Formation, uptake and bioaccumulation of

methylmercury in coastal seas – a Baltic Sea

case study

Aleksandra Skrobonja

Department of Chemistry

Umeå 2019

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

Dissertation for PhD

ISBN: 978-91-7855-177-4

Cover: Sampling cruise, incubation, and mesocosm experiment by Aleksandra Skrobonja

Electronic version available at http://umu.diva-portal.org/

Printed by: The service centre in KBC, Umeå University, Sweden 2019

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i

Table of Contents Abstract............................................................................................................................... ii

List of publications ......................................................................................................... iii

Enkel sammanfattning på svenska ...................................................................................... v

1. Introduction ................................................................................................................ 1

1.1. Mercury as a pollutant ............................................................................................. 1

1.2. Formation of MeHg .................................................................................................. 2

1.3. Uptake of MeHg ....................................................................................................... 3

1.4. Bioaccumulation of MeHg ....................................................................................... 4

1.5. Aims of the thesis..................................................................................................... 6

2. Materials and Methods............................................................................................... 7

2.1. Study sites ................................................................................................................ 7

2.2. Experimental approaches ........................................................................................ 8

2.2.1. Field sampling ................................................................................................... 8

2.2.2. Determination of total Hg and MeHg in aqueous samples and biota .............. 8

2.2.3. Methylation and demethylation rate constants ............................................... 9

2.2.4. DOC, humic matter content and ancillary data .............................................. 10

2.2.5. Microalgal cultivation ..................................................................................... 10

2.2.6. Mesocosm experiment ................................................................................... 11

3. Results and discussion .............................................................................................. 14

3.1. Organic matter input impacts MeHg formation and cycling (Paper I) ................... 15

3.2. The role of water column redoxclines on MeHg formation and cycling (Paper II) 18

3.3. Uptake kinetics of MeHg in a freshwater alga exposed to MeHg-thiol complexes

(Paper III) ...................................................................................................................... 21

3.4. Multiple impacts of humic-rich dissolved organic carbon on methylmercury

accumulation in heterotrophic pelagic food webs (Paper IV) ...................................... 27

3.5. Future research remarks ........................................................................................ 33

4. Conclusions ............................................................................................................... 35

5. Acknowledgements .................................................................................................. 37

6. Literature .................................................................................................................. 39

ii

Abstract

Methylmercury (MeHg) is a potent neurotoxin which can bioaccumulate to harmful levels in aquatic food webs. Methylmercury formation is a predominantly biotic process which involves phylogenically diverse microorganisms (e.g. iron- or sulfate-reducing bacteria). The formation of MeHg is related to the presence of organic matter (OM) which contains substrates essential for methylating microbes and reduced sulfur ligands (thiols, RSH) that form strong bonds with inorganic mercury (HgII) and affect its bioavailability. In aquatic systems, MeHg is bio-concentrated from the water column to the base of the food web and this step is crucial for MeHg levels found at higher trophic levels. Trophic transfer processes of MeHg in the food web are also of great importance. Discharge of OM in coastal areas affects light conditions needed for phytoplankton growth, and promotes heterotrophy, i.e. bacteria production. This may lead to a shift from the phytoplankton-based to the longer bacteria-based (microbial loop) food web and influence the amount of bioaccumulated MeHg in higher trophic levels. Methylmercury levels in predatory biota is thus affected by the bioavailability of HgII for methylation (studied in Paper I & II), MeHg speciation in the water column, crucial for MeHg incorporation at the base of the food web (Paper III), and the structure of the pelagic food web (Paper IV).

In this thesis, it is shown that OM can act as a predictor of dissolved MeHg levels in estuarine and coastal systems. It impacts MeHg levels both by affecting HgII bioavailability (through Hg complexation with humic matter) and the activity of methylating microbes (providing metabolic electron donors) (Paper I). Moreover, elevated concentrations of particulate and dissolved HgII and MeHg, are associated with the presence of pelagic redoxclines in coastal seas. The redoxcline affects HgII speciation in the water column and its bioavailability for methylation (Paper II). It is further shown that the molecular structure of ligands in MeHg complexes affects the kinetics of MeHg uptake in phytoplankton. Rate constants for association of MeHg to the cell surface of a green algae were higher in treatments containing smaller thiol ligands of simpler structure than in treatments with larger thiols and more “branched” structure (Paper III). Finally, it is demonstrated that MeHg bioaccumulation in zooplankton can increase in systems with highly heterotrophic food webs and enhanced loadings of terrestrial OM (Paper IV). Such conditions are expected to occur in northern latitude coastal systems following climate changes.

Key words: Mercury, methylmercury, bioaccumulation, mesososm, isotope tracers, methylation,

demethylation, stability constant, kinetic model, coastal sea, ICPMS, LC-MS/MS

iii

List of publications

Publications included in this thesis

I. Organic matter drives high interannual variability in methylmercury

concentrations in a subarctic coastal sea

Anne L. Soerensen, Amina T. Schartup, Aleksandra Skrobonja and Erik Bjorn

Environmental Pollution, 2017, 229, 531-538

II. Deciphering the Role of Water Column Redoxclines on Methylmercury Cycling Using Speciation Modeling and Observations from the Baltic Sea Anne L. Soerensen, Amina T. Schartup, Aleksandra Skrobonja, Sylvain Bouchet, David Amoroux, Van Liem-Nguyen and Erik Björn Global Biogeochemical Cycles, 2018, 32, 1498-1513

III. Uptake kinetics of methylmercury in a freshwater alga exposed to methylmercury complexes with environmentally relevant thiols Aleksandra Skrobonja, Zivan Gojkovic, Anne L. Soerensen, Per-Olof Westlund, Christiane Funk and Erik Björn Environmental Science & Technology, 2019, 53, 13757-13766

IV. Multiple impacts of humic-rich dissolved organic carbon on methylmercury accumulation in heterotrophic pelagic food webs Aleksandra Skrobonja, Sonia Brugel, Anne L. Soerensen, Evelina Griniene, Agneta Andersson and Erik Björn Manuscript in preparation

Paper I was reprinted with permission from Environmental Pollution. Copyright © 2017, Elsavier Ltd. All rights reserved. Paper II was reprinted with permission from Global Biogeochemical Cycles. Copyright © 2018, American Geophysical Union. All rights reserved. Paper III was reprinted with permission from Environmental Science & Technology.

Copyright © 2019, American Chemical Society. All rights reserved.

iv

Author contribution:

Paper I. The author participated in the planning and execution of one of the field

campaigns used in the study, performed the experimental analysis from samples

collected during several field campaigns and commented on the manuscript.

Paper II. The author participated in the planning and execution of one of the field

campaigns used in the study, conducted most of the experimental work, participated in

the data evaluation and commented on the manuscript.

Paper III. The author formulated the scientific research objectives and led the planning of

the study, performed most of the experiments and data processing and was lead author

on the manuscript.

Paper IV. The author was involved in formulating the scientific research objectives and

approaches, led the planning of the study, performed the experiments related to the

behavior of mercury in the system, processed the data and contributed significantly to

the writing of the manuscript.

v

Enkel sammanfattning på svenska

Metylkvicksilver (MeHg) är ett potent neurotoxin som kan bioackumuleras till skadliga nivåer i akvatiska näringsvävar. Bildning av metylkvicksilver är en övervägande biotisk process som involverar olika mikroorganismer (t.ex. järn- eller sulfatreducerande bakterier). Bildningen av MeHg är relaterad till förekomst av organiskt material (OM) som innehåller substrat som är viktiga för metylerande mikrober och som innehåller reducerade svavelligander (tioler, RSH) som bildar starka bindningar med oorganiskt kvicksilver (HgII) och påverkar biotillgängligheten. I akvatiska system biokoncentreras MeHg från vatten till näringsvävens bas och detta steg är avgörande för MeHg koncentrationer på högre trofiska nivåer. Överföring av MeHg mellan trofinivåer i näringsvävar är också av stor betydelse. Tilförsel av OM i kustområden påverkar ljusförhållanden som behövs för växtplanktons tillväxt och främjar heterotrofi, dvs. bakterieproduktion. Detta kan leda till ett skift från den växtplanktonbaserade till den längre bakteriebaserade näringsväven och påverka mängden av MeHg som bioackumuleras i högre trofiska nivåer. Metylkvicksilvernivåer i rovdjursbiota påverkas av biotillgängligheten av HgII för metylering (studerad i artiklarna I & II), speciation av MeHg i vatten, avgörande för MeHg inkorporering vid basen av näringsväven (artikeln III) och strukturen av den pelagiska näringsväven (artikeln IV).

I denna avhandling visas det att OM kan fungera som en prediktor för halten av löst MeHg i estuarier och kustsystem. Organiskt material påverkar halterna av MeHg både genom biotillgänglighet av HgII (genom Hg-komplexering med humusämnen) och aktiviteten för metylerande mikrober (genom att tillhandahålla metaboliska elektrondonatorer) (artikeln I). Förhöjda koncentrationer av partikulärt och löst HgII och MeHg är dessutom kopplade till förekomst av pelagiala redoxkliner i kustnära hav. Redoxkliner påverkar speciationen av HgII i vatten och biotillgängligheten för metylering (artikeln II). Det visas vidare att molekylstrukturen för ligander i MeHg-komplex påverkar kinetiken för upptag av MeHg i växtplankton. Konstanter för associering av MeHg till cellytan i en grönalg är högre i behandlingar med små tiolligander med enklare struktur än i behandlingar med större tioler och mer "grenad" struktur (artikeln III). Slutligen visas det att MeHg bioackumulering i djurplankton kan öka i system med mycket heterotrofiska näringsvävar och ökad tillförsel av terrest OM (artikeln IV). Sådana förhållanden förväntas öka i kustnära system på nordliga latituder i och med klimatförändringar.

