METAL and METALLOID ACCUMULATION in SEDIMENT, WATER and FISH in LAKE TITICACA (BOLIVIA) Number of Words: 42 058

METAL AND METALLOID ACCUMULATION IN SEDIMENT, WATER AND FISH IN LAKE TITICACA (BOLIVIA) Number of words: 42 058

Fonteyne Arthur Student number: 01509225

Promotors: Prof. dr. ir. Gijs Du Laing, Prof. dr. ir. Geert Janssens Tutors: dr. Arturo Muñoz Saravia, Bernd Mees

Master’s Dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Bioscience Engineering: Chemistry and Bioprocess Technology

Academiejaar: 2019 – 2020

Deze pagina is niet beschikbaar omdat ze persoonsgegevens bevat. Universiteitsbibliotheek Gent, 2021.

This page is not available because it contains personal information. Ghent University, Library, 2021.

Acknowledgements Now, after five years of hard work and perseverance, finishing this master thesis, feels like finalising my career as a student. This project was a unique and interesting experience, which wouldn’t have been the same without the help of some important people. Now, I would like to thank everyone who made it possible to have such a wonderful time during my career as a student, my field work in Bolivia, for the guidance in the laboratory, for the advice whilst writing this thesis or just helped with a warm smile and some motivating words.

First of all I want to express my gratitude to Prof. dr. ir. Gijs Du Laing and Prof. dr. ir. Geert Janssens for the opportunity to work on this project and for their support. Thank you for all the tips, suggestions and critical reviews on the right moment. Furthermore I would like to thank my tutors Bernd Mees and Arturo Muñoz Saravia. Bernd, thank you for the guidance in the lab, for answering all my questions and for the critical reviews regarding this master thesis. Arturo, thank you for all the help and advice during the field work in Bolivia, the guidance throughout the year and the critical reviews.

Secondly I would like to thank the Limnology department of the Universidad Mayor de San Andrés (UMSA), for their warm welcome, all their help, advise and the possibility to use their equipment. A special thanks to Erick Loayza Torrico, without you, this thesis project would not have been possible. Your knowledge and experience at the lake, were of vital importance to accomplish our goals set in this master thesis. During the fieldwork we worked extremely hard, we could work from 5 a.m. till 4 p.m. without lacking enthusiasm. I’m very grateful for all your help, information, guided tours in La Paz, Oruro, Copacabana, the bouldering and so much more. Also a special thanks to Sara Neyrot and Vivi Cruz for their support at the lab, the lake and the nice times at La Paz. Don Ricardo, thank you for all the safely driven kilometres which brought us to our sampling locations. At last, also a special thanks to Yara Fernandez, when Eric and I needed a motivational boost, you knew exactly what could help us. I’m very grateful for all the support I received from everyone in Bolivia involved in this project. Due to all of you, this project resulted in an unforgettable experience.

In addition, I would like to thank all the people working in the ECOCHEM laboratory of the department of Green Chemistry and Technology. Especially Roseline Blanckaert, who guided me throughout the year, provided me information regarding digestion methods applied in the laboratory and answered all my questions with full enthusiasm. Thank you, and I wish all the best for you and your baby.

Finally, I’m very grateful to my friends and family, for all the support they gave me. Thank you mom and dad, Louise and Ester for giving me the opportunity to study, the endless support, motivational speeches, warm hugs and so much more. These five years were intensive but I only succeeded because of your support. Jolien, the last three years were amazing together, I’m sorry I went for 6 long weeks to the other side of the planet for my thesis. I admire your unlimited support even when I’m in the need of undertaking such adventures. I’m looking forward to the adventures the future may bring for both of us. At last, a thank you, to all my friends who made my time as a student unforgettable! These five years were extraordinary and the time flew by at the speed of 299 792 458m/s.

Preambule In this preambule, the impact of the corona measures in Belgium on this master thesis will be discussed. Due to the measures taken by Ghent University to ensure the safety of students and employees, the laboratory operations had to be stopped prematurely. Following analyses could not be carried out:

 Second measurement of the water samples to obtain better recoveries  Second measurements of the muscle samples to obtain recoveries for Pb  Analysis of muscle samples from O.agassizii at lake Uru Uru  Analysis of liver tissue from fish samples  Analysis of the amphipod samples  Analysis of the Diffusive Gradient in Thin film (DGT) samplers

During the summer of 2019, field work was carried out in Bolivia: prospection, sample collection and preparation. During the first semester a suitable digestion method for sediment, water, fish and amphipod samples was developed. During the second semester the digestion and analyses of the samples were carried out. Thirty sediment and water samples and 175 fish samples were digested and analysed. The analyses given above were scheduled 3 weeks before the eastern holidays (11/03-27/03/2019) and could not be carried out. Not all analyses could be conducted which affected the amount of obtained results. Due to this hindrance a more extensive literature study was done and the work needed to finish this project, including analytical methods, are described in “Future perspective”. This preambule was drawn up after consultation between the student and the supervisor and is approved by both.

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Resume Lake Titicaca has a valuable but vulnerable ecosystem and close monitoring of its pollution is very important to avoid ecologic disasters. In South America, high arsenic geogenic background concentrations of surface and groundwater are present. Besides, As, Hg, Cd and Pb are occurring in abnormally high concentrations near to urban areas, metalliferous mines or major road works. Elevated As, Hg, Cd and Pb could pose a substantial threat to the vulnerable ecosystem of Lake Titicaca and pose a risk to the health of the local communities. It is proven that the elevated intake of these elements could have a negative effect on plant, animal and human life. The prevention of increasing metal and metalloid concentrations by mining activity, agriculture and untreated urban waste water is of great importance.

This master thesis focuses on the pollution and the accompanying risk of As, Hg, Cd and Pb at Lake Titicaca. The aim is to assess the quality of water and fish as a food source for the people who are living around the Bolivian part of lake Titicaca and to assess the water and sediment quality for animal life in this ecosystem. In order to obtain the As, Hg, Cd and Pb concentrations in sediment and biota, a method was selected to digest the sample matrix and to analyse the metals and metalloids via ICP-MS. Aqua regia or nitric acid were used for microwave-assisted digestion in closed vessel. This technique was selected because it is fast, results in complete digestion of the organic matter under high pressure, and shows high repeatability and better recoveries. Recoveries were obtained within a deviation of maximum 20% from full recovery for Cd, Hg and Pb. For As, elevated recoveries were found, which could have been caused by the interference of 40Ar35Cl+ and 40Ca35Cl+, formed in the plasma of the ICP-MS. Analysing As in dynamic reaction mode (DRC) instead of collision cell mode on the ICP-MS may resolve this problem.

In this study, the As, Hg, Cd and Pb concentrations were measured in sediment, water and seven fish species occurring in Lake Titicaca. It could be concluded that for As in sediment, adverse effects on the aquatic environment can be expected. The obtained results for the water samples taken at Lake Titicaca showed elevated As concentrations which are situated near to the safety threshold value (10 g/L). At lake Uru Uru, the As concentrations in water are approximately six times higher than the safety threshold level. Negative effects can be expected for aquatic and terrestrial life due to the chronic exposure to (drinking) water contaminated with As. Regarding the As, Hg, Cd and Pb bioaccumulation in the seven different fish species, As and Hg may pose a threat to the health of the local communities. The As concentrations exceeded the safety threshold level (0.1 mg/kg) in all seven fish species. For Hg, O. bonarienses had an average Hg concentration above the safety threshold level (0.5 mg/kg) and Trichomycterus spp. showed concerning elevated concentrations. Furthermore, the in captivity grown O. mykiss, had the lowest As and Hg concentrations, which could be the result of the captive breeding. If the in captivity bred up O. mykiss has the potential to accumulate lower metal and metalloid concentrations, this could possibly have a similar impact on O. bonariensis and the other fish species. Additional research is needed to investigate the impact of the captive breeding on the metal and metalloid bioaccumulation in the fish species from Lake Titicaca. Finally, consumption limits were calculated. From this evaluation it seems advisable to replace the water of Lake Titicaca by treated or non-contaminated water for consumption by children under the age of 12 years. Besides, recommended maximum daily intakes of fish from Lake Titicaca were calculated. However, in order

to conduct a full risk analysis, the consumption behaviour of the local population and assessment of the metal and metalloid concentrations in other, non-aquatic food sources are still needed.

Samenvatting Het Titicacameer heeft een zeer waardevol maar kwetsbaar ecosysteem waardoor het opvolgen van vervuiling zeer belangrijk is om ecologische catastrofes te vermijden. In Zuid-Amerika komen hoge As concentraties voor in grond- en oppervlaktewater door natuurlijke processen. Daarnaast werden verhoogde As, Hg, Cd en Pb concentraties waargenomen in de omgeving van steden, metaalhoudende mijnen of grote wegenwerken. Een verhoging van As, Hg, Cd en Pb concentraties in het meer kan een bedreiging zijn voor het kwetsbaar ecosysteem en de plaatselijke bevolking. Het is aangetoond dat verhoogde absorptie van deze elementen een negatief effect kan hebben op plant, dier en mens. Dit toont het belang aan om de toename in concentraties in het meer, veroorzaakt door mijnactiviteit, landbouw en onbehandeld afval water, tegen te gaan.

Deze masterproef beoogt de verontreiniging en bijhorende risico’s van As, Hg, Cd en Pb in het Titicacameer te beoordelen. Het doel is de kwaliteit van water en vis als voedingsbron na te gaan voor de lokale bevolking aanwezig in het Boliviaanse deel van het Titicacameer. Daarnaast zal de kwaliteit van water en sediment nagegaan worden alsook de impact op de biota in dit ecosysteem. Om de As, Hg, Cd en Pb concentraties na te gaan in sediment en biota, werd een methode geselecteerd om de matrix van de samples af te breken en de metaalconcentraties te analyseren via ICP-MS. Een behandeling met koningswater of salpeterzuur werd toegepast om de matrix van de stalen af te breken in combinatie met de “Microwave assisted digestion in closed vessel” techniek. Deze techniek werd gekozen omwille van de snelheid, de volledige digestie van het organisch materiaal onder hoge druk, en toont hoge herhaalbaarheid en betere recuperatie van de referentiestalen. Recuperaties binnen een maximumafwijking van 20% ten op zichte van volledige recuperatie werden bekomen voor Cd, Hg en Pb. Voor As werden verhoogde recuperaties van de referentie stalen waargenomen. Dit kan te wijten zijn aan de vorming van ArCl+ en 40Ca35Cl+ in de plasma van de ICP- MS. Analyseren in “Dynamic reaction mode” in plaats van “Collision cell mode” zou een oplossing kunnen bieden voor dit interferentie probleem.

In deze studie werden de As, Hg, Cd en Pb gehaltes nagegaan in sediment, water en zeven vis soorten voorkomend in het Titicacameer. Het kan geconcludeerd worden dat de As concentraties in sediment hoog zijn en negatieve gevolgen op het aquatische leven kan verwacht worden. Ook het water in het Titicacameer vertoonde verhoogde As concentraties, deze waarden liggen dicht bij de maximale toegestane concentraties in drinkwater (10 g/L). In het meer Uru Uru werden hogere concentraties gemeten, tot zes keer de toegestane concentratie in drinkwater. Negatieve gevolgen kunnen verwacht worden voor het aquatische en terrestrische leven door de chronische blootstelling aan (drink) water met As. Ook in vis werden de As, Hg, Cd en Pb concentraties geanalyseerd. Daaruit bleek dat de As en Hg concentraties risico’s kunnen vormen voor de gezondheid van de lokale bevolking van het meer. De As concentraties waren in alle vissoorten boven de opgestelde veiligheidsconcentratie (0.1 mg/kg). Voor Hg, hadden O. bonarienses en Trichmycterus spp. verontrustend hoge concentraties. Bovendien, had de in gevangenschap gekweekte O. mykiss de laagste As en Hg concentraties. Dit kan te wijten zijn aan de manier van kweken. Als de gekweekte O. mykiss lagere concentraties aan As en Hg accumuleert, kan het zelfde effect optreden bij de andere vissoorten. Hiervoor is extra onderzoek noodzakelijk om de impact van het kweken op de metaal en metalloïde accumulatie na te gaan in de verschillende vissoorten. Als laatste werden consumptie

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limieten berekend. Uit deze evaluatie bleek het raadzaam om kinderen onder de 12 jaar geen water van het Titicacameer te laten gebruiken als drinkwater. Daarnaast werd ook uitgerekend wat de aanbevolen maximale inname van vis uit het Titicacameer is. Om een volledige risico analyse uit te voeren, is bijkomend onderzoek noodzakelijk. Extra onderzoek, naar het voedingspatroon van de lokale bevolking, waarbij de metaal en metalloïde concentraties in de andere niet-aquatische voedingsbronnen worden bepaald, kan meer inzicht geven in het potentieel risico voor de lokale bevolking.

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Table of content Acknowledgements ...... I Preambule ...... III Resume ...... V Samenvatting ...... VII List of abbreviations ...... XIII Introduction...... 1 1. VLIR project ...... 1 2. The master thesis ...... 1 2.1. Background...... 1 2.2. Overview...... 2 2.3. Objectives ...... 2 2.4. Relevance ...... 2 Literature review ...... 5 1. Study Area ...... 5 1.1. Location ...... 5 1.2. Climate ...... 9 2. Metals and metalloids ...... 10 2.1. Introduction ...... 10 2.2. Bioavailability ...... 12 2.3. Arsenic ...... 16 2.4. Mercury ...... 19 2.5. Cadmium ...... 21 2.6. Lead ...... 23 3. Diffusive gradients in thin films ...... 25 3.1. General principle ...... 25 3.2. Applied samplers ...... 26 3.3. Advantages ...... 27 4. Fish in Lake Titicaca ...... 27 4.1. Fishery ...... 27 4.2. Species ...... 27 4.3. Morphology ...... 29 4.4. Trophic interaction ...... 30 5. ICP-MS ...... 31 5.1. Introduction ...... 31 5.2. Isotopes ...... 32

5.3. Interferences ...... 32 5.4. Modes ...... 33 5.5. Internal standards ...... 34 5.6. Mercury analysis ...... 34 Materials and Methods ...... 35 1. Reagents ...... 35 2. Sampling locations ...... 35 3. Sampling method ...... 36 3.1. Water ...... 36 3.2. Sediment ...... 38 3.3. Fish ...... 39 3.4. Amphipods ...... 40 4. Determination of metal and metalloid concentration in water, sediment and biota ...... 40 4.1. Selection of digestion method biota and sediment ...... 40 4.2. Digestion method biota and sediment samples from Bolivia ...... 44 4.3. ICP-MS analysis ...... 44 5. Determination of water properties ...... 45 6. Statistical processing ...... 45 Results ...... 46 1. Dissection results: O. gilsoni, O. ispi and O. bonariensis ...... 46 2. Comparison of digestion methods ...... 47 3. Selection digestion method ...... 48 3.1. Screening ...... 48 3.2. Fine tuning ...... 50 4. Samples Lake Titicaca and Uru Uru...... 54 4.1. Sediment ...... 54 4.2. Water ...... 56 4.3. Fish ...... 59 5. Multi-way ANOVA analysis ...... 61 5.1. ANOVA: O. luteus ...... 61 5.2. ANOVA: O. bonariensis...... 62 Discussion ...... 63 1. Morphology O.ispi, O. gilsoni and O. bonariensis ...... 63 2. Digestion method ...... 63 3. Sediment from Lake Titicaca ...... 64 4. Water from Lake Titicaca ...... 65

5. Fish from Lake Titicaca ...... 66 5.1. Risk of arsenic in fish ...... 66 5.2. Risk of mercury in fish ...... 67 5.3. Bioaccumulation in the different species ...... 67 6. Daily consumption limits ...... 70 7. Improvements field work ...... 73 Conclusions ...... 74 Future laboratory work ...... 75 1. Water samples ...... 75 2. Recovery of Pb ...... 75 3. Fish: whole fish, muscle and liver tissue ...... 75 4. Amphipods...... 76 5. Analysis of the DGT samplers ...... 76 Addendum ...... 78 Tables ...... 78 Figures ...... 85 Calculations ...... 90

Calculations digestion procedure: hotplate with H2O2 ...... 90 Calculations daily consumption limits ...... 91 Bibliography ...... 94

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List of abbreviations

AB Arsenobetaine aBW Average body weight BW Body weight CRlim Allowable fish consumption rate [kg/day] CRmm Allowable fish consumption rate [meals/month] DMA Dimethylarsinate DGT Diffusive gradients in thin film technique DGT sampler Diffusive gradients in thin film sampler DRC Dynamic reaction mode EC European Commission Eh Redox potential HgP Hg associated with particulate matter ITCZ Intertropical convergence zone ICP-MS Inductively coupled plasma mass spectrometry KED Kinetic energy discrimination (Collision mode) LC50 Concentration of a chemical that kills 50% of a group of test animals. LOD Limit of detection LOQ Limit of quantification MS Meal size MA Methylarsonate m/z Mass/charge ratio NOAEL No observed adverse effect level PEC Probable effect concentration RfD Reference dose Std Standard deviation TEC Threshold effect concentration Tprofile Temperature profile WW Wastewater WWTP Wastewater treatment plant VLIR Flemish Interuniversity Council UV Ultraviolet radiation UMSA Universidad Mayor San Andres

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Introduction 1. VLIR project This master thesis is part of a project between the Limnology department of the Universidad Mayor de San Andrés (UMSA) and the department of veterinary medicine (Gent University), funded by the Flemish Interuniversity Council (VLIR). The aim of this cooperation is to evaluate the water and fish quality meant for human consumption and to create a healthier aquaculture at Lake Titicaca. The project is conducted in three research areas: ecotoxicology, aquaculture and trophic and nutritional ecology. This study focusses on the ecotoxicology area, in order to identify the correlation and accumulation of metals and metalloids in sediments, water, macroinvertebrates and seven fish species. These seven fish species are commonly used as food source: Orestias agasizii, Orestias luteus, Orestias ispi, Orestias gilsoni, Trychomycterus spp., Odontesthes bonariensis and Oncorhynchus mykiss. Furthermore the VLIR-UOS project focusses on sustainable alternatives to trout farming in cooperation with the PACU research center situated in Tiquina and the captive breeding of the Orestias species for reintroduction and to encourage its consumption at the local level.

2. The master thesis This master thesis is the first venture in this VLIR-UOS project. The aim is to assess the quality of water and fish as a food source for the people who are living in the Bolivian part of lake Titicaca and to assess the water and sediment quality for animal life in this ecosystem. Focus is mainly laid on the occurrence of toxic trace elements. Lake Titicaca has a valuable fauna and flora. It is a crucial breeding and wintering area for up to sixty bird species, and it is inhabited by seven amphibian species and twenty six fish species1–4. Over the last thirty years there has been a reduction of forty five percent in native fish biomass and many species are now listed as threatened1. This is disturbing because of their ecological value and the economic importance of fishery as a source of income and food for local communities. Furthermore, the lake water is of great value for agricultural purposes and as a source of drinking water.

2.1. Background Studies have revealed abnormally high concentrations of metals and metalloids, especially lead (Pb), cadmium (Cd), mercury (Hg) and arsenic (As), in ecosystems near to urban areas, metalliferous mines or major road works5. In South America, As is of great interest because of the naturally high As geogenic background of surface and groundwater. The prevention of increasing metal and metalloid concentrations and eutrophication by mining activity, agriculture and untreated urban waste water is of great importance. Lake Titicaca has a vulnerable ecosystem and close monitoring of its pollution is very important to avoid ecologic disasters. For example, a comparison can be made with lake Poopó, where a decreasing water level and increasing levels of salts and heavy metals in the lake caused a collapse in fishery6. Also a recent event shows the ecological vulnerability of Lake Titicaca. In 2015, eutrophication occurred in the smaller part of the lake, leading to an algae bloom. This had an enormous ecological impact on the lake, resulting in a massive death of pelagic organisms e.g. fish and frogs7,8. Because of the previously given reasons, this

master thesis will focus on the accumulation of metals and metalloids (Pb, Cd, Hg and As) in sediment, water, macroinvertebrates and seven fish species.

2.2. Overview Firstly the study area will be discussed in this paper followed by the general behaviour of metals and metalloids in the environment, with specific focus on As, Hg, Cd and Pb. Secondly, the biota important for this investigation and the techniques (DGT samplers and ICP-MS) applied in this thesis will be discussed. The Diffusive gradients in thin films sampler (DGT sampler) is used to selectively accumulate metal species and the inductively coupled plasma mass spectrometer (ICP-MS) is required to analyse the metal and metalloid concentrations present in water, sediment and biota. At last the methods and the results obtained during this research will be discussed and a proposal for further investigation and improvements of this project will be given.

2.3. Objectives Firstly, in order to obtain the As, Hg, Cd and Pb concentrations in sediment, water and biota, a method has to be selected to digest the sample matrix and to analyse the metals and metalloids via ICP-MS. In this thesis a method will be set up to analyse simultaneously As, Hg, Cd and Pb. Secondly, with the data obtained from sediment and water, the metal and metalloid distribution will be evaluated at Lake Titicaca. Furthermore, the As, Cd, Hg and Pb bioaccumulation in the seven different fish will be examined and the influence of the environmental parameters will be studied. Based on the fact that the main rivers represent the most important inflow of metal pollution, it can be expected that the sediment, water and biota at the discharge sites would have the highest metal concentrations1. Besides, the metal concentration in the fish species, will also depend on environmental factors (bioavailability: pH, soil characteristics, etc.) and the fish profile ( size, age, species, etc)1,9–11. At last a risk assessment will be conducted and an advice will be given about the daily water and fish intake.

2.4. Relevance Previous studies regarding metals (Hg, Pb, Cu, Zn, Cd, Pb, Co, Fe) and metalloids (As) at the Lake are available1,8,12,13. This master thesis is relevant because additional aspects are introduced. The study of D. Archundia et al. (2016) investigated the pollution entering the lake via the Katari watershed (mainly polluted by El Alto city)12. This master thesis gives a more general picture regarding the metal distribution at the lake (especially Lago Menor) and focuses on the impact on the fish intended for human consumption. The study of G. Sarret et al. ( 2019) investigated the As distribution at Lago Menor and Uru Uru with its main focus on the possible bioaccumulation and its applications8. This master thesis focuses on the impact of metal and metalloid concentrations (As, Hg, Cd, Pb) in the fish intended for human consumption and the distribution between the trophic levels. The study of S. Guedron et al. (2017) investigated the Hg contamination (Hg2+ and MeHg+) at the Lake Titicaca and Uru Uru and the bioaccumulation and biomagnification occurring between the different trophic levels13. Compared to this study, this thesis provides extra information about the As, Cd and Pb concentrations occurring at the different trophic levels. The study of M. Monroy et al. (2014) focussed on the metal contamination in the Peruvian part of the lake and the bioaccumulation occurring in five different fish species1. This thesis gives more insights in the

contamination and the bioaccumulation in fish occurring in the Bolivian part of the Lake and two extra fish species were investigated. Another extra aspect is the use of the DGT samplers. Speciation analysis will be performed by the use of DGT samplers. This technique has not been applied previously at the Lake and will provide more information regarding the bioavailability of the metals and metalloids occurring in Lake Titicaca and Uru Uru.

Literature review 1. Study Area 1.1. Location Lake Titicaca is the largest lake (approximately 8.5 km²) in South America. It is located on the border of Peru and Bolivia in the Andes Mountains at 3810 m above sea level1,12,14. The lake is divided in a northern and a southern part. The northern part, the biggest side of the lake, is called Lago Mayor, El Grande or Lake Chucuito and has a mean dept of 135 m. The southern part, Lago Menor or Huinaymarca, has a mean dept of 9m. In this paper the terms Lago Mayor and Lago Menor are used. The lake is situated on the northern side of the Altiplano, a high endorheic basin with limited drainage system15–17. At the Altiplano three lacustrine areas occur. The first area is formed by the Lake Titicaca, the second area contains the Lakes Poopo and Uru Uru and most southerly of the Altiplano, Coipasa and Uyuni. Approximately 12 000 years ago the Altiplano reached its highest water levels and Lake Tauka was formed, covering lake Poopo, Coipasa and Uyuni. The Lake received water from Lake Titicaca. Due to evaporation Lake Poopo turned into a shallow lake and Caipasa and Uyuni turned into salt flats16.

Figure 1: The Altiplano with tree lacustrine areas: the first containing the Titicaca Lake encircled in red, the second with the Poopó and Uru-Uru Lake encircled in green and the third Coipasa and Uyuni salt pan encircled in blue.16,18 Country borders are indicated in yellow. Illustration made with Google Earth Pro®.

1.1.1. Lake Titicaca Value of the lake Ecologically the Lake Titicaca is of great importance. It is a crucial breeding and wintering area for up to sixty bird species, and it is inhabited by seven amphibian species and twenty six fish species1–4. Over the last thirty years there has been a reduction of forty five percent in native fish biomass and many species are now listed as threatened1. This is disturbing because of their ecological value and the economic importance of fishery as a source of income and food for local communities, especially because Bolivia has no marine resources. Furthermore, the lake water is of great value for agricultural purposes and as a source of drinking water for cattle and the local population.

Threats Currently, the ecosystem in and around the lake is threatened by the decreasing water level, the introduction of exotic species, intensive fishery, urbanization, agriculture and mining activity13,19–21. The decreasing water level is a threat on the long term, which can be influenced by mining activity, intensive irrigation of agricultural lands, reduced wet seasons and by reduced melt water inflow from the glaciers17,21– 23. Lower water levels would negatively affect the reed beds, fishery and the regional wild life. A threat on short term is the discharge of agricultural, mining and urban waste water into rivers. This is often the main input of pollution in large lakes1. Polluted streams with high growth stimulating nutrients can cause eutrophication and lead to algae bloom. Eutrophication is defined by the European Commission, in the Urban Waste Water Treatment Directive as: the enrichment of water by nutrients, especially compounds of nitrogen and phosphorus, causing an accelerated growth of algae and higher forms of plant life to produce an undesirable disturbance to the balance of organisms and the quality of the water concerned24. For example, in 2015, eutrophication occurred in the smaller part of the lake, leading to an algae bloom. Due to intensive rainfall large amounts of nutrients from the Katari watershed became available and caused the bloom. This had an enormous ecological impact on the lake: acidification occurred in the eutrophic shallow areas of the lake, the oxygen concentration decreased (drop in redox potential) and hydrogen sulphide increased. This resulted in a massive death of pelagic organisms e.g. fish and frogs, showing the fragile balance in the ecosystem and the need of closely monitoring the lake to prevent future tragedies7,8.

Metals and metalloids in the Lake Metals and metalloids pose a high environmental risk caused by their long-term persistence in nature. Few metals can be removed through elimination reactions out of the body (e.g. Aluminium), and this gives a high risk for bioaccumulation and biomagnification (e.g. As(III), methylmercury)25,26. For example many invertebrates (e.g. amphipods) process sediment as a food source and can bioaccumulate toxic metals during their life. This bioaccumulation in the trophic level at the beginning of the food chain can potentially threaten the health of many species at the top of the chain (birds, fish, humans) by bio-accumulation or biomagnification of the toxic elements.

Sources of pollution As previously explained the main input of pollution comes via waste discharge in rivers1. However, agricultural areas surrounding the lake could also be a potential source of contamination by the discharge

of pesticides and fertilizers1,20,27,28. At Lake Mayor three rivers represent the main source of metal pollution due to the location of mining areas upstream: the Ramis, Coata and Ilave Rivers (Figure 2)1,13,29. A second major source of pollution at El Grande is via wastewater disposal of the city Puno. This City has 118 000 inhabitants and only twenty percent of the wastewater is treated by a wastewater treatment plant (WWTP)13,20.

