters A and B represent the river inflows into the harbour, and numbers 1 to 13 represent the stormwater inlets into the harbour (Cited from Preston-Whyte, F., Silburn, B., Meakins, B., Bakir, A., Pillay, K., Worship, M., Paruk, S., Mdazuka, Y., Mooi, G., Harmer, R. and Doran, D., 2021. Meso-and microplastics monitoring in harbour environments: A case study for the Port of Durban, South Africa. Marine Pollution Bulletin, 163, p.111948.) 22 yacht clubs, industrial development and effluent discharge, which result in contaminants entering the harbour (Adeleke et al., 2020' Preston-Whyte et al., 2021). Anthropogenic activities occurring nearby to the harbour include petrochemical processing and vehicular traffic (Vogt et al., 2019). Shipping activities also form a major contributor which could lead to microplastic pollution in the environment (Vanapalli et al., 2021a). Durban Bay undergoes active dredging for the upkeep of the harbour’s operational channels and the sediment is discharged at an offshore site nearby the edge of the continental shelf. Identifying the accumulation zones and distribution patterns of microplastics within sediments will benefit understanding the scale of their potential impact on the environment post dredging activities (Preston-Whyte et al., 2021). The potential inputs of pollution from shipping activities around Durban Bay include the maintenance of container vessels, bulk and breakbulk cargo, automotive carriers, cruise ships, and smaller fishing and recreational boats (Preston-Whyte et al., 2021). Microplastic pollution can be derived from the activities and land uses typical of the area around Durban Bay (Adeleke et al., 2020). The extensive amounts of pollution directed towards the harbour are a result of inadequacies in the environmental maintenance and management of surrounding areas. Recreational activities (bathing, surfing and fishing) were banned in Durban in 2019 as a consequence of the inflow of untreated sewage into the aquatic environment (Heyden, 2019). An investigation by the Department of Water and Sanitation faulted mechanical pump failures due to malfunctional rakes and the entry of foreign objects into the pump system at the Mahatma Gandhi Pump Station, which resulted in uncontrolled raw sewage leakages into the sea (Heyden, 2019). 3.1.2 Hennops River The Hennops River, located in the Gauteng Province, is an urban riverine system under pronounced stress driven by heavy pollution from domestic solid waste, industrial effluent and untreated sewage, which is severely impairing its ecological function. The catchment is located between Johannesburg and Tshwane and flows through three large municipal jurisdictions, namely the City of Johannesburg, City of Tshwane and Ekurhuleni Metropolitan Municipality. The Hennops River flows from Kempton Park, through Centurion, into the Crocodile River, and drains into the Hartbeespoort Dam catchment located in a landlocked area that receives urban runoff from densely populated urban regions. Rapid urbanisation, informal https://0-www-sciencedirect-com.innopac.wits.ac.za/topics/agricultural-and-biological-sciences/urban-runoff 23 settlements, industrial developments and business areas as well as residential areas have led to the deterioration and loss of aesthetic appeal of the Hennops River. The urban regions consist of informal and formal residential-, industrial-, commercial- and recreational areas such as the Irene Country Club, Irene Dairy Farm, Centurion Golf Estate, Centurion Lake and the Hennops Park. Rapid deterioration of the river has occurred since the beginning of its urbanisation in the 1970s. The river cannot sustain aquatic life and appears in a poor environmental state with high levels of pollution and strong sewage odours due to poor environmental management, and the lack of basic sanitation available for residents dwelling in informal settlements. Poor water quality issues and high levels of pollution in the river have raised concerns about the health risks posed to the inhabitants of surrounding communities. The South African Human Rights Commission (SAHRC) Gauteng Provincial Office undertook an inquiry into the sewage pollution of the City of Tshwane’s rivers, after receiving several documented complaints regarding the levels of pollution and the release of untreated sewage into the Hennops River from malfunctioning WWTPs (SAHRC, 2021). The Olifantsfontein Wastewater Treatment Plant (WWTP) and Sunderland are situated in the Hennops River catchment. Treated effluent from the Sunderland Ridge WWTP is discharged into the Hennops River. The deteriorating water quality has been attributed to the densely populated informal settlements of Tembisa, Ivory park and Rabie Ridge, located in the upper catchment area of the Hennops River. The river receives heavy pollution along these settlements due to the lack of technical facilities, such as inadequate sanitation facilities, poor solid waste management and inefficient storm water drainage. Habitants of informal settlements lack access to sanitation services subsequently causing the build-up and overflow of solid waste into river systems. The sudden rise in population density that is associated with the setup of informal settlements places increasing pressure on the WWTPs which service the area. There are significant amounts of waste and vegetation debris present along the river. The vegetation debris consists of branches and dead trees which collect alongside the river. Other solid waste debris includes urban litter ranging from cans, bottles, plastic bags, clothing, shoes and tyres. The urban litter that builds up from informal settlements is due to the lack of proper waste collection, handling and removal services. A majority of this debris is discarded directly into the river but may also be flushed into the river during periods