1

1. Introduction

1.1. Mercury as a pollutant

Mercury (Hg) is a global pollutant which causes adverse health effects to humans and

wildlife. It is a naturally occurring element in the environment, comprising the Earth’s

crust with an average mass abundance of 0.08 parts per million (ppm).1 The Hg ores may

contain up to 12-14% of Hg, most commonly found as cinnabar (HgS) ore.2,3 Sources of Hg

release into the atmosphere can be of both natural (e.g. volcanic eruptions) and

anthropogenic origins (processes involving burning of fossil fuels, mining and other

industrial activities).4–6 Mercury is also present in the atmosphere, mainly in the form of

gaseous elemental mercury (Hgo) in a relatively low range of concentrations (1-170 ng m-

3).7,8 Both of these types of sources are predominantly emitting inorganic forms of Hg,

however, the more toxic organic form of Hg, methylmercury (MeHg), is the dominant Hg

species found in rice and fish.9–11

Excessive exposure to Hg can result in damage to the central nervous system (CNS) and

in severe cases lead to death. Higher risk for Hg exposure was linked to people working in

mines under poor conditions and insufficient knowledge.7 However, the main source of

Hg to humans involves ingestion of fish and rice originating from Hg contaminated waters

and rice paddies.9,12–14 The first incident involving consumption of MeHg contaminated

fish and shellfish was officially documented in 1956 in Minamata city, Japan.15 With

thousands of patients diagnosed, and a high mortality rate, the disease including MeHg

intoxication was named “The Minamata disease”.15,16

During the 1950s and 1960s, the use of organic Hg compounds as fungicides in agriculture

and pulp and paper industry was widespread in Sweden. Moreover, high amounts of Hg

were discharged into the water and air from chlorine-alkali industries as well as from

incineration of waste containing Hg.17 Today, one of the most serious concerns is the

elevated concentration of Hg in piscivorous fish (e.g. pike), as it exceeds the levels

considered safe for human consumption (0.1 µg of Hg/kg body weight/day).18

Furthermore, it has been reported that elevated hair concentration of Hg (0.58 µg g-1) was

detected in about 10% of children born in Sweden and this could be linked to the mother’s

diet affected by Hg levels surpassing health guidelines and regulations.7,19

2

Considering the bioaccumulating, biomagnifying properties of MeHg and its effect on the

population and the environment, it is of great importance to improve the understanding

of MeHg formation, entrance into the base of the aquatic food web, bioaccumulation and

biomagnification in the food web.

1.2. Formation of MeHg

Methymercury is formed from inorganic mercury (HgII) predominantly via biological

reactions which take place in suboxic and anoxic environments. The methylation process

has been coupled with the presence of sulfate reducing bacteria (SRB), iron reducing

bacteria (IRB), and more recently, methanogens and other microbes.20–25 In the oxic water

column, HgII can be converted to MeHg in the water column during the process of organic

matter (OM) remineralization (microbes break down the OM) and within particulate

organic matter (POM, fraction of OM that does not pass through a 0.2 µm filter)

aggregates with low oxygen micro-environments.26–30 Bioavailability of HgII is an

important factor for Hg methylation as it can limit the amount of available HgII for uptake

by methylating microogranisms. In nature, HgII availability and speciation are controlled

by pH and redox conditions, concentration of inorganic sulfide (S(-II)) due to the strong

affinity to Hg, and properties of organic matter (OM). Precursor material, source origin,

maturity, composition and other characteristics of OM can affect HgII methylation.

Namely, OM can carry substrates necessary for methylation microbes and promote

methylation.31 On the other hand, OM can contain ligands which form strong bonds with

HgII, such as organic ligands with a sulfhydryl (thiol) group (RS-) in their structure, and

repress methylation.32–37 Dissolved organic matter (DOM) is defined in this work as the

fraction of OM which passes through the 0.2 µm filter, and HgII and MeHg in marine

environments may be present complexed with OM in the dissolved form, depending on

the binding affinity to ligands and ligand distribution between the dissolved and

particulate phase.35,38 Redox potential is another important factor controlling MeHg

production in water columns as elevated concentrations of total mercury (THg) and MeHg

have been reported in hypoxic (<2 mL O2 L-1) and anoxic (total depletion of O2) zones.39–

41

3

In this thesis work, research on MeHg formation was conducted based on observations

from the Baltic Sea. In the northern Baltic Sea, freshwater input creates a north-south

salinity gradient and causes a decrease in the fraction of terrestrially discharged

(allochthonous) to in situ produced (autochthonous) DOM along this gradient.42,43 During

spring and fall, loading of allochthonous DOM to the estuarine and nearshore sites

increases.44,45 In the southern basins of the Baltic Sea, phytoplankton blooms caused by

excess nutrient and OM runoffs increase the system’s biological oxygen demand (BOD)

and contribute to the formation of permanently hypoxic and anoxic zones (35-40% of the

surface area).46–48 Mercury in the surface waters binds to allochthonous and

autochthonous particulate organic matter (POM) and sinks into the deeper water where

it is released during the microbial OM remineralization process. Furthermore, increases

in dissolved THg at the hypoxic-anoxic interface have been reported in other systems with

hypoxic and anoxic conditions and these observations were linked to the dissolution of

iron (Fe) aggregates which trap OM and Hg.49,50 Redox potential can further induce

different distributions between Hg-thiol and Hg-sulfide complexes and Hg partitioning

between the dissolved and particulate phases which all affect Hg bioavailability and

reactivity.

One of the objectives in this thesis work was to explore how OM composition and

concentration as well as hypoxic/anoxic conditions impact MeHg production and fate in

the water column of coastal seas (Paper I & II).

1.3. Uptake of MeHg

Once MeHg is produced in the water column, it can be bio-concentrated to the base of

the food web. This step is crucial for MeHg levels observed higher up the food web as the

concentration from the water to the basal producers is increased by a factor of 104 to

107.38,51–56 Cellular uptake of metals by microorganisms from the aqueous phase involve

diffusion of the metal (or metal complex) to the cell surface which is then followed either

by passive diffusion through the cell wall, or formation of a metal complex with ligands

located on the cell wall or biological membranes and subsequent active transport into the

cytoplasm.57–59 The association of MeHg to the cell surface would be regulated by the

competition between its binding to the ligands in the dissolved phase and at the cell

4

surface.This is hypothesized in the Biotic Ligand Model (BLM) which has been previously

established for several trace metals, but not yet for Hg.60,61 In natural waters, MeHg forms

strong complexes with organic ligands which contain reduced sulfur groups in the DOM

pool, and the chemical speciation of MeHg thus controls its bioavailability and governs

the uptake rate by phytoplankton or bacteria.32–34,62 Various studies have investigated the

relation between aqueous speciation of MeHg and cellular uptake as well as the

mechanistic aspect of the MeHg uptake process.52,62–67 While some studies suggested

passive diffusion as the main uptake mechanism for MeHg uptake in unicellular

algae63,66,68, evidence for active transport being involved in this process has been found in

several other cases.52,54,62

In Paper III, we investigate how the molecular structure and size of six thiol compounds

commonly present in the environment affect cellular uptake kinetics of MeHg in the green

freshwater alga Selenastrum capricornutum. The uptake of MeHg was determined in

short-term exposure experiments on the laboratory scale for whole cells and the

cytoplasmic fraction. This data was further applied to two kinetic models which were used

for determination of the rate constants involved in the cellular uptake process of MeHg.

1.4. Bioaccumulation of MeHg

Following the bioconcentration step from the dissolved phase to the base of the pelagic

food web, MeHg is mainly accumulated in zooplankton and higher trophic levels via

grazing and less through direct uptake of MeHg from the water column.67 Herbivorous

zooplankton feed on phytoplankton and microbes whereas the diet of omnivorous

zooplankton in addition includes ingestion of smaller zooplankton. Trophic transfer up

the food web is more efficient for MeHg species and this results in higher percentage of

MeHg in the THg pool as the trophic level increases.53,69,70 The concentration of MeHg in

top consumers is influenced by the total length of the food web and the bioaccumulation

factor (BAF) for each step (usually varying between 0.5 to 1.0 log units).71,72 Increase in

MeHg from one trophic level to another is defined as biomagnification factor (BMF). It

has been proposed that longer food webs such as those found in heterotrophic systems

result in elevated concentrations of MeHg in zooplankton.73 Moreover, factors like growth

5

rate (biodilution), trophic level of the prey and lifespan and body size of the consumer

may impact and control MeHg levels in these predators.70,72,74,75

The Baltic Sea is a coastal, semi-enclosed sea with spatial gradients in salinity,

temperature, proportions of allochthonous to autochthonous OM and, consequently,

share of bacteria versus phytoplankton as primary producers. These factors could be

important controllers for MeHg formation, bioaccumulation and biomagnification in

different areas of the Baltic Sea. Recently, it has been suggested that MeHg production

and bioavailability are reduced in marine systems with higher influence of allochthonous

over autochthonous OM.27,76 However, aqueous concentration of MeHg is most often

higher in the estuarine coastal areas than in the offshore waters due to the transport of

MeHg by river discharge. Higher content of allochthonous OM can promote heterotrophy

and cause changes in the structural changes at the base of the pelagic food web,

potentially enhancing MeHg bioaccumulation.77 To test this hypothesis, we have

performed a mesocosm experiment (Paper IV).

6

1.5. Aims of the thesis

This thesis work was conducted to improve the current understandings of MeHg

formation, incorporation in the base of the food web and bioaccumulation. The individual

aims of the thesis work are (1) Deciphering the role and impact of OM and water column

redoxclines on MeHg formation and cycling in a subarctic coastal sea (using observations

from the Baltic Sea) (Paper I & II). (2) Establishment of comprehensive kinetic models

describing MeHg uptake rate constants in phytoplankton, based on MeHg speciation in

the medium (Paper III). (3) Exploration of the OM and food web structure influence on

bioaccumulated MeHg in higher trophic levels (zooplankton) (Paper IV). The summary

figure showing contributions of each Paper to address MeHg formation, uptake and

bioaccumulation is shown in Figure 1.

Figure 1. A summary figure illustrating used approaches and studied processes of this thesis work.