Figure 2: Main inflow of metal pollution at Lago Mayor indicated in blue: Puno, the Ramis, Coata and Ilave Rivers1,13,29 County borders are indicated in yellow. Illustration made with Google Earth Pro®.

The main water inflow at Lago Menor is via Tiquina (Figure 4), a small passage which connect Lago Menor with Lake Major, and via Cohana Bay. At Cohana bay the water from the Katari watershed enters the lake (Figure 3). Four rivers flow through the watershed who reach the lake at Cohana bay: the Seco, Seke, Pallina and the Katari rivers. The Seke and Seco river receive water from Milluni Lake, which receives mine drainage and serves as the main reservoir of drinking water for La Paz city. Next, both rivers flow through El Alto city and receive treated and untreated urban wastewater12. Thereafter, both rivers flow into the Pallina river which will flow into the Katari river. This river passes through agricultural areas and discharges ultimately at Lago Menor (Figure 3) . The city of El Alto has a population of 1.1 million and is located at a height of approximately 4000m. Despite the height, cold and high solar radiation, El Alto has become one of the fastest-growing cities in Latin America20,30. This city has a lot of industry with more than 5000 enterprises and most of them have no WWTP. This causes the discharge of untreated sewage from the food processing, leather, cement and timber industries into the lake’s tributaries12,20. El Alto has a small WWTP and has the capacity to treat the wastewater (WW) of 300 000 inhabitants ( 500 L_WW/s)12,20. Figure 4 gives a simplified model of the in and out flow at the Titicaca lake.

Figure 3: The Katari watershed (encircled in red) with the main rivers (in blue): Katari, Seke, Seco and Pallina12. Illustration made with Google Earth Pro®.

Figure 4: Simplified model of the in- and outflow at the Lake Titicaca. County borders are indicated in yellow. Illustration made with Google Earth Pro®.

Improvements However, there are signs of improvements. Two new WWTP’s will help enhance the water quality, an educational program is started to inform the people about climate change and its consequences, etc20. The introduction of extra wastewater treatment plants has a positive impact on the removal of biodegradable organics, growth stimulating nutrients and the elimination of disease-causing pathogenic micro-organisms, but sufficient removal of toxic and trace compounds (e.g. metals) is still a challenge31. Cost effective technologies are in high demand. Conventional processes for metal removal include chemical precipitation, coagulation and flocculation, carbon adsorption, ion exchange, evaporation, electrochemical treatment and membrane filtration processes31–33. Beside the conventional methods the use of biological materials has gained popularity due to increased performance, availability and low cost of raw materials31,32. Wastewater stabilization ponds, membrane bioreactors, activated sludge processes, biosorption, trickling filters and phytoremediation technics are examples of possible biological treatments used for metal removal from wastewater8,31–35.

1.1.2. Lake Uru Uru As shown in Figure 1, the water from Lago Menor flows into the Desaguadero river which discharges its water at the Lakes Uru Uru, Poopo and Salar de Coipasa16–18. Lake Uru Uru is a man-made reservoir supplied mainly by the river Desaguadero and is located in the central part of the Bolivian Altiplano (Figure 1)13. Lake Titicaca and Lake Uru Uru are part of the same endorheic system. Both of the lakes are subjected to high 8 UV, low O2 conditions and slight to moderate salinity . The lake is very shallow ( depth: 0.25-1 m) and the surface varies between 120 and 150 km² 8,13. Due to the semi-arid climate and the shallow depth it is subjected to extensive evaporation. The city Oruro is located north of the lake where mining and smelting activities are concentrated13. There are 2 main rivers who are causing pollution. The Tagarete River discharges the untreated wastewater from Oruro to the lake and the Huanuni River is impacted by acid main drainage8. The lake is also ecologically and economically of importance. A lot of species thrives well in these shallow lake waters and fishery is a source of food and income for the local communities13. Because of the higher metal concentrations and pollution of this lake compared to lake Titicaca, a link can be made of what the future may bring for Lake Titicaca. If the water level of lake Titicaca keeps decreasing and the growing pollution at the lake does not stagnate (increase of metal concentrations). The ecosystem of Lake Titicaca could be impacted in the same way as in Lake Uru Uru.

1.2. Climate The climate is determined by the intertropical convergence zone (ITCZ) and by its high altitude. Due to the altitude, high daily variations in temperature occur varying from -1 to 16°C, controlled by high solar radiation. The amplitude of daily temperature variation is higher than the annual amplitude. Average annual fluctuation in temperature is approximately 7.7°C (at Tiwanaka near Lake Titicaca) 12,18. The northern part of the altiplano is semiarid and cold. The Titicaca area has a short wet season from December until March and a long dry season (Apr-Nov). Seventy percent of the rainfall occurs in the wet season when humid air from the Amazon region enters the Altiplano, resulting in small variations of the water level of the lake Titicaca. The average annual precipitation is 532 mm/year12,13,36. Furthermore, the precipitation is influenced by the El Niño-Southern Oscillation. During this event anomalously warm water appears along

the coast of Ecuador and Peru, causing a rising water surface temperature which influences the climate around the equatorial Pacific region. During El Niño the rainfall is lower at the Altiplano causing a decrease in the water level of lake Titicaca17,37.

2. Metals and metalloids 2.1. Introduction This project focuses on the contamination of metals and metalloids in the Titicaca Lake, especially: arsenic (As), lead (Pb), mercury (Hg) and cadmium (Cd). Studies have revealed abnormally high concentrations of metals and metalloids in ecosystems near to urban areas, metalliferous mines or major road works. Soils in such areas were polluted especially with Pb, Cd, Hg and As5. In the context of the chemical hazards and the safe use of metals and metalloids the term “heavy metals” is frequently used in research, books, publications and legislation. Although it is often used as a group name for metals and metalloids having a specific density of more than 5 g/cm³, knowledge of the specific density gives little information about the biological effect of metals and is meaningless for living organisms38,39. Furthermore the term is mostly used in a negative manner assuming that all heavy metals have highly toxic or ecotoxic properties, this must be nuanced26,38,39. The inconsistent use has led to confusion regarding the significance of the term. Because of the previously given reasons the terms metals and metalloids will be used instead of heavy metals. Metals may be defined as elements with high electrical conductivity, metallic luster, the capacity to lose electrons and to form cations (e.g. Cd, Pb, Hg) in its metallic form26,38. In solution metals can appear as metal ions and metal salts. Metalloids are elements having the physical appearance and properties of a metal but behaving chemically like a non-metal (e.g. As). Firstly the toxicity of metals and metalloids and their essentiality for human life will be discussed. Secondly the aspects influencing the bioavailability will be explained after which the elements of interest (As, Hg, Cd and Pb in this thesis) will be discussed more in detail.

2.1.1. Toxicity The toxicity depends on the concentration of the metals and metalloids. Only when a certain threshold concentration is exceeded, the elements will exert toxic effects on the organism39. Furthermore, metals or metalloids can have an essential function. A distinction can be made between essential and non-essential elements. There are several definitions regarding essential elements. In general an element is essential when its removal from a diet results in the change of a physiological function from optimal to suboptimal40. In a more restricted vision essential elements are required for the life cycle of the organism, are not replaceable by another element and must be required for a specific physiological function41,42. When to low concentrations are occurring, the organism will have deficiency problems and when a threshold concentration is exceeded toxicity will occur. Non-essential elements are not required for the life-cycle of the organism and will be toxic when a threshold concentration is exceeded (Figure 4)42,43. Although the essential metals and metalloids have vital biological functions, their speciation (e.g. chemical coordination and oxidation state) will determine their essentiality or toxicity26.

Figure 4: Influence of metal and metalloid concentration for an essential (a) and non-essential (b) element. O stands for the functioning of a certain process/metabolic pathway in an organism.41–43 When too low concentration appear, deficiency problems will occur. Too high concentrations will result in toxicity and when the plateau is reached the functioning is optimal.

2.1.2. Essential elements There are three classes of essential elements for human and animal life: the bulk structural elements, macro elements and trace elements (Table 1)40,41. The elements of interest are situated in the class of the trace elements. Trace elements are the elements having an average concentration of less than 100 mg/kg44. There are 20 essential trace elements in the human body and the most abundant ones are the metals: iron, zinc and copper40,41. Deficiency diseases were proven for iron (Fe), cupper (Cu), chromium, zinc (Zn), selenium (Se), iodine and fluor40. The trace elements can be divided in three classes based on their essentiality in the human body: essential for humans, essential for humans with a low risk of deficiency and essential in extremely low concentrations (Tabel 1)40. Arsenic, Pb and Cd are essential trace elements in extremely low concentrations, Hg is a non-essential element40,41. Each metal or metalloid has its own physicochemical characteristics which determine its biological and toxicological properties. Furthermore they can form a wide range of compounds with diverse properties (different speciation forms, species)38. In order to assess the toxicity or ecotoxicity of the elements of interest (As, Hg, Cd and Pb), dividing them into metal or metalloid and information on their essentiality will not be sufficient. Individual characterisation of the occurring species is necessary to predict its behaviour and toxicity (Section 2.3, 2.4, 2.5, 2.6)

Table 1. Classification of the essential elements40,41 Classification Element 1. Bulk structural elements: H, C, N, O, P, S 2. Macro elements: Na, K, Mg, Ca, Cl 3. Trace elements: Essential Fe, I, Cu, Zn, Se, Cr, Mo, F Low chance of deficiency Mn, Si, V, Ni, Sn, Co Essential in low concentrations B, Br, As, Pb, Cd, Li

2.2. Bioavailability The bioavailable fraction of an element is the total mass of that element that has the potential of being absorbed by an organism9. It is influenced by many complex mechanisms. It is for example influenced by the substance properties, matrix properties, the biology of the organism, climate, etc. which have an impact on the mass transfer and uptake of the elements into the organism.9 In aquatic environments, sediments are a concentrated pool of metals and metalloids, and play an important role in the uptake of these elements in organisms1,10. Ingestion of sediments and uptake from solution may both be important ways of metal bioaccumulation for benthic or deposit feeding species10. Furthermore these species are mostly at the bottom of the food chain so bioaccumulation and biomagnification trough the food web is a potential risk. Metal or metalloid bioaccumulation is the net result of uptake and elimination processes of the element via all possible exposure routes (from the abiotic and biotic environment). Biomagnification occurs when food is the major source of bioaccumulation. It is the transfer of a xenobiotic element from food to organism resulting in a higher concentration within the organism compared with the food source45,46. This leads to increasing concentration along the food chain. Regarding metals, organic Hg (e.g. methyl mercury) has a great potential to show biomagnification. Most of the other metals and metalloids are regulated and excreted45. An important factor influencing the bioavailability and bioaccumulation in aquatic environments, is the variation in geochemistry of the sediment. Because of different sediment geochemistry, bioaccumulation by plants and animals may vary from one environment to another, independent of concentration in sediment or water. Differences in metal bioavailability can give an enhanced or a reduced vulnerability of the biota to metal contamination.10

2.2.1. Sediment characteristics As previously explained the geochemistry of sediment has an important impact on the bioavailability of metals and metalloids in an aquatic environment. Numerous processes can occur which changes metal and metalloid concentration and speciation such as: settling, precipitation/dissolution, co-precipitation, occlusion sorption/desorption, complexation/decomplexation, cation-exchange, phytoaccumulation, microbial-activity, etc.8,11,47–49 During these processes, the distribution between metal or metalloid species is a critical factor, which is largely determined by the redox conditions and pH of the environment11,50. The redox conditions have an effect on the availability of active sites, counter ions and ligands for sorption, precipitation and complexation, respectively11. Figure 4 shows possible interactions between water-soluble metals and sediment.

Figure 4: Interaction between the soluble metals and sediment in an aquatic environment11,42,47

Cation exchange capacity The cation exchange capacity (CEC) is the total capacity of a soil or sediment to hold exchangeable cations51. Cations (e.g. positively charged metal species) are retained by negatively charged surfaces by electrostatic attraction11,50,51. The adsorption of a certain metal or metalloid is determined by the physico-chemical environment of the medium, the properties of the metal of interest and the properties of other cations and ligands occurring in the medium50. The physico-chemical environment of the medium can be characterised by the clay, organic matter and Fe/Mn/Al-hydroxides content of the sediment. Depending on the pH, these substances can contain negatively or positively charged surfaces.11,50,51 When the pH is higher than the iso- electric point of the substance, the surfaces will mostly contain negative charges. Increasing clay, organic matter, Fe/Mn/Al-hydroxides content or increasing pH, result in a CEC increase. A high CEC reduces the bioavailability and mobility of positively charged metals in solution11,50. Furthermore the adsorption on or accumulation into microbial or fungal biomass of metals and metalloids can alter the bio-availability8,11. The previously presented processes are very complex. As example, organic matter (OM) can also cause an enhanced availability and mobilisation of metals and metalloids. Decaying plant material, animal tissue and micro-organisms are a source of organic material in the aquatic environment. The high molecular weight organic material acts as a sink for metals and metalloids but also dissolved organic ligands (e.g. amino acids, fulvic acids,…) can be formed. These dissolved organic ligands can form metal complexes and causes an increase in metal mobility and availability in the water phase. This can also be applied to small suspended particles containing: Fe/Mn oxides, carbonates and clay minerals. These particles can also act as metal and metalloid carriers and will increase their mobility.11

Redox conditions and microbial activity Redox processes occurring in sediments, soil and surface waters are catalysed by micro-organisms and are determined by the available electron donors and acceptors. Redox reactions that generate a maximum amount of available free energy will first occur by combining the most efficient electron donors and acceptors. As electron donor, organic carbon is preferred but the use of reduced forms of sulphur, iron and - nitrogen is also possible. Oxygen (O2) is preferred as electron acceptor followed by nitrate (NO3 ), 2- manganese(IV) (Mn(IV)), ferric iron (Fe(III)), sulphate (SO4 ) and finally carbon dioxide (CO2) during methanogenesis11,49,50,52. This preference in electron acceptors creates zonation in the sediment layer and is characterised by the redox potential (Eh)11,48. The redox potential is the tendency of an inert electrode connected to a reference electrode, to either give up electrons to the solution or take electrons from it. The redox potential is determined against the standard electrode, which has a half cell potential of 0.0mV53. This tendency or potential difference is determined as the voltage. Oxidizing conditions are occurring above an Eh of 300mV11,52. During oxidising conditions, Fe(III) oxides and Mn(IV) oxides will precipitate, which will result in adsorption and co-precipitation of metals48,50. At moderately reducing conditions, between an Eh - of 300mV and -50mV, facultative reducing microbes will use NO3 , Mn(IV) and Fe(III) as electron acceptor. - - When NO3 is used, nitrogen gas and ammonium will be formed. After the reduction of NO3 , Fe(III) and Mn(IV) will be reduced to Fe(II) and Mn(II) whose oxides are soluble. Together with reduced iron and manganese, the adsorbed and co-precipitated metals will dissolve11,48. This solubilisation reaction is also influenced by the pH. With increasing proton (H+) concentration (decreasing pH), the solubilisation of Mn(IV) and Fe(III) will be increased according to Le Chatelier’s principle. The reduction reaction is given below (reaction 1 and 2)11,50. The solubility of metals and metalloids can be represented by pourbaix diagrams as shown in Figure 5. Besides the pH and Eh, the persistency of the Fe- and Mn-oxides is also influenced by the crystallinity. Newly formed amorphous crystalline minerals (e.g. Fe(OH)3: ferrihydrite) are easily reducible, but ‘aged’ forms have a more crystalline character (e.g. Fe2O3: hematite) and are more resistant11. − + 2+ 1) MnO2 + 2e + 4H → Mn + 2H2O − + 2+ 2) Fe2O3 + 2e + 6H → 2Fe + 3H2O

2- SO4 and CO2 will be used by the obligate reducing microbes producing sulphides (H2S) and methane (CH4) at Eh levels below -50mV. CH4 is produced by the Methanogens out of acetate or CO2 and H2. Sulphides have 11,48,52 an important role in the bioavailability of metals. H2S is produced by sulphate reducing bacteria from 2- 52 SO4 (as electron acceptor) and an electron donor (e.g. H2 or volatile fatty acids) . With increasing sulphides, metals precipitate as metal sulphides in both sediment and surface water. Mainly Fe forms sulphide precipitates (FeS and FeS2) because Fe is often available in higher concentration and has fast reaction kinetics. But during the precipitation, metals and metalloids can absorb, co-precipitate or can also form metal sulphides (reaction 3)11,48. In a simplified model the sediment can be divided in an oxidised and a reduced layer. In this simplified system, metal and metalloid concentrations in pore water is determined by sorption on Al/Fe/Mn oxides in the oxidised layer, and controlled by sulphide precipitated in the reduced layer48. 3) M2+ + FeS(s) ↔ MS(s) + Fe2+

Beside sulphides, carbonates are important during adsorption and precipitation reactions11,47. Carbonates are important in decreasing the availability of metals and metalloids but are less stable than sulphides. Especially the precipitation of Pb and Ni is very effective.47 A second very important feature is the buffering capacity of the carbon dioxide-bicarbonate system. According to the Hederson-Hasselbach Equation the pH - - of a CO2/HCO3 buffered solution is given by: pH= 7.74+ log ([HCO3 ]/atmsphere CO2 pressure). This system buffers a solution from pH 6.74-7.7454. The stability in pH, can be important for the availability of metals and metalloids in sediment and solution (figure 5). A threat to this stabilization mechanism is decalcification. Complete decalcification can for example result in acidification, causing metals (e.g. Pb, Hg, Cd) going in solution. Decalcification is a slow process but can ultimately have a very strong environmental impact.11

Figure 5: Pourbaix diagram: schematic presentation of major trends for increasing element mobility as a function of redox and pH changes55.

2.2.2. Salinity Increasing salinity can result in cation mobilization and the flocculation of particles. The first process, cation mobilization, is caused by the complexation of cations by negatively charged ions (e.g. Cl-) and competition between different cations for adsorption sites, both leading to desorption of weakly bound metals and mobilization of these cations from sediment.11 Especially Cd is sensitive to increasing salt concentrations due to its weak sorption affinity to the solid phase and due to the formation of stable and soluble cadmium chloride complexes11,56. In the second process, flocculation, the charge of negatively (resp. positively) charged particles is neutralized by cations (resp. anions), the particles can approach one another more closely and larger aggregates are formed which results in flocculation11. During flocculation, metal ions bounded on the particles will be removed from solution together with the aggregates and co-precipitation can happen as well.

2.3. Arsenic 2.3.1. Sources Arsenic is a metalloid which occurs naturally in the earth’s crust8,26,57. More than 200 mineral species have been identified, especially species including sulfide26,57,58. The geogenic background of As in surface and ground-water is naturally high in South America. About 4.5 million people in South America are chronically exposed to high levels of As. This is due to the release of the weathering products of young volcanic rocks in arid oxidizing conditions8. Besides the natural geologic processes As contamination can be caused by human activities. Anthropogenic activities causes the release of As into the air, water and soil58. Anthropogenic sources of As include: industrial emissions, mining, processing of ores, combustion of fossil fuels, use of pesticides, herbicides, insecticides, fertilizers, drugs, wood preservatives and feed additives (poultry) 26,27,57–59. At the Altiplano, mining operations are frequently occurring (Section 1.1.1). Arsenic is obtained as a by-product of e.g. copper, lead, cobalt and gold57,58. Furthermore mining operations produce large quantities of unusable mine tailings58. These tailings pose an important threat to contaminate ground and surface water with metals and metalloids. Sulphur is often present in these tailings, and exposed to the atmosphere sulphur will oxidize, releasing As from sulphidic minerals and creating acidic conditions. These acidic conditions can further dissolve many elements, including As58.

2.3.2. Physical and chemical properties of Arsenic Arsenic has only one stable isotope, 75As, has an atomic mass of 74.9215966u and atomic number 3360. Arsenic is situated in group 15 of the Periodic table along with nitrogen and phosphorus. Arsenic exists in the oxidation states –III, 0, +III, +V, the same as phosphor. Because of these similar properties, the chemistry of As will be in many aspects the same of these two essential elements57.

2.3.3. Speciation and availability Arsenic is mostly found in its inorganic form in natural waters26,61,62. The most abundant inorganic forms are 3- 3- 26,59 arsenite (AsO3 ) and arsenate (AsO4 ) . Arsenite is a As(III) species and arsenate contains As(V). Most stable arsenic species found under normal natural conditions contain As(V)57. The most important species in the environment and food are: As(III), As(V), methylarsonate (MA), dimethylarsinate (DMA), 61 3- arsenobetaine (AB), arsenosugars and arsenolipids . Arsenic occurs as an oxyanionic species (e.g. AsO3 ), and tends to become more soluble as the pH increases (Figure 5). As a result oxyanionic species are some of the most common trace contaminants in natural waters59. Arsenic speciation is most importantly controlled by the redox potential (Eh) and pH, with As staying in solution over a wide range of redox conditions59. Arsenic release can be triggered via pH fluctuations57. pH fluctuations in Lake Titicaca can for example occur due to seasonal changes. Figure 6 shows the pourbaix diagram for aqueous As species. Besides pH and Eh, elevated chloride concentrations, aridity and mixing with geothermal water can cause As going into solution50. The As speciation in fresh water fish is variable according to the fish species, feeding, habitat and the inorganic As contamination of water62,63. In some cases arsenobetaine (AB) is the main As species, but often other compounds are abundant, including inorganic species63. AB or 2- trimethylarsoniumylacetate is considered as a non-toxic compound, being rapidly excreted via urine when ingested64. It is thought to represent the terminal As metabolite in marine fish65. In rainbow trout (Oncorhynchus mykiss), it is found that up to 80% of the extractable As compounds can be composed out

of AB66. Following As species can occur in fresh water fish: AB, MA, DMA, trimethylarsine oxide, arsenosugars, As(III), As(V), etc.62,63. Table 2 gives an overview of the most important As species in the environment, food and fish.

Figure 6: Pourbaix diagram for aqueous As species at 25°C and 1 bar total pressure59.

Table 2: The most important As species in the environment and fish and their oxidation state (OS)57,61–63 Name Abbreviation OS Chemical structure 3- Arsenite / +III AsO3 3- Arsenate / +V AsO4 2- Methylarsonate MA +V CH3AsO3 - Dimethylarsinate DMA +III (CH3)2AsO2

Trimethylarsine oxide TMAO +V (CH3)3AsO + - Arsenobetaine AB(V) +V (CH3)3As CH2CO2 Arsenosugars AS(V) +V

e.g. Arsenolipids AL(V) +V

e.g.

2.3.4. Human health As previously explained, the speciation has an impact on the physicochemical properties and bioavailability of As. The absorption by the human body will differ for each As species and between organisms. The inorganic forms of As are found to be more dangerous to human health, because of their 26,61,62 67 3- 3- carcinogenity . As(III) is sixty times more toxic than As(V) . AsO3 has a LC50 of 5.5M, AsO4 a LC50 of

61 571M in contrast with AB (organic species) which has a LC50 above 56000M . The LC50 is the lethal concentration of a compound in air or water that kills 50% of the test population. The inorganic species can cause malfunctioning of cell respiration, cell enzymes and mitosis by interactions with sulphydryl groups of enzymes or by replacing phosphate in crucial pathways26. Arsenic is well absorbed in the small intestine67. The inorganic forms are extensively biotransformed and are excreted in urine. Biomethylation is a detoxification process, methylated As species in urine are an indication of the chronic exposure of As. DMA is the primary urinary metabolite (60-70%), followed by inorganic As (10-30%) and methylarsonate (10- 20%).57 As previously mentioned, about 4.5 million people are chronically exposed to As in the Altiplano8. Chronic exposure to As is called by the term arsenicosis68. Pigmentation and keratosis are a typical indication of arsenicosis (Figure 7). Long term health effects are: skin lesions, skin cancer, internal cancers (bladder, kidney, lung), liver disease, neurological effects, gastrointestinal disease, pulmonary disease, diabetis mellitus, hypertensia and cardiovascular disease 57,67–70. In order to protect human health the European union has set threshold values for contaminants in drinking water and foodstuffs57,71. The Council Directive 98/83/EC states that Member States shall set limits of 10 g/L for As in water intended for human consumption71. For As, the European Commission did not set maximum levels for fish intended for human consumption72. Up till now, only safety concentrations are set for As in rice based foodstuffs, with 0.1 mg/kg being the lowest threshold value (in rice destined for the production of food for infants and young children). In 2016-2018 a monitoring campaign was organized, to evaluate the need for setting additional maximum levels for As in foodstuffs (e.g. fish)70. Several studies used the international standard of 0.1 mg/kg for the total amount As in fish as the threshold level73–75. For As, the no observed adverse effect level (NOAEL) is 0.8 mg/kg body weight (BW)/day. This NOAEL was based on a human toxicity study57. When divided by a safety factor of 3, the reference dose (RfD) of 0.3 g/kg BW/day can be used to estimate the total amount of drinking water and fish that can be consumed per day without a risk57. Quality guidelines for As in sediments were derived from the study of MacDonald et al. (2000). In that study, threshold effects concentrations (TEC) and a probable effect concentration (PEC) were developed. Below the TEC no adverse effects are to be expected, above the PEC enhanced adverse effects on the aquatic environment can be expected especially for the sediment dwelling organisms. For As a TEC of 9.79 mg/kg was constructed and the PEC of 33 mg/kg can be used as a maximum concentration for the sediment samples from Bolivia76.

a) b) Figure 7: a) Arsenical pigmentation affecting the front of the chest, b) arsenical keratosis68.

2.4. Mercury 2.4.1. Sources Mercury is a naturally occurring metal. In liquid form it has a silver-white colour, whereas a gas it is odourless and colorless26,77. Mercury is released into the environment via natural and anthropogenic sources. Mercury is naturally mobilized from the earth via volcanic and geologic activity. When released, complex transformation and cycles between atmosphere, land and aquatic systems are occurring: the biogeochemical cycle of Hg77,78. This cycle is disturbed due to anthropogenic inputs such as agriculture, municipal waste water discharges, mining, waste incineration, coal burning and discharges of industrial waste water26,67,78. At the altiplano mining is an important input of Hg, especially the artisanal gold mining operations. These operation use amalgamation to extract gold, which is illegal in most countries. During amalgamation, mined sediment or crushed rock is mixed with Hg and a semi-solid Hg-Au amalgam is formed. Next, the amalgam is roasted and Hg is vaporised, leaving the purified Au behind. This results in the release of Hg in the invironment29.

2.4.2. Physical and chemical properties of Mercury Mercury has seven stable isotopes, 202Hg is the most abundant one and has an atomic mass of 201.97063u and its atomic number is 80.60 Table 3 shows the different isotopes and their abundancy. Mercury is situated in group 12 of the Periodic table, along with Zn and Cd. It has three oxidation states: 0, +I and +II.