of heavy rainfall and 24 flooding. During dry seasons, litter collects in the streets and stormwater drains, therefore, after periods of heavy rainfall and flooding the debris flushes into the river, and plastic builds up along the riverbanks. The Kaal Spruit is one of the most contaminated tributaries of the Hennops River. The stream borders along the densely populated informal settlements of Tembisa and Ivory Park. There is often accumulation of waste from the informal settlements due to poor waste collection from weak organisational structures, poor infrastructural maintenance, the lack of municipal management and funds. Raw sewage is released directly into the Kaal Spruit which feeds into the Hennops River (SAHRC, 2021). The densely populated informal settlements located in the catchment area of the Hennops River possibly influence microplastic pollution and should be noted because wastewater in the surrounding areas often runs directly into the river. 3.2 Sample Collection 3.2.1 Durban Bay Sampling Surface water and sediment sampling was conducted across 15 sites at Durban Bay on the 18th August 2020 during the dry, winter season. A combination of water and sediment samples were collected at each site in the harbour to provide a robust assessment of microplastic contamination. Sediment and surface water samples were collected using a vessel provided by the Council for Scientific and Industrial Research (CSIR). The sampling sites were selected to cover as much of the harbour as possible to gain an adeqaute spatial understanding of the distribution of microplastics, as well as identify the potential microplastic pollution inputs from different land-based activities. The land-based activities surrounding the area include effluent of wastewater treatment plants, road drainage, urban runoff, river inlets and agricultural drainage. The table below in conjunction with the map in Figure 3 provide insights into the nearby activities which serve as potential sources of microplastic pollution associated with the various sampling sites. 25 Table 1. Sampling site coordinates and depths with their locations in Durban Bay and associated anthropogenic activities nearby. Sampling Site Coordinates Water Depth (m) Location Nearby Activity Latitude Longitude Durban Bay 1 -29.903863 31.008163 1.5 Bayhead River inflow; Yacht Club Durban Bay 2 -29.898478 31.004679 3 Bayhead Canal River inflow; Stormwater Dain Durban Bay 3 -29.892686 31.006605 7 Bayhead Canal River inflow; Stormwater Dain Durban Bay 4 -29.885709 31.006780 7 Bayhead Canal Stormwater Drains; Shell Petrol Station; Kind Edward Hospital Durban Bay 5 -29.879045 31.007044 14 Maydon Wharf Stormwater Drain Durban Bay 6 -29.871302 31.014386 16 Harbour Stormwater Drain Durban Bay 7 -29.869218 31.027294 14 Harbour Shipping Activity; Cruise Passenger Terminal Durban Bay 8 -29.874960 31.018762 13 Harbour Shipping Activity Durban Bay 9 -29.881358 31.025232 13 Durban Container Terminal & Pier 1 Container Terminal Durban Bay 10 -29.879390 31.033074 13 Durban Container Terminal & Pier 2 Container Terminal Durban Bay 11 -29.880742 31.042174 12 Island View Naval Station Durban Bay 12 -29.874669 31.048850 12 Durban Point Terminal Mahatma Gandhi Sewage Pump Station Durban Bay 13 -29.874823 31.041952 12 Durban Point Terminal Mahatma Gandhi Sewage Pump Station Durban Bay 14 -29.865470 31.032881 13 T Jetty & Cato Creek Quayside Road Durban Bay 15 -29.864234 31.024148 5 Boatman's Road Stormwater Drain; Yacht Clubs; Laundry Service Water Sampling Surface water samples were collected from each of the 15 sites by towing a conical plankton net alongside the vessel across a 50 meter transect (Figure 4). Sample collection was conducted at sea surface-level using a plankton net (50 m mesh size, 0.5 m opening size, 1 m length) towed at constant speed alongside the boat vessel. An approximate volume of 10 000 to 20 000 L of water was sampled along each transect. A calibrated flowmeter attachment (Hydrobios mechanical flow meter) was fixed in the centre of the mouth of the net and used to calculate the exact volumes of water that were sampled. The particulates inside the net tube and those retained in the net were sample reduced by washing into a 500 mL glass jar for laboratory analysis. The glass jars with metal lids that were used for sample storage were pre-rinsed with filtered reverse osmosis water. Samples were preserved using filtered 100 % ethanol for transportation and further analysis at the University of the Witwatersrand. 26 Sediment Sampling Sediment sampling was performed at each site (n = 15) using a grab sampler in conjunction to the water samples collected on the vessel. At a selected number of sites, two sediment samples were collected to look at small scale variability. The second sediment grab was mixed to uniform consistency in a glass bowl using a stainless steel spoon to determine if mixing had any effect on microplastic results. Approximately 150 g of sediment samples collected were from the top layer of sediment and were placed in Ziploc bags for transportation and storage. Particle size analysis for the sediment at each site was conducted by the CSIR to investigate whether microplastic abundances varied according to the differences in sediment compositions. A) B) Figure 4. Sampling at Durban Bay A) Deployment of the plankton net used for microplastic surface- water sampling in Durban Bay. B) Photograph of sampling site 7 nearby to a cruise passenger terminal within Durban Bay. 27 3.2.2 Hennops River Sampling Sampling in the Hennops River in the area of Centurion, Gauteng province was carried out on the 6th – 7th of July 2020. The Hennops River site selection was based on the subjection to various land use activities and anthropogenic perturbance in the area. Sampling was conducted across two sites along the Hennops River (Figure 