7

2. Materials and Methods

2.1. Study sites

Water and seston samples were collected from different areas of the Baltic Sea during

two individual cruises for three consecutive years (2014, 2015 and 2016), i.e. in total six

cruises. The field samplings included cruises with Umeå Marine Science Center crew

onboard the Swedish Coast Guard’s ship KBV181 in the northern basin of the Baltic Sea

(Bothnian Bay and Bothnian Sea) whereas samples from the southern areas of the Baltic

Sea (the Baltic Proper, Belt Seas) were collected onboard R/V Aranda research ship in

collaboration with Swedish Meteorological and Hydrological Institute (SMHI). The map,

locations and sampling transect are shown in Figure 2.

Figure 2. Map of the Baltic Sea showing the locations of sampling stations as well as the sampling

transect. Station names in black color indicate stations where anoxic conditions were not detected,

stations marked in red illustrate the locations with anoxic conditions in deep water while the names

in purple indicate stations where anoxia was present only some years. (Reproduced from Paper II

with permission from American Geophysical Union)

8

2.2. Experimental approaches

2.2.1. Field sampling

Water and seston samples were collected for three consecutive years, during six cruises

in the Baltic Sea (data published in Paper I & Paper II): September 2014 (SEP14-S); July

2015 (JUL15-S) and July 2016 (JUL16-S) in the Southern Baltic (Baltic Proper and Belt Seas),

and September 2014 (SEP14-N), August 2015 (AUG15-N) and August 2016 (AUG16-N) in

the Northern Baltic Sea (Bothnian Bay and Sea). All water samples were collected using 2

L Niskin bottles attached to a rosette or 5 L Teflon-lined General Oceanic (GO-FLO) bottles.

Total Hg samples were collected in 125 mL amber glass (I-CHEM certified™) or PFA and/or

FEP (Nalgene) bottles while the samples for MeHg analysis (monomethylmercury (MMHg)

+ dimethylmercury (DMHg)) were collected in 125 or 250 mL Teflon® or HDPE bottles pre-

cleaned with trace metal grade HCl (Suprapur HCl, Merck). A subset of MeHg samples in

2015 and 2016 were filtered through 0.22 µm hydrophilic PTFE filters using Teflon or

Sterivex 0.22 µm filter units. All samples were preserved with hydrochloric acid (1% v/v,

Suprapur® HCl 30%, Merck Millipore) and stored in the dark at 4°C until they were further

processed.

2.2.2. Determination of total Hg and MeHg in aqueous samples and biota

Total Hg in natural aqueous samples was detected using atomic fluorescence

spectrometry following USEPA 1631.78 All Hg species in the samples were oxidized to HgII

using bromine monochloride (BrCl) which was followed by HgII reduction to volatile

elemental Hg (Hg0) using tin(II) chloride (SnCl2) solution.78 In the mesocosm water

samples, elemental Hg was purged and the analysis was done using an on-line cold vapor

generation (HGX-200, Cetac) system connected to a PerkinElmer/Sciex ELAN DRCe ICPMS.

Total Hg in biota samples was measured using a Direct Mercury Analyzer (Leco DMA 80)

following the manufacturer’s method specifications.

Methylmercury content in water samples was analyzed based on previously described

methods.79,80 In short, water samples were pH adjusted to 4.8 using a 2 M acetate buffer

and sodiumhydroxide (NaOH). Further, MeHg species were ethylated by addition of

9

sodium tetraethylborate (STEB) and purged and trapped onto Tenax adsorbent matrix.

Ethylated mercury species were then thermal-desorbed to gas chromatography coupled

with inductively coupled plasma mass spectrometry (TDGC-ICPMS, 6890 Agilent GC and

7700 ICPMS).81 Biota samples for MeHg analyses were digested for 2 h at 60 °C using

tetramethylammonium hydroxide (TMAH) and sonication.82 The pH value was

subsequently adjusted to 4.8 by adding concentrated acetic acid, after which the samples

were ethylated, purged and trapped, and analyzed in the same way as the water samples.

2.2.3. Methylation and demethylation rate constants

The rate constants for inorganic HgII methylation and MeHg demethylation (km, kd (d-1),

respectively) in Paper II and IV were determined using enriched stable isotope tracers and

followed the experimental and data treatment described by Rodriguez-Gonzales et al.83

The rate constants were determined in incubation experiments which involved both light

exposed and samples kept in the dark. In Paper II, the light incubations were performed

on water samples collected from 5-10 m and in a few cases from 20 m, during the 2015

and 2016 field sampling onboard. The dark incubations were performed in 2014, 2015

and 2016 for water collected from three different depths (<15 m, 30-40 m and >60 m).

Each sample was spiked with isotopically enriched Me204Hg and 198HgII and the

incubations were stopped by acidification with 0.1 M HCl. For 2014 and 2016 samples,

the incubations were stopped at t=0 and 24 h while the incubations for samples collected

in 2015 were stopped at t=0, 4, 8 and 24h. In the mesocosm experiment (Paper IV), the

incubations were stopped at t=0 and 14 h and only the dark incubations were performed.

Following acidification, the samples were spiked with isotopically enriched Hg standards

for isotope dilution analysis and the samples were analyzed as previously described.

10

2.2.4. DOC, humic matter content and ancillary data

Concentrations of oxygen, sulfide, nitrate, ammonium, phosphorous, silicate, chlorophyll

a, DOC and humic matter content are measured on a monthly basis as part of the Swedish

National Monitoring Program. The samples are analyzed following HELCOM guidelines84

and it is possible to download the quality controlled data from the SMHI’s website (Paper

I & II).85 Humic matter is measured using fluorescence spectroscopy at 350/450 nm

excitation/emission wavelength using quinine sulfate as a calibration standard (Papers I,

II & IV).84 Concentrations of DOC were determined using a Shimadzu TOC-5000 high

temperature catalytic oxidation instrument with NDIR detection after sample

acidification and purging.86 Carbon concentration was then calculated using potassium

hydrogen phthalate as a standard.

2.2.5. Microalgal cultivation

The green unicellular microalga Selenastrum capricornutum (Culture Collection of Algae,

Goettingen University, SAG) was used to assess MeHg uptake in assays with six different

MeHg-thiol complex treatments and a control (Paper III). All glassware was autoclaved

and acid washed following trace metal clean protocols. In the pre-inoculum, 30 ml of algal

culture was grown axenically in 100 mL Erlenmeyer flasks. The medium used in this study

was sterile Bold’s basal medium, depleted in Cu, Zn and EDTA, with addition of 30 mM

sodium nitrate (3N BBM)87. The culture was kept in a closed orbital shaker at 115 rpm,

25°C and 100 µmolph m-2 s-1 of white light for seven days. The pH was maintained at 6.6 ±

0.2 by the balance between CO2 addition (acidifying the medium) and gradual nitrate

assimilation by the alga (uptake of protons).88,89 The medium was prepared without Cu

and Zn to avoid their competition with MeHg for cellular binding sites, and EDTA was left

out to avoid competitive binding of MeHg. The culture was transferred to 100 ml

Erlenmeyer flasks with 70 ml of the same BBM medium, maintained for 5 days at 20°C,

under 100 µmolph m-2 s-1 of white light and bubbled with 3% CO2 in air at a constant flow

of 1 L min-1. No contamination of the culture was detected under the light microscope

(Leica DMi1, 40× magnification). Population density was measured each day using

Beckman Coulter Multisizer 3 cell counter (aperture size 70 µm, analytical volume 100 µL,

sample dilution 200×) following manufacturer’s method specifications. Optical density

11

was measured at 750 nm wavelength on a UV/Vis Spectrophotometer (Varian Carry 50

Bio) using a 10 mm light path polystyrene cuvette. The culture was inoculated and further

cultivated in a 5 L Duran® borosilicate glass bottle with a GL 45 screw neck. The bottle was

filled up to 4.8 L with depleted BBM. The culture was illuminated with a white LED panel

(650 µmolph m-2 s-1) and bubbled with 3% CO2 in air at a constant flow rate of 1 L min-1.

After ~48 h, the cell density reached ~ 4 000 000 cell ml-1 and the content from the 5 L

bottle was evenly divided into seven different trace metal clean and autoclaved 1 L

Duran® borosilicate bottles. The control (MeHgOH) and six MeHg-thiol complexes were

then added to separate bottles to the final concentration of 1.5 nM MeHg and 90 nM

thiol. Each solution was further split into two replicates and the flasks were maintained

for 8 h at 20°C using a white LED panel (650 µmolph m-2 s-1) bubbled with a constant flow

of 1 L/min of 3% CO2 in air during the exposure.

2.2.6. Mesocosm experiment

Brown, humic rich soil was sampled from a location close to the Öre River (coordinates

63° 59.5469′ N, 19° 80.9809′ E). The soil was homogenized manually and sieved through

4-6 mm sieve nets. Following, 9.8 kg of the soil was mixed with 2.3 kg of chelating resin

(Amberlite™ IRC748 and Diaion® CR11, sodium form, Sigma Aldrich) and 65 L of MQ water

(18.2 MΩ cm-1, at 25°C and 5 ppb total organic carbon (TOC)) for the extraction of DOC. A

mechanical pump was immersed into the extraction tank to allow vigorous stirring during

48 h of extraction. The extract was sequentially passed through 100 µm and 15 µm nets,

centrifugated (10000 rpm, 10min, rotor JA-14, 6*250ml bottles) and stepwise filtered

through a filtration system compiled of 25, 5, 3 and 1 µm filters. In Paper IV we refer to

the <1 µm extract fraction as DOC, even though it has not passed through a 0.2 µm filter.

The TOC content of the extract was 2.4 g L-1 and total Hg and MeHg concentrations were

8.9 ± 1.4 pmol g-1 (d.w) and 32 ± 4.8 fmol g-1 (d.w), respectively.