Table 3: Isotopes of mercury and their abundancies60. Isotope Mole fraction 196Hg 0.0015 198Hg 0.0997 199Hg 0.1687 200Hg 0.2310 201Hg 0.1318 202Hg 0.2986 204Hg 0.0687

2.4.3. Speciation and availability 0 2+ + Mercury has three chemical forms: elemental or metallic Hg (Hg ) , inorganic Hg (Hg ,Hg2 ) and organic Hg + + 77,79 (e.g. methyl mercury (CH3Hg ) and ethyl mercury (C2H5Hg )) . Atmospheric deposition is an important source of enhanced Hg concentrations80. It is emitted in its metallic form via geological sources, land and water surfaces and via anthropogenic activities. Via anthropogenic sources, the emission of Hg2+ and Hg associated with particulate matter (HgP) is also possible78. The exact speciation of Hg2+ in the air is not 0 2+ known but it could be present as HgCl2. In the atmosphere Hg is most abundant, Hg and HgP are less volatile and tend to deposit through wet and dry deposition78. Through the deposition of Hg2+ and HgP, Hg enters soil, ground and surface waters. Main factors contributing to human exposure are through inhalation of elemental mercury vapours during industrial processes and through the consumption of methyl mercury, mainly in fish and shellfish78,79. In solution the Hg species are taken up by microorganisms and are transformed into methyl mercury within the organism26,67. Different groups of bacteria catalyse Hg

methylation such as: sulphate-reducing bacteria, iron-reducing bacteria and methanogens7,78. The methyl mercury production is affected by presence of sulphur, organic matter, sediment structure, pH, etc. (Section 2.2) by changing the bioavailability of inorganic Hg or by stimulating microbial activity78. Methyl mercury is strongly bound to muscle tissue, and it accumulates with increasing duration of exposure13,81. The concentrations increase with fish age and size because of the slow rate of elimination of methyl mercury compared to its uptake81. This makes the aquatic life vulnerable for the bioaccumulation and biomagnification of methyl mercury7,45,67,77,78. The highest trophic levels in the food chain contain the greatest concentrations of methyl mercury. Due to the consumption of fish at a higher trophic level, humans and animals are exposed to elevated Hg concentrations through the consumption of fish. Also the use of fish meal as animal feed can lead to elevated Hg concentrations in meat and other animal based products67. Table 4 presents the Hg species discussed in this section.

Table 4: Elemental mercury and mercury ions/species with their oxidation state (OS). Chemical form Name OS Chemical structure Elemental mercury Elemental mercury 0 Hg0 2+ Inorganic mercury ions Mercurous ion +I Hg2 Mercuric ion +II Hg2+ + Organic mercury Methyl mercury +II CH3Hg + Ethyl mercury +II CH3CH2Hg

2.4.4. Human health Mercury is considered the most toxic trace metal in the environment26. It has a wide spectrum of adverse health effects, but the impact on the nervous system is the most important one26,64. The nervous system is very sensitive to all types of Hg. Increased exposure can alter brain function and lead to memory problems, tremors, shyness, changes in vision, etc.26 Methyl mercury is of major concern because its bioaccumulation and possible biomagnification in aquatic lifeforms and because the organic species can cross the placental barrier between the mother and the unborn baby64. Organic forms of Hg can pose an important threat for unborn babies. The uptake can result in neurological disturbances from impaired learning to obvious brain damage. Methyl mercury is a neurotoxic compound which causes microtubule destruction, mitochondrial damage, lipid peroxidation and accumulation of neurotoxic molecules26. The developing brain should be considered the most sensitive target organ for methyl mercury toxicity, but any organ can be affected. It leads for example to the malfunctioning of nerves, kidneys and muscle26,64. The toxicity of methyl mercury and mercuric mercury is caused by their interaction with soft nucleophilic groups, mainly thiols and selenols26,77. Mercury vapours can cause temporary respiratory problems, asthma and bronchitis26. In order to protect the human health the European Commission has also set threshold mercury concentrations for contaminants in drinking water and foodstuffs71,72,77. The threshold values for total mercury concentrations in drinking water and fish are 1 g/L and 0.5 mg/kg, respectively71,72. For Hg, the (NOAEL) is 0.23 mg/kg BW. This NOAEL was based on a toxicity study on rats77. To take into account the interspecies (safety factor 10) and intraspecies (safety factor 10) variation this value should be divided by a safety factor of 100 (safety factor 10* safety factor 10). A value of 2.3 g/kg BW/day can be used to make an estimation of the total amount of drinking water and fish that can be consumed per day without a risk. The study of M. Raissy and

M. Ansari used a RfD of 0.1 g/kg for the consumption of fish73. From the study of MacDonald et al. (2000), a TEC of 0.18 mg/kg can be derived and the PEC of 1.06 mg/kg can be used as a maximum Hg concentration for the sediment samples from Bolivia76.

2.5. Cadmium 2.5.1. Sources Cadmium is a metal naturally occurring in the earth’s crust and in the environment. It can be released by natural processes such as volcanic emissions, weathering of rocks and via anthropogenic inputs. Cadmium occurs naturally in Zn ores and to a lesser extent in Pb and Cu ores26,82–84. Due to this co-occurrence, Cd is a by-product of the metallurgy of these elements26,82,83. It is used in various applications such as alkaline batteries, rechargeable batteries, coatings, pigments, plasticizers26,82,83. It is released into the environment, by anthropogenic means, via metallurgical processes, the burning of fossil fuels, incineration of waste materials and the agricultural use of phosphate and sewage sludge82–84.

2.5.2. Physical and chemical properties of Cadmium Cadmium has eight stable isotopes, 114Cd is the most abundant one. It has an atomic mass of 113.90336u and its atomic number is 48.60 Table 5 shows the different isotopes and their abundancy. Cadmium is situated in group 12 of the periodic table and has two oxidation states: 0 and +II, the same as zinc (Zn). Because of these similarities, the chemistry of Cd will be in many aspects the same as Zn, and Cd will be able to replace Zn in certain chemical reactions26. Cadmium and Zn occur together naturally in the environment in a ratio (Cd:Zn) of 1:100-1:1000.

Table 5: Isotopes of cadmium and their abundancies60 Isotope Abundancy 106Cd 0.0125 108Cd 0.0089 110Cd 0.1249 111Cd 0.1280 112Cd 0.2413 113Cd 0.1222 114Cd 0.2873 116Cd 0.0749

2.5.3. Speciation and availability The species of Cd in the environment are dominated by its inorganic compounds in the +II oxidation state. Elemental Cd (Cd0) is relative volatile, but in gas phase it is rapidly oxidised56,82. In fresh waters, Cd is most abundant as Cd2+ and can form a number of inorganic salts and complexes: Cd halides (bond between metal and halogen) and nitrates are very soluble, while others such as Cd oxides, hydroxides and carbonates are less soluble82. The formed species depend on generally the pH, Eh and salinity. At pH 6, Cd may begin to 56 precipitate with carbonates (CdCO3), decreasing the Cd availability . As explained in Section 2.2.2, with increasing salinity the competition for binding sites will occur and complexation with Cl- ions will increase, + - 11,56,61,82 which can cause adsorbed Cd going in to solution as CdCl , CdCl2, CdCl3 . This increase in soluble Cd due to elevated salt concentrations, does not necessarily result in an increase of Cd bioaccumulation in

organisms. This is due to the fact that Cd is most bio-available to organisms in its free ionic form (Cd2+)82. When sulphur is present Cd will precipitated as sulphides (CdS), but high sulphide concentrations, in anoxic 11,56 conditions, can cause the formation of a soluble bisulfide complex with Cd (Cd(HSO3)2) . Figure 7 shows ___ + the pourbaix diagram for Cd in --- seawater and in freshwater. In seawater CdCl and CdCl2 predominates and in freshwater Cd2+.56 Table 6 shows the most important species in aquatic environments. Aquatic lifeforms can accumulate Cd to concentrations hundreds times higher than the water concentrations82. Cadmium in soil is readily absorbed by plants, compared to Hg and Pb84. Plants can tolerate higher concentrations of Cd compared to animals and it is accumulated in the leaves of the plants82,84. Therefore leafy vegetables grown on Cd contaminated land pose a substantial treat82.

Figure 7: Pourbaix diagram for cadmium in: --- seawater and in ___ freshwater, with Cd=10-8.35M, total S=10-4 M (freshwater) and 10-1.4M (seawater), total Cl-=10-3.54M (freshwater) and 10-0.26M (seawater)56.

2.5.4. Human health Cadmium is a known carcinogen82,85. It is poorly absorbed into the body but once absorbed it is slowly excreted. It has an estimated biological half-life of 10-30 years64,82. Once absorbed through the lungs or the gut it is transported via the blood to other parts of the body, but it is mainly stored in liver and kidney. The placenta is effective against penetration of Cd into the unborn baby.83 However, premature birth and reduced weights are effects caused by high Cd exposure during pregnancy26. As mentioned, Cd is similar to Zn and disturbs the zinc metabolism. Cadmium enters the human cells via metal transporters and once inside it binds with high affinity to metallothioneins, replacing zinc26,82,83. Cadmium has toxic effects on the kidney and is accumulated in higher concentrations in the proximal tubular cells of the kidney26,64. Furthermore it has an impact on the lungs. Tobacco plants can accumulate Cd in their leaves, this is why smokers are more susceptible to Cd intoxication26,83. It has been shown through animal experiments that 50% of Cd gets adsorbed via the lung and less in the gastrointestinal tract26. Another important aspect is the impact on the bone structure (e.g. osteoporosis, bone mineralisation, etc.)26,82,83, reflected in the Itai- Itai disease. This disease was caused by Cd exposure and a shortage of vitamin D which caused skeletal

deformations. The symptoms are dominated by pain in the back and legs83. In order to protect the human health the European union has set threshold Cd concentrations for contaminants in drinking water and foodstuff71,72,82. The threshold values for total Cd concentrations in drinking water and fish are 5 g/L and 0.05 mg/kg, respectively71,72. For Cd, the NOAEL is 3 mg/kg BW86. This NOAEL was based on a toxicity study on rats86. To take into account the interspecies and intraspecies variation this value should be divided by a safety factor of 100. A value of 30 g/kg BW/day (RfD) can be used to make an estimation of the total amount of drinking water and fish that can be consumed per day without a risk. The study of MacDonald et al. (2000) reported a TEC of 0.99 mg/kg and a PEC of 4.98 mg/kg for sediments in freshwater76.

2.6. Lead 2.6.1. Sources Lead is a naturally occurring metal and its most abundant mineral is galena (PbS)87. Its main inputs in the environment is via anthropogenic sources such as mining, smelting, soldering, battery manufacturing, ammunition, Pb water pipes, electricity production, vehicles exhaust, fertilizers, pesticides, etc.26,88. Lead is normally obtained as sulphide ores, mostly in combination with other elements such as Zn, Cu and Ag89. The knowledge about the adverse health effects of Pb has led in the 1970s to the implementation of regulations regarding Pb release. For example the use of leaded paints, leaded petrol and lead solder in food cans and pipes were regulated or banned88. Since the phase-out of leaded petrol in many countries, a significant reduction in background Pb levels occurred88.

2.6.2. Physical and chemical properties of Lead Lead is a soft, dense (11.3g/cm³), malleable metal and a poor conductor of electricity88–90. It is very resistant against corrosion but begins to discolour on contact with air to a dull grey metal26,88,90. It has four stable isotopes from which 208Pb is the most stable one. Lead has an atomic mass of 207.976636u and its atomic number is 82.60 Table 6 shows the different isotopes and their abundancies occurring near Lake Titicaca (Matilde, Bolivia: 15°45’S: 68°58’W)91. The Pb isotope ratio may vary, unlike the other discussed elements91,92. Natural occurring Pb originates from two sources: Pb naturally occurring since the formation of the earth and Pb formed by decay of radioactive materials. Depending on the source of Pb the isotope ratios may vary91,93. Lead is situated in group 14 of the periodic table and has three oxidation states: 0, +II and +IV. To illustrate the variance in Pb abundancies the 206Pb/204Pb ratios can be compared. For example, the 206Pb/204Pb ratios at Missouri (US), Colorado (US), Matilde (Bolivia) and Taebaegesan (Korea) are 20.5- 21.5, 17.7, 18.3 and 19.0-20.0, respectively.

Table 6: Isotopes of lead and the abundancies occurring near Lake Titicaca (Matilde, Bolivia: 15°45’S: 68°58’W)91 Isotope Abundancy 204Pb 0.0135 206Pb 0.2499 207Pb 0.2127 208Pb 0.5238

2.6.3. Speciation and availability The oxidation state of Pb which is most abundant in the environment is +II. Inorganic compounds predominate over organic forms and the inorganic species usually contain Pb(II) (e.g. lead phosphate, lead carbonate)88. In the aquatic environment the free bivalent ionic form is most abundant and highly mobile, and thus the most bioavailable form88. Pb2+ can act as an Ca2+ analogue. This can be important in the uptake of Pb via the gills of fish, because the Pb uptake by the freshwater fish presumably takes place through the Ca2+ uptake mechanisms at the gills94. Pb2+ occurs also in complexation with dissolved humic materials, attached to colloidal particles or attached to solid particles (e.g. clay, organic matter, etc). Due to the strong bond of Pb in these forms, its bioavailability is limited11,88. The speciation and bioavailability of Pb compounds is dependent on many factors such as the pH, salinity, adsorption (organic matter, clay, Fe/Mn/Al-hydroxides) and biotransformation processes. Lead is effectively removed from the water column by adsorption to organic matter, clay and Fe/Mn/Al-hydroxides87. Sorption of Pb (decrease in bioavailability) increases with increasing pH (Section 2.2 and Figure 5). Furthermore, increasing pH results 87,89 2+ in the precipitation of PbCO3 and Pb(OH)2 . In acidic freshwater environment Pb is most abundant as Pb 89 and in alkaline environment as PbCO3 . In seawater (high salinity), Pb is most abundant as PbCl2 and PbCO3. In reducing conditions where sulphur is present, lead sulphide is formed and will precipitate87. An additional factor of Pb speciation in the aquatic environment is the enrichment of anthropogenic Pb species.

Tetraethyl lead (Pb(C2H5)4) and tetramethyl lead (Pb(CH3)4) are the most common alkyl-Pb compounds and can be present in freshwater due to anthropogenic activities. (Pb(CH3)4 can also be formed by biological methylation. Both species are highly volatile and soluble lipids. Most alkylated Pb compounds are composed out of Pb(IV). In aquatic environment, the tetra alkyl Pb species undergo degradation or volatilization. The species degrade from tetra- to tri- to dialkylated species and eventually inorganic species are formed.88,89 Groundwater contains mostly low concentrations of Pb due to the strong binding capacity (Section 2.2.1) of the soil under normal environmental conditions87–89. Table 7 gives an overview of the discussed species in the aquatic environment.

Table 7: Lead species occurring in aquatic environment Chemical form Name OS Chemical structure Elemental lead Elemental lead 0 Pb0 Inorganic lead Ionic form +II Pb2+

Lead carbonate +II PbCO3

Lead hydroxide +II Pb(OH)2 Lead sulfide +II PbS

Organic lead species Tetra methyl lead +IV Pb(CH3)4

Tetra ethyl lead +IV Pb(C2H5)4

2.6.4. Human health Lead is considered as a carcinogen26,88. It is toxic to plants and animals and it bioaccumulates in most of the organism89. The mechanism of toxicity is due to the ability of Pb ions to replace other bivalent cations (e.g. Ca2+, Mg2+, Fe2+) and monovalent cations (e.g. Na+), this disturbs the biological metabolism of the cell. This mechanism causes changes in various important biological processes such as cell adhesion, ionic

transportation, release of neurotransmitters, etc.26 Humans can be exposed to Pb via contaminated food, water, soil, dust and air. The organic and inorganic forms differ in toxicity, the organic forms (e.g. Pb(CH3)4 and Pb(C2H5)4) are most toxic. When Pb is absorbed, it enters various tissues via the blood stream and accumulates primarily in the skeleton26,88. It is deposited as insoluble phosphate in the skeletal bones26. From the skeleton in can be released again, particularly during periods of bone demineralization such as pregnancy, lactation, osteoporosis, etc. This poses an important risk because Pb can be transferred from mother to foetus/child in utero or via breast milk88,95,96. Lead is one of the most dangerous metals for children due to the critical negative effect on the developing nervous system of young children95,96. Beside the important impact on central nervous system, Pb has an negative effect on the blood, cardiovascular, renal, endocrine, gastrointestinal, immune and reproductive systems26,87,88,96. The threshold values for total Pb concentrations in drinking water and fish are 10 g/L and 0.3 mg/kg, respectively71,72. For Pb, the benchmark dose lower confidence level (BMDL) of 12 g/L can be used to estimate the safe daily intake of Pb. This value was based on a human toxicity study88. The same as for the NOAEL a safety factor of 10 is necessary to take into account the intraspecies variation. A value of 1.2 g/kg BW/day (RfD) can be used to make an estimation of the total amount of drinking water and fish that can be consumed per day without a risk. The study of MacDonald et al. (2000) reported a TEC of 35.8 mg/kg and a PEC of 128 mg/kg for sediments in freshwater ecosystems76.

3. Diffusive gradients in thin films 3.1. General principle The diffusive gradients in thin film technique (DGT) is made to in situ sample labile trace metals in seawater, freshwater, estuarine waters and sediment pore waters in a passive way97–99. This device is composed out of three layers: a filter, a diffusion layer and a binding agent (Figure 8)100,101. Conventionally, a polyacrylamide hydrogel is used as a diffusion layer and the Chelex-100 resin as a binding agent98,100. Ions can diffuse through the filter and diffusive gel, and bind to the binding layer. It measures the species in solution which are available to biota and excludes those that are incorporated inside mineral particles, large particles and colloids99,100. It is possible to use chelating resin to bind labile trace metals such as Hg, Cd, Pb, Co, Al and Cr. Using alternative binding sites it is even possible to bind selective certain metal species such as As(III), As(V), Hg2+ and methyl mercury.99 The technique is based on Fick’s first law of diffusion100. If the deployment time (t), the mass (M) of the accumulated compound on the resin and the physical area of the exposed filter membrane is known, the flux of the compound can be calculated via Equation 4. Figure 8 shows the steady-state concentration gradient (___) of the compound when the device is deployed in solution. Fick’s first law of diffusion states that the flux, J, in this system is equal to the diffusion coefficient (D) multiplied by the concentration gradient, dC/dx. C is the concentration of the compound and x 101 represents the distance. It can be assumed that dC/dx can be approached by Csol/∆g . With Csol the concentration of the compound in solution and ∆g the total thickness of the materials in the diffusion layer, this results in Equation 5. The standard DGT Equation (Equation 6) can be formed by rearranging Equation 4 and 5. This equation can be used in situations where the DGT is deployed in water that is flowing or is subjected to convection currents99,101. A more detailed explanation regarding the use and principles of the DGT is given in the book “Diffusive Gradients in Thin-films for Environmental Measurements”, edited by William Davison101.

푀 4) J= A∗t 퐷∗퐶푠표푙 5) J= ∆푔 푀∗∆푔 6) CDGT= D∗A∗t

Equation 6: M (ng) is the mass of the metal accumulated on the resin . CDGT (ng/mL) is the time average concentration of the analyte in the deployment medium. dG (cm) is the total thickness of the materials in the diffusion layer (diffusive gel + filter membrane (= 0.094 cm, applied samplers in this paper)). D (cm²/s) is the diffusive coefficient of the analyte in the diffusion layer (diffusive gel, filter membrane and diffusion boundary layer). A (3.14 cm², applied samplers in this paper) is the physical area of the exposed filter membrane. t (s) is the deployment time.99

Figure 8: Composition of the diffusive gradients in thin film sampler and the steady-state concentration gradient (___) 101

3.2. Applied samplers 3.2.1. Loaded DGT sampler for metals in solution (LSNX-NP) This device can measure up to 40 metals including As, Cd, Pb and Se. It contains a 0.8 polyacrylamide hydrogel diffusive gel, polyethersulphone filter membrane and a mixed binding layer of Chelex and titanium oxide.99 Chelex can be used for the measurement of trace metals and the titanium oxide-based adsorbent to measure oxyanions of, for example, As and Se102.

3.2.2. Loaded DGT sampler for mercury and As(III) in solution (LSNB-AP) This device can measure labile Hg present as Hg2+ and methyl mercury and it can measure As(III). It contains a 0.8 mm agarose diffusive gel, a polyethersulphone filter membrane layer and a 3-mercaptofunctionalised silica gel binding layer.99 It has been proven that the sulfhydryl groups present on the functionalized silica gel can efficiently bind Hg103–105. Gao et al. developed a method to determine the methyl mercury concentration using the DGT samplers with a 3-mercapto functionalized silica gel binding layer104. In the

paper of W. Bennet et al. it is shown that the 3-mercapto functionalized silica gel binding layer can also selectively bind As(III). As(V) can be measured by taking the difference of the measured As (via the LSNB- AP sampler) and the measured As(III) (via the LSNB-AP).106

3.3. Advantages This technique has several advantages: it is easy to use; it can selectively measure certain metal species 2+ + (Hg , CH3Hg ) and bio-availability (labile metals); it can be deployed in situ; many trace elements can be measured simultaneously; it is independent of the matrix and flow and it yields time-average concentrations over the length of the deployment period. This time-average concentrations makes it possible to deal with peak concentrations.97,99

4. Fish in Lake Titicaca 4.1. Fishery Fishing occurs nationwide at the three major basins: the Amazon basin, Del Plata basin and the Altiplano basin6. Bolivia has no marine resources, so fishery and aquaculture in rivers and open waterbodies is of great importance as a source of food and income. Human activity has an important impact on the fishery resources. For example, (I) negative impacts by pollution of the lake, overharvesting, the introduction of exotic species and (II) positive impacts are possible by conservation activities, reducing pollution and sustainable use of the resources6,13,19–21. At the Titicaca Lake the estimated fish production is 10 000 ton fish/year resulting in negative trends in fishing population. There is a clear need for legislation to control fisheries and support their sustainable management and a need for the sensitization of the local fishermen. On Lake Titicaca fishery continues without control. The Aymmaras, Uros and Quechuas are the local ethnic groups who forms the fisher communities at the altiplano6.

4.2. Species Two genera of native fish are abundant in Lake Titicaca: Orestias (local name: Carachi) and Trichomycterus (local name: Mauri and Suche). The Trichomycterus is a bentic genus, the genera Orestias includes benthic, benthopelagic and epipelagic species107. The well thriving introduced fish species are Odontesthes bonariensis (local name: pejerrey, English: silverside) and Oncorhynchus mykiss ( local name: trutcha, English: rainbow trout). In this paper the scientific names are used. Oncorhynchus mykiss (O. mykiss) and Odontesthes bonarienses (O. bonarienses) are pelagic species 1,3,29,108. In 1942, O. mykiss was introduced in the lake and supported a commercial fishery. Later on the population of the O. mykiss decreased due to overfishing and due to the introduction of the O. bonarienses. O. bonarienses was introduced from Argentina into the Altiplano and goes in competition with the local fish fauna (e.g. decrease of O. ispi population)6,108. The O. mykiss is still available in plenty of fishing markets, most of them come from farms and are grown in cages in the lake. In contrast with O. mykiss, the wild O. bonarienses has thrived in Lake Titicaca and has become an important source of food. O. bonarienses has a varied diet and can spawn throughout the lake, unlike the O. mykiss who needs the tributary rivers to reproduce.1,6,29,108 Amphipods represent an important fraction in the diet of all fish species (including O. bonariensis and O. mykiss), except for larger O. bonariensis and O. mykiss who switch their diet to small fishes4,109. The amphipod genus Hyalella has a high diversity in the lake. Up till now, eleven endemic species and one non-endemic species

are found in the lake110. The Hyalella are an important food source for many other species including the critically endangered Titicaca water frog Telmatobius culeus111. An investigation of the metal and metalloid concentration in Hyalella is important to verify the metal intake at all higher trophic levels. Currently, the O. bonarienses population decreased in the lake and the O. mykiss population increased (internal communication). All other species are omnivorous feeding on plant material, plankton and macroinvertebrates. Highest metal concentration are occurring in species living close to the sediment or in species feeding on high trophic levels. Therefore bentic (e.g. Trychomycterus spp.) and fish predators (e.g. O. bonarienses) could possibly have the highest metal concentrations1. Anthropogenic activities such as the introduction of new species, overfishing, sewage discharges, mining activities have made many species vulnerable, critically endangered or even going extinct107. In the Addendum (Table 1) pictures are given of the different fish species.

4.2.1. Trychomycterus spp. In lake Titicaca two Trichomycterus species have been described: T. dispar and T. rivulatus3,108. They are a scaleless, benthic filter feeding species3,112. Their size ranges from 12-20 cm.3 In this study these species will be described on a genus-level: Trichomycterus spp. The diet of Trichomycterus spp. is given in Table 8. These benthic species come in close contact with the sediment and feed also on detritus. Due to the fact that sediment acts as a sink for metals and metalloids these benthic species are likely to be exposed to elevated concentrations (Section 2.2)1,10.

4.2.2. Orestias In the lake, 28 different Orestias species are reported108. Orestias occurs throughout the high altitude lakes of Peru, Chili and Bolivia29. They have a high genetic diversity and are specialized to specific microhabitats within the lake3. In this study O. luteus, O.agassizii, O. ipsi and O. gilsoni will be examined. The Orestias species are mature within the year and spawn on macrophytes in shallow coastal zones112. Its size ranges from 5-20 cm, depending on the species108. Its depends on the species, but also on factors such as ontogeny, size and availability of the food source3,112. The diet of the four Orestias species is presented in Table 8. Orestias can be benthic, benthopelagic or epipelagic (Table 8).

4.2.3. Odontesthes bonariensis The pelagic O. bonariensis (silverside) was introduced in the Lake from Argentina in the 1950s. It is a fast growing fish and can reach lengths of approximately 50 cm29. Males reach maturity at 17cm, females at 25cm. Spawning occurs when the water temperature reaches 14-17°C, in shallow areas with macrophytes112. The diet of O. bonariensis is given in Table 8.

4.2.4. Oncorhynchus mykiss O. mykiss (rainbow trout) is a pelagic introduced species and is mostly farmed in cages deployed in the lake. Lengths up to 65.8 cm are reported112. It migrates in the rainy season or in the autumn to tributary rivers to spawn. Unlike the other species it does not spawn at the lake112. The diet of O. mykiss is given in Table 8.

Table 8: The habitat and diet of the fish species discussed in this paper107. Species Habitat Diet Orestias gilsoni Epipelagic Zooplankton Orestias ispi Pelagic Zooplankton, eggs Orestias agassizii Benthic, pelagic, littoral Amphipods, zooplankton, insects, plant detritus Orestias luteus Benthic Amphipods, molluscs, insects, algae, eggs, fish Trichomycterus spp Benthic Amphipods, molluscs, insects, eggs, algae, plant detritus Odontesthes bonariensis Pelagic, littoral Amphipods, zooplankton, insects, fish Oncorhynchus mykiss Pelagic, littoral Amphipods, zooplankton, insects, fish

4.3. Morphology In this master thesis, O. luteus, O. agassizii, O. gilsoni, O. ispi, O. bonariensis, Trychomycterus spp. and O. mykiss are of interest. In the study conducted by Monroy et al. (2014), muscle tissue was selected to determine the risk posed by metal pollution to humans and liver because it is a key organ in detoxification processes1. Furthermore, whole fish samples will be collected, to obtain a general overview of the fish. In this study three kinds of tissue will be collected: muscle tissue, liver tissue and whole fish samples. Based on an internal study of the Laboratory of Animal Nutrition, the different fish species can be dissected. Below an overview is given of the different fish species and their morphology (Figure 9, 10, 11). No data was available of O. bonariensis and the morphology of the different Orestias species were described on a genus- level in this internal study.

Figure 9: Left-lateral of O. mykiss: Ph=Pharynx, H=Heart, O = Oesophagus, L = Liver, CS = Cardiac part of the stomach, PS = Pyloric part of the stomach, PC = Pyloric caeca, SB = Swim bladder, R = Rectum. Photos taken by Andy Vervaet.

Figure 10: Left-ventrolateral view of an Orestias luteus: Ph = Pharynx, H = Heart, L = Liver, SB = Swim bladder, I = Intestine = Hepatic loop. Photos taken by Andy Vervaet.

Figure 11: Left-lateral view of Trichomycterus spp.: L = Liver, St = Stomach, T = Testes, I = Intestine, R = Rectum. Photos taken by Andy Vervaet.

