1

CHEMICAL CHARACTERIZATION OF

DISSOLVED ORGANIC MATTER IN

RELATION WITH HYDROGRAPHY IN

THE ARCTIC OCEAN

A Thesis submitted to the Committee on Graduate Studies

in Partial Fulfillment of the Requirements for the Degree of Master of

Science in the Faculty of Arts and Science.

TRENT UNIVERSITY

Peterborough, Ontario, Canada

(c) Copyright by Zhiyuan Gao 2016

Environmental and Life Science M.Sc. Graduate Program

January 2017

ii

ABSTRACT

Chemical characterization of dissolved organic matter in

relation with hydrography in the Arctic Ocean

Zhiyuan Gao

In this thesis, water mass distribution of dissolved organic matter (DOM)

characteristics (i.e. molecular weight, fluorescent components, thiols and humic

substances concentration) was observed in the Arctic Ocean. For the first time, DOM

molecular weight (MW) in Beaufort Sea was assessed using asymmetrical flow field-

flow fractionation, as well as the first monitoring of thiols and humic substances (HS)

using cathodic stripping voltammetry (CSV) in the Arctic Ocean. Based on

fluorescence property, DOM characterization was carried out using parallel factor

analysis – excitation-emission matrices. Pacific winter waters in the Canada Basin

showed higher MW DOM associated with higher fluorescence intensity. High HS was

associated with the Arctic outflow waters in top 300 m of the Canadian Arctic

Archipelago. Interestingly, maximum thiol concentration was associated with the

subsurface chlorophyll-a maximum at most sites, but not universal along the study area.

Comparable distributions of CSV-based HS and humic-like fluorescent components

suggest similar sources/ processes in the Arctic Ocean. The findings in this thesis

suggested DOM characteristics could be used as fingerprints in tracing water masses in

the Arctic Ocean.

iii

Keywords

DOM, Cathodic stripping voltammetry, Asymmetrical flow field-flow fractionation,

PARAFAC-EEMs, Molecular weight, Thiols, Humic substances

iv

Acknowledgements

I would like to express my gratitude to my supervisor Dr. Céline Guéguen for the

wonderful graduate project opportunity to explore the north as well as her patient

guidance and helpful engagement through the learning process of this master thesis. I

would like to give special thanks to my committee member Dr. Peter Lafleur and Dr.

David A. Ellis and other faculties at Trent University for the help and assistant in this

thesis. Furthermore, I would like to thank Northern Scientific Training Program,

Canada Research Chair Program (CG), National Sciences and Engineering Research

Council of Canada (CG), Geotraces and ArcticNet programs for funding my project

and the assistantship from crew member and fellow scientists of CCGS Amundsen and

Louis S. St-Laurent. Also, I would like to thank my family and my friends, who have

supported me throughout entire process, both by keeping me harmonious and helping

me putting pieces together. I will be grateful forever for your love.

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Table of Content

ABSTRACT……… ...................................................................................................... ii

Acknowledgements ...................................................................................................... iv

Table of Content……………………………...………………………………………. v

List of figures and tables ............................................................................................. vii

List of Abbreviations and Symbols .............................................................................. ix

Chapter 1. The chemical nature of dissolved organic matter and its current situation

in the Arctic Ocean ........................................................................................................ 1

1.1 Introduction .....................................................................................................................1

1.2 Importance of DOM and monitoring methods ................................................................2

1.3 The hydrology in the Arctic Ocean ...................................................................................5

1.4 Objectives of my study .....................................................................................................7

Reference ...............................................................................................................................9

Figures and tables ............................................................................................................... 16