Twelve double-mantled high-density polyethylene (HDPE) indoor mesocosms (5 0.74 m

in diameter) were used, located at the Umeå Marine Sciences Center facility, Umeå

University (Paper IV). Using a pumping system, the mesocosms were simultaneously filled

with 1.2 mm filtered brackish water (average salinity 3.79 ± 0.01 PSU, DOC 3.83 ± 0.07 mg

L-1) from two inlets located 800 m from land (in the Öre River estuary) at water depths of

12

2 and 8 m which allowed even distribution of planktonic communities. For each DOC

treatment (Level 1: DOC 5.5 mg L-1, Level 2: DOC 6.5 mg L-1 and Level 3: DOC 8.0 mg L-1)

and the Reference, three replicate mesocosms were prepared and maintained. As light

sources, 150 W metal halogen lamps were used (MASTER Colour CDM-T 150W/942 G12

1CT) and the light:dark cycle was set to 12:12 hours. The photosynthetically active

radiation level of the lamps was adjusted to be 350 ± 17.5 µmol s-1 m-2 at 1 cm below the

water surface. To achieve a high rate of convective stirring of the water column, the

bottom sections of the mesocosms were continuously heated with a hot (40°C) 40%

propylene glycol solution, while a cold (-4°C) solution was added to the top parts. The

middle sections were set to maintain 15°C and thus controlled the overall temperature in

the water column (average 15.2 ± 0.3 °C), while upholding convective stirring with a

complete mixing for 20 min.90

Nitrate (NO3-), phosphate (PO4

3-) and ammonium (NH4+) solutions were prepared from

pure salts of NaNO3, NaH2PO4 H2O and NH4Cl, respectively and added to the mesocosms

as nutrients (detailed addition scheme is presented in Table S2 (Paper IV).

The following HgII enriched isotopes, purchased as HgO or HgCl2 from Oak Ridge National

Laboratory (TN, USA) were used in the experiment: 199Hg (91.09%), 200Hg (96.41%) and

201Hg (96.17%). The Me200Hg and Me201Hg enriched stock solutions were prepared

following the procedures described elsewhere.91 Isotope standards 199Hg (10 µM) and

Me201Hg (1 µM) were pre-equilibrated with 5 mL of the DOC extract in 5 L of MQ water

by being left to mix on a magnetic stirrer overnight prior to the mesocosm additions

(details specified in Table S3, Paper IV). Water samples were regularly analyzed for total

Hg (HgT) and MeHg concentration after sampling and the additions would be accordingly

adjusted the next sampling day to maintain a constant level of HgT (2 pM) and MeHg (0.2

pM).

Water samples were taken from the mesocosms at 1 m depth twice a week (Mondays

and Thursdays) for determination of total Hg and MeHg concentrations, bacteria and

primary production rates, and concentrations of DOC, humic matter, nutrients and

chlorophyll (Chl a). Once a week, 50 mL and 25 L of water were collected for taxonomic

classification and counting of nano- and microplankton, and zooplankton, respectively.

13

Water for total Hg and MeHg determination was sampled in acid-washed bottles of 125

mL (I-Chem Boston Round Narrow-Mouth Amber Glass Bottles) and 250 mL (Nalgene

Fluorinated Narrow-Mouth HDPE Bottles), respectively, using a peristaltic pump with

online filtration (0.2 µm PTFE membrane filters) and Teflon tubing. Total Hg samples were

oxidized by adding of 1.25 mL of a BrCl solution78 prior to acidification. MeHg samples

were acidified using hydrochloric acid (1% v/v, Suprapur® HCl 30%, Merck Millipore).

Following, 200HgII or Me200Hg were added as internal standards for isotope dilution

analysis and the samples were stored at 4°C until analysis.

Vertical irradiance profiles of photosynthetically active radiation were measured using a

LICOR-193SA light sensor. Light measurements were carried out at the water surface and

depths of 0.1, 0.2, 0.4, 0.6, 1, 1.5, 2, 2.5, 3, 3.5 and 4 m. The diffuse light attenuation

coefficient (Kd) was calculated from the slope of the linear regression of the natural

logarithm of down-welling irradiance versus depth. After each water sampling, the

mesocosm tanks were refilled with 0.2 µm filtered seawater to compensate for the

volume lost during the sampling procedures (~25 and ~10 L for sampling days including

and not including plankton taxonomic determination, respectively).

Four seston size fractions were collected at the end of the experiment (day 36) from the

entire water column above 0.77 m from the mesocosm bottom, using a series of 200-, 90-

, 50- and 20 µm mesh size plankton nets for water filtration. The plankton samples were

frozen and stored at -20°C until they were freeze-dried and analyzed for δ13C, δ15N and

MeHg concentration.

14

3. Results and discussion

It has been established that OM is a driver of HgII methylation and that OM composition

as well as concentration have an impact MeHg levels measured in a certain system.27,37,92–

95 However, not many studies have looked into how interannual concentration and

composition of OM affect MeHg formation and its biogeochemical cycling within systems.

In Paper I, we used the Baltic Sea as our study system and explored how characteristics

and concentration of OM affect aqueous MeHg concentrations. We used Hg and OM

measurements in a range from 2014 to 2016 and also discussed how interannual

variability of OM parameters may impact future MeHg concentrations, especially under

predicted climate change scenarios. Furthermore, excess of nutrients and OM in coastal

systems such as the Baltic Sea, transported by runoff from e.g. sewage treatment facilities

or agricultural lands, cause phytoplankton blooms which, eventually, results in reduced

oxygen levels or complete depletion in certain zones. A number of studies has evaluated

the importance of chemical speciation of Hg on its cycling and bioavailability to biota,

however, transformation pathways of Hg in coastal seas with water column redox

gradients are understudied.36,96,97 Hence, in Paper II we used concentrations of THg and

Hg species (e.g. Hg0, HgII, MeHg, DMHg) to assess MeHg production and its fate under

hypoxic and anoxic conditions. Paper II uses the complete dataset from the field cruises

2014-2016 whereas in Paper I, a subset of this data is used since the focus of the paper is

placed on the Bothnian Bay and Sea basins of the Baltic Sea. In Paper III we studied MeHg

uptake by a green algae model organism in a microcosm-scale experiment. Contrasting

results from previous work on MeHg uptake processes bring to attention the fact that

more detailed studies are needed to understand MeHg bioavailability and uptake

mechanisms in phytoplankton when MeHg is complexed with low molecular mass (LMM)

thiols.52,62–67 In Paper III we evaluated how the molecular structure of six LMM thiols,

detected in the Baltic Sea, affects cellular uptake kinetics of MeHg in the freshwater alga

Selenastrum capricornutum when exposed to MeHg-thiol complexes. In paper IV, we

studied MeHg bioaccumulation under increased loadings of terrestrial organic matter as

it has been predicted that terrestrial runoff may increase in large regions, particularly in

the northern hemisphere, due to the predicted global warming scenarios. It has been

15

previously demonstrated that increased input of terrestrial OM can enhance the

bioaccumulation factor (BAF) of MeHg in zooplankton by shifting the pelagic food web

from phytoplankton-based to more bacteria-based.73 We conducted a mesocosm

experiment with three treatments containing different levels of increased DOC

concentrations, mimicking hypothetical climate change scenarios, and a reference system

with no additions of terrestrially derived OM. Using concentrations of MeHg and various

biological parameters (e.g. primary and bacteria production rates, taxonomic

classification), we calculated the BAF of MeHg in zooplankton, described the food web

structure in the four treatments and discussed the outcome with regards to the predicted

increase in precipitation followed by enhanced input of terrestrial OM to coastal systems,

induced by climate change.

3.1. Organic matter input impacts MeHg formation and cycling (Paper I)

Organic matter contains strong binding ligands for HgII such as reduced sulfur groups, and

substrate molecules which are essential for metabolism of heterotrophic microbes.31,35

Since methylating organisms require both bioavailable HgII and metabolic supplements to

produce MeHg, OM composition and concentration are one of the most important factors

that control MeHg ambient concentrations. In the Northern Baltic Sea, most of the

freshwater enters the system through the Bothnian Bay and creates north to south

gradient in salinity and proportions of allochthonous and autochtonous DOM (fraction of

organic matter in solution that passes through a 0.2 µm filter). The amount of terrestrially

derived DOM decreases from the coastal regions over the Bothnian Bay and Sea, however

the total DOM levels are relatively constant in the offshore waters.42 Furthermore, it has

been shown that there is a ~300 µM refractory level of DOC (concentration of organic

carbon in the DOM fraction) which is not readily utilized by microbes, thus the remaining

portion of labile DOM (concentration >300 µM) from the total DOM pool has potential to

be metabolized by microbes and cause OM remineralization in this area.45,98

The trend showing decrease in total Hg following the salinity gradient from the Råne

estuary (5.95 pM, station RA1 in Figure 1, inner estuary, and 2.00 pM, station RA2, outer

estuary), over the Bothnian Bay (1.2 ± 0.30 pM, stations A5 and A13) and the Bothnian

Sea (0.84 ± 0.24 pM, stations C3, C14, GA1) is presented in Figure 3A. Figure 3B illustrates

16

the same decreasing trend for MeHg from the maximum measured concentration in the

inner estuary (306 fM) to the average offshore concentrations (80 ± 25 fM in 2014, <LOD

in 2015 and 21 ± 9 fM in 2016). Figures 3C and 3D present the relationship between the

labile DOC pool and MeHg concentrations showing that labile DOC may be used as a proxy

for OM remineralization rate and HgII methylation potential. Possessing strong binding

sites, DOM binds and affects bioavailability of MeHg, but also reduces MeHg

photodemethylation due to decreased light attenuation. We propose that MeHg

demethylation outcompetes MeHg production when labile DOC pool is exhausted, which

results in MeHg depletion from the water column. Humic matter content shown in Figure

3D suggests that humic matter has a negative correlation with MeHg concentrations.

Presence of humic matter could enhance MeHg levels by decreasing light attenuation and

MeHg bioavailability resulting in reduced MeHg photodemethylation and microbial

demethylation, respectively. On the other hand, humic matter can form stable complexes

with HgII making it less susceptible for microbial methylation, and it has also been shown

that humic matter can abiotically reduce HgII to Hg0.27,99,100 Our results point to reduced

HgII bioavailability which can either be explained by the formation of stable complexes

between the humic matter and HgII, or HgII reduction to Hg0.