4.4. Trophic interaction In an ecosystem a complex web of interactions occurs between different trophic levels. Traditional approaches to analyse these complex links between the different trophic levels are observational studies of fish species behaviour and gut content analysis107,113. In the lake, the fish species belong to a highly diverse community and most species appear to have relatively similar feeding behaviour. In this situation, niche overlap will occur and subtle modifications in the structure of the food web can be hard to detect with the traditional approaches107. A solution can be given by analysing the stable isotope profiles of the different fish species. The analysis of 13C and 15N can lead to useful insights regarding the position in the food web and foraging habitat of the species. 15N can be used to predict the species its trophic position because 15N is enriched throughout the trophic levels81,107. The ratio between 15N and 14N (15N) increases approximately 3‰ in each trophic transfer and gives an indication of the trophic position of the organism81.13C content can be used to predict the foraging habitat of the fish, because of the similarities in 13C composition between the diet (prey) and the consumer (predator)81,107,113. The study of M. Monroy et al. (2014) investigated the trophic interactions between the native and introduced fish species in Lake Titicaca based on the 13C and 15N composition of the fish species and gut content analysis. Figure 12 shows the 13C-15N bi-plot for the different fish species. Based on this bi-plot and findings in previous studies the fish were classified in six groups: (I) epipelagic and strictly zooplanktophagous species, (II) pelagic and mainly zooplanktophagous species, (III) benthopelagic and mainly omnivorous species, (IV) littoral species that mainly feed on insects and amphipods. Introduced fish species were independently sorted into two groups corresponding to O. bonariensis (V) and O. mykiss (VI)107. Table 9 shows the content 15N (‰) in species of interest in this study and the classification based on the study of M. Monroy et al. (2014). Regarding metal accumulation throughout the different trophic levels the 15N (‰) can be plotted towards the metal concentrations of the different species.

Figure 12: Mean 훿15N and 훿 13C values of 15 native fish species from Lake Titicaca, the two introduced species and potential invertebrates used as food by these fish species. The fish species of interest were classified into groups: (I) composed of Orestias gilsoni (gil); (II) containing Orestias ispi (isp); (III) Orestias luteus (lut), Orestias agassizii (aga), Trichomycterus rivulatus (riv); Represent food sources such as chironomids (chi), amphipods (amp) and zooplankton (zoo). Note that large and small (<90mm total length) individuals of Odontesthes bonariensis (group V) are shown ( , ), and that Oncorhynchus mykiss (group VI) is shown ( ).

Table 9: 15N (‰) content in the species of interest in this study107.

Species 15N (‰) Classification O. gilsoni 8.88 (I) O. ispi 10.24 (II) O. agassizii 8.8 (III) O. luteus 7.3 (III) Trichomycterus spp. 7.94 (III) O. bonariensis 10.75 (V) O. mykiss 10.40 (VI)

5. ICP-MS 5.1. Introduction Inductively coupled plasma (ICP) mass spectrometry (MS) analyses liquid samples. Figure 13 shows the basic components that make up an ICP-MS system. First the liquid sample is pumped into the nebulizer where an aerosol is formed with argon gas. In the spray chamber the fine droplets are selected after which the fine aerosols are transported into the argon plasma torch93,114. In the torch positively charged ions are formed

and are directed into the mass spectrometer via the interface region. The interface region is the most critical area. Here the ions are injected in the mass spectrometer while attempting to maintain perfectly stable conditions. Fluctuating potential differences, pressure, etc. affect the data obtained via the mass spectrometer114. Three main types of mass spectrometers are commercially used: the quadrupole, time-of- flight and sector field61,93. Most commercial ICP-MS systems apply a quadrupole mass spectrometer93. In the mass spectrometer the ions can be selected based on their mass-to-charge ratio (m/z). A quadrupole has four rods, where static and alternating potentials can be applied on. These voltages create a fluctuating electrical field between the rods wherein only ions of a given m/z remain stable and can pass through the detector. Ions with different m/z are unstable and are rejected (Figure 13, ii)61,93. At last, the selected ions who reach the detector are converted into an electrical signal with an ion detector114. The software will compare the intensities of the measured pulses to the intensity of a calibration curve, to determine the concentration of the element93. The ICP-MS is very sensitive to undesirable matrix components, such as excessive ion concentrations (Na, K, Ca, Mg, etc.), high dissolved solids and organic matter from incomplete digested samples. A solution to reduce the matrix components effects is to employ sample dilution in preparing samples for ICP-MS analysis115.

i) ii)

Figure 13: i) Basic instrumental components of an ICP mass spectrometer114 and ii) principle of the single collector quadrupole ICP-MS61.

5.2. Isotopes As mentioned before (Section 2.3, 2.4, 2.5 and 2.6) different isotopes occur in nature. As mentioned in Section 2.6, the ratio of the isotopes is fixed in nature, except for Pb. Lead originates from two sources: Pb naturally occurring since the formation of the earth and Pb formed by decay of radioactive materials. Depending on the source of Pb, the isotope ratios may vary (Section 2.6). To accurately measure Pb concentration it is necessary to measure the sum of the isotopes available.93

5.3. Interferences Three types of interferences can be expected: physical, matrix and spectroscopic interferences. Physical interferences are generally caused by viscosity effects, aerosol transport from the nebulizer, changes in spray formation, differences in ionization, etc116,117. Matrix interferences can be defined on how the matrix

affects the energy of the plasma and what happens when the sample enters the vacuum system116. It causes analyte signal suppression or enhancement compared to the signal expected from the same analyte concentration in a matrix free solution118. The cones, lens and detector components of the ICP-MS are especially sensitive to undesirable matrix components (e.g. excessive ion concentrations)115. Spectroscopic interferences are caused by atomic or molecular ions that have the same mass-to-charge as the analyte of interest. This category can be divided in to isobaric (elemental), double charged and polyatomic (molecular) spectroscopic interferences116,118. ICP-MS instrumental software can correct for interferences caused by isobaric interferences and overlapping isotopes of different elements but it is not able to correct for the double charged and most of the polyatomic interferences119. Sources of polyatomic ions are the sample matrix, reagents used for preparation, plasma gases, entrained atmospheric gases, etc.119 Table 10 gives a list of the possible polyatomic ions which can cause interference when analysing As, Pb, Cd with the ICP- MS. The most common interference related to this investigation is the one from 40Ar35Cl+ and 40Ca35Cl+ 119,120. The ICP-MS uses argon to create aerosols and to create a plasma. When chlorides are present in the sample, 40Ar35Cl and 40Ca35Cl+ (if Ca is present) can be formed in the plasma torch. 40Ar35Cl and 40Ca35Cl+ has a m/z value of 75, the same as As. To deal with these interferences the ICP-MS can measure the analyte in different modes.

Table 10: Polyatomic ions which cause interference on ICP-MS when analysing As, Pb, Hg, Cd119,120. Isotope Interference 75As 40Ar35Cl+, 40Ca35Cl+, 59Co16O+, 36Ar38Ar1H+, 38Ar37Cl+, 36Ar39K, 43 16 23 12 40 12 31 16 + Ca O2, Na C Ar, C P O2 110 39 16 + Cd K2 O 111 95 16 94 16 1 + 39 16 1 + Cd Mo O+, Zr O H , K2 O2 H 112 40 16 40 16 96 16 + Cd Ca2 O2, Ar2 O2, Ru O 113 96 16 1 + 40 16 1 + 40 16 1 + 96 17 + Cd Zr O H , Ca2 O2 H , Ar2 O2 H , Ru O 114Cd 98Mo16O+, 98Ru16O+ 116Cd 100Ru16O+ 206Pb 190Pt16O+ 207Pb 191Ir16O+ 208Pb 192Pt16O+

5.4. Modes Three modes can be used whilst analysing with an ICP-MS: the standard mode, the kinetic energy discrimination (KED) or collision cell mode and the dynamic reaction cell mode (DRC). The standard mode is shortly described above. The collision mode and the reaction mode are modifications to the standard mode to deal with interferences93,117. The KED mode is firstly based on the principle that the interfering ion is physically larger compared to the analyte ion. If both ions pass through a cloud of inert gas molecules (e.g. He), the larger molecule (interfering ion) will collide more frequently with the inert gas molecules. Secondly when an ion collides with an inert molecule in the gas cloud, an amount of kinetic energy will be transferred from the ion to the inert molecule. As result, at the end of the collision cell, the analyte ion (smallest) will retain more kinetic energy compared to the interfering ion. At last, at the end of the collision

cell an energy barrier can be adjusted so that the higher energy-analyte ions are allowed to pass though and the interfering ions are retained. When the DRC mode is applied, a reactive gas is used (e.g. ammonia,

O2). This gas can react with the analyte or with the interfering ions. When the gas reacts with the analyte and not with the interferent, the analyte can be measured at another m/z ratio. When the gas reacts with the interfering ion, the m/z ratio of the interferent will have changed, causing the removal of the interfering ion when it goes through the quadrupole.93,117

5.5. Internal standards The use of KED or DRC mode can be a solution for spectroscopic interferences, while the use of internal standards can offer a solution for physical interferences117. The concept is as follows: a constant amount of the internal standard is added to every sample. The signal of the internal standard should be a constant, but due to physical interferences the signal of the internal standard and the analyte can fluctuate. Ideally, any factor that affects the analyte signal will also affect the signal of the internal standard. Via the fluctuation of the internal standard, the fluctuation of the analyte can be corrected. A good internal standard should: not be present in the samples, not be interfered by the sample matrix, not interfere on analytes, not be common environmental contaminants and match the masses of the analytes116. Commonly used internal standards are: Li, Sc, Ga, Ge, Y, Rh, In , Tb, Tm, Lu, Ir116.

5.6. Mercury analysis As mentioned at Section 2.4, metallic Hg has a low vapour pressure and will be easily lost from the environmental samples if it can undergo reduction to the elemental species via natural biochemical pathways. By adding gold (Au) to the liquid sample, Hg will stay in solution. Au acts as a strong oxidizing agent that ensures the stability of Hg in its mercuric ionic form. Hg, as its mercuric ionic form will stay in solution. This Au-Hg interaction will cause no difficulties analysing Hg with the ICP-MS because the energies involved will cause the formation of the separate cations in the plasma torch.121,122

Materials and Methods 1. Reagents

The bulk chemicals (HNO3, HCl, H2O2), were bought from Chem-Lab NV (Belgium). Hydrogen peroxide 30 weight% (p.a. solution), Nitric acid 67-69% (p.p. solution), Hydrochloric acid 34-37% (p.p. solution) were used during laboratory handlings. The certified (ISO 17025) reference solutions were bought from Merck KGaA (Germany) and Chem-Lab (Belgium). The As, Hg, Pb and Cd standard solution are: 1000 mg/L,

Certipur®, traceable to SRM from NIST (arsenic as H3AsO4 in HNO3 0.5 mol/L, mercury as Hg(NO3)2 in HNO3 ® 2 mol/L, lead as Pb(NO3)2 in HNO3 0.5 mol/L and Cd as Cd(NO3)2 in HNO3 0.5 mol/L). The Milli-Q -reference system from Merck (Germany) was used to produce ultrapure water. This pure water is necessary to execute analyses on ICP-MS.

2. Sampling locations In order to map the metal and metalloid distribution in water and fish and to investigate the bioaccumulation and biomagnification in the different trophic levels in Lago Menor, the metal concentration in the sediment, water, macro-invertebrates and different fish species was examined. In order to gain an overall picture of the metal pollution, samples were collected at six locations in Lago Menor, two locations in Lago Mayor and two locations in Lake Uru Uru. The locations can be seen on Figure 14. In the addendum a larger figure of the Titicaca Lake is added with all sampling points (Addendum: Figure 1) and in Section 1.1 of the literature review a general map of the Altiplano is given with the location of Lake Titicaca and Uru Uru. The first location in the bigger part of Lake Titicaca is situated at Isla de La Luna (marked as IDL), an island in the middle of the Bolivian part of Lago Mayor. The second location at Lago mayor is near to Escoma (marked as E). This is located near to the estuary of the Huaycho river and upstream this river there a mine is located. The last two sampling points were located at Lake Uru Uru. At Lago Menor, the 6 locations were chosen to map the in- and outflow. A simplified illustration of the in- and outflow at Lago Menor is given at Section 1.1.1, Figure 4 (Literature review). The main sources of water in the minor part, are the inflow from Lago Mayor at Tiquina (TQ) and from the Katari river shed at Cohana Bay (C). Samples were taken at these two locations. At Tiquina there is fishing activity, but at Cohana Bay no or low fishing activity occurs because of the low dept in those parts of the lake. The third spot was chosen at the outflow of the Lake. This is situated at Desaguadero (D), the border between Bolivia and Peru, where the Desaguadero river leaves the lake. The other 3 locations were chosen in consultation with the limnology institute of UMSA and with the local fishermen. The locations reflect the water flow and are taken at places with an abundancy of fishing activities (except the spot at Cohana Bay). The fourth location is situated at Huatajata a region in the East of Lago Menor and near to the shore, and the fifth further away from the shore near to a sampling device of the university of San Andrés. The last sampling location is situated at Taraco, a site between Cohana Bay and Desaguadero were fishing activity is abundant. Coordinates of each sampling point can be found in the addendum (Tabel 2).

i) ii) Figure 14: i) Sampling locations at Lake Titicaca: Escoma (E), Isla de la Luna (IDL), Huatajata (H), Research platform UMSA (X), Cohana bay (C), Taraco (T), Desaguadero (D). ii) Sampling Locations at Lake Uru Uru: UU1 and UU2. Country borders are indicated in yellow. Illustration made with Google Earth Pro®.

3. Sampling method At every sampling location close cooperation with the local fishermen was necessary. To deal with possible anomalies and outliers 3 different spots were selected with a distance of approximately 15m at each sampling location to collect materials. On each spot, sediment, water and amphipods were collected and the 2 different kind of DGT samplers (LSNB-AP and LSNX-NP) were installed in the water (Section 3, Literature review). To collect the Fish samples, the nets of the local fishermen were checked and the desired fish species were collected.

3.1. Water Water samples were taken at a depth of 4 m on each sampling spot with a water sampler (type: LaMotte 1077 Model JT-1 Water Sampler). At each spot 10mL of the collected water was stored in graduated 10mL polypropylene tubes. In Bolivia no nitric acid was available at the sampling location so proper cleaning of the equipment was not possible. Because of this reason, closed tubes were brought from Belgium to Bolivia. In Bolivia, the water was stored in a freezer until transportation to Belgium. The second type of water analysis was conducted with two different kinds of DGT samplers (Section 3, Literature review). These samplers were attached on a self-made holder (Figure 15). The LSNB-AP device was always placed on top, and the LSNX-NP samplers at the bottom of the holder. The DGTs were stored in a refrigerator prior to their use and 5 to 10 minutes before deployment of the samplers in the lake, the sealed plastic back was taken off. The DGT samplers at the Lake Titicaca stayed for approximately 25 days in the lake and the DGTs at Lake Uru Uru 12 days. The deployment period per DGT sampler is given in Table 11. The deployment period was based on the use of DGT samplers in previous studies97,99,102,123. An agreement was made with the local fishermen to keep an eye on the samplers. During the deployment time the DGTs were checked to verify if

they were still on the correct location and to make sure there was not too much biofilm formation on top of the filters. No biofilm formation occurred. After the deployment period the DGTs were collected and rinsed with Milli-Q water, sealed in (labelled) plastic bags and afterwards stored in a refrigerator until transportation to Belgium. Not all the samplers could be recovered. On certain spots the DGTs were replaced by gillnets. The lost samplers are included in Table 11. In Belgium the samplers were stored in a refrigerator and the water samples in a freezer until analysis. The executed handlings and use of the DGTs are described at the website of ‘DGT research’99.

Figure 15: Deployment mechanism DGT samplers.

Table 11: Location and deployment time of the DGT samplers, “/” marks the lost DGT samplers. Spot Code Deployement Time Retrieval time Deployement period (s) Isla de la Luna IDL1 1/08/19 14:26:00 28/08/19 / / IDL2 1/08/19 14:31:00 28/08/19 10:30:00 2318340 IDL3 1/08/19 14:41:00 28/08/19 / / Huatajata H1 3/08/19 9:46:00 29/08/19 / / H2 3/08/19 14:00:00 29/08/19 12:11:00 2239860 H3 3/08/19 14:17:00 29/08/19 11:55:00 2237880 Cohana C1 3/08/19 11:42:00 29/08/19 9:46:00 2239440 C2 3/08/19 11:52:00 29/08/19 / / C3 3/08/19 12:04:00 29/08/19 9:16:00 2236320 Platform UMSA X1 3/08/19 12:52:00 29/08/19 11:13:00 2240460 X2 3/08/19 13:12:00 29/08/19 10:31:00 2236740 X3 3/08/19 13:30:00 29/08/19 / / Taraco T1 4/08/19 10:58:00 30/08/19 12:58:00 2253600 T2 4/08/19 11:07:00 30/08/19 12:44:00 2252220 T3 4/08/19 11:24:00 30/08/19 12:31:00 2250420 Desaguadero D1 4/08/19 12:08:00 30/08/19 11:22:00 2243640 D2 4/08/19 12:14:00 30/08/19 / / D3 4/08/19 12:21:00 30/08/19 11:43:00 2244120 Escoma E1 4/08/19 18:08:00 31/08/19 8:30:00 2298120 E2 4/08/19 18:15:00 31/08/19 8:59:10 2299440 E3 4/08/19 18:21:00 31/08/19 8:44:25 2298205 Tiquina TQ1 5/08/19 11:56:00 28/08/19 11:05:00 1984140 TQ2 5/08/19 12:12:00 28/08/19 / / TQ3 5/08/19 12:34:00 28/08/19 11:36:00 1983720 Uru Uru UU1_1 19/08/19 11:03:00 1/09/19 11:11:00 1123680 UU1_2 19/08/19 11:48:00 1/09/19 11:01:15 1120380 UU1_3 19/08/19 12:24:00 1/09/19 11:23:00 1119540 UU2_1 19/08/19 13:28:00 1/09/19 / / UU2_2 19/08/19 14:12:00 1/09/19 / / UU2_3 19/08/19 14:55:00 1/09/19 10:23:00 1106880

3.2. Sediment Sediment was taken using a Van Veen grab sediment sampler. Only the top layer was collected because benthic fish species and the macro-invertebrates come in contact with this sediment layer10. The samples were stored in a 40 mL polypropylene container. Water was (if possible) sampled at a dept of 4 m and a height of 1 m above the bottom, resulting in a desired sediment sampling depth of 5 m. Depts of the sediment taken samples are shown in Table 12. The samples were dried in an oven for 2 days at 70°C at the PACU research center situated in Tiquina. When the sediment samples were dry, the flasks were sealed and brought to Belgium. In Belgium the dried samples were grinded with a mortar into a fine powder and stored

in new 40 mL containers. After each sample the mortar was cleaned with tap water, 1% HNO3 in Milli-Q water, Milli-Q water and dried with cleaning paper.

Table 12: Location and depth sediment samples. Spot Code Depth lake (m) Spot Code Depth lake (m) Isla de la Luna IDLS1 2 Desaguadero DS1 8,6 IDLS2 3,8 DS2 8,5 IDLS3 6,6 DS3 8,3 Huatajata HS1 4 Escoma ES1 13,5 HS2 5 ES2 11,5 HS3 5 ES3 13,1 Cohana CS1 2,6 Tiquina TQS1 6,2 CS2 2,6 TQS2 6,38 CS3 2,6 TQS3 6,31 PlatformXavier XS1 19 Uru Uru UU1S1 1,1 XS2 22 UU1S2 1,2 XS3 10 UU1S3 0,8 Taraco TS1 3,1 UU2S1 1,1 TS2 5,5 UU2S2 0,9 TS3 6,1 UU2S3 0,9

3.3. Fish The different fish species were collected at two different moments, the day of sediment and water sampling and the day of the retrieval of the DGTs. Fish was collected from the nets of the cooperating fishermen and fish was bought from local fishermen who were fishing in the same area as the sampling spots. At the altiplano only two types of nets are used: gillnets (to catch deep-water fish such as Orestias and Trichomycterus) and trawls6. All fish was caught via gillnets, a wall of netting made out of nylon that hangs in the water column124. With these nets, the head of the fish can go through the mazes but the fish cannot go back anymore because the net gets stuck behind their gills. O. luteus and O. agassizii were caught with nets having a mesh size of approximately 40 mm and Odonthestes bonariensis with nets having a mesh size of approximately 60 mm. The smaller fish species (O. ispi and O. gilsoni) were caught with gillnets having a mesh size around 20 mm. The fish were freshly caught and killed by the fisherman, stored in polystyrene boxes and brought to the PACU research centre. The goal was to collect at every location 15 fishes of the desired species, 10 fishes meant for dissection and 5 fishes to dry entirely. During the dissection liver and muscle tissue were taken. In Section 4.2.3 (Literature review), the localization of liver and other organs were presented. The dissection of the different organs was based on the information presented in Section 4.2.3. Firstly the dimensions of the fish were measured during the dissection: the length from the top of the head to the end of the caudal fin, the length without the caudal fin (standard length), the body height, body width and the body weight. Secondly an incision was made from right before the anal tract to the beginning of the head, after which the intestines were removed from the body. The gonads were measured and

weighted, the intestinal tract was stored in an 75% alcohol solution and the liver was stored in a 40 mL polypropylene straight container. The intestinal tract was stored for an additional study on the diet of the fish. At last a muscle sample was taken (ranging from 0.05-0.5 g) below the dorsal fin. After the dissection, the liver tissue, muscle tissue and the whole fish were dried in an oven at 70 degrees Celsius for 2 days. It was not possible to dissect the smaller fish species (O. ispi and O. gilsoni) properly so the head and guts were removed and the remaining parts of the fish were dried for 2 days at 70°C. After drying the samples, the tubes were sealed and the fish were wrapped in household paper and plastic foil. All samples were stored in dry conditions until transportation to Belgium. In total 347 fish were collected, 206 fish were used for dissection (muscle, liver, gut content, gonads) and 141 were dried entirely. Table 13 shows the sampling locations and the total amount of fish dissected.

Table 13: Fish sampling locations and total amount of dissected fish, V: Fish was sampled, /: No fish caught during sampling campaign. IDL: Isla de la Luna, E: Escoma, TQ: Tiquina, H: Huatajata, D: Desaguadero, UU: Lake Uru Uru . Lake Plaats O. luteus O. agasizii O. gilsoni O. ispi Trychomycterus sp O. bonariensis O. mykiss Mayor IDL V / V V V / V E V V / / V V / Menor TQ V / / / / V V H V V V / / V / D V V / / / / / Uru Uru UU V V / / / / / Amount 61 23 16 14 20 33 20

3.4. Amphipods Amphipods were collected by means of three different methods. When vegetation was available two methods were applied. Using the first method, vegetation was collected at the sampling spot and the amphipods were afterwards collected at the research facility of PACU. Using the second method, the amphipods were captured by filtering the water and vegetation using a sieve. When no vegetation was available the amphipods were collected by filtering the water and sediment. The amphipods were stored in 40 mL polypropylene containers and dried in the sun. The dried samples were taken back to Belgium for further analysis. In Belgium the samples were grinded with a mortar into a fine powder and stored in new

40 mL containers. After each sample the mortar was cleaned with tap water, 1% HNO3 in Milli-Q water, Milli-Q water and dried with cleaning paper.

4. Determination of metal and metalloid concentration in water, sediment and biota 4.1. Selection of digestion method for biota and sediment For every digestion procedure the recovery rates were calculated on reference material so the accuracy of the digestion procedure could be checked. The reference material was chosen to have a similar matrix as the samples taken in Bolivia. To select the most suitable digestion method, different methods were taken

from literature as well as the current digestion methods deployed at the Department of Green Chemistry and Technology of the faculty of Bioscience Engineering, Ghent University1,12,125–127. For the sediment samples, 2 methods were applied on lake sediment (BCR-701): i) Microwave digestion, closed vessel with aqua regia, and ii) Microwave digestion, closed vessel with nitric acid. For the fish samples, six methods were applied on fish muscle (ERM-BB422) and on mussels tissue (ERM-CE278k). These six methods are: i) Dry ashing and ash dissolution with nitric acid, ii) Microwave digestion, open vessel with aqua regia, iii) Microwave digestion, closed vessel with aqua regia iv) Microwave digestion, closed vessel with nitric acid, v) Hotplate digestion with nitric acid and hydrogen peroxide, low dose and vi) Hotplate digestion with nitric acid and hydrogen peroxide, high dose. Three reagents were used: HCl, HNO3 and H2O2. HCl is a strong acid and in combination with HNO3 (3:1 ratio), aqua regia is formed. Aqua regia has strong oxidising properties, due to the formation of Cl2 and NOCl. HNO3 is a strong oxidising acid, but less reactive compared to aqua - regia. The advantage of HNO3 is that no HCl is used. Cl is very reactive and during ICP-MS analysis interfering - 40 35 + polyatomic species can be formed with Cl (e.g. Ag Cl ). The combination of HNO3 and H2O2 also does not have to deal with these interferences and has strong oxidising properties. In the fish muscle reference material no certified Pb concentration was given, so mussels tissue (with certified Pb) was selected to evaluate accuracy of the Pb analyses. For the amphipoda, plankton was chosen as reference material (BCR- 414) and 5 methods were applied. All used materials were 3 times rinsed before use with a solution of 10% nitric acid in ultra-pure Milli-Q water and 3 times with Milli-Q water. A second manner to verify the accuracy of the digestion method and the analysis is the addition of a spike of As, Cd, Hg and Pb to the desired matrix. For all analyses indium, gallium and thorium were measured as internal standards. The ICP-MS was operated in two modes: the standard and KED mode (Section 5.4, Literature review).

4.1.1. Digestion of sediment Microwave digestion, closed vessel with aqua regia (MW + Closed vessel + aqua regia)

6 mL HCl and 2 mL HNO3 were added to 0.3 g sample in a microwave vessel. The microwave vessels were tightly sealed and immediately subjected to a temperature profile (Tprofile) in the microwave. This Tprofile consists of three steps. First a ramp time of 20-25 minutes to reach 180°C after which this temperature was held constant for 20 minutes. At last the samples were cooled for approximately 20-25 minutes until a temperature of 70°C was reached. After the digestion, the mixture was poured into a 50 mL volumetric flask and the vessel used for the digestion was 3 times rinsed with a 1% Milli-Q solution. After each rinse the solution remaining in the vessel was added to the 50 mL flask. Thereafter, the volumetric flask was diluted up to 50 mL with Milli-Q water. At last, this diluted solution was poured into a certified 50 mL polypropylene centrifuge tube and stored until analysis. If residue (silicates) remained, this settled in the centrifuge tubes. Before analysis 10 mL sample was decanted in 10 mL polypropylene test tubes.

Microwave digestion, closed vessel with nitric acid (MW + Closed vessel + HNO3)

10 mL HNO3 was added to 0.3 g sample in a microwave vessel. The microwave vessels were tightly sealed and immediately subjected to the above mentioned Tprofile in the microwave. After the digestion, the mixture was poured into a 50 mL volumetric flask and the vessel used for the digestion was 3 times rinsed with a 1% Milli-Q solution. After each rinse the solution remaining in the vessel was added to the 50 mL flask. Thereafter, the volumetric flask was diluted up to 50 mL with Milli-Q water. At last, this diluted

solution was poured into a certified 50 mL polypropylene centrifuge tube and stored until analysis. Before analysis 10mL sample was decanted in 10 mL polypropylene test tubes.