5) in the dry winter season with no rainfall activity. Approximately 15 – 17 m3 of surface water samples (n = 2) and 150 g of sediment samples (n = 6) were sampled at each site. Table 2. Sampling site coordinates and depths with their locations in Hennops River and associated anthropogenic activities nearby. Sampling Site Coordinates Water Depth (m) River Width (m) Nearby Activity Latitude Longitude Hennops 1 -25.86228 28.194360 1 21 Sewage Outfall, Informal Settlement, Railway and Road Hennops 2 -25.86877 28.207010 1 17 Residential Area, Golf Park The coordinates and surrounding anthropogenic activities associated with the sampling sites in the Hennops River are summarised in Table 2. Sampling site 1 presents evidence of illegal Figure 5. Map showing the sample collection locations, indicated by red markers, along the Hennops River located in Centurion, Gauteng. The red pins indicate where the plankton nets were deployed in the river, sediment samples were collected in the surrounding areas. Samples were collected in July 2020. The base map is produced in ArcGIS Pro. 28 dumping of solid waste along the river. The site is situated in close proximity to the informal settlements and receives pollutants from surface run-off during seasonal rain periods. Informal settlements have inadequate sanitation facilities and minimal access to clean water sources with the build-up of solid waste being common in the area. The site is located nearby to a road and railway with sewage plumes leading directly into river (Figure 6). Water quality and clarity in the Hennops river is poor and carries a strong sewage odour to the extent that no aquatic life can be detected in the area, indicative of raw sewage being pumped directly into the freshwater system. Site 1: Land-based activities nearby: metro track, informal settlement, road, two sewage plumes leading directly into the river. Sampling site 2 (Figure 7) at the Hennops River was conducted over a bridge located near a residential suburb area and a golf estate nearby to a road. A significant hydrological problem in the area is the large amount of debris present (Figure 7.A). A) B) C) D) Sed 1B Sed 1A Sed 1C Figure 6. Photographs depicting Hennops sampling site 1. The site in the river has sewage pipes discharging directly into the river with many solid waste materials and discarded debris (Figure A and B). Sed 1A, 1B and 1C depict the points where sediment samples were collected. 29 Water Sampling Surface water samples were collected using a 50 m mesh plankton net (0.5 m mouth diameter, 1 m length) deployed in the river for approximately two hours at each site. Microplastics were collected under normal river flow conditions by suspending the plankton net from a bridge over the centre of the river. The upper portion of the mouth of the net was kept at surface level by adjusting the length of the rope. The exact volume of water sampled through the net was calculated by using a flowmeter (Hydrobios mechanical flow meter) attached to the centre of the net mouth during water sampling. All of the material collected in the end of the net after the sampling period was transferred into pre-rinsed 500 mL glass jars using filtered water. The collected samples were transported to the laboratory to initiate sample processing in order to separate the microplastics from other suspended materials. Sediment Sampling A total of three surface sediment samples were collected at each site (n = 2) in order to account for small scale variability. Sediments were collected using a stainless steel spatula in approximately 250 g portions along the river bank, and transferred into pre-rinsed glass jars for collection. The collected sediments were tightly packed and transported to the University of the Witwatersrand for further analysis. A procedural blank was collected at each site, which consisted of pre-rinsed glass jars subject to atmospheric deposition of particles by being positioned unclosed for the duration of sediment collection. The jars were fastened afterwards and transported in the same conditions as the collected sediments to measure any contamination that may have occurred during sampling. A) B) C) Figure 7. Photographs depicting Hennops sampling site 1. A) Plastic and organic debris along the river bank. B) Bridge over the river where net was suspended from C) Plankton net suspended in the centre of the river with normal river flow conditions. 30 3.3 Sample pre-treatment The wet sediment samples were stored in glass jars and kept in a freezer until subjected to sample processing. The sediment samples were freeze-dried for approximately 48 hours prior to sample processing to obtain the dry weights, and standardise the differences in moisture content between the samples. Approximately 100 g of dry sediment was weighed into triplicate subsamples and transferred into 50 mL centrifuge tubes. The exact weight of each triplicate subsample was recorded. All equipment and glassware were prewashed with filtered reverse osmosis water three times prior to use during sampling and sample processing to avoid contamination. 3.4 Grain size Grain size compositions of the collected sediment samples were determined by sieving the dried and weighed sediments (100 g), and determining their percentage grain compositions according to the following grain size classes: mud (< 0.063 mm), very fine-grained sand (0.063 – 0.125 mm), fine-grained sand (0.125 – 0.25 mm), medium-grained sand (0.25 – 0.50 mm), coarse-grained sand (0.5 – 1.0 mm), very coarse-grained sand (1.0 – 2.0 mm) and gravel (> 2.0 mm). 31 3.5 Sample processing Surface water and sediment samples underwent sample processing and analysis as outlined in Figure 8 below. 3.5.1 Sediment sample processing Density separation was used for the separation of microplastics from sediment samples by flotation, followed by vacu