17

Figure 3. Water MeHg and DOC concentration. A) solid lines indicate significant linear regressions

for the Bothnian Bay and Bothnian Sea; B) The color of squares indicates the concentration of

humic matter content. (Reproduced from Paper I with permission from Elsevier Ltd)

From aqueous phase in estuarine systems, Hg can be removed either via evasion or

settling with particles. Moreover, significant amounts of DOC may be lost through

flocculation and settling in areas where freshwater and water with higher salinities mix.101

In our study, the loss of total Hg due to the conservative mixing of salinities 1 to 2 causing

DOM flocculation and settling, is estimated to be 49% (Figure 3A and Figure S1, Paper I).

Looking into the effect of POC, DOC, suspended particulate matter (SPM) and particle

18

partitioning coefficient, we calculate that 1 – 11% of total Hg is lost through particle

settling in the Råne estuary.102–104 We furthermore estimate 8 – 17% of dissolved Hg to

be lost through DOM flocculation and settling, and prescribe the rest of the remaining

loss of total Hg from the estuary (43-82%) to be due to Hg0 evasion from the

system.101,105,106 Even though some fraction of the MeHg pool undergoes the same

removal transformation as HgII (i.e. particle and flocculation settling), HgII methylation and

MeHg demethylation processes also come into play, affecting the proportions of HgII and

MeHg when comparing estuarine and offshore water columns.

We propose that the DOC and humic matter content can be used to explain the variability

in MeHg concentration in the Northern Baltic Sea. We further suggest that this variability

is controlled by the OM remineralization rate (with labile DOC used as a proxy) and HgII

availability (explained by the humic matter content effect), both contributing to net HgII

methylation.

3.2. The role of water column redoxclines on MeHg formation and cycling (Paper II)

The importance of redoxclines in water columns has been shown through elevated THg

and MeHg concentrations reported for hypoxic (<2 mL O2 L-1) and anoxic (total depletion

of O2) zones.39–41 To be able to predict the Hg fate following increased spread of oxygen

depleted zones in marine ecosystems, better understanding of Hg chemical speciation

and cycling across the redoxclines is needed.47,107 The Baltic Sea is a shallow, semi-closed

system with a large inflow of freshwater from runoff.108,109 The runoff usually contributes

to an increase in the amount of nutrients and OM (e.g. from water treatment facilities or

agricultural soils), which induces phytoplankton blooms and eventually causes oxygen

depletion in certain areas.47,107 Since the majority of methylating microbes thrive in

hypoxic or anoxic oxygen conditions, these oxygen depleted zones present potential

hotspots for MeHg formation. In the surface water, Hg can bind to the particulate organic

matter (POM, fraction of OM that does not pass through a 0.2 µm filter) transported by

runoff or formed from decaying biological material, and sink to deeper waters where it

can be released during remineralization of OM. The released HgII is potentially available

19

for methylating microbes at or below the redoxcline. Previous studies conducted in the

Black sea found elevated concentration of THg at the interface between the hypoxic and

anoxic zone, and it was suggested that dissolution of iron (Fe) oxides with the capability

to trap Hg and OM in their aggregates is causing these increases in THg levels.49,50

Methylmercury concentrations measured in the Baltic Sea were higher in the hypoxic

layer than in the surface water, and highest in the anoxic zone.39,110 Furthermore, it has

been suggested that HgII and MeHg in coastal seas will mainly bind to reduced organic

sulfur ligands and form complexes which could partition between the dissolved and the

particulate phase depending on the redox conditions, and this could impact Hg

bioavailability and reactivity.36 Spatial distribution of averaged THg and MeHg for all

cruises is presented in Figures 4A and 4B. To summarize, total and methylmercury

concentrations were all significantly higher in the hypoxic and anoxic layer compared to

the surface one (range of 0.5 to 10.7 pM for THg and <LOD to 1640 fM for MeHg) whereas

Hg0 only showed statistically significant difference between the surface and anoxic water

(range of 0.02 to 0.77 pM). We interpret the two THg peaks at the normoxic-hypoxic and

hypoxic-anoxic interfaces as the result of : 1) Fe(III) aggregates formation when Fe is

transported from the anoxic to the hypoxic zone and oxidized to Fe(III)oxyhydroxides

which can trap DOM and metals, comprised of mainly particulate THg and 2) dissolution

of Fe(III)oxyhydroxide-DOM aggregates and/or HgS(s) particles below the reductive

dissolution zone, and that this peak of THg is mainly composed of released HgII during the

dissolution processes.111–114

20

Figure 4. Spatial distribution of A) total Hg, B) MeHg C) HgII methylation rate constants and D) dark

MeHg demethylation rate constants averaged across all sampling campaigns. Grey lines indicate

the normoxic-hypoxic interface and white lines the hypoxic-anoxic interface. (Reproduced from

Paper II with permission from American Geophysical Union)

Spatial distribution of HgII methylation and MeHg demethylation rate constants in

unfiltered water samples is presented in Figure 4C and 4D. The methylation rate constants

in two samples obtained from the normoxic zone were detected (2.4 x 10-4 – 4.8 x 10-4 d-

1) and in four samples from the anoxic water (16 x 10-4 – 48 x 10-4 d-1). Methylation rate

constants for HgII were 4-20 times higher in the anoxic water than in the normoxic layer.

The range of MeHg dark demethylation rate constants was <LOD – 0.34 d-1 and these rate

constants were around 3 times higher in the anoxic water compared to both the hypoxic

and normoxic water. Methylmercury photodemethylation rate constants ranged from

0.07 to 0.96 d-1. Using biogeochemical model simulations, we suggest that the elevated

MeHg concentrations found in the anoxic zone of the Baltic Sea is most likely due to the

high in situ MeHg production.39 Given the high levels of dissolved sulfide with an average

value of 19.7 and a maximum of 56 µM in the anoxic zone, and the predominance of delta-

proteobacteria in the Baltic Proper,115 we propose that HgII methylation in the anoxic zone

is mainly carried out by sulfate reducing bacteria. The dark demethylation rate constant

was higher in the anoxic than in the normoxic zone for a factor of 3 as opposed to the

factor of 10 difference observed for HgII methylation rate constant. This contributes to an

overall increase in MeHg net production and MeHg concentrations in the anoxic zone. We

propose that the increase in MeHg demethylation under anoxic conditions can be

21

explained by the presence of the same bacteria responsible for HgII methylation and

higher bioavailability of the dissolved MeHg-sulfide complexes.116–118 Furthermore, most

of the MeHg variability across the anoxic zone is found close to the hypoxic-anoxic

interface and we suggest three possible explanations for this observation: 1) direct mixing

of water mass between the two zones, 2) a change in chemical speciation of HgII which

affect its bioavailability for methylation and 3) the activity of methylating microbes

depending on the concentration of electron donors or acceptors (OM composition,

Fe(III)/SO42-). Results from our speciation model suggest that, for the rest of the anoxic

zone, chemical speciation of HgII does not considerably vary and will not drive variability

in MeHg concentrations or HgII methylation.

Exposure of MeHg to biota in water columns with redoxclines would primarily be

impacted through the organisms feeding in the hypoxic zone. As phytoplankton are less

likely to be found in this zone, zooplankton can take up MeHg directly from the water or

through ingestion of OM and smaller zooplankton. The 2-6 time increase in MeHg

concentration could thus result in significant exposure for zooplankton and the effect of

grazing in the hypoxic zone remains to be addressed, especially considering the spread of

oxygen depleted areas in coastal zones worldwide.

3.3. Uptake kinetics of MeHg in a freshwater alga exposed to MeHg-thiol complexes (Paper III)

Methylmercury is bio-concentrated from water to the base of the food web by a factor of

104 to 107 and this step is thus crucial for MeHg concentrations found at higher trophic

levels in aquatic ecosystems.38,51–53 Uptake of MeHg involves diffusion from the medium

to the cell surface which is followed either by diffusion through the cell wall or binding to

reactive sites at the cell wall or the membrane and transport into the cytoplasm.57–59 The

bioavailability of MeHg is controlled by its chemical speciation in the medium and this is

a key factor when determining cellular uptake rate and accumulation by bacteria or

phytoplankton.32,62 However, previous studies have reported contrasting results with

regards to the type of mechanism involved in the cellular uptake process.52,54,62,63,66,119

We evaluated how the chemical structure and stability constant of MeHg complexed with

six environmentally relevant low molecular mass thiols affect cellular uptake kinetics of

22

MeHg in a freshwater green microalga Selenastrum capricornutum. We tested the

assumption that MeHg bound to hydroxide ligands in the control, or to thiols with simple

structure (cysteine (Cys), mercaptoacetic acid (MAC) and 2-mercaptopropionic acid

(2MPA)) would have higher uptake rate by our model organism than MeHg complexed

with thiol ligands of larger size and structure complexity (glutathione (GSH), N-acetyl-L-

cysteine (NACCys) and N-acetyl-Penicillamine (NACPEN)). We further developed two

kinetic models and applied them to different algal fractions to calculate the rate constants

for the processes involved in cellular uptake of MeHg.

Methylmercury mole fractions in whole cells for individual MeHg-thiol treatments and

the control during the entire exposure period (8 h) are presented in Figure 5, and the

points represent measured values while the solid lines are the modeling results (Model

1). The measured MeHg fractions and the corresponding concentrations are shown in

Figures S4 and S5 (Supporting Information, SI, Paper III). Already after 15 minutes, the

fraction of MeHg associated with the algal cells increased rapidly reaching 0.33 ± 0.03 –

0.50 ± 0.09 in the MeHg-thiol treatments and 0.77 ± 0.03 in the control. At a time point

between 2 and 4 h the cell associated MeHg fraction plateaued in the MeHg-thiol

treatments, and after approximately 1 h in the control. We identified three groups of the

tested complexes with an increasing rate of MeHg association with the cells (GSH, 2MPA,

NACPEN, NACCys < MAC, Cys < Control). This observation is in line with the hypothesis

that cellular uptake of MeHg bound to ligands with lower complex stability (such as

hydroxide) or to small thiols of simple structure (MAC, Cys) is higher than for MeHg

complexes with larger and/or branched-chain thiols (NACCys, NACPEN, 2MPA and GSH).