Spike To evaluate the accuracy of the obtained Pb concentrations in the sediment samples, a spike of 10g/L Pb was added to the sediment digest from Lake Uru Uru (sampling spot 1, replicate 1). The standard solution contains 1000 mg/L Pb. Firstly a diluted stock solution was made of 100 g/L. 100 L of the standard solutions was diluted in a volumetric flask of 1 L, creating a stock solution of 100 g/L Pb. In the vessels for the microwave digestion, 0.3 g of the sediment sample and 5 mL of the stock solution was added. The same steps were applied as mentioned above during the digestion procedure. After dilution to 50 mL, a concentration (added through the spike) of 10 g/L Pb was added to the final digest. However the sample from Bolivia is contaminated with Pb. Another sample from Uru Uru (same spot: sampling spot 1, replicate 1) was treated in the same way as described above without the addition of a spike (a blanc sample). The obtained concentration from this blanc sample was subtracted from the spiked concentration in order to correct for the naturally occurring Pb concentrations in the sediment sample. At last, the recovery can be calculated by dividing the obtained concentration (after subtraction of blanc) by 10 g/L.

4.1.2. Digestion of biota

Dry ashing and ash dissolution with nitric acid (Dry ashing + HNO3) In the first step 0.2 g of sample was added in a porcelain crucible and ashed in a furnace. The Tprofile consists of three steps. Fist a ramp time of 40 min to reach 450°C after which this temperature was held constant for 2 hours. At last the samples were cooled for approximately 1 hour, so a safe temperature to handle the samples was reached. Subsequently, the ash was poured in to a 100 mL Erlenmeyer flask and 5 mL of HNO3 were added. The Erlenmeyer was covered with a watch glass and the mixture was heated on a hotplate for 2 hours. The T was slowly increased from 90°C to 120°C during the first 30 minutes and then kept steady for 90 minutes at 120°C. When the mixture was almost evaporated a solution of milliQ water with 1% nitric acid was added. Subsequently, the mixture was filtered and washed 3 times with a 1% nitric acid solution after cooling down. At last the mixture was diluted in a 50 mL flask with milliQ water and stored in 50 mL polypropylene centrifuge tubes until analysis.

Microwave digestion, open vessel with aqua regia (MW + open vessel + aqua regia)

3.75 mL HCl and 1.25 mL HNO3 were added to 0.5 g sample in a 50 mL centrifuge tube. Next, the mixture was placed in a fumehood for 24 h with the centrifuge tube not fully closed. Secondly the centrifuge tubes were place in the microwave and subjected to a Tprofile. The Tprofile consists of four steps. First a ramp time of 20 minutes to reach 70°C, this temperature was held constant for 30 minutes. Next, the temperature was increased (ramp time: 10 minutes) to 100°C, and this temperature was held constant for 45minutes. At last the samples were cooled for approximately 20 minutes until a temperature of 70°C was reached. After the digestion, the mixture was poured into a 50 mL volumetric flask and the centrifuge tube, used during digestion, was 3 times rinsed with a 1% Milli-Q solution. Thereafter, the volumetric flask was diluted up to 50 mL with Milli-Q water. At last, this diluted solution was poured into a certified 50 mL

polypropylene centrifuge tube and stored until analysis. If there is still residue remaining an extra filtration step was performed.

Microwave digestion, closed vessel with aqua regia (MW + Closed vessel + aqua regia)

7.5 mL HCl and 2.5 mL HNO3 were added to 0.3 g reference sample in a microwave vessel. Secondly the microwave vessels were placed in a sonicator for 15 min at 40°C. Thirdly the microwave vessels were tightly sealed and immediately subjected to a Tprofile in the microwave. The Tprofile consists out of three steps. First a ramp time of 20-25 minutes to reach 180°C after which this temperature was held constant for 20 minutes. At last the samples were cooled for approximately 20-25 minutes until a temperature of 70°C was reached. After the digestion, the same steps as describe for the MW + open vessel + aqua regia treatment was applied.

Microwave digestion, closed vessel with nitric acid (MW + Closed vessel + HNO3)

10 mL HNO3 were added to 0.3 g reference sample in a microwave vessel. Secondly the microwave vessels were placed in a sonicator for 15 min at 40°C. Thirdly the microwave vessels were tightly sealed and immediately subjected to the above mentioned T profile in the microwave (MW + Closed vessel + aqua regia). After the digestion, the same steps as describe for the MW + open vessel + aqua regia treatment was applied.

Hotplate digestion with nitric acid and hydrogen peroxide (Hotplate + HNO3 + LD or HD H2O2) Based on the study of Wilson, R. P. and Cowey C.B. (1985), an estimation was made on the amino acid composition of rainbow trout128. Via the study of Haliloǧlu, H. I. et al. (2004), an estimation was made on the fatty acid composition of rainbow trout129. A digestion procedure was constructed based on the oxidation of the amino acids and fatty acids by hydrogen peroxide (H2O2). The theoretical amount of H2O2 was calculated to oxidise the organic matter occurring in the O. mykiss tissue. Based on this amount, two digestion procedures were designed: a first one with an excess factor of 5 times the required amount of

H2O2 (Low dose: LD) and a second one with 10 times (High dose: HD) the amount of H2O2. This resulted in the addition of 4.5mL and 8.5mL respectively of a 30% H2O2 solution. The calculations are given in the Addendum.

Procedure

For procedure 1 (low dose (LD)) 4.5 mL H2O2 and 2 mL HNO3 were added and for procedure 2 (High dose

(HD)) 9 mL H2O2 and 2 mL HNO3 were added to 0.2 g reference sample in a 100 mL Erlenmeyer flask. Secondly the mixture was placed with a watch glass on top of the Erlenmeyer in a fumehood for 24h. The Erlenmeyer was covered with a watch glass and the mixture was heated on a hotplate for 2 hours. The temperature was slowly increased from 90°C to 120°C during the first 30 minutes and kept steady for 90 minutes at 120°C. When the mixture was almost evaporated a solution of Milli-Q water with 1% nitric acid was added. At last the mixture was filtered and washed 3 times with a 1% nitric acid solution after cooling down. Then the mixture was diluted in a 100 mL volumetric flask with Milli-Q water.

4.1.3. Digestion water The water samples were not turbid. Before analysis, 10mL of sample was acidified with three drops of HCl and 1 drop of HNO3. After 1h the samples were analysed on ICP-MS.

Spike To assess the accuracy of the obtained concentrations in the water samples, spikes of 10 g/L As, Hg, Cd and Pb were added to the water sample from Tiquina (replicate 1). The standard solutions contain 1000 mg/L As, Hg, Cd and Pb. Firstly a diluted stock solution was made of 100 g/L. 100 L of all standard solutions were diluted in a volumetric flask of 1 L, creating a stock solution of 100 g/L As, Hg, Cd and Pb. In a volumetric flask of 50 mL, 25 mL of the water sample and 5 mL of the stock solution was added after which it was diluted up to 50 mL with Milli-Q water. After dilution to 50 mL, a concentration (added through the spike) of 10g/L Pb was added to the final digest. Before analysis, 10 mL of sample was acidified with three drops of HCl and 1 drop of HNO3. After 1 h the samples were analysed on ICP-MS. Simultaneously a blanc from Tiquina (replicate 1) treated in the same way (without addition of a spike) was analysed. The obtained concentrations from this blanc sample was subtracted from the spiked concentrations, in order to correct for the naturally occurring As, Hg, Cd and Pb concentrations in the water sample. At last, the recovery can be calculated by dividing the obtained concentration (after subtraction of blanc) by 10 g/L.

4.2. Digestion method biota and sediment samples from Bolivia After the optimisation, the most optimal method was selected to digest the sediment and biota samples. The microwave digestion, closed vessel with nitric acid was selected to digest the sediment and fish samples. The microwave digestion, closed vessel with aqua regia was selected to digest the amphipod samples. In all batches, samples and reference material were digested and analysed in the same manner. This was done to check if nothing went wrong during the digestion and analysis on ICP-MS. As internal standards, Indium, Gallium and Thorium were analysed. When a sample was below the detection limit, the assumption was made that no contaminant was present in the sample46. The water samples were acidified with three drops of HCl and 1 drop of HNO3.

4.3. ICP-MS analysis All analysis were conducted on a NexION® 350 S ICP Mass Spectrometer (PerkingElmer). During the optimisation of the analysis process, both standard as KED mode were applied. Per analysed sample 3 replicates were analysed, with three readings per replicate and 3 sweeps per reading. As internal standards indium, thorium and gallium were used. Indium appeared to be the most suited internal standard. The recovery of indium stayed within a range of 20% from full recovery. The samples from Bolivia were analysed in KED mode. The most suited isotopes for analysis are discussed in the Results, Section 3: As can only be measured on 75As; the signal for Pb was measured as the sum of the signals of the three most abundant isotopes: 206Pb, 207Pb and 208Pb; Hg was measured on 200Hg and 202Hg and for Cd, the isotopes 112Cd and 114Cd were selected.

5. Determination of water properties General water quality variables were determined at each sampling point using a Hanna HI-9828 multi- parameter probe to determine the temperature (°C), conductivity (S/cm), dissolved oxygen (mg/L) and pH. To measure the depth at the different sampling spots the Hondex portable digital depth probe was used. At Lake Menor it was not possible to recalibrate the pH probe so pH data, obtained by another research group of UMSA who was sampling at Lake Menor on 29/08/2019, were used. This research group measured the pH in the vicinity of the sampling spots used in this thesis: Huatajata, Tiquina, Cohana bay and Desaguadero. The pH at Taraco and Desaguadero was considered the same.

6. Statistical processing Water, sediment and fish variables were tested for normality (normal distribution) and homogeneity of variances using Shapiro-Wilk and the Levene test130. When the data was normal distributed, one way ANOVA was applied to compare differences between mean metal concentrations occurring at a certain sampling point or in a certain fish species. When there was a significance difference, the ANOVA analysis was followed by Tukey HSD post-hoc test for pair-site comparisons. When the data showed a non-normal distribution, the non-parametric Wilcoxon signed-rank test with bonferonni adjusted p-values was applied. When multiple averages are compared with each other, the type 1 error increases. The bonferonni correction adjusts the p-values to minimise the chance on a type 1 error. This is conservative method, a negative aspect is the decrease in power in rejecting the null hypothesis130. Correlations were calculated and tested for its significance with the Pearson correlation test. To explore the influence of the parameters on the metal or metalloid concentration in the fish tissue, a multi-way linear model was constructed. This multi-way ANOVA analysis was conducted on the 2 fish species with the largest samples and with the most sampling locations. The concentrations of As and Hg occurring in the fish samples were fitted against the weight of the fish, the gutted weight, the length of the fish (without the caudal fin), gender, T, DO, pH, salinity, conductivity, the concentration of As, Hg, Cd and Pb in sediment and the As, Hg, Cd and Pb concentration in water, carousel of analysis, mass analysed. The carousel and mass analysed are variables obtained during analysis of the samples. These variables where included in this multi-way ANOVA analysis, to check if there occurred analysis induced variability. Secondly the ANOVA function was used to determine how much variability in the outcome variable (As and Hg in fish) is explained by one of the parameters. Further selection was manually done or via a backward selection based on the AIC criterion130. At last the ANOVA function was applied at the selected model to determine which parameters could explain significantly the variance in the model.

Results 1. Dissection results: O. gilsoni, O. ispi and O. bonariensis In the internal study of the Laboratory of Animal Nutrition, the morphology of the genus Orestias, Trichomycterus and Oncorhynchus was discussed in detail, but detailed information and documentation were not given about the species Odonthestes bonariensis, Orestias ispi and Orestias gilsoni. Based on the documentation given in the literature review (Section 4.2.3) we located the internal organs of O. ispi, O. gilsoni and O. bonariensis. The following figures (Figure 16,17 and 18) can be used to complete the database of the Laboratory of Animal Nutrition.

Figure 16: Orestias Gilsoni: A=Operculum, B=Caudal fin, C=Anal fin, D=Dorsal fin, G=Gills, H=Heart, L=Liver, E=hindgut. Photos taken by Arthur Fonteyne and Eric Loayza.

Figure 17: Orestias ispi: A=Operculum, B=Caudal fin, C=Anal fin, D=Dorsal fin, E=Pectoral fin, G=Gills, H=Heart, L=liver, F=Hindgut. Photos taken by Arthur Fonteyne and Eric Loayza.

Figure 18: Odonthestes bonariensis: A=Operculum, B=Caudal fin, C=Anal fin, D=Dorsal fin, E=Pectoral fin, F=Hindgut, G=Gills, H=Heart, I=Intestine, J=Adipose fin, L=Liver, V=Gonads. Photos taken by Arthur Fonteyne and Eric Loayza.

2. Comparison of digestion methods A first comparison can be made based on the time necessary to analyse a batch of samples. The faster a method, the more samples can be treated in the same time span. The closed vessel technique takes the least time. The hotplate technique combined with dry ashing takes approximately eight hours before the hotplate destruction can be applied, all other techniques should rest 24h before a heat treatment is applied on the samples. The digestion procedures are given in Section 4 (Materials and Methods). A second comparison is based on the oxidation of organic matter. After treating the samples with the closed vessel digestion technique, no residue remained, so the closed vessel technique efficiently destroys the organic matter57,131. During this technique higher pressures and temperatures are possible. When the other methods were applied, residue remained in the solute and an additional filtration step was necessary. This additional step can be an extra source of contamination or loss of the analyte can occur. Nowadays, closed systems are preferred to open systems because digestions can be performed at temperatures above the boiling point of the digestion mixture, which increases the oxidation power57,131. Higher temperatures are, for example, required to digest AB131. During digestion of the sediment samples residue remained. This can 132 be explained by the fact that an HNO3 or aqua regia treatment cannot dissolve silicates . In order to fully 42,61,132 digest the sediment samples HF in combination with a strong oxidising acid (e.g. HNO3) can be used . Thirdly, the microwave, closed and open vessel techniques are very easy to apply and the repeatability is high, compared with the hotplate techniques. For example, during digestion on the hotplate it is difficult to maintain an uniform Tprofile in all samples. However, an advantage of the hotplate method is the higher amount of samples possible per batch. Using the microwave closed and open vessel techniques, 40 samples and 52 samples, respectively, can be digested at once. Negative aspects of the closed and open vessel techniques are the higher cost (expensive vessels should be replaced regularly) and limited availability of the equipment in the ECOCHEM research group (internal communication). Table 15 gives an overview of the digestion methods and the different aspects.

Table 15: Comparison of the microwave (MW) closed vessel technique, microwave open vessel technique and hotplate technique. The treatments are described in Section 4 (Materials and Methods) Parameter Closed vessel Open vessel Hot plate Time + - - Completeness digestion ++ + - Repeatability + + - Cost -- - + Availability - - + Samples per batch 40 52 >40

3. Selection of digestion method 3.1. Screening To select a digestion method for the biota samples, 5 methods were applied on reference material. Fish muscle (ERM-BB422) and plankton (BCR-414) were used as a reference for the fish samples and amphipod samples, respectively. These methods are extensively described in Section 4 (Materials and Methods). The simplified names given in Section 4 will be used in the following sections: i) Dry ashing + HNO3 ii) MW +

Open vessel + aqua regia, iii) MW + Closed vessel + aqua regia, iv) Hotplate + HNO3 + HD H2O2, v) Hotplate+

HNO3 + LD H2O2.

3.1.1. Amphipods Figure 19 shows the result from the first digestion attempt on the plankton reference material. It is clear that all methods have low recovery rates for Pb and for Hg, but the closed vessel method has for both elements a recovery which approached closest 100% recovery. All measured concentrations were above the detection limits (LOD) and above the limit of quantification (LOQ). The concentrations were at least a factor 80 above the LOQ, except As was a factor 10 above the LOQ. For Cd analysed in standard mode, all methods are in a range of 20% deviation from 100% recovery, except the MW + Open vessel + aqua regia method on 114Cd. In KED mode, all methods are in a range of 20% deviation from 100% recovery, except the MW + Open vessel + aqua regia method. For As, only the open vessel and closed vessel are in the range of 20% deviation from 100% recovery. A solution to get better results for Pb, is to measure the signal of Pb as the sum of the signals of the three most abundant isotopes (Pb206, Pb207 and Pb208) (Section 5.2 (Literature review), Section 4.3 (Materials and Methods))93. A solution to obtain better results for Hg is to make sure that metallic mercury is not volatilized (Section 5.6, Literature review). For Hg, the graph shows a difference in recovery rate for the MW + Closed vessel + aqua regia technique and the other techniques. The MW closed vessel technique retains better the Hg. The other techniques are open digestions which can explain the loss of Hg via volatilization. To retain Hg in solution Au can be added before the digestion of the sample as explained in Section 5.6 (Literature review). As a result the closed vessel method was selected for further fine-tuning because it has acceptable results for Hg, Cd and As. For Pb and Hg previous solution will be applied during the fine tuning and checked if the problem is solved.

1.4

1.2

1 MW+Closed vessel+aqua regia

0.8 MW+open vessel+aqua regia Dry ashing+HNO3 0.6 Hotplate+HNO3+HD H2O2

0.4 Hotplate+HNO3+LD H2O2

0.2 100% recovery

0 112Cd 114Cd 208Pb 202Hg 75As 202Hg 112Cd 114Cd (Standard) (Standard) (Standard) (Standard) (KED) (KED) (KED) (KED)

Figure 19: Recovery rates for plankton reference samples treated with 5 different methods: (i) MW + closed vessel + aqua regia, (ii) MW + open vessel + aqua regia, (iii) Dry ashing + nitric acid, iv) Hotplate digestion +

HNO3 + HD H2O2 and v) Hotplate digestion + HNO3 + LD H2O2.

3.1.2. Fish Figure 20 shows the result from the first digestion attempt on the fish muscle reference material. All measured concentrations were above the LOD and the LOQ. The concentrations were at least a factor 80 above the LOQ, except the As concentrations were a factor 10 above the LOQ. When 112Cd is analysed, all methods are in a range of 20% deviation from 100% recovery in standard and KED mode. MW + Open vessel 114 + aqua regia and Dry ashing + HNO3 are not in the range of 20% for Cd in standard mode. In KED mode, 114Cd obtains good recovery rates for all methods except for the MW + Closed vessel + aqua regia. This closed vessel technique has a deviation above 80% relative to full recovery. Important to mention is the

high standard deviations (std), in particular when the hotplate methods (Hotplate + dry ashing + HNO3 and Hotplate + HNO3 + HD/LD H2O2) are applied in KED mode. This high standard deviation can be explained due to the fact that residue remained after the hotplate destructions. The non-uniform conditions during destruction could have caused the variation in recovery rates between the replicates. When 202Hg was analysed the best recovery rate was obtained applying Hotplate + HNO3 + LD H2O2 in standard mode and the best recovery rate was obtained by the MW + Closed vessel + aqua regia method in KED mode. In standard mode, this closed vessel method was also in a range of 20% from 100% recovery. For 202Hg in standard mode an abnormal deviation can be seen when the Hotplate + HNO3 + LD H2O2 was applied, here a deviation is obtained above 120%. For As only the MW + Closed and Open vessel methods had acceptable recovery rates. No recovery rates for Pb were calculated because the reference material did not have a certified concentration of Pb. The MW + Closed vessel + aqua regia had acceptable recovery rates for 112Cd, 114Cd and 202Hg in standard mode and acceptable recovery rates for 75As, 202Hg and 112Cd in KED mode. As a result this closed vessel method was selected for further fine-tuning. Only when 114Cd was analysed with the ICP-MS a deviation above 80% occurred from the optimal recovery rate. A possible explanation could be the presence of molybdenum or ruthenium in the Fish muscle reference material which can result in the

formation of 98Mo16O+, 98Ru16O+. These polyatomic species have a m/z of 114, the same as the m/z of 114Cd. 114Cd in standard mode has also elevated values for the recovery rates and has a high std of 0.4 but the deviation from the optimal recovery rate is lower compared to 114Cd analysed in KED. This is strange because the KED mode should lower the interference of polyatomic ions. Because of this reason, the elevated recovery of 114Cd will possible have another cause than the interference of a polyatomic species.

2.4 2.2 2 1.8 MW+Closed vessel+aqua regia 1.6 1.4 MW+Open vessel+aqua regia 1.2 Dry ashing+HNO3 1 Hotplate+HNO3+HD H2O2 0.8 0.6 Hotplate+HNO3+LD H2O2 0.4 100% recovery 0.2 0 112Cd 114Cd 202Hg 75As 202Hg 112Cd 114Cd (Standard) (Standard) (Standard) (KED) (KED) (KED) (KED) Figure 20: Recovery rates for fish muscle reference samples treated with 5 different methods: (i) MW + closed vessel + aqua regia, (ii) MW + open vessel + aqua regia, (iii) Dry ashing + nitric acid, iv) Hotplate

digestion + HNO3 + HD H2O2 and v) Hotplate digestion + HNO3 + LD H2O2.

3.2. Fine tuning During fine tuning, digestions were carried out on plankton, fish muscle, mussels tissue and lake sediment reference material. The reference samples of lake sediment and fish muscle did not contain certified Pb concentrations. To obtain recovery rates for Pb, mussels tissue reference material was selected as an alternative for the fish muscle reference material. To obtain recovery rates for Pb in sediment, a spike of Pb (0.01 mg/L) was added to the lake sediment sample from Lake Uru Uru (sampling spot 1, replicate 1). As an improvement 20 L Au was added and the sum of the different Pb signals was used to calibrate and calculate the total amount of Pb occurring (Total Pb) in the reference material (Section 5.2 and Section 5.6, Literature review). Furthermore two closed vessel procedures were applied on the reference samples. The first, a MW closed vessel method with aqua regia treatment and the second a MW closed vessel with only nitric acid as oxidising agent (Section 4, Materials and Methods). This was done to check if it would be possible to digest the samples with only nitric acid as digestion agent and to prevent interferences caused by 40Ar35Cl+, 38Ar37Cl+. These two polyatomic species have a m/z ratio of 75, the same as 75As. This can cause spectroscopic interferences (Section 5.3, Literature review).

3.2.1. Plankton Figure 21 shows the recovery rates of the plankton reference samples, digested with the closed vessel treatment: aqua regia and HNO3. All measured concentrations were above the LOD and the LOQ. For Hg, both the HNO3 and the aqua regia treatment have recovery rates in the range of 20% deviation from the 200 optimal value in KED. Hg has the smallest deviation from the optimal value in KED (recovery: 0.92 (HNO3) and 0.89 (aqua regia)). When analysing Hg, the optimal treatment would be the HNO3 treatment in KED on 200Hg. It has the best recovery rate and the smallest std (std: 0.03). The recovery rates for Pb are in the range of 20% deviation from the optimal value for both treatments, but the HNO3 treatment has a smaller std for the total Pb and the recovery rate has the smallest deviation from 100% recovery. When analysing

Pb, the optimal digestion would be the HNO3 treatment in standard mode. Analysing As, as well the HNO3 as the aqua regia treatment have elevated recovery rates, but the aqua regia treatment has still a recovery rate between the range of 20% deviation from the optimal value. For Cd, the aqua regia treatment has the best recovery rates but has larger std’s compare to the HNO3 treatment. The recovery rates for the aqua 114 regia treatment are all within range of 20% and the HNO3 treatment is in range of 20% deviation for Cd in standard mode and for 112Cd and 114Cd in KED mode. Tabel 16 gives a summary of the methods and their deviation from full recovery. Based on this summary, the aqua regia treatment gives a solution to analyse Cd, Pb, Hg and As within the range of a 20% deviation from full recovery, using only one method. 1.6 1.4 1.2 1 HNO3 0.8 0.6 Aqua regia 0.4 100% recovery 0.2 0 112Cd 114Cd Total Pb 202Hg 200Hg 75As 202Hg 200Hg 112Cd 114Cd (Standard)(Standard)(Standard)(Standard)(Standard) (KED) (KED) (KED) (KED) (KED)

Figure 21: Recovery rates for plankton reference samples, digested via MW closed vessel method: HNO3 treatment and aqua regia.

Table 16: Summary of methods applied on plankton reference samples and their deviation from full recovery: deviation in range of 20% (V), deviation out of range (X) and no data (/).

Treatment Aqua regia HNO3

Mode Standard KED Standard KED

Cd V V X V Pb V / V / Hg X V V V As / V / X

3.2.2. Fish Figure 22 shows the recovery rates of the fish muscle reference material digested with the MW closed vessel treatment: HNO3 and aqua regia. All measured concentrations were above the LOD and the LOQ.

When the HNO3 treatment is applied, recovery rates are obtained within a range of 20% for all elements except for 75As and 112Cd. When the aqua regia method is applied, 112Cd and 114Cd in standard mode and 75As, 112Cd and 114Cd in KED mode are not in the range of 20% from the optimal value. The low recovery rates of Cd were not expected, in the previous analysis good recovery rates for Cd were obtained using the aqua regia technique. Table 17 gives a summary of the methods and their deviation from full recovery.

Based on this summary, the HNO3 treatment gives a solution to analyse Cd, Pb and Hg within the range of a 20% deviation from full recovery, using only one method. The aqua regia method is not selected for further analyses, because of the low recoveries for Cd in standard as in KED mode. For As, recovery rates with a deviation above 20% are obtained for the HNO3 and the aqua regia method. When the aqua regia or

HNO3 method is applied it has to be mentioned that the obtained concentrations for As could be an overestimation. 1.6

1.4

1.2

1 HNO3 0.8 Aqua regia 0.6 100% recovery 0.4

0.2

0 112Cd 114Cd Total Pb 202Hg 200Hg 75As 202Hg 200Hg 112Cd 114Cd (Standard)(Standard)(Standard)(Standard)(Standard) (KED) (KED) (KED) (KED) (KED)

Figure 22: Recovery rates for fish muscle reference samples, digested via MW closed vessel method: HNO3 treatment and aqua regia.

Table 17: Summary of methods applied on fish muscle reference samples and their deviation from full recovery: deviation in range of 20% (V), deviation out of range (X) and no data (/).

Treatment Aqua regia HNO3 Mode Standard KED Standard KED

Cd X X V V

Pb V / V / Hg V V V V As / X / X

3.2.3. Sediment Figure 23 shows the recovery rates of the lake sediment reference material and the recovery of the spike of 0.01 mg/L Pb added to the sediment sample from Lake Uru Uru. All measured concentrations were above the LOD and the LOQ, except for Pb analysed with the MW + Closed vessel + HNO3 method. The samples were digested with the MW closed vessel treatment: HNO3 and aqua regia. When the HNO3 treatment is applied, recovery rates are obtained within a range of 20% for all elements, except for Pb. When the aqua regia method is applied, 112Cd in standard mode and 75As, 112Cd and 114Cd in KED mode are not in the range of 20% from the optimal value. Tabel 18 gives a summary of the methods and their deviation from full recovery. Based on this summary, the HNO3 treatment gives a solution to analyse Cd, Hg and As within the range of 20% deviation from full recovery, using only one method. For Pb no data were obtained for the

HNO3 treatment. The reference samples did not contain certified Pb so a spike (0.01 mg/L) was added before digestion. When Pb was analysed, negative values were obtained (due to a detector overflow) via the HNO3 method, these values are not plotted in Figure 23. When the aqua regia method is applied this method can be used to analyse Cd, Hg, and Pb, but for As the recovery rate deviates more than 20% from the optimal value. The MW + Closed vessel+ HNO3 method is selected for further analyses, because of the better recovery for As.

1.6 1.4 1.2 1 0.8 HNO3 0.6 Aqua regia 0.4 100% recovery 0.2 0 112Cd 114Cd Total Pb 202Hg 200Hg 75As 202Hg 200Hg 112Cd 114Cd (Standard) (Standard)(standard) (Standard) (Standard) (KED) (KED) (KED) (KED) (KED) Spike

Figure 23: Recovery rates for lake sediment reference samples, digested via closed vessel: HNO3 treatment and aqua regia.

Table 18: Summary of methods applied on sediment reference samples and their deviation from full recovery: deviation in range of 20% (V), deviation out of range (X) and no data (/).

Treatment Aqua regia HNO3 Mode Standard KED Standard KED Cd X V V V Pb / V / / Hg V V V V As / X / V

4. Samples Lake Titicaca and Uru Uru 4.1. Sediment 4.1.1. Recovery rates

To digest the sediment samples, the MW + Closed vessel + HNO3 treatment was applied. To analyse the samples, KED-mode was used on the ICP-MS. Figure 24 gives the recoveries of the reference samples digested and analysed at the same time and manner as the samples from Bolivia. The reference samples contained no Pb, so no information is available about the recoveries and accuracy could be judged for Pb analysis. Figure 24 shows the recoveries for the lake sediment reference samples. Based on these results, the concentrations analysed via 75As, 202Hg and 114Cd in KED mode were selected to use in further data analysis. 75As has an elevated recovery (1.26). When using these data it has to be considered that in reality the As concentration could possibly be lower. 1.6 1.4 1.2 1 0.8 Recovery rate sediment 0.6 100% recovery 0.4 0.2 0 75As 202Hg 200Hg 112Cd 114Cd

Figure 24: Recovery rates for reference lake sediment samples, digested and analysed together with the sediment samples from Bolivia.

4.1.2. Metals and metalloid concentration in the sediment samples In Figure 25, the sediment metal and metalloid concentrations are given for each sampling spot. In the Addendum (Figure 4) the metal and metalloid concentrations are visualised per sampling spot on a map. For As, Hg, Cd and Pb the LOQ was 0.8 mg/kg, 0.06 mg/kg, 0.009 mg/kg and 0.06 mg/kg. All measured concentrations were above the LOD and theLOQ, except the Hg concentrations measured at Isla de la Luna, Cohana bay and Huatajata were close to the LOQ. The As concentrations in sediment did not differ significantly between the locations from the lake Titicaca itself (ANOVA, p>0.05). However, the As concentrations measured in Lake Uru Uru differed significantly from those measured in lake Titicaca (ANOVA, p<0.05). Because the As concentrations in sediment from lake Titicaca did not vary significantly, the As concentrations of Lake Titicaca are plotted in Figure 26. The mean As concentration of all sampling points at Lake Titicaca (29.27 mg/kg) is just below the PEC value of 33 mg/kg but above the TEC value of 9.8 mg/kg. Below the TEC no adverse effects are to be expected, above the PEC enhanced adverse effects on the aquatic environment can be expected. The mean As sediment concentration from Lake Uru Uru (101,6 mg/kg) is above the PEC level. Figure 25 (i) shows concentrations exceeding the As PEC level at the locations: Escoma (E), Tiquina (TQ), Taraco (TA) and at Lake Uru Uru (UU). For Hg, Cd and Pb, the concentrations in all sediment samples were below the TEC level. Average sediment concentrations in the Titicaca Lake are: 0.05 mg/kg for Hg, 0.13 mg/kg for Cd and 6.08 mg/kg for Pb. The Tuckey adjusted p-values for the As, Hg, Cd and Pb concentrations are given in the Addendum: Table 3.

i) ii)

iii) iv) Figure 25: Metal and metalloid concentrations (i) As, ii) Hg, iii) Cd and iv) Pb) in sediment, sampled at Escoma (E), Isla de la Luna (IDL), Tiquina (TQ) Huatajata (H), a platform of UMSA (X), Cohana bay (C), Taraco (TA), Desaguadero (D) and Uru Uru (UU). The TEC (….) and PEC (---) for As, Hg, Cd and Pb in sediment is based on the study of MacDonald et al. (2000)76.

Figure 26: Arsenic concentrations in sediment sampled at Lake Titicaca (T) and Uru Uru (U). The TEC (….) and PEC (---) for As in sediment is based on the study of MacDonald et al. (2000)76.

4.2. Water 4.2.1. Recovery rates To digest the water samples, the treatment described in Section 4.1.3 (Materials and Methods) was used. Figure 27 gives the recoveries of the spikes (0.01 mg/L: As, Pb, Cd, Hg) applied in the water samples. It is important to mention that during this digestion method p.a. quality grade HCl was used and no Au was added before digestion and analysis. An improvement would have been to use p.p. graded HCl and to add 40 35 + 20L Au (Discussion: Section 4). To avoid interferences of Ar Cl , only HNO3 could be added as oxidising agent (no addition of HCl). Figure 27 shows the abnormal low recovery for Hg, Cd has a recovery in the range of 20% deviation from full recovery and Pb and As have abnormal high recoveries. It is important to keep in mind that the Hg concentrations normally should be higher and the concentrations of As and Pb lower compared to the real Hg, As and Pb concentrations occurring in Lake Titicaca.

2.2 2 1.8 1.6 1.4 1.2 Recovery rate water 1 100% recovery 0.8 0.6 0.4 0.2 0 208Pb 202Hg 75As 112Cd (standard) (standard) (KED) (KED) Figure 27: Recovery rates spiked water sample analysed in parallel with the water samples from Bolivia.

4.2.2. Results water Lake Titicaca Water properties At every sampling spot the salinity, conductivity, pH, T and dissolved oxygen were measured. Values are given in Table 19. The salinity and conductivity are very high in Lake Uru Uru compared with Lake Titicaca. A possible explanation is the high evaporation occurring in the lake which causes the rise in salinity and conductivity. The redox potential was not measured during the sampling campaign. An estimation of the redox potential can be made based on the results of G. Sarret et al. (2019)8. The mean redox potential of two sampling locations located at the middle of Lago Menor was approximately -100mV8. The same study 2- reported relatively high sulphur content (mostly as SO4 ) in the lake ranging from 131-277mg/L at Lake Titicaca. In Lake Uru Uru concentrations up to 500 mg/L were measured. At Lake Titicaca alkaline conditions are occurring (pH>7), the highest pH was measured at Lake Uru Uru (pH: 8.94).

Table 19: Properties of the water at the Lake Titicaca and Uru Uru. Water was sampled at Isla de la Luna (IDL), Huatajata (H), Cohana bay (C), a platform of UMSA (X), Taraco (TA), Desaguadero (D), Escoma (E), Tiquina( T), Uru Uru (UU). Site Salinity(g/kg) Conductivity(S/cm) pH T(°C) DO(mg/L) IDL 0,69 1370 8,76 13,18 6,72 H 0,73 1427 8,80 11,73 7,89 C 0,71 1397 8,40 11,47 7,50 X 0,73 1431 8,86 11,25 7,66 TA 0,89 1747 8,68 11,55 8,62 D 0,88 1727 8,68 11,25 7,93 E 0,71 1394 8,56 12,02 7,96 TQ 0,68 1343 8,40 13,38 6,55 UU 3,07 5610 8,94 12,81 7,32

Metal and metalloid concentrations in water samples In Figure 28 the metal and metalloid concentrations in water are given for each sampling spot. In the addendum (Figure 5) the metal and metalloid concentrations are visualised per sampling spot on a map. For As, Hg, Cd and Pb the LOQ was 0.2 g/L, 0.04 g/kg, 0.01 g/kg and 0.03 g/kg. All measured concentrations were above the LOD and the LOQ, except for the Cd and Pb concentrations. The Cd concentrations measured at Cohana bay and Huatajata. These concentrations are close or just below the LOQ. For Pb, the concentrations are below the detection limit (LOD: 0.01 g/L), so Pb concentrations are below a concentration of 0.01 g/L, which is 1000 times below the threshold value set by the European Commission (EC) for drinking water (threshold value EC: 10 g/L). The mean Hg and Cd concentrations are below the threshold set by the EC (Hg: 1 g/L, Cd: 5 g/L), but at Lago Mayor (sampling spot: Escoma and Isla de la Luna) elevated Cd concentrations are detected compared to the other sampling spots (p values just above 0.05, Addendum Table 4). For Hg no significant differences were detected between the sampling points at Lake Titicaca and Uru Uru except between Desaguadero and Taraco (ANOVA, p: 0.045) and between Desaguadero and Uru Uru (p: 0.024). Only for As, the threshold value of 10 g/L was exceeded at Huatajata (12 g/L), Cohana bay (11 g/L), Taraco (12 g/L), Desaguadero (12 g/L) and at Lake Uru Uru (64 g/L). The As concentrations in Lake Uru Uru are the highest with a mean concentration of 64 g/L and a median of 58 g/L, this is six times higher compared with the allowed As concentration. There was no significant difference between the As concentrations at Lake Titicaca (ANOVA, p>0.05). Only the As concentrations from Lake Uru Uru differed significantly from all As concentrations measured at Lake Titicaca (ANOVA, p<0.05). Because the As concentrations in sediment at lake Titicaca did not differ significantly a boxplot was constructed for all As concentrations measured at Lake Titicaca (Figure 29). The average concentration at Lake Titicaca is 11 g/L, which is above the safety threshold level of 10 g/L.

(i) (ii)

(iii) Figure 28: Metal and metalloid concentrations (i) As, ii) Hg and iii) Cd) in water sampled at Escoma (E), Isla de la Luna (IDL), Huatajata (H), Cohana bay (C), Taraco (TA), Desaguadero (D) and Uru Uru (UU). The safety threshold values (…...) for metal concentrations in drinkingwater are based on the European directive 98/83/EG71.

Figure 29: Arsenic concentrations in water at Lake Titicaca (T) and Uru Uru (U). The safety threshold value (…..) for As in drinkingwater is based on the European directive 98/83/EG71.

4.3. Fish 4.3.1. Recovery rates Table 20 gives the recoveries of the reference samples digested and analysed together with the samples from Bolivia. The reference samples contained no Pb, no information is available about the recoveries and accuracy could be judged for Pb analysis. Table 20 shows the recoveries for the fish muscle reference samples and the isotopes with the best recoveries. Based on these results, the concentrations obtained via the selected isotopes (Table 20) in KED mode were used in further data analysis. Important to mention is that the samples digested in caroussel 1 will probably have to low Hg concentrations and the samples in carousel 4 too low Cd concentrations. Furthermore, As has for all carousels elevated recoveries. This could be expected, as this trend also occurred during the optimization of the digestion method.

Table 20: Recoveries of the fish muscle reference material for 75As, 200Hg , 202Hg, 112Cd, 114Cd in KED mode. Green: in the range of 20% deviation, Orange: 20-50% deviation, red: >50% deviation from full recovery. Carousel 75As 202Hg 200Hg 112Cd 114Cd Selected isotopes 1 1.35 0.56 0.60 1.47 0.86 75As, 200Hg, 114Cd 2 1.38 1.08 1.09 1.00 1.39 75As, 202Hg, 112Cd 3 1.28 1.03 1.05 0.87 0.71 75As, 202Hg, 112Cd 4 1.28 0.99 0.97 -0.70 0.23 75As, 202Hg, 114Cd 5 1.29 0.95 0.95 0.79 0.93 75As, 200Hg, 114Cd 6 1.28 1.0 1.04 0.69 1.11 75As, 202Hg, 114Cd

4.3.2. Metals and metalloid concentrations in fish samples In Figure 30, the metal and metalloid concentrations for As, Hg, Cd and Pb are given for the seven different fish species from lake Titicaca. All measured As and Hg concentrations were above the LOD and above the LOQ. Some of the Cd and Pb concentrations were below the LOD, it was assumed that in these fish samples no Pb occurred. The LOD of Pb was 0.04 g/kg and the LOD of Hg 5 g/kg. These results approximate the distribution of the metal or metalloid concentration in fish meant for human consumption from lake Titicaca. All fish were caught in nets used by the local fishermen who sell their fish at the local markets and at the markets at El Alto and at La Paz. Normality and homogeneity of variances of the dataset were tested and could not be accepted for As, Hg, Cd and Pb, so one way ANOVA cannot be applied. Instead the Wilcoxon signed-rank test was applied. For Cd and Pb all mean concentrations are below the threshold values set by the EC (threshold values: Cd=0.05 mg/kg , Pb=0.3 mg/kg). Table 5 in the Addendum gives the average concentrations of all seven fish species caught in Lake Titicaca. The adjusted p-values are given in Table 6 of the Addendum. For Pb, the benthopelagic O. agassizii (0.052 mg/kg) and the epipelagic O. gilsoni (0.045 mg/kg) had the highest concentrations. For Cd, the epipelagic O. ispi (0.016 mg/kg) and the benthopelagic O.agassizii (0.015 mg/kg) and O. luteus (0.010 mg/kg) had the highest concentrations. For As and Hg elevated concentrations in the fish species occur. For the As concentrations, all species have average concentrations above the threshold value (0.1 mg/kg). O. ispi, O. agassizii, Trichomycterus spp. and O. luteus have the highest metalloid concentrations: 1.0 mg/kg, 1.0 mg/kg, 0.88 mg/kg and 0.54 mg/kg, respectively. The lowest mean concentrations are measured in O. bonariensis and O. mykiss: 0.34 and 0.25 mg/kg, respectively (Table 5, Addendum). O. Ispi has significantly higher As concentrations compared with O. bonariensis, O. gilsoni, O. luteus and O. mykiss. O. agassizii has significantly higher As concentrations

compared with O. bonarienis and O. mykiss. For the Hg concentrations, only O. bonariensis has a mean concentration (0.61 mg/kg) above the threshold level set by the EC (0.5 mg/kg). The Hg concentrations in O. bonariensis are significantly higher compared with the mean Hg concentrations occurring in all other fish species except in Trichomycterus spp. The Hg concentrations in Trychomcterus spp. varies over a large range and the first quadrille is situated above the threshold concentration. The lowest mean Hg concentrations occur in O. Luteus (0.12 mg/kg), O. gilsoni (0.083 mg/kg) and O. mykiss (0.046 mg/kg). Only O. luteus was caught at Lake Uru Uru and analysed. Both the median As and Hg concentrations in O. luteus caught at Lake Uru Uru are above the threshold values and showed a significant difference in concentration (Wilcoxon, p<0.05) (Figure 31). At Lake Uru Uru, the mean As and Hg concentration are 2.3 mg/kg and 0.58 mg/kg, respectively. This is approximately 4 times higher compare to the mean concentration at Lake Titicaca.

i) ii)

iii) iv) Figure 30: Metal and metalloid concentrations (i) As, ii) Hg, iii) Cd and iv) Pb ) in seven fish species from Lake Titicaca: Trichomycterus spp. (Trycho), O. ispi (ispi), O. gilsoni (gilsoni), O. agassizii (agassizii), O. luteus (luteus), O.bonariensis (bonar) and O. mykiss (mykiss). The safety threshold values (___) for metal concentrations in fish are based on the European directive 1881/2006/EG for Hg, Cd and Pb, for As the international standard is used as a threshold value72,73

i) ii) Figure 31: As (i) and Hg (ii) concentrations in O. luteus sampled in Lake Titicaca (T) and Uru Uru (UU). The safety threshold values (___) for metal concentrations in fish are based on the European directive 1881/2006/EG for Hg, for As the international standard is used as a threshold value72,73

5. Multi-way ANOVA analysis Most fish samples were collected from O. luteus (61 dissected fish) and O. bonariensis (33 dissected fish), O. luteus was collected at all fish sampling locations and O. bonariensis at three different sampling locations (Section 2.3, Materials and Methods.). Because of the large amount of data and the fact that these species are sampled at multiple locations these two species were selected to undergo a multiway ANOVA analysis. O. luteus will represent a model for the benthopelagic, mainly omnivorous species and O. bonariensis as a pelagic carnivore fish species. This analysis will be conducted on the two elements who pose the biggest thread at the lake: As and Hg. Firstly a multi-way linear function was constructed, as explained in Section 5 (Materials and Methods), after which the ANOVA function was used to determine how much variability in the outcome variable (As and Hg) is explained by one of the parameters.

5.1. ANOVA: O. luteus 5.1.1. Mercury in O. luteus When the ANOVA function was applied on the linear function with Hg as output variable, the weight and gutted weight, T, pH and salinity showed significant interaction (ANOVA, p<0.05). The length of the fish is significantly correlated with the weight of the fish ( r= 0.689, p= 8.346e-9). When two parameters are correlated, is better to take one of these parameters out of the function. Also carousel (p: 0.59) and mass of the sample (p: 0.39) were taken out of the function, as these are parameters related to the applied digestion method. This is beneficial for the obtained results, there is no significant analysis induced variability. The factor variable gender was also taken out of the function (p: 0.34). Secondly, an optimal function was selected, using a backward selection based on the AIC-criterion. When ANOVA was applied on this optimised function, it showed that most of the variance could be explained by the mean conductivity (ANOVA, p: 6.4e-12), gutted weight (ANOVA, p:0.000133), the T (ANOVA, p: 0.01) and the weight (ANOVA, p:0.018).

5.1.2. Arsenic in O. luteus When the ANOVA function was applied on the linear function, only the DO, pH and mean salinity showed significant interaction (ANOVA, p<0.05). The same manual selection was applied as done for Hg: the length (p: 0.55), gender (p:0.62), carousel (p:0.91) and mass (p: 0.31) were removed from the first model. The same three parameters showed significant impact on the As concentrations. At last the weight and gutted weight were manually removed in this model only the mean salinity (ANOVA, p: 0.0003), DO (ANOVA, p: 0.009) and pH (ANOVA, p: 0.031) could significantly explain the variance of As in O. luteus.

5.2. ANOVA: O. bonariensis 5.2.1. Mercury in O. bonariensis The length, T and DO showed significant interaction (ANOVA, p<0.05), when the ANOVA function was applied on the linear function. The length of the fish is significantly correlated with the weight ( r= 0.965, p= 2.2e-16) and gutted weight of the fish (r= 0.966, p=2.2e-16). As previously mentioned, it is better to take these parameters out of the function. Similar to the model selection of O. luteus, carrousel, mass of the sample and the factor variable gender were taken out of the function. There appears to be no significant analysis induced variability. Secondly, an optimal linear function was selected using a backward selection based on the AIC-criterion. When ANOVA was applied on this optimised function it showed that most of the variance could be explained by the DO (ANOVA, p: 0.0003), the length (ANOVA, p:0.0018) and T (ANOVA, p:0.0336).

5.2.2. Arsenic in O. bonariensis At last, when the ANOVA function was applied on the model for As, the length, the weight, the gutted weight, T and DO showed significant interaction (ANOVA, p<0.05). The gutted weight of the fish (ANOVA, p: 0.0008) was selected to maintain in the model, the weight (ANOVA, p: 0.0437) and length of the fish (p: 0.0607) were left out. Also the gender, carousel and massa of the analysed sample were excluded from the new model (ANOVA, p>0). Furthermore, an optimal model was selected using a backward stepwise selection based on the AIC-criterion. An optimal function was selected based on the gutted weight (p: 0.503), T (p:0.03849) and DO (p: 0.008). The T and DO could significantly explain the variance in the output variable.

Discussion 1. Morphology O.ispi, O. gilsoni and O. bonariensis In this study the liver was dissected because it is a key organ in detoxification processes1. The extra documentation (Section 1, Results) gives an extension on the study of the Laboratory of Animal Nutrition and can be used as a template to dissect O. bonariensis, O. gilsoni and O. Ispi and to locate the liver in future studies. Extra pictures taken during dissection are given in the Addendum (Figures 2 and 3). During dissection an O. ispi was discovered infected with flatworms (Figure 2). Besides the pressure on the fish by extensive fishery and metal and metalloid pollution, the drop in the Orestias population can also be caused by parasite infections112,133. For example the introduction of O. mykiss infected with a protozoarian parasite (Ichthyophthirius multifiliis) caused the death of 18 000 000 fish of the genus Orestias112. Extra investigation into the different parasites and the infected species in the lake could provide new insights into the vulnerability of the fish species occurring in Lake Titicaca.

2. Digestion method In this thesis a procedure is developed to digest and analyse As, Hg, Cd and Pb with one method. The closed vessel, microwave technique was selected because of its fastness, the complete digestion of the organic matter, high pressures being possible, high repeatability, better entrainment of the gasses and better recoveries during the optimisation process. Negative aspects are the higher cost and the relatively low amount of samples that can be processed per batch compared to the other techniques. At the ECOCHEM laboratory of the Department of Green Chemistry and Technology (Ghent University), the microwave is extensively used, which resulted in a lower availability of the equipment. During all digestions, reference samples were analysed to check the accuracy of the obtained results. To digest the sediment samples, the closed vessel technique with nitric acid as oxidising agent provides a solution to analyse Cd, Hg and As within a range of 20% deviation. For Pb negative recovery rates were obtained. This negative value can be explained by the fact that 0.5 g sediment instead of 0.2 g (aqua regia method) was used to digest. This could have caused the detector overflow error. To resolve this problem the sample should be diluted. The closed vessel technique with the aqua regia treatment provides a solution to analyse Cd, Hg and Pb in sediment, but for As the recovery deviated more than 20% from the optimal value. To digest fish muscle tissue, the closed vessel HNO3 treatment gives a solution to analyse Cd, Pb and Hg within the range of a 20% deviation from full recovery, using only one method. For As, recovery rates with a deviation above 20% are obtained when the HNO3 closed vessel technique is applied. When the HNO3 method is applied it has to be taken into account that the obtained concentrations for As could be higher than the real concentrations. To digest amphipods samples, aqua regia treatment combined with a closed vessel microwave treatment gives a solution to analyse Cd, Pb, Hg and As within the range of 20% deviation. The HNO3 treatment had even better recoveries for Cd, Hg and Pb but the recoveries for As were above 20% deviation from full recovery.

Both the aqua regia (recovery: 119%) as the HNO3 treatment (recovery: 140%) had elevated recoveries for - As. The advantage of the HNO3 treatment is that no HCl is used. Cl is very reactive and during ICP-MS analysis interfering polyatomic species can be formed (e.g. 40Ar35Cl+ and 40Ca35Cl+). It is strange that the 75 HNO3 treatment had a higher recovery for As compared with the aqua regia treatment. To deal with the polyatomic interferences for As, the DRC mode could be applied on the ICP-MS. Using CH4 as reactive gas, the interference of ArCl+ is eliminated and the background signal reduced134. To analyse samples with a complex matrix and high salinity, the use of oxygen as reactive gas provides better accuracy. In the study of

M. Grotti and R. Frache (2007), it appeared that the use of CH4 as reactive gas was not able to reduce the spectral interferences on the m/z ratio of 75120. However, using oxygen, 75As+ is efficiently converted to 75As16O+, with a m/z of 91. The use of oxygen as reactive gas gives a solution to deal with interferences caused by 40Ar35Cl+ and 40Ca35Cl+ 120,134. An extra improvement could be the addition of 4 v/v% methanol to the analysis solution134. It has been reported that carbon addition (e.g. methanol) to the plasma can improve the signal background ratio by decreasing the background signal and increasing the signal of As+ or AsO+ 134,135. The study of W. Guo et al. (2011) reported a 4-fold improvement for the signal intensity of 75As16O+ and an improvement of the signal to background ratio from 0.18 to 205136. To conclude, DRC mode with O2 as reactive gas and the addition of 4 v/v% methanol could improve the accuracy of the As analyses.

3. Sediment from Lake Titicaca

To digest the sediment samples, the HNO3 digestion was applied. During the digestion of the samples, the reference samples did not contain certified Pb, so no information is available about the recoveries and accuracy could be judged for Pb analysis. The sediment Pb concentrations obtained in this thesis, can be used to get an indication of the order of magnitude of the Pb concentrations occurring in sediment from Lake Titicaca. To check the reliability of the obtained concentrations in sediment in this study, we compared the As sediment concentrations with results published in the study of G. Sarret et al. (2019) and the Hg, Cd and Pb concentrations in sediment with results published in the study of M. Monroy et al. (2014). These values were in the same order of magnitude. For Pb, the sediment concentrations in the study of M. Monroy et al. (2014) ranged from 2.2-75.0 mg/kg (mean: 20 mg/kg) at the Bolivian part of Lago Mayor1. The high concentration of 75 mg/kg was measured at the inlet of the river Ramis, one of the most polluted rivers at the lake (Section 1.1.1, Literature review), which can explain the high concentration. The mean concentration measured at the Peruvian side of the lake (20 mg/kg), is in the same order of magnitude as the results obtained in this thesis (Pb concentrations: 0.31-19 mg/kg). The sediment quality guidelines constructed in the study of MacDonald et al. (2000) can be used to identify contaminated areas and determine the potential of injury to sediment-dwelling organisms (e.g. bentic fish species and amphipods) in freshwater ecosystems. It is important to use these sediment guidelines in combination with bioaccumulation tests and tissue residue guidelines76. In this thesis, besides the sediment characteristics, also the water metal and metalloid concentrations and the metal and metalloid bioaccumulation in fish are evaluated. Below the TEC no adverse effects are to be expected, above the PEC enhanced adverse effect on the aquatic environment can be expected especially for the sediment dwelling organisms. The Hg, Cd and Pb concentrations in sediment from Lake Titicaca and Uru Uru were below the TEC values, so no adverse effects are to be expected. For As, elevated recoveries in sediment (Results: 4.1.1. Recovery rates) were obtained. It has to be considered that in reality the As concentration could possibly be lower. Similar results for As in sediment were obtained in the study of G. Sarret (2019), which indicates that the results in this master thesis are of the right order of magnitude. The mean As sediment concentration in lake Titicaca is just below the PEC and above the TEC, but the upper quartile of the data is located above this PEC. It can be concluded, that elevated As sediment concentrations occur in the lake and adverse effects on the aquatic environment can be expected especially at Escoma, Tiquina, and Lake Uru Uru. These locations have As concentrations above the PEC value. At Taraco, an As concentration above the PEC was measured but info about this site is not reliable. At Taraco it was only possible to take one sediment sample because of the rock formations at the bottom of the lake. This concentration could just as well be an outlier due to a point source contamination. To be sure, at least three sampling locations at one spot are essential to see the

variation occurring at one sampling spot. The mean As, Hg, Cd and Pb concentrations in sediment from Escoma (Lago Mayor) are higher compared to the sediment concentrations from Isla de la Luna (Lago Menor) and the sampling points from Lago Menor (except the Cd concentrations at Tiquina). This can be explained by the fact that Escoma is located at the instream of the river Huaycho. As explained in Section 1.1.1 (Literature review), the main input of pollution comes via waste discharge through rivers. According to information giving by the department of Limnologia, UMSA University, upstream this river, mining activity is taking place. This can explain the elevated metal and metalloid concentrations at Escoma. For Cohana Bay the same trend was expected because of the inflow of the Katari River but this did not occur. The sampling point at Cohana Bay was located further away from the shore and at the instream of the Katari river wetland conditions are present. It has been shown that wetlands can act as a filter and they can efficiently remove pollutants such as metals from water137. The study of G. Sarret et al. (2019) showed that the totora plants and periphyton present in the Lake Titicaca (abundant at the instream of the Katari river) can accumulate As8. These conditions can explain the relative low concentrations in the sediment at the sampling location near to Cohana Bay.