During the first 60 min of the exposure, MeHg concentrations was measured in the

cytoplasmic fraction of S. capricornutum and MeHg mole fractions were calculated.

Modeling results (Model 2) for these experimental data are presented in Figure 6 while

the corresponding concentrations are found in Figure S6 (SI, Paper III). At 1 h of exposure,

the cytoplasmic fraction of MeHg ranged from 0.070 ± 0.007 to 0.150 ± 0.003 in the

MeHg-thiol treatments, and was 0.107 ± 0.008 for the control. The cytoplasmic

accumulation of MeHg was slow when compared to MeHg association to the cell

membrane. Rapid association of MeHg to the cells was observed by Gorski et al.65 The

proportions of cytoplasmic MeHg in our study are lower than what has previously been

23

reported, however, it cannot be excluded that the cytoplasmic mole fraction of MeHg

would continue to increase beyond the fraction measured at 1 h and steady-state

conditions predicted by Model 2.

Two kinetic models for rate constant determination were used in this study. The two-site

Model 1 was used to describe the exchange kinetics of MeHg between the bulk medium

and whole cells and two rate constants were calculated: MeHg cellular association (kas)

and clearance (kc) rate constants. The kas values for the thiol treatments ranged from [29.3

± 2.9] x 10-3 (MeHg-GSH) to [42.5 ± 2.7] x 10-3 (MeHg-Cys) and kas for the control was

determined to be [97.6 ± 4.1] x 10-3 min-1. The three-site model (Model 2) was used to

describe the MeHg exchange between the bulk medium, cell membranes and cytoplasmic

fraction via rate constants for MeHg adsorption (kad) and desorption (kd) to cell

membranes and cellular internalization (ki) and efflux (ke). Using the rate constants

determined by Model 1 and 2, we calculated thermodynamic constants between the

whole cells and the medium (kBA=kas /kc), cell membranes and the medium (kCA=kad /kd)

and cell membranes and the cytoplasmic fraction (kDC=ki /ke). The rate and

thermodynamic constants for all treatments and the control are presented in Table 1. The

calculated kas indicated that association of MeHg to the cell was slower when MeHg is

bound to thiol ligands with larger size and/or branched structure (GSH, NACCys, NACPEN,

2MPA) than when it is bound to hydroxide or smaller thiols with simpler structure (Cys,

MAC). Results reported by Lee and Fisher67 and our results suggest that MeHg has lower

bioavailability when it is bound to thiol ligands (log K = 16.7 – 17.5)120–122 than to less

thermodynamically stable ligand such as hydroxide (log K = -4.5). However, the relative

cellular uptake rate differs depending on the phytoplankton species.

24

Figure 5. Methylmercury (MeHg) mole fractions in whole cells and medium. The curves represent

the fitted data wilt Model 1 with a 95% confidence interval while the points are the experimental

data. (A) GSH, (B) 2MPA, (C) NACPEN, (D) NACCys, (E) MAC, (F) Cys, (G) Control. (Reproduced from

Paper III with permission from American Chemical Society)

25

Figure 6. Methylmercury (MeHg) mole fractions associated with cell membranes, cytoplasmic

fraction and medium. The curves represent the fitted data with Model 2 with a 95% confidence

interval while the points are the experimental data. (A) GSH, (B) 2MPA, (C) NACPEN, (D) NACCys, (E)

MAC, (F) Cys, (G) Control. (Reproduced from Paper III with permission from American Chemical

Society)

26

Table 1. Calculated MeHg rate constants (association kas, clearance kc, adsorption kad, desorption kd, internalization ki, efflux ke) for MeHg-thiol treatments

and the control using Model 1 and Model 2, equilibrium values (KBA, KCA, KDC), model merits of fit and statistical p value for kas and kc. (Reproduced from Paper

III with permission from American Chemical Society)

MODEL 1 MODEL 2

kas

(×10-3 min-1)

KBA kc

(×10-3 min-1)

Merit of fit (%)

kad

(×10-3 min-1)

kd

(×10-3min-1)

KCA ki

(×10-3 min-1)

ke

(×10-3 min-1)

KDC

Merit of fit (%)

GSH 29.3 ± 2.9 A*, a 26.4 ± 0.6 1.1 ± 1.1 0.75 29.4 1.8 16.3 14 100 0.14 1.72

2MPA 30.0 ± 1.9 A, a 15.5 ± 0.2 1.9 ± 0.8 0.27 30.1 2.7 11.2 18.6 170 0.11 0.41

NACPEN 30.8 ± 2.3 A, a 22.2 ± 0.4 1.4 ± 0.9 0.42 32.1 1.7 19.0 101 700 0.14 2.68

NACCys 33.2 ± 2.7 A, a 18.8 ± 0.4 1.8 ± 1.0 0.41 33.0 2.5 13.2 98 700 0.14 0.73

MAC 39.0 ± 4.6 A, b 30.7 ± 0.9 1.3 ± 1.6 0.24 40.1 1.8 21.7 23.8 140 0.17 0.65

Cys 42.5 ± 2.7 A*, b 43.5 ± 0.7 1.0 ± 1.0 0.17 42.6 1.3 33.0 28 200 0.14 0.42

Control 97.6 ± 4.1 B 35.2 ± 0.4 2.8 ± 0.8 0.03 98.7 3.3 29.9 16.5 150 0.11 0.11

Different capital letters indicate statistically significant (p < 0.05, one-way ANOVA, Tukey HSD post-hoc test) differences between individual treatments while

the asterisk symbol (*) marks that ka for GSH is close to being statistically significant from Cys (p = 0.057). Different lowercase letters indicate statistically

significant (p < 0.05, one-way ANOVA, Tukey HSD post-hoc test) differences between the average ka of GSH, 2MPA, NACPEN and NACCys and the average ka

of Cys and MAC.

27

We propose that the observed higher cellular uptake rate for thermodynamically less

stable complexes and complexes with small ligands can be explained by uptake

mechanisms involving metal-binding to the cell surface ligands and formation of a new

complex with the cell surface biotic ligand prior to internalization (FIAM or BLM).

However, we do not exclude passive diffusion as a transport mechanism. Although the

fraction of MeHg adsorbed to the cell membrane was higher in the control than the thiol

treatments, the cytoplasmic fraction of MeHg was not and this suggests that the rapid

adsorption to the cell surface ligands may not necessarily lead to internalization. Overall,

our results point to the potential importance of thiol ligand composition controlling MeHg

incorporation in the food web in aquatic ecosystems.

3.4. Multiple impacts of humic-rich dissolved organic carbon on methylmercury accumulation in heterotrophic pelagic food webs (Paper IV)

Changes in climate is predicted to lead to regional increases in e.g. precipitation and

snowmelt which is accompanied by enhanced input of terrestrially derived

(allochthonous) OM to coastal systems.123–127

It has been suggested that increased loading of terrestrial OM may increase total Hg input

to the systems by promoting MeHg formation in the water column.81,128 As another

consequence of enhanced loading of humic rich OM, brownification of water decreases

light penetration and suppresses photosynthetic primary production.129,130 However, OM

may promote bacterial growth and increase their share in the pelagic food web structure

thereby decreasing the energy transfer efficiency from the base of the food web to top

predators.125,131–136

While bacteria are unable to be directly consumed by mesozooplankton due to their size

as opposed to phytoplankton, they are readily grazed on by protozoans.137,138

Mesozooplankton also feed on protozoans and therefore larger share of bacteria as the

base in heterotrophic food webs (microbial loop food webs) results in a higher number

trophic levels and lower energy transfer efficiencies to top consumers.135,139 As MeHg

28

biomagnifies with each trophic level, higher MeHg levels can be hypothesized in top

predators in systems favoring a bacteria-based food web compared to a phytoplankton-

based one. In support of this hypothesis, Jonsson et al73 reported 2-7 times enhanced

MeHg bioaccumulation factor (BAF) in zooplankton in a mesocosm experiment following

a shift in food web structure from net autotrophic (46% bacteria production of the total

basal production) to net heterotrophic (72% bacteria production). The shift in food web

structure was induced by loadings of terrestrial OM relevant for regional climate

scenarios for northern Scandinavia. In Paper IV we studied the effect of humic-rich DOC

loading on the pelagic lower food web structure and bioaccumulation of MeHg in an

experimental mesocosm study. We investigated if MeHg bioaccumulation is further

increased, or if it levels-off, in food webs with higher proportions of bacteria production

than previously studied. An improved understanding of the quantitative relationship

between food web structure and MeHg bioaccumulation is important for reliable

predictions of MeHg in aquatic biota for different environmental change scenarios. We

used four treatments with different levels of DOC: Reference (4.0 mg L-1), Level 1 (5.5 mg

L-1), Level 2 (6.5 mg L-1) and Level 3 (8.0 mg L-1) simulating the current average DOC level

in the Baltic Sea (Reference) and increases by 38%, 63% and 100%, respectively (DOC and

humic matter content results are presented in Figures S15 and S16, Paper IV).

The primary production rate (PP) followed the expected treatment effects whlie the

bacteria production rate (BP) was high in all treatments (Table 2, Figure 7). The

zooplankton production rate (ZP) was low in all treatments and it was used to calculate

the theoretical food web efficiency (FWE) (Eq.S1, SI, Paper IV) which resulted in low values

across treatments (Table 2). The δ15N results for the seston size fractions were used for

calculation of the trophic level distance between the smallest (20-50 µm) fraction and

other three (Eq. S2, Paper IV). The δ15N baseline in the systems is not known, and likely

differed among treatments due to the added DOC and the total number of trophic levels

up to zooplankton could therefore not be calculated. Therefore, we could not establish if

there were any differences among treatments in the number of trophic levels for the part

of the food web constituted by organisms <20 µm.

29

Phytoplankton species were classified into four groups based on their trophicity:

heterotrophic (HT) organisms larger and smaller than 20 µm, and the sum of autotrophic

and mixotrophic organisms (referred to as AU) larger and smaller than 20 µm (Figure 2,

Figure S5, Paper IV). The taxonomic compositions of the AU and HT groups were overall

very similar among treatments (Figures S6-7, Paper IV). Moreover, the abundance of

heterotrophic nanoflagellates (HNF), serving as an indicator of heterotrophy within a

system, was similar in all treatments (~1.0 mg C m-3) suggesting similar heterotrophy for

all treatments. (Figure S8, Paper IV). However, our data indicate that the autotrophic

pathway was more prominent for the Reference. Due to the high BP and the low

abundance of edible AU phytoplankton in all treatments, we suggest that the major part

of the energy went through the heterotrophic food web (1: bacteria, 2: flagellates, 3:

ciliates, 4: mesozooplankton). In addition, we propose that a larger fraction of energy flow

was channeled through the more efficient autotrophic food web (1: phytoplankton (<20

µm), 2: mesozooplankton) in the Reference than in the Level 1-3 treatments.

The additions of humic DOC increased concentrations of both dissolved total Hg and

MeHg (Table 1; Figures S1 and S2, Paper IV). This result was likely a consequence of

decreased partitioning of Hg to particulate matter (and mesocosm walls) due to formation

of dissolved Hg complexes with thiol groups in the added DOC. Decreased

photodemethylation of MeHg caused by water brownification also could have

contributed to higher dissolved MeHg levels. While these observations corroborate with

what was previously reported by Jonsson et al.73, it is important to note that high

concentrations of humic DOM may also decrease MeHg bioavailability.27,62,64,76,140

The concentration of MeHg measured in seston showed increased from the smallest (20-

50 µm) to the largest fraction (>200 µm). This result is explained by their different trophic

position (based on δ15N) in the pelagic food web (Table 1). The corresponding log MeHg

BAF values ranged from 4.5 to 4.7 (20-50 µm) and from 5.7 to 6.0 (>200 µm )which is at

the upper end of what has been found in previous studies.27,38,53,69,141 This suggests that

changes in allochthonous DOC loadings can impact the range of BAFs in estuarine and

coastal systems.

30

The addition of humic DOC in our study could contribute to large differences in MeHg

bioavailability. We attempted to correct for these differences in a separate uptake

incubation experiment with the green alga Selenastrum capricornutum. The highest

uptake was observed for the Reference system and the lowest uptake for the Level 3. Our

results indicated that across the large quantitative DOC ranges found in coastal to open

ocean systems, the bioavailability of MeHg may have a large impact on BAFs, as previously

observed by Schartup et al.27 The corrected BAF (BAF’) resulted in a maximum increase of

68% compared to the uncorrected BAF values for the Level 3 treatment (Figure 3, Paper

IV). These results suggest that bioavailability of dissolved MeHg is affected by the

presence of humic DOC and that this can further impact MeHg BAFs. Furthermore, the

correction resulted in new statistical differences between the Level 3 and the Level 1-2

treatments in MeHg bioaccumulation, illustrating the contribution of differences in the

food web processes.

The slope of the regression of log[MeHg] versus δ15N in a food web has been used to

evaluate the biomagnification potential within a system.142 The biomagnification slopes

within the lower food web (20−50 µm to >200 µm seston size fractions) ranged between

0.35-0.50 (Table 1). This is in the higher end of slopes found for higher levels of the food

webs (plankton <200 µm and up to fish and birds, usually ranging from 0.2-0.4)143 and

suggests efficient transfer of MeHg in the lower food web. The higher slope of the

Reference system (Table 1) suggests a more efficient transfer of MeHg per trophic level

than in the other treatments and indicates that higher accumulation of MeHg (due to

more trophic levels) can be counteracted by a lower MeHg transfer efficiency per trophic

level in a heterotrophic compared to autotrophic food web.

The results from our study and what was reported by Jonsson et al. suggest that an

increase in BP proportion from 45% to 90% induced by enhanced humic DOC discharges

may enhance MeHg bioaccumulation by one order of magnitude in zooplankton (Figure

8). Moreover, these results are relevant for coastal systems such as the Gulf of Bothnia,

and other systems in the northern hemisphere, expected to be affected by increased

humic DOC loadings following climate scenarios.

31

Figure 7. Primary and bacterial biomass production rates averaged for the whole duration of the

mesocosm experiment for the four treatments (Reference, Level 1, Level 2 and Level 3). Bars

present the mean from three mesocosom replicate treatments with one SD error.

Figure 8. Average of logarithmic values of methylmercury (MeHg) bioaccumulation factor (BAF) in

seston > 50 μm as a function of the % bacteria production rate of the total (bacteria + primary

production) basal production. Red data points are average for each of the four different treatments

in the current studies, and blue data points are reproduced from Jonsson et al. The data points are

expressed as the mean of three mesocosom replicate treatments shown with one horizontal and

one vertical standard error bar.

0

100

200

300

400

500

600

Reference Level 1 Level 2 Level 3

PP (mgC m-2 d-1) BP (mgC m-2 d-1)

4.5

4.7

4.9

5.1

5.3

5.5

5.7

5.9

0 10 20 30 40 50 60 70 80 90 100

log

of

aver

age

Me

Hg

BA

F in

ses

ton

>5

0 µ

m

% Bacterial production of the total basal production

32

Table 2. Averaged (± SD) values of measured parameters for the whole duration of the experiment. For log BAF and log BAF’, the statistical tests were run on the data before the logarithm was applied.

Reference Level 1 Level 2 Level 3

DOC mg L-1 3.79 ± 0.03A 4.48 ± 0.03 B 5.09 ± 0.07C 5.83 ± 0.06D

Humic matter content µg L-1 12.85 ± 0.01 A 15.98 ± 0.15 B 17.84 ± 0.05 C 20.57 ± 0.16 D

HgT pM 1.61 ± 0.11A 3.14 ± 0.06BC 3.60 ± 0.15CD 3.69 ± 0.23D

MeHg pM 0.17 ± 0.01A 0.21 ± 0.04AB 0.28 ± 0.03B 0.35 ± 0.05c

MeHg 20-50 µm pmol g-1 d.w. 8 ± 7 10 ± 2 9 ± 5 12 ± 1

MeHg 50-90 µm pmol g-1 d.w. 154 ± 33 138 ± 12 140 ± 35 147 ± 7

MeHg 90-200 µm pmol g-1 d.w. 118 ± 5 59 ± 34 78 ± 21 129 ± 38

MeHg >200 µm pmol g-1 d.w. 172 ± 32 138 ± 55 185 ± 70 203 ± 117

log BAF MeHg 20-50 µm 4.58 ± 0.34 4.68 ± 0.08 4.49 ± 0.18 4.52 ± 0.10

log BAF MeHg 50-90 µm 5.95 ± 0.14A 5.81 ± 0.05A 5.69 ± 0.15A 5.587 ± 0.003B

log BAF MeHg 90-200 µm 5.84 ± 0.02A 5.40 ± 0.15B 5.43 ± 0.17B 5.55 ± 0.09AB

log BAF MeHg >200 µm 6.00 ± 0.12 5.79 ± 0.09 5.80 ± 0.22 5.71 ± 0.32

log BAF’ MeHg 20-50 µm 4.58 ± 0.34 4.74 ± 0.08 4.53 ± 0.18 4.74 ± 0.10

log BAF’ MeHg 50-90 µm 5.95 ± 0.14 5.88 ± 0.05 5.73 ± 0.15 5.813 ± 0.003

log BAF’ MeHg 90-200 µm 5.84 ± 0.02A 5.47 ± 0.15B 5.47 ± 0.17B 5.78 ± 0.09A

log BAF’ MeHg >200 µm 6.00 ± 0.12 5.85 ± 0.09 5.84 ± 0.22 5.93 ± 0.32

TDN µM 18.4 ± 0.5 A 22.8 ± 0.8 B 26.0 ± 0.3 C 30.7 ± 0.6 D

TDP µM 0.45 ± 0.03 A 0.59 ± 0.07 B 0.66 ± 0.01 C 0.75 ± 0.01 D

NO3- µM 4.9 ± 0.2A 4.9 ± 0.9A 5.2 ± 0.4AB 5.7 ± 0.3B

NH4+ µM 0.37 ± 0.01AB 0.26 ± 0.03A 0.32 ± 0.03AB 0.49 ± 0.03B

PO4 3- µM 0.31 ± 0.02 0.32 ± 0.06 0.33 ± 0.03 0.35 ± 0.02

Chl a mg m-3 2.5 ± 0.2A 2.3 ± 0.2A 1.8 ± 0.2B 2.0 ± 0.1B

pH

7.89 ± 0.06A 7.80 ± 0.07B 7.69 ± 0.05C 7.68 ± 0.03C

Kd m-1 1.19 ± 0.02A 1.62 ± 0.02 A 1.92 ± 0.08 B 2.12 ± 0.01 C

PP mg C m-2 d-1 81.6 ± 11.5A 59.3 ± 14.9B 47.6 ± 6.9BC 40.1 ± 1.2C

BP mg C m-2 d-1 207 ± 12A 301 ± 87B 346 ± 148B 307 ± 32B

ZP mg C m-2 d-1 2.8 ± 1.2 2.1 ± 0.3 1.9 ± 1.0 2.2 ± 1.3

BP/(PP+BP) 0.68 ± 0.03A 0.81 ± 0.02B 0.84 ± 0.05BC 0.87 ± 0.01C

FWE % 0.9 ± 0.4A 0.5 ± 0.1B 0.4 ± 0.1B 0.5 ± 0.3AB

δ15N 20-50 um ‰ 2.9 ± 0.1A 2.6 ± 0.4AB 2.6 ± 0.1B 2.4 ± 0.2AB

δ15N 50-90 um ‰ 5.4 ± 0.3A 5.2 ± 0.2AB 4.8 ± 0.2B 5.1 ± 0.1AB

δ15N 90-200 um ‰ 5.2 ± 0.2 5.1 ± 0.7 5.1 ± 0.4 5.1 ± 0.5

33

δ15N >200 um ‰ 5.9 ± 0.2 5.7 ± 0.3 5.6 ± 0.2 5.6 ± 0.2

δ13C 20-50 um ‰ -19.4 ± 0.8A -22.2 ± 1.7AB -22.7 ± 1.4AB -24.0 ± 1.5B

δ13C 50-90 um ‰ -16.0 ± 0.8 -17.6 ± 2.2 -17.9 ± 2.8 -17.5 ± 1.4

δ13C 90-200 um ‰ -16.7 ± 1.1 -17.8 ± 2.6 -18.4 ± 1.5 -17.8 ± 1.5

δ13C >200 um ‰ -16.2 ± 1.1 -16.9 ± 2.3 -17.5 ± 2.8 -17.3 ± 2.1

k (δ15N) 0.49 ± 0.05 A 0.35 ± 0.05B 0.42 ± 0.06C 0.37 ± 0.04BC

The following abbreviations were used in the Table: Dissolved organic carbon (DOC), total mercury (HgT), methylmercury (MeHg), non-corrected bioaccumulation factor (BAF), corrected bioaccumulation factor (BAF’), total dissolved nitrogen (TDN), total dissolved phosphorous (TDP), nitrate (NO3