4. Water from Lake Titicaca It is important to mention that during the digestion method p.a. quality grade HCl was used and no Au was added before digestion and analysis. During analysis the water samples were spiked with 10 g/L Pb, Hg, As and Cd to check the recoveries. Hg had too low recoveries, but a possible solution can be to add Au (Section 5.6 Mercury analysis). Pb and especially As obtained abnormally high recoveries. A first possible explanation is the use of p.a. HCl which could have caused contamination of Pb and As. Secondly the elevated recovery of As could be caused by the formation of polyatomic species: 40Ar35Cl+ (38Ar37Cl+, less abundant) and 40Ca35Cl+. Thirdly, something could have gone wrong during the addition of the spike to the water sample. Despite the lower quality of the recovery rates, the data obtained via this method were used. It is of high importance to interpret the obtained values critically and to keep in mind that the Hg concentrations normally should be higher and the concentrations of As and Pb lower. In order to make a good evaluation it is necessary to analyse the water samples a second time and as an improvement p.p. graded HCl should be used and 20 L Au should be added. To avoid interferences of 40Ar35Cl+ and 40Ca35Cl+, only HNO3 could be added as oxidising agent (no addition of HCl). Furthermore, the use of DRC mode on

ICP-MS with O2 as reactive gas and the addition of 4 v/v% methanol could improve the accuracy of the As analyses. To verify the accuracy of the obtained As concentrations in the water samples, a comparison was made with the values from the study of G. Sarret et al. (2019)8. In this study, the As concentrations in lake Titicaca ranged from 5.0-14.9 gL with a mean of 9.8 gL. At lake Uru Uru a concentration of 78.5 gL was measured. This is similar to our findings. In Lake Uru Uru even higher As concentration were measured in the study of G. Sarret et al. (2019) compared to our results. This indicates that our results are not necessarily too high. It is possible that something went wrong during the addition of the spike which caused the elevated recoveries and the accurate As concentrations measured in the lake. Due to the similar As concentrations measured in both studies we can use the obtained values in this study as an indication of the real As concentration in both lakes. This shows that the As concentrations at Lake Titicaca are high and are situated near to the safety threshold value for drinking water (10 gL). In lake Uru Uru, the As concentrations are approximately six times higher compared with the safety threshold level. Based on these findings negative effects can be expected in aquatic, terrestrial life due to the chronic exposure of animals and humans to contaminated water with As at Lake Uru Uru and Lake Titicaca. To get a better understanding

of the potential risk at the lake, speciation analysis is of great importance. As explained in Section 2.3 (Literature review), the toxicity of As is related to the occurring As species. Inorganic As species are the most toxic. Furthermore, differences in toxicity between different inorganic species are high. Arsenite, as example, is sixty times more toxic compared to arsenate. So if arsenite would be the major species occurring in the lake the threat would be much greater than if there were mostly arsenate. In lake Titicaca a mean pH of 8.64 occurred and a redox potential can be assumed of -100mV (Section 4.2, Results). When these 0 2- conditions are applied on the pourbaix diagram for aquatic As species (Figure 32, i), H3AsO3 and HAsO4 can be expected as the most abundant forms (i.e. both As(III) as As(IV) species). The results to be obtained from the DGT samplers will give useful insights in the As speciation in Lake Titicaca. The low availability of Hg, Cd and Pb and the high availability of As can be explained by the pH and redox potential as shown in Figure 32,ii. Arsenic will be more soluble compared to Hg, Cd and Pb in the slightly alkaline environment occurring in Lake Titicaca. It can be concluded that Hg, Cd, Pb pose no substantial threat regarding the safety of drinking water.

i) iii) Figure 32: i) Pourbaix diagram for aqueous As species at 25°C and 1 bar total pressure59. The circle indicates the pH-Eh conditions occurring in Lake Titicaca. ii) schematic presentation of major trends for increasing element mobility as a function of redox and pH changes55

5. Fish from Lake Titicaca Several studies used the international standard of 0.1 mg/kg for the total amount of As in fish as the threshold level73–75. In order to protect the human health the European commission has set threshold Hg, Cd and Pb concentrations for contaminants in foodstuff such as fish.71,72,77 The threshold limit set by the EC for Hg, Cd and Pb and the international standard for As can be used as maximum tolerable concentration in fish to ensure safe consumption of fish by the local communities. Based on these threshold values, it can be concluded that As and Hg may pose a threat to the health of the local communities.

5.1. Risk of arsenic in fish The As concentrations in fish are above the threshold value in all species. O. ispi, O. agassizii and Trichomycterus spp. have the highest As concentrations and O. bonariensis and O. mykiss have the lowest As concentrations (Addendum: Table 5) . This maximum level of As in fish is a guideline. In order to assess the real risk that As poses to the local population an investigation into the abundancy of the different As

forms is important. As explained in the literature review Section 2.3 (Literature review) the As speciation in fresh water fish is variable according to the fish species, feeding, habitat and the inorganic As contamination of water62,63. In some cases arsenobetaine is the main As species in fish, but often other compounds are abundant, including inorganic species63. As example, in rainbow trout (O. mykiss), it is found that up to 80% of the extractable As compounds can be composed out of arsenobetain (AB). AB is considered as a non- toxic compound64. If, for example, the As speciation in all fish species from Lake Titicaca have a high abundancy of AB, the risk on negative effects due to As contamination will be rather low. An extra investigation into the As species occurring in the fish of Lake Titicaca would be a useful addition to this thesis. It can be concluded that the As concentrations in Lake Titicaca are high, and safe consumption of the fish cannot be guaranteed without As speciation data. So in order to assess the magnitude of the treat of As, the abundancy of the different As species occurring in the fish from lake Titicaca should be checked. A better safety threshold value should be based on the toxic species of As (e.g. arsenite and arsenate) and not on the total As concentration in fish. But in this case, when only total As concentration in fish were measured it can be used as a safety guideline.

5.2. Risk of mercury in fish Regarding the bioaccumulation of Hg in the fish species, the consumption of O. bonariensis and Trichomycterus spp. poses a threat on the short and the long term for the local population. Mercury tends to bioaccumulate in muscle tissue and can biomagnify through the foodweb as methyl mercury (Section 2.4, Literatuur review). On the long term chronic Hg exposure (especially the organic forms such as methyl mercury) can cause different human health problems (Section 2.4, Literature review). On the short term, methyl mercury poses an important thread during pregnancy. As explained in Section 2.4, methyl mercury can cross the placental barrier and can have negative effects on the developing brain of the unborn child.

5.3. Bioaccumulation in the different species Elevated metal and metalloid concentrations in the fish species were expected especially in O. bonariensis, O. mykiss and Trychomycterus spp: in O. bonariensis and O. mykiss because they are at a high trophic level and in Trychomycterus spp. because it is a sediment dwelling fish species. Besides, the metal concentration in the fish species, will also depend on environmental factors (bioavailability: pH, soil characteristics, etc.) and the fish profile (size, age, species, etc)1,9–11. In the following sections the metal and metalloid bioaccumulation in the four different genera: Ondonthestes (bonarienses), Oncorhynchus (mykiss), Trichomycterus (spp.) and Orestias (ispi, gilsoni, agassizii and luteus) will be discussed.

5.3.1. O. bonariensis and O. mykiss O. bonariensis and O. mykiss showed no elevated As concentrations. For Hg, O. bonariensis showed elevated Hg concentrations but O. mykiss not. Hg can biomagnify through the food web as methyl mercury, which could explain the elevated concentrations in O. bonariensis. Interestingly, the lowest As and Hg concentrations are occurring in O. mykiss. An explanation could be the fact that O. mykiss was cultivated in captivity and was exposed to lower As and Hg concentration via food intake during its life. This could also have the same impact on O. bonariensis and the other fish species. During breeding in captivity the intake of metals and metalloids via feed can be regulated. To verify this impact on O. mykiss, wild occurring O.

mykiss should be captured and the metal and metalloid concentration should be checked and compared with O. mykiss which are cultivated in captivity. An extra analysis of the food pellets given to O. mykiss in the cages could provide useful information about the metal and metalloid intake. To check the impact of the trophic interaction on the metal and metalloid accumulation, the 15N content in the fish muscle samples could be analysed and compared with the metal and metalloid concentrations. As explained in the Section 4.3 (Literature review), 15N can be used to predict trophic position of species. In the paper of M. Monroy et al. (2014), the same link was made with the bioaccumulation pattern of O. bonarienses and its higher trophic level.

5.3.2. Trichomycterus spp. Trichomycterus spp. is a benthic species and feeds on detritus, so this species comes in close contact with the sediment (Section 4.2, Literature review). It has been shown in this thesis that the sediment concentrations exceeded the PEC value, so enhanced adverse effects on the aquatic environment can be expected especially for the sediment dwelling organisms such as Trichomycterus spp. This study showed great variation in the As and Hg concentrations in Trychomycterus spp. The average As concentration is above the threshold level and for Hg the upper quartile is situated above the safety threshold value set by the EC. This indicates the potential threat in the consumption of this species. An explanation for this variance in concentration can be the fact that the length and weight of the captured fish were not uniform. Methyl mercury is strongly bound to muscle tissue and accumulates with increasing duration of exposure13,81. However, as the weight, gutted weight and standard length showed no correlation with the Hg bioaccumulation (r0.05), this is unlikely. A second explanation could be the spatial variations in sediment and water characteristics. When the metal and metalloid concentrations of Trichomycterus spp. are compared based on the location of captivity, significant differences (ANOVA, p<0.05) for all metal and metalloids concentrations could be observed (Figure 33). Trichomycterus spp. was only caught at Lago Mayor, in the area of Escoma and the area of Isla da la Luna. Figure 33 shows elevated As, Hg, Cd and Pb concentration at the area of Isla de la Luna in Trychomycterus spp. This is strange because the highest metal and metalloid concentrations in sediment are situated at Escoma. The differences in bioaccumulation in this benthic species could be impacted by spatial characteristics such as sediments characteristics (OM, clay content, Fe/Mn oxides,etc), and water characteristics (pH, sulphur concentration, Eh, DO, salinity, etc.) which will influence the metal and metalloid bioavailability (Section 2.2, Literature review). In this paper, only water characteristics could be measured. In order to get better insights into the impact of the sediment on the benthic organisms, it could be useful to check also sediment properties such as the OM content, the CEC, clay content, redox potential, metal an metalloid concentrations in pore water, etc. (Section 2.2.1, Literature review). In a following study these measurements could provide interesting extra information. In the paper of M. Monroy et al. (2014), the benthic and benthopelagic fish species (O. luteus, O. agassizii and Trichomycterus) had the highest Pb and Cd concentrations. This is in line with the obtained results of this master thesis, the Pb and Cd concentrations were the highest in the benthic and benthopelgic species.

Figure 33: As, Hg, Cd and Pb concentrations in Trychomycterus spp. depending on the sampling location.

5.3.3. Orestias Orestias shows in the case of Hg accumulation no significant differences between the four different species. This similar bioaccumulation pattern can be explained by the fact that all four Orestias species has a similar feeding behaviour1. For the As bioaccumulation in the Orestias species, O. ispi had significant higher concentrations compared with O. luteus and O. gilsoni (Addendum: Table 6, ANOVA, p<0.05). An explanation could be that the diet of O. ispi could be enriched in As. The study of M. Monroy et al. (2014) reported elevated 15N in O. ispi compared with O. agassizii, O. luteus and O. gilsoni. This can indicate that O. ispi is situated on a higher trophic level. The elevated 15N could be attributed to the occasional consumption of eggs (Section 4.2.2, Literature review, Table 8). An additional reason could be, that the pelagic conditions enables O. ispi to feed on prey leftover items, which may lead to the higher 15N concentrations. Accordingly, the different feeding pattern of O. ispi could be the reason for its elevated As bioaccumulation107. In the Orestias species, differences in metal and metalloid concentrations in function of their location of capture could be shown, except for O. ispi which was only collected at Isla the la Luna. In the addendum the p-values are given for the Orestias species (Addendum: Table 7, 8 and 9). The pelagic species, O. bonariensis and O. mykiss showed no significant difference in metal or metalloid concentration in function of their location (ANOVA, p>0.05). To check the parameters which influence the Orestias species a multi-component analysis was conducted on O. luteus.

Impact of environmental parameters The most important parameters showing impact on Orestias are the weight or length, T, DO, pH, and salinity. The weight or length could be explained by the accumulation of As and Hg with increasing duration of exposure. An increase in temperature will cause an increase in solubility of the metals and metalloids. The pH, dissolved oxygen and salinity will impact the solubility of the metals and metalloids as explained in the literature review Section 2.2. These parameters have an influence on the metal and metalloid availability. An increase in bioavailability, could cause an increase in metal and metalloid accumulation in the fish species. In order to get an insight of what the future (worst case scenario: increasing metal, metalloid and salt concentration) might bring for Lake Titicaca, a comparison can be made with lake Uru Uru, as explained in Section 1.1.2 (Literature review). Only O. luteus was caught in Lake Titicaca and Uru Uru. The As and Hg concentrations in fish from both lakes showed a significant difference in concentration based on their location (ANOVA, p<0.05).

Arsenic in Lake Uru Uru O. luteus captured in lake Uru Uru has significantly higher As concentrations compared with the O. luteus from Lake Titicaca (Wilcoxon, p<0.05). The elevated As concentrations in O. luteus from lake Uru Uru (Section 4.3: Figure 31, Results) can be explained by the elevated As concentrations in water and sediment compared to Lake Titicaca. The sediment concentrations are three times higher and As concentrations in water are sixty times higher in Lake Uru Uru. Around Lake Uru Uru mining and smelting activities are concentrated which causes the inflow of metals and metalloids in the lake (Section 1.1.2, Literature review). Via the Multi-way ANOVA analysis, it appears that the DO, pH and salinity could significantly explain the variance of the As concentrations in O. luteus. In lake Uru Uru the measured DO was 7.32 mg/L, the pH 8.94 and the salinity 3.07 g/kg. This pH and salinity were higher compared with Lake Titicaca. Figure 32 ii, shows that As will become more soluble with increasing pH and redox potential. The increase in DO can result in an increase of the redox potential (Eh). With an increasing salinity the elevated ion concentrations can go in competition with binding sites on sediment of as well cations as anions, this could cause the As oxyanion species going in solution. The higher availability in water, could explain the elevated As concentrations in O. luteus.

Mercury in Lake Uru Uru The Hg concentrations in water and sediment are not significantly higher in Uru Uru compared with Lake Titicaca (Addendum: Table 3 and 4). It is of high importance to be critically about the Hg concentration in this study. As during water analysis low recoveries of spikes were obtained for Hg, in reality the Hg concentration in water could be higher. The results of the DGT samplers will give additional information about the bioavailability of Hg in Lake Titicaca and Uru Uru. There might be significant spatial differences in bioavailable Hg. Via the Multi-way ANOVA analysis, it appears that the length, DO and T could significantly explain the variance of the Hg concentrations in O. luteus. As previously explained, Hg accumulates with increasing duration of exposure in fish. Increasing length can lead to an increase in Hg concentrations. The increase in DO can result in an increase in Eh. As shown in Figure 32 ii, Hg will become more soluble with increasing Eh. At last, increasing temperature can increase the solubility and bioavailability of the metal ions. These observations could give an insight of what the future may bring for Lake Titicaca. The decreasing water level, increasing salinity and increasing metal and metalloid contamination at Lake Titicaca could increase the metal and metalloid bioaccumulation in fish intended for human consumption. Especially As and Hg tend to bioaccumulate more under conditions occurring in lake Uru Uru. Based on the results from this study, it can be concluded that As and Hg pose a considerable threat to the health of the local community at Lake Uru Uru due to the elevated As concentrations in water and elevated As and Hg concentrations in fish meant for human consumption.

6. Daily consumption limits Based on the RfD (reference dose in g/kg BW/day), the average body weight (aBW in kg) and the measured metal or metalloid concentration (Cm in g/kg) an estimation can be made of the maximum allowable fish consumption rate per day (CRlim in kg fish/day). This can be calculated based on Equation 7. The detailed calculations are given in the Addendum (Calculations daily consumption rate). The CRlim will be calculated

for As and Hg, because these two elements pose the greatest threat at Lake Titicaca. Equation 7 has a disadvantage, as this equation does not take into account the As intake via other food sources such as water. To correct for the As intake via water, Equation 7 is adjusted resulting in Equation 8. The RfD of 0.3 g/kgBW/day and 0.1 g/kgBW/day can be used for As and Hg, respectively (Literature review, Section 2.4 and Section 2.5). In this analysis the population will be divided in 4 subgroups according to their age: toddlers, children, adolescents and adults. Table 21 gives an overview of the mean weight, the mean water, As and Hg intake for the 4 subgroups. First the remaining safe As and Hg intake (RSintake) after the consumption of the advised amount of water can be calculated via Equation 9 (Table 22). If a negative value is obtained for a subgroup the As or Hg intake is exceeded and the safe consumption of the daily water intake cannot be assured for this subgroup. Based on the results given in Table 22, it can be concluded that the daily As intake via water exceeds the safe intake for toddlers and children. As a result, no extra As intake via food is recommended and it would be advisable to replace the water of Lake Titicaca by treated or non- contaminated water for children under the age of 12 years. Secondly the daily amount of fish which safely can be consumed (CRlim) (taking into account the As and Hg intake via water), can be calculated via Equation 8. If an assumption is made that the local communities use bottled/clean, non-polluted water as drinking water, the safe daily intake of fish will increase. Based on the mean CRlim of the different fish species and the assumption of a fish meal size (MS) weighing 0.2 kg, the monthly fish consumptions (Consumption rate meals per month: CRmm) can be calculated (Equation 10). Table 23 gives the maximum average allowable fish consumption rate according to the Hg and As content taking into account the daily As and Hg intake from water out of Lake Titicaca. Table 24 gives the maximum average allowable fish consumption rate according to the Hg and As content assuming no extra As and Hg intake via other food sources at Lake Titicaca. Based on the results obtained in Table 23 and 24, a recommendation can be given regarding the maximum amount of that can be consumed per month without a risk. An extra study regarding the consumption behaviour of the local population is required to assess the amount of fish consumed per month. If this amount is higher compared to the CRmm, the chronic exposure can pose a risk for the local population at Lake Titicaca. In Table 23 and 24, the CRmm’s based on the As concentration in fish are lower compared with the CRmm’s based on the Hg concentrations. This is because the As concentrations in fish and water are higher. These results show, if water and fish from the lake are consumed, the intake of As and Hg is high. As previously explained (Section 5.3, Discussion), an extra investigation into the As species distribution occurring in the different fish species, could provide interesting extra information. If the fish species from Lake Titicaca contain mostly organic As species, the CRmm could be higher because the organic forms of As are less toxic compared with the inorganic forms (Section 2.3, Literature review). The results of the DGT samplers will give additional information about the bioavailability of As and Hg in water from Lake Titicaca and Uru Uru. These values should be used critically because only As and Hg intake via fish and water are incorporated. In a real scenario other As and Hg food sources will be present such as vegetables (tomatoes, potatoes, rice,…) and meat ( chicken, pork, beef,…). In order to conduct a full risk analysis an extra investigation into the consumption behaviour of the local population is necessary and an investigation into the metal and metalloid concentrations occurring in the other food sources. Monitoring the monthly intake of water and fish is important for the local population, especially for children under 12 years old.

푅푓퐷∗푎퐵푊 7) CRlim = 퐶푚 푅푓퐷∗푎퐵푊−푐표푛푡푎푚푖푛푎푛푡 푖푛푡푎푘푒 푣푖푎 푤푎푡푒푟 8) CRlim = 퐶푚 9) RSinktake = 푅푓퐷 ∗ 푎퐵푊 − 푐표푛푡푎푚푖푛푎푛푡 푖푛푡푎푘푒 푣푖푎 푤푎푡푒푟 퐶푅푙푖푚∗30.44 10) CRmm = 푀푆

Table 21: Water intake, As and Hg intake via water and the average weight for the 4 subgroups: toddlers, children, adolescents and adults73,138. Subgroup Age Mean weight Water intake As intake via water Hg intake via water (years) (kg) (L/day) (g/day) (g/day) Toddlers 3-5 11.6 1 11 0.17 Children 6-12 30.5 1.5 16 0.26 Adolescents 12-18 55.9 1.5 16 0.26 Adults >19 71.88 1.5 16 0.26

Table 22: The remaining safe intake (RSintake) of As and Hg, after the advised water intake. Subgroup RSintake As RSintake Hg Toddlers < 0 0.99 Children < 0 2.8 Adolescents 0.42 5.3 Adults 5.2 6.9

Table 23: Maximum average allowable fish consumption rate according to the Hg and As content taking into account the daily As and Hg intake from water out of Lake Titicaca: the CRlim (daily consumption rate limit) and the CRmm (consumption rate limit meals per month). Subgroup Average CRlim (kg fish/day) Average CRmm (meals/month) Compound Arsenic Mercury Arsenic Mercury Toddlers / 0.008 / 1.3 Children / 0.024 / 3.6 Adolescents 0.001 0.045 0.13 6.9 Adults 0.011 0.059 1.7 8.9

Table 24: Maximum average allowable fish consumption rate according to the Hg and As content assuming no extra As and Hg intake via other food sources at Lake Titicaca: the CRlim (daily consumption rate limit) and the CRmm (consumption rate limit meals per month). Subgroup Average CRlim (kg fish/day) Average CRmm (meals/month) Compound Arsenic Mercury Arsenic Mercury Toddlers 0.007 0.010 1.1 1.5 Children 0.019 0.026 2.9 3.9 Adolescents 0.035 0.047 5.3 7.2 Adults 0.045 0.061 6.8 9.3

7. Improvements field work In Bolivia no nitric acid was available so clean material was brought from Belgium to Bolivia. In a future project regarding trace metal analysis, the availability of nitric acid to rinse al the used materials is important. Furthermore, to check if no contamination occurred during transport of the samples, blanks should have been checked to verify the contamination during export, this isn’t done in this project. Next, in order to create a better view on the metal and metalloid distribution in sediment and water in the lake, an improvement would be to take more sediment and water samples. At lake Titicaca 8 sampling spots were selected, of which six at Lago Menor. This is enough to get a rough estimation of the metal and metalloid distribution at Lago Menor, but in order to select the best location to set up new fish pots a more extensive sampling campaign is necessary in Lago Menor and Lago Mayor. This was not possible due to the limited time of the sampling campaign. It would be beneficial to have an agreement between Bolivia and Peru, so sampling at both parts of the lake would be possible. Also the measurement of extra sediment characteristics (clay content, CEC, Eh, etc.) could have provided extra interesting information regarding the availability of the metals and metalloids in sediment. Here, DGT samplers could also be a useful tool to analyse the bioavailable concentrations in sediment99. Another improvement is the capture of all fish species at all the sampling spots. Only O. luteus was caught at al sampling spots. In order to get a more accurate representations of the metal and metalloid bioaccumulation in the fish species from Lake Titicaca, more fish samples should be obtained from the other fish species. Our sampling campaign was conducted in the dry season of the Altiplano. During the wet season, seventy percent of the rainfall will occur. This will increase the inflow of water. The rivers will take elevated amounts of nutrients and pollutants to the lake and the run-off from agricultural and mining areas will increase. It would be interesting to analyse what impact the seasonal variation has on the metal and metalloid concentrations occurring in the lake.

Conclusions In this master thesis, the As, Hg, Cd and Pb concentrations in water, sediment and fish from Lake Titicaca and Uru Uru and the accompanying risks were investigated. In order to obtain the As, Hg, Cd and Pb concentrations in sediment and biota, a HNO3 treatment in combination with the microwave-assisted digestion in closed vessel method was selected to digest the sample matrix. This microwave technique was selected because of its fastness, the complete digestion of the organic matter under high pressure, and shows high repeatability and better recoveries. The HNO3 treatment is preferred above an aqua regia 40 35 + 40 35 + treatment (HCl + HNO3), so the formation of polyatomic species (e.g. Ar Cl and Ca Cl ) in the plasma of the ICP-MS is minimised. Recoveries were obtained within a deviation of maximum 20% from full recovery for Cd, Hg and Pb. For As, elevated recoveries were found, which could have been caused by the interference of 40Ar35Cl+ and 40Ca35Cl+, formed in the plasma of the ICP-MS. Analysing As in dynamic reaction mode instead of collision cell mode on ICP-MS may resolve this problem.

In sediment from Lake Titicaca and Uru Uru, the Hg, Cd and Pb concentrations were below the TEC values, so no adverse effects are to be expected. For As in sediment it can be concluded that adverse effects on the aquatic environment can be expected especially at Escoma, Tiquina, and Lake Uru Uru. At these locations As concentrations above the PEC value (9.8 mg/kg) were measured. The obtained results from the water samples taken at Lake Titicaca showed elevated As concentrations which are situated near to the safety threshold value for drinking water (10g/L). At lake Uru Uru, the As concentrations in water are approximately six times higher compared with the safety threshold level. Based on these findings, negative effects can be expected in aquatic and terrestrial life, due to the chronic exposure of animals and humans to water contaminated with As from Lake Uru Uru and Titicaca. To get a better understanding of the potential risk at the lake, speciation analysis is of great importance. Regarding the As, Hg, Cd and Pb bioaccumulation in the seven different fish species, As and Hg may pose a threat to the health of the local communities. The As concentrations exceeded the safety threshold level (0.1mg/kg) in all seven fish species. This maximum level of As in fish is a guideline. In order to assess the real risk that As poses to the local population an investigation into the abundancy of the different As forms is important. For Hg, O. bonarienses had an average Hg concentration above the safety threshold level (0.5mg/kg) and Trichomycterus spp. showed concerning elevated concentrations. Furthermore, the in captivity grown O. mykiss had the lowest As and Hg concentrations, which could be the result of the captive breeding. This could have the same impact on O. bonariensis and the other fish species.

At last, consumption limits were calculated in this investigation. From this evaluation it seems advisable to replace the water of Lake Titicaca by treated or non-contaminated water for children under the age of 12 years. These values should be interpreted carefully because only As and Hg intake via fish and water were incorporated. In a real scenario other As and Hg food sources will be present. In order to conduct a full risk analysis an extra investigation into the consumption behaviour of the local population and an investigation into the metal and metalloid concentrations occurring in the other food sources is necessary.

Future laboratory work As mentioned in the Preambule, due to the measures taken by Ghent University to ensure the safety of students and employees, the laboratory operations had to be stopped prematurely. In this section of the master thesis, the procedures to be followed to finish all analyses will be presented.

1. Water samples As explained in the discussion, the obtained recoveries for the water samples were not optimal. Possible causes for these abnormal recovery rates are the usage of p.a. quality grade HCl and the fact that no Au was added to the samples. When the results were compared with other research conducted at the lake the measured concentrations in this study were in the same order of magnitude, so no abnormal concentrations were obtained. In order to find out what went wrong, the water analyses should be carried out a second time. The water samples are not turbid. As digestion, the samples should be acidified with 3 drops of HNO3 and 20 L Au solution (1000 mg/L) should be added. The HNO3 is of p.p. quality and HCl is left out to minimise the interference caused by the formation of 40Ar35Cl+ and 40Ca35Cl+. Spikes should be added as explained in Section 4.1.3 (Materials and Methods). On the ICP-MS, standard and KED mode can be applied to investigate which mode will give the best recoveries. To deal with the high recoveries for As, the DRC mode could be applied on the ICP-MS as previously mentioned (Discussion: 2. Digestion method). + Using CH4 as reactive gas, the interference of ArCl is eliminated and the background signal reduced. An 75 + alternative for CH4 is the use of oxygen as reactive gas. Using oxygen, As is efficiently converted to 75As16O+, with a m/z of 91. The use of oxygen as reactive gas gives a solution to deal with interferences caused by 40Ar35Cl+ and 40Ca35Cl+ 120,134. An extra improvement could be the addition of 4v/v% methanol to the analysis solution, which could increase the signal to background ratio134,135.

2. Recovery of Pb During the digestion of the sediment samples and fish samples, the used reference samples were not certified for Pb content. To obtain more reliable results regarding the Pb concentrations analysed in the samples, these samples should be analysed a second time. For the fish samples, mussels tissue (ERM- CE278k) can be used as reference material. For sediment, spikes can be added to the samples as explained in Section 4.1.1 (Materials and Methods). During this investigation it became clear that Pb poses no risk to the aquatic life or fish intended for human consumption, so no extra analyses are required.