-), ammonium (NH4+), phosphate (PO4

3-), chlorophyll a (Chl a), vertical diffuse light attenuation coefficient (Kd), primary production (PP), bacterial production (BP), zooplankton production (ZP), food web efficiency (FWE), stable δ15N and δ13C isotope signatures and linear regression slope between δ15N and BAF’. Bold italic text and different capital letters indicate statistically significant (p < 0.05, Repeated Measure One-way ANOVA or One-way ANOVA, Tukey HSD post-hoc test) differences between individual mesocosm treatments.

3.5. Future research remarks

While this thesis work contributes to better understanding of processes and

transformation pathways involved in Hg biogeochemical cycling and exposure to biota in

systems with pronounced redoxclines such as in the Baltic Sea (Paper II), impacts of direct

uptake of MeHg into the food web in these zones remain to be addressed. This is an

important question to answer since a 2 – 6 times increase in MeHg levels has been

observed in hypoxic compared to normoxic water. Considering the fact that hypoxic zones

spread in coastal aquatic ecosystems worldwide,107 there is a potentially increased risk of

MeHg entrance into the food web by direct consumption by organisms who either

constantly or temporarily reside in these areas.

Moreover, new mechanistic information on uptake processes of MeHg in various

phytoplankton species under different conditions, e.g. oxygen and DOC concentrations

which affect MeHg speciation in the dissolved phase and availability, would improve

assessment of MeHg potential for incorporation in the food web. Considering MeHg

affinity to reduced sulfur groups and uncertainties in concentration and composition of

thiol and sulfide ligands in aquatic environments, improved understanding of these

34

parameters would contribute as well to an overall better prediction and risk assessment

of MeHg in top predators.

Lastly, our results from Paper IV indicate that the effect of a longer food web, i.e. higher

number of trophic levels, on MeHg accumulation can be counteracted by lower MeHg

transfer efficiency per trophic level in bacteria-based food webs compared to webs where

phytoplankton is the base. Improved knowledge on MeHg transfer efficiency in the

bottom section of aquatic food can thus be important and help to assess and predict the

potential for MeHg transfer and MeHg levels in fish or other higher trophic levels.

Therefore, further studies focusing on understanding these processes in both autotrophic

and heterotrophic food webs are needed.

35

4. Conclusions

To conclude, this thesis work contributes some important aspects to improve the current

understanding regarding MeHg formation, uptake and bioaccumulation in coastal aquatic

natural environments, where increased runoff of terrestrial OM is expected due to the

predicted changes in climate, and to refine assessment and prediction of MeHg

biogeochemical cycling and bioaccumulated levels in higher trophic level organisms in

such systems.

Paper I provides information with regards to the effect of organic matter in coastal

aquatic systems on MeHg formation and interannual variability. Namely, the impact of

two investigated parameters (DOC and humic matter content) may be used to assess

MeHg variability in this type of environments. Using concentration of DOC as a proxy, HgII

methylation potential and overall MeHg levels may be estimated. Furthermore, due to

the strong binding affinity of Hg to OM, the effect of humic matter content may be used

to assess HgII availability and therefore its potential for methylation in the water column.

Important aspects of pelagic redoxclines, often present in coastal systems due to the

expansion of oxygen depleted zones, on MeHg formation and measured concentrations

were evaluated in Paper II. We found that net MeHg production is one of the main drivers

of MeHg concentrations in normoxic, hypoxic and anoxic zones. Moreover, turbulent

diffusion is an important factor along the redox gradient because it affects the

methylation potential of HgII, depending on the Hg chemical speciation in water zones

with different oxygen concentrations. To exemplify, HgII may occur in the form of stable

complexes with thiol groups in the DOM pool or in mineral form as HgS(s) or be associated

with Fe-DOM aggregates in the hypoxic zone, and when moving into the reductive

dissolution zone, HgII is released from these complexes and becomes readily available for

methylation. The formed MeHg could then be transferred by direct mixing of water into

the hypoxic zone and enter the food web via organisms which actively feed in this zone.

Impact of molecular structure of different thiol ligands on uptake kinetics of MeHg in a

model phytoplankton organism (green alga) was evaluated in Paper III. Lower MeHg

association and adsorption rate constants were found in cases when the alga was exposed

36

to MeHg complexes with larger thiols with more complex and “branched” structure than

in treatments with thiol ligands with simpler structures. In addition, concentrations of

MeHg associated to the cells and highest association rate constant was found in the

control without added thiols. The results demonstrated that both the chemical structure

of MeHg complexes and thermodynamic stability are important controlling factors for

MeHg interactions with the cell surface, but not necessarily for MeHg exchange across

the membrane. These findings are in line with uptake mechanisms which involve

formation of MeHg complexes with ligands located at the cell surface prior to

internalization and point to the importance of thiol ligand composition in the

environment for MeHg incorporation in the food web.

Paper IV provides new information regarding capacities for MeHg bioaccumulation in

zooplankton in systems pronounced heterotrophy. Our results are in compliance with

previous findings which showed high MeHg bioaccumulation in highly heterotrophic food

webs, and demonstrate that MeHg bioaccumulation further increases at proportions of

BP higher than 72%. Together with the results from a study by Jonsson et al.73, an increase

in MeHg bioaccumulation in zooplankton by one order of magnitude is expected under

conditions of enhanced proportions of BP from 45% to 90%. The enhancement in BP and

its effect on the structure of the aquatic pelagic food webs, induced by higher discharges

of humic DOC to coastal systems, are expected following climate change scenarios.

37

5. Acknowledgements

First and foremost, I would like to thank my principal supervisor, professor Erik Björn, for

choosing me among all other applicants for this position, and allowing me to be a part of

this wonderful project. Erik, I would like to immensely thank you for being the best

mentor a PhD student could possibly ask for, for making me a better employee, a better

scientist, and most importantly, a better person. Thank you for helping me every step of

the way, for always being willing to listen and help me overcome all issues that would

arise. Thank you for being my inspiration for the past four and a half years, and for a good

share of both happy and devastating memories which I will cherish forever. I can only

hope that you do not regret your choice and that I have not been a disappointment or not

lived up to your expectations.

Secondly, I would like to thank my assistant supervisors, Anne Soerensen and Agneta

Andersson for their mentorship, valuable inputs and help with analyses and

interpretation of piles among piles of acquired data. I would like to thank Anne for her

help with the manuscripts and crucial comments which helped me develop better writing

skills and for expanding my knowledge when it comes to data analyses. Thank you

sincerely for a good time and good laughs we shared in the lab and during the sampling

cruise 2016.

Further, I would like to acknowledge my co-authors, Živan Gojković and Sonia Brugel.

Žiko, hvala najlepše na pomoći oko uzgajanja algi i što si samim svojim prisustvom sprečio

da poludim ovde na dalekom severu. Hvala na druženju i na svemu kroz šta smo prošli,

jedva čekam da te ponovo vidim! Drago mi je što smo uspeli da ostvarimo prijateljstvo

potpuno nenadano i izvan Srbije. Sonia, thank you for keeping me sane during the

exhausting 36 days of the mesocosm experiment, we all made a really good team. Thank

you for all the cocktail nights and interesting conversations, for being my friend in lonely

Umeå and for listening to my personal problems.

I would also like to thank my colleagues and ex-colleagues Caiyan, Wei, Eric, Ulf, Tower,

Mareike, Lars, Richard, Temi, Liem, Sofi and most of all, Rain. Thank you Rain for being

my close and sincere friend ever since I moved to Sweden, for all the help during the years

38

at work as well as in my private life. Thank you for visiting me and my family in Serbia! I

would like to thank Lars for being my go-to person when I started my PhD studies and for

all that he has taught me during the years we shared an office. I would also like to thank

Richard for all his help in the lab, interesting conversations, mutual encouragement when

no instruments would work properly and successful “tube fishing” from the

malfunctioning TD. I would like to thank the rest of the Hg team for their help and support,

interesting discussions and critical evaluations/feedbacks.

Finally, I would like to thank my loved ones, my family and my closest friends. Mama,

tata, Teodora i Devone, hvala vam na svemu što ste učinili za mene i na podršci. Hvala na

učestalim posetama, na pomoći u borbi protiv samoće i ludila. Pre svega, hvala vam na

ljubavi. Babama, dedi, Dušanu i Ljilji takođe hvala na vremenu provedenom zajedno, bilo

u Švedskoj, bilo u Srbiji. Hvala svemu što ste učinili za mene I hvala SVIMA što se ponosite

mnome. Devonu hvala na velikoj pomoći tokom jako iscrpljujućeg perioda kada smo radili

mesocosm eksperiment pošto bih najverovatnije umrla od gladi da njega nije bilo. Hvala

najboljim prijateljima, Momi i Saši na druženju kao da smo i dalje komšije iako smo

kilometrima udaljeni. Hvala na razgovorima i pomoći u najtežim trenucima.

Hvala i VOLIM VAS!

39

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