3. Fish: whole fish, muscle and liver tissue Not all muscle tissue was digested and analysed. The remaining muscle samples should be analysed via the closed vessel microwave digestion with nitric acid. This procedure is given in Section 4.1.2 (Materials and Methods). During digestion it is recommended to digest and analyse in each batch also reference samples of fish muscle (ERM-BB422) and mussels tissue (ERM-CE278k) to obtain recovery rates for As, Hg, Cd and Pb. On ICP-MS the KED mode should be applied and the settings stated in Section 4.3 (Materials and Methods) should be used. To deal with the elevated recoveries of As, this element should be analysed in

DRC, using CH4 or O2 as reactive gas. To analyse the dissected liver tissue and whole fish samples the same protocol as for the fish muscle tissue is suggested (with the modification for the As analysis). It could be interesting to analyse also the 15N concentration in the whole fish tissues so the impact of the trophic level of the fish on the metal bioaccumulation can be verified. As explained in Section 4.3. (Literature review),

15N can be used to predict the species its trophic position because 15N is enriched throughout the trophic levels. Furthermore the Se concentration occurring in the different fish species could be analysed. The Se concentration in fish and water could be interesting because it is an essential element: Se functions as a component of antioxidant enzymes (e.g. gluthathione peroxidases and thioredoxin reductases); it has been suggested to be a cancer chemo-preventive agent; it is necessary for the thyroid hormone system; it is required for a good immune response; etc139–141. Besides its essential function in the human body, it could also play an important role in the uptake of Hg. It has been shown that the Hg and Se interactions are important in the toxicology of both elements. Se can act as a detoxifying agent for Hg142. Assessing the Se concentrations in water and fish could provide information about the possible beneficial impact of Se on the uptake and toxicity of Hg for the local population and fish occurring in the lake142,143. To analyse 80Se

(most abundant isotope), the DRC mode on ICP-MS should be used with CH4 as reactive gas. By using CH4 40 + 135,144 during analyses in DRC mode, the predominant interferent Ar2 is successfully eliminated . Interferences caused by oxides and double charged ions are also decreased with 50%. An extra addition of 4% methanol to the analytical solution could further improve the reductions of background interferences135. 78 To analyse Se the DRC mode using NH3 as a reactive gas could also be applied. It appears that interference 2+ 2+ 78 + of double charged ions (Gd , Dy ) on Se could not be sufficiently minimised using CH4 as reactive gas. 144 The use of NH3 as reactive gas in DRC mode appeared to be more effective .

4. Amphipods

After the optimisation, the closed vessel microwave digestion with HNO3 was selected to digest the amphipod samples. This procedure is given in Section 4.1.2 (Materials and Methods). On ICP-MS the KED mode should be applied and the settings stated in Section 4.3 (Materials and Methods) should be used. As previously explained, for the As analyses, DRC mode should be applied with O2 as reactive gas. Here it could also be interesting to analyse the 15N concentration in the amphipods samples so the impact of the trophic level on the metal bioaccumulation can be verified.

5. Analysis of the DGT samplers As explained in Materials and Methods, DGT samplers were deployed in Lake Titicaca and Uru Uru for approximately 3 weeks and 2 weeks, respectively. The date and time of deployment and retrieval is given in Table 11 (Section 3.1, Materials and Methods). These DGT samplers are used to verify the bioavailable As, Cd, Pb and Se concentrations with the LSNXP DGT sampler and the bioavailable Hg and As(III) with the LSNB-AP DGT sampler (Literature review: Section 3, Materials and Methods).

Sample treatment Firstly, to retrieve the resin gel, a screw driver should be inserted into the grove in the cap whilst twisting it. The cap will be broken at the most fragile point. The broken cap, the filter and diffusive layer should be removed so the bottom resin-gel will be revealed. Secondly, the resin gel should be placed in a clean sampling tube (for example a 1.5 mL centrifuge tube) and 1mL of 1M p.p. HNO3 should be added for at least 24h before analysis. Make sure the resin gel is fully immersed.

Sample analysis

After the treatment with HNO3, the sample should be diluted 5 times before analysis with ICP-MS. The same setting can be used as stated in Section 4.3 (Materials and Methods). Firstly, the mass (M) of the metal accumulated in the resin gel layer has to be calculated, using Equation 11. Ce is the concentration of the metals in the 1M HNO3 elution solution, VHNO3 is the volume of HNO3 added to the resin (1 mL), Vgel is the volume of the resin gel (typically: 0.126 mL) and fe, the elution factor, is the fraction of the metals released. 99,101 An fe of 0.8 can be used. Secondly the occurring time averaged metal and metalloid concentrations in Lake Titicaca can be calculated via the standard DGT equation given at Section 3, Equation 6 (Literature study). This equation is repeated below as equation 12. M (ng) is the mass of the metal accumulated on the resin. CDGT (ng/mL) is the time average concentration of the analyte in the deployment medium. ∆푔 (cm) is the total thickness of the materials in the diffusion layer (diffusive gel + filter membrane (0.094 cm, applied samplers in this paper)). D (cm²/s) is the diffusive coefficient of the analyte in the diffusion layer (diffusive gel, filter membrane and diffusion boundary layer). A (3.14 cm² for applied samplers in this paper) is the physical area of the exposed filter membrane. t (s) is the deployment time.99 The deployment periods (in seconds) are reported in Section 3.1 (Materials and Methods), the diffusion coefficient for the agarose crosslinked polyacrylamide can be retrieved from the DGTresearch site99,101 and the diffusion coefficients using an agarose diffusive gel can be retrieved from the studies of W. Bennet et al. (2011) and Y. Gao et al. (2011)105,106. For the agarose crosslinked diffusive gel a D for: As of 3.76e-6 cm2/s, Cd of 4.16e-6 cm2/s, Pb of 5.49e-6 cm2/s and for Se of 4.92e-6 cm2/s can be used. For the agarose diffusive gel a D can be used for Hg of 8.44e-6 cm2/s and for As(III) a D of 9.04e-6 cm2/s.

퐶푒∗(푉퐻푁푂3+푉푔푒푙) 11) M = 푓푒 푀∗∆푔 12) CDGT= D∗A∗t

Addendum Tables

Table 1: Investigated species from Lake Titicaca. Pictures taken by Arthur Fonteyne and Erick Loayza Torrico. O. gilsoni

O. ispi

O. luteus

O. agassizii

Trichomycterus spp.

O. bonariensis

O. mykiss

Tabel 2: Overview of the coordinates from the different sampling spots. spot code replicate Latitude Longitude Altitude (m) Isla de la Luna IDL IDL1 -16,0452 -69,0682 3807 IDL2 -16,0452 -69,0682 3807 IDL3 -16,0452 -69,0682 3807 Huatajata H H1 -16,2148 -68,692 3807 H2 -16,2158 -68,6908 3807 H3 -16,2157 -68,6918 3807 Cohana C C1 -16,3171 -68,7373 3807 C2 -16,3167 -68,7366 3807 C3 -16,3159 -68,7357 3807 PlatformXavier X X1 -16,2464 -68,6808 3807 X2 -16,246 -68,6809 3807 X3 -16,2466 -68,6799 3807 Taraco TA TA1 -16,4837 -68,9207 3807 TA2 -16,4861 -68,9251 3807 TA3 -16,4874 -68,9283 3807 Desaguadero D D1 -16,5198 -68,9652 3807 D2 -16,5216 -68,9655 3807 D3 -16,5229 -68,9637 3807 Escoma E E1 -15,674 -69,1967 3807 E2 -15,6741 -69,1961 3807 E3 -15,6747 -69,1966 3807 Tiquina T TQ1 -16,2234 -68,8351 3807 TQ2 -16,2231 -68,8333 3807 TQ3 -16,2241 -68,8327 3807

Table 3: Tuckey adjusted p-values for the mean metal and metalloid concentrations (As, Hg, Cd and Pb) in sediment, sampled at Escoma (E), Isla de la Luna (IDL), Tiquina (TQ), Huatajata (H), a platform of UMSA (X), Cohana bay (C), Taraco (TA), Desaguadero (D) and Uru Uru (UU). Significant p-values are marked in bold. Spot p-value As p-value Hg p-value Cd p-value Pb D-C 1.000 0.002 0.0368 0.878 E-C 0.837 0.233 0.9933 1.96E-04 H-C 1.000 0.006 0.0208 0.999 IDL-C 1.000 0.072 0.0673 0.161 TA-C 0.898 0.009 0.0062 0.219 TQ-C 0.995 0.750 1.0000 0.768 UU-C 3.90E-06 0.771 0.2693 1.30E-06 X-C 0.955 0.972 0.0019 0.996 E-D 0.826 0.288 0.0062 0.004 H-D 1.000 0.999 1.0000 0.995 IDL-D 1.000 0.651 1.0000 0.867 TA-D 0.892 0.994 0.9933 0.752 TQ-D 0.994 0.587 0.3036 1.000 UU-D 3.70E-06 0.048 0.9700 0.000 X-D 0.959 3.26E-05 6.00E-07 0.999 H-E 0.709 0.662 0.0034 0.001 IDL-E 0.554 0.999 0.0118 0.078 TA-E 1.000 0.755 0.0010 0.769 TQ-E 0.998 1.000 0.9982 0.006 UU-E 1.13E-04 0.982 0.0589 0.753 X-E 0.220 0.014 0.0155 0.001 IDL-H 1.000 0.952 0.9995 0.415 TA-H 0.826 1.000 0.9996 0.416 TQ-H 0.976 0.851 0.2229 0.976 UU-H 2.40E-06 0.170 0.8989 4.70E-06 X-H 0.988 1.54E-04 3.00E-07 1.000 TA-IDL 0.730 0.979 0.9577 0.999 TQ-IDL 0.919 0.999 0.4106 0.944 UU-IDL 1.40E-06 0.766 0.9962 0.001 X-IDL 0.999 0.003 1.00E-06 0.508 TQ-TA 0.996 0.896 0.1101 0.836 UU-TA 0.023 0.223 0.6026 0.162 X-TA 0.449 2.26E-04 1.00E-07 0.481 UU-TQ 2.22E-05 1.000 0.7360 4.59E-05 X-TQ 0.580 0.292 0.0592 0.992 X-UU 4.00E-07 0.134 5.20E-06 6.60E-06

Table 4: Tuckey adjusted p-values for the mean metal and metalloid concentrations (As, Hg, Cd and Pb) in water, sampled at Escoma (E), Isla de la Luna (IDL), Huatajata (H), a platform of UMSA (X), Cohana bay (C), Taraco (TA), Desaguadero (D) and Uru Uru (UU). Significant p-values are marked in bold. Spot p-value As p-value Hg p-value Cd IDL-E 1.000 1.000 0.539 H-E 1.000 1.000 0.052 X-E 1.000 1.000 0.120 C-E 1.000 1.000 0.051 TA-E 1.000 0.951 0.051 D-E 1.000 0.300 0.052 UU-E 0.001 0.961 0.019 H-IDL 1.000 1.000 0.946 X-IDL 1.000 1.000 0.979 C-IDL 1.000 1.000 0.944 TA-IDL 1.000 0.989 0.945 D-IDL 1.000 0.352 0.947 UU-IDL 0.004 0.994 0.914 X-H 1.000 1.000 1.000 C-H 1.000 1.000 1.000 TA-H 1.000 0.985 1.000 D-H 1.000 0.216 1.000 UU-H 0.002 0.991 1.000 C-X 1.000 1.000 1.000 TA-X 1.000 0.962 1.000 D-X 1.000 0.460 1.000 UU-X 0.003 0.973 1.000 TA-C 1.000 0.972 1.000 D-C 1.000 0.253 1.000 UU-C 0.002 0.981 1.000 D-TA 1.000 0.045 1.000 UU-TA 0.002 1.000 1.000 UU-D 0.002 0.024 1.000

Table 5: Average As, Hg, Cd and Pb concentrations in seven different fish species from Lake Titicaca Species As (mg/kg) Hg (mg/kg) Cd (mg/kg) Pb (mg/kg) Trichomycterus spp. 0.8746 0.3488 0.0041 0.0115 O. agasizii 0.9993 0.2337 0.0150 0.0521 O. luteus 0.5415 0.1162 0.0098 0.0079 O. gilsoni 0.3868 0.0829 0.0064 0.0450 O. ispi 1.0078 0.1240 0.0161 0.0171 O. mykiss 0.2478 0.0462 0.0091 0.0149 O. bonariensis 0.3353 0.6131 0.0042 0.0336

Table 6: Bonferroni adjusted p-values from the Wilcoxon signed-rank test for the mean metal and metalloid concentrations (As, Hg, Cd and Pb) in the seven different fish species from lake Titicaca: Trychomycterus spp. (Trycho), O. gilsoni (gilsoni), O. ispi (ispi), O. agassizii (agassizii), O. luteus (luteus), O. bonarienses (bonar), O. mykiss (mykiss). Significant p-values are marked in bold. Species p-value As p-value Hg p-value Cd p-value Pb bonar-agassizii 0.0002 0.003 0.008 1.000 gilsoni-agassizii 0.0878 0.680 0.276 0.656 ispi-agassizii 1.0000 1.000 0.448 1.000 luteus-agassizii 0.0998 1.000 0.250 0.669 mykiss-agassizii 5.60E-06 0.005 0.613 1.000 Trycho-agassizii 1.000 1.000 0.034 1.000 gilsoni-bonar 1.000 5.40E-07 0.233 0.685 ispi-bonar 5.50E-07 1.50E-05 3.90E-05 1.000 luteus-bonar 1.000 3.20E-09 0.057 0.291 mykiss-bonar 1.000 6.90E-12 0.012 1.000 Trycho-bonar 1.000 0.193 0.796 1.000 ispi-gilsoni 0.00162 0.312 0.085 1.000 luteus-gilsoni 1.000 1.000 0.837 0.003 mykiss-gilsoni 1.000 1.000 0.444 0.305 Trycho-gilsoni 1.000 0.894 0.420 0.101 luteus-ispi 0.00759 1.000 0.015 0.006 mykiss-ispi 3.60E-07 4.50E-04 0.133 0.156 Trycho-ispi 1.000 1.000 0.006 1.000 mykiss-luteus 0.677 0.814 0.057 1.000 Trycho-luteus 1.000 0.236 0.213 1.000 Trycho-mykiss 1.000 0.243 0.059 1.000

Table 7: Multiple comparison of averages with holm corrected p-values, for Orestias luteus at sampling location: Escoma (E), Isla de la Luna (IDL), Tiquina (TQ), Huatajata (H), Platform UMSA (X), Cohana Bay (C), Taraco (T), Desaguadero (D). Spot p-value As p-value Hg p-value Cd p-value Pb E-D 1 1 1 1 H-D 1 1 1 1 IDL-D 1 1 1 0.59 TQ-D 1 1 1 1 UU-D 0.369 3.7e-7 1 1 H-E 1 0.138 1 1 IDL-E 1 0.790 1 0.28 TQ-E 1 0.041 1 0.82 UU-E 0.0007 1.6e-11 1 1 IDL-H 1 1 1 1 TQ-H 1 1 1 1 UU-H 3.7e-4 1.3e-7 1 1 TQ-IDL 1 1 1 1 UU-IDL 0.023 1.5e-7 1 0.32 UU-TQ 0.006 6.4e-7 1 0

Table 8: Multiple comparison of averages with holm corrected p-values, for Orestias agassizii at sampling location: Escoma (E), Huatajata (H), Desaguadero (D). Spot p-value As p-value Hg p-value Cd p-value Pb E-D 0.56 0.227 0.949 0.92 H-D 0.99 0.227 0.001 0.84 H-E 0.27 6.6e-4 8.5e-6 0.73

Table 9: Multiple comparison of averages with holm corrected p-values, for Orestias gilsoni at sampling location: Isla de la Luna (IDL), Huatajata (H). Spot p-value As p-value Hg p-value Cd p-value Pb IDL-H 0.005 0.33 0.78 0.22

Figures

Figure 1: Sampling points Lake Titicaca. Illustration made with Google Earth Pro®.

Figure 2: Extra pictures taken during dissection of O. Ispi: a) internal organs b) O. ispi infected with a platworm. Pictures taken by Arthur Fonteyne and Erick Loayza Torrico.

Figure 3: Extra pictures regarding the dissection of O. bonariensis. An O. ispi in the gut of an O. bonariensis. Pictures taken by Arthur Fonteyne and Erick Loayza Torrico.

i) ii)

iii) iv)

Figure 4: Metal and metalloid concentration in sediment at Lake Titicaca: i) As, ii) Hg, iii) Cd and iv) Pb. Illustraded with QGIS®.

i) ii)

iii)

Figure 5: Metal and metalloid concentration in water at Lake Titicaca: i) As, ii) Hg and iii) Cd. Illustraded with QGIS®.

Calculations

Calculations digestion procedure: hotplate with H2O2 Based on the study of Wilson, R. P. and Cowey C.B. ‘Amino acid composition of whole body tissue of rainbow trout and Atlantic salmon’ an estimation was made on the amino acid composition of rainbow trout128. Via the study of Haliloǧlu, H. I. et al. ‘Comparison of fatty acid composition in some tissues of rainbow trout (Oncorhynchus mykiss) living in seawater and freshwater’ an estimation was made on the fatty acid composition of rainbow trout129. An estimation was made that the fatty acid composition of rainbow trout exist out of 12.5% fat with 22.5% oleic acid (18:1 n-9, C18H34O2), 17.3% palmitic acid( 16:0, C16H32O2) and

12,4% docosahexaenoic acid (22:6 n-C22H32O2) and out of 16.6% amino acids with 14.22% glutamic acid

(C5H9NO4), 9.94% aspartic acid (C4H7NO4) and 8.49% Lysine (C6H14N2O2). A digestion procedure was constructed based on the oxidation of the amino acids and fatty acids by hydrogen peroxide (H2O2).

Following redox reaction will occur when H2O2 is added and we assume that the rainbow trout is composed out of the three main amino acids and fatty acids.

1) Redox reactions fatty acids

C18H34O2 + 51 H2O2 → 18 CO2 + 68 H2O

C16H32O2 + 46 H2O2 → 16 CO2 + 62 H2O

C22H32O2 + 58 H2O2 → 22 CO2 + 74 H2O

2) Redox reactions amino acids - + C5H9NO4 + 13 H2O2 → 5 CO2 + 17 H2O + NO3 + H - + C4H7NO4 + 10 H2O2 → 4 CO2 + 13 H2O + NO3 + H - + C6H14N2O2 + 22 H2O2 → 6 CO2 + 28 H2O + 2 NO3 + 2 H With this information the amount of peroxide necessary to oxidize 2g of fish (rainbow trout) can be calculated (Table 14).

Table 1: Amount H2O2 necessary to oxidize 2g of fish sample Formule MM (g/mol) Amount (g) in 2g Amount (mol) Amount (mol) Amount (mol)

fish sample in 2g sample H2O2 per mol FA H2O2

C18H34O2 282,5 0,0108 3,81E-05 51 0,00195

C16H32O2 256,4 0,0083 3,23E-05 46 0,00149

C22H32O2 0,0059 328,5 1,81E-05 58 0,00105 C5H9NO4 147,1 0,0145 9,83E-05 13 0,00128

C4H7NO4 133,1 0,0101 7,59E-05 10 0,00076

C6H14N2O2 146,2 0,0086 5,91E-05 22 0,00130

Som 0,00782

As shown in Table 14, 0.00782 mol or 0.266 g H2O2 should be added. Two excess factors will be applied (two different methods), 5 times the required volume and 10 times the required volume. This resulted in the addition of 4.5mL and 8.5mL respectively of a 30% H2O2 solution.

Calculations daily consumption limits Based on the RfD, the average body weight (aBW) and the measured metal or metalloid concentration (Cm) an estimation can be made of the maximum allowable fish consumption rate per day (CRlim). This can be calculated based on Equation 5. Only the CRlim for As and Hg are calculated because these elements had the highest concentrations in fish and exceeded the safety threshold values (Results: 3.1. Fish). But this Equation does not take in to account the As intake via other food sources such as water and vegetables. To correct for the As intake via water, Equation 1 is adjusted so the As and Hg intake via water is incorporated ( Equation 2). The RfD of 0.3 g/kgBW/day and 0.1 g/kgBW/d can be used for As and Hg, respectively. In this analysis the population will be divided in 4 subgroups according to their age (Table 1): todlers, children, adolescents and adults. The daily water intake is estimated based on the advised amount of water intake according to the “Wetenschappelijk instituut volksgezondheid”138. The average weight per subgroup is based on the study of M. Raissy and M. Ansari (2014)73. If the assumption is made, that the local community uses the water from Lake Titicaca as drinking water, the daily intake of As and Hg via water can be calculated (Table 1). The average As and Hg concentration in Lake Titicaca is 10.9g/L and 0.174g/L, respectively. The remaining safe As and Hg intake (RSintake) after the consumption of the advised amount of water can be calculated via Equation 3. If a negative value is obtained for a subgroup the As or Hg intake is exceeded and the safe consumption of the daily water intake cannot be assured for this subgroup. Based on the results given in Table 2, it can be concluded that the daily As intake via water exceeds the safe intake for toddlers and children. So no extra As intake via food is recommended, it would also be advisable to replace the water of Lake Titicaca by filtered or non-contaminated water for children under the age of 12 years. Furthermore the daily amount of fish which safely can be consumed (CRlim) (taking into account the As and Hg intake via water), can be calculated via Equation 3. The results are given in Table 3 for As and in Table 4 for Hg. No values are given for toddlers and children because the intake via water was already exceeded. If an assumption is made that the local communities use bottled/clean non polluted water as drinking water, the safe daily intake of fish can increase. Table 5 and 6 show the CRlim calculated via Equation 1. Based on the mean CRlim of the different fish species and the assumption of a meal size (MS) weighing 0.2 kg, the monthly fish consumptions (Consumption rate meals per month: CRmm) can be calculated via Equation 4. The value of 30.44 was used as the average amount of days in a month. Table 7 gives the maximum average allowable fish consumption rate according to the Hg and As content taking into account the daily As and Hg intake from water out of Lake Titicaca. Table 8 gives the maximum average allowable fish consumption rate according to the Hg and As content assuming no extra As and Hg intake via other food sources at Lake Titicaca. 푅푓퐷∗푎퐵푊 1) CRlim = 퐶푚 푅푓퐷∗푎퐵푊−푐표푛푡푎푚푖푛푎푛푡 푖푛푡푎푘푒 푣푖푎 푤푎푡푒푟 2) CRlim = 퐶푚 3) RSinktake = 푅푓퐷 ∗ 푎퐵푊 − 푐표푛푡푎푚푖푛푎푛푡 푖푛푡푎푘푒 푣푖푎 푤푎푡푒푟 퐶푅푙푖푚∗30.44 4) CRmm = 푀푆

Table 1: Water intake, As and Hg intake via water and the average weight for the 4 subgroups: toddlers, children, adolescents and adults73,138. Subgroup Mean weight Water intake As intake via water Hg intake via water (kg) (L/day) (g/day) (g/day) Toddlers (3-5 years) 11.6 1 11 0.17 Children (6-12 years) 30.5 1.5 16 0.26 Adolescents (12-18 years) 55.9 1.5 16 0.26 Adults (>19 years) 71.88 1.5 16 0.26

Table 2: The remaining safe intake (RSintake) of As and Hg after the advised water intake. Subgroup RSintake As RSintake Hg (g/day) (g/day) Toddlers < 0 0.99 Children < 0 2.79 Adolescents 0.42 5.33 Adults 5.2 6.93

Table 3: The As CRlim (kg/day) for the fish species at Lake Titicaca taking into account the daily As and Hg intake from water out of Lake Titicaca: T= Trichomycterus spp., A= O. agassizii, L= O. luteus, G= O. gilsoni, I= O. ispi, M= O. mykiss, B= O. bonariensis. Subgroup CRlim T CRlim A CRlim L CRlim G CRlim I CRlim M CRlim B Mean CRlim Toddlers / / / / / / / / Children / / / / / / / / Adolescents 0.00048 0.00042 0.00078 0.00109 0.00042 0.00170 0.00125 0.00088 Adults 0.00596 0.00522 0.00963 0.01348 0.00517 0.02104 0.01555 0.011

Table 4: The Hg CRlim (kg/day) for the fish species at Lake Titicaca taking into account the daily As and Hg intake from water out of Lake Titicaca: T= Trichomycterus spp., A= O. agassizii, L= O. luteus, G= O. gilsoni, I= O. ispi, M= O. mykiss, B= O. bonariensis. Subgroup CRlim T CRlim A CRlim O CRlim G CRlim I CRlim M CRlim B Mean CRlim Toddlers 0.0028 0.0042 0.0085 0.012 0.008 0.021 0.0016 0.0083 Children 0.0080 0.012 0.024 0.034 0.023 0.060 0.0046 0.024 Adolescents 0.015 0.023 0.046 0.064 0.043 0.115 0.0087 0.045 Adults 0.020 0.030 0.060 0.084 0.056 0.150 0.0113 0.059

Table 5: The As CRlim (kg fish/day) for the fish species at Lake Titicaca assuming no extra As intake via other food sources at Lake Titicaca: T= Trichomycterus spp., A= O. agassizii, L= O. luteus, G= O. gilsoni, I= O. ispi, M= O. mykiss, B= O. bonariensis. Subgroup CRlim T CRlim A CRlim O CRlim G CRlim I CRlim M CRlim B Mean CRlim Toddlers 0.0040 0.0035 0.0064 0.010 0.0035 0.014 0.010 0.0073 Children 0.011 0.0092 0.017 0.024 0.0091 0.037 0.027 0.019 Adolescents 0.019 0.017 0.031 0.043 0.017 0.068 0.050 0.035 Adults 0.025 0.022 0.040 0.058 0.021 0.087 0.064 0.045

Table 6: The Hg CRlim (kg fish/day) for the fish species at Lake Titicaca assuming no extra Hg intake via other food sources at Lake Titicaca: T= Trichomycterus spp., A= O. agassizii, L= O. luteus, G= O. gilsoni, I= O. ispi, M= O. mykiss, B= O. bonariensis. Subgroup CRlim T CRlim A CRlim O CRlim G CRlim I CRlim M CRlim B Mean CRlim Toddlers 0.0033 0.0050 0.010 0.014 0.0094 0.025 0.0019 0.0098 Children 0.0087 0.013 0.026 0.037 0.025 0.066 0.0050 0.023 Adolescents 0.016 0.024 0.048 0.067 0.045 0.12 0.0091 0.047 Adults 0.021 0.031 0.062 0.087 0.058 0.16 0.012 0.061

Table 7: Maximum average allowable fish consumption rate according to the Hg and As content taking into account the daily As and Hg intake from water out of Lake Titicaca: the CRlim (daily consumption rate limit) and the CRmm (consumption rate limit meals per month). Subgroup Average CRlim (kg fish/day) Average CRmm (meals/month) Compound Arsenic Mercury Arsenic Mercury Toddlers / 0.0083 / 1.3 Children / 0.024 / 3.6 Adolescents 0.00088 0.045 0.13 6.9 Adults 0.011 0.059 1.7 8.9

Table 8: Maximum average allowable fish consumption rate according to the Hg and As content assuming no extra As and Hg intake via other food sources at Lake Titicaca: the CRlim (daily consumption rate limit) and the CRmm (consumption rate limit meals per month). Subgroup Average CRlim (kg fish/day) Average CRmm (meals/month) Compound Arsenic Mercury Arsenic Mercury Toddlers 0.0073 0.0098 1.1 1.5 Children 0.019 0.026 2.9 3.9 Adolescents 0.035 0.047 5.3 7.2 Adults 0.045 0.061 6.8 9.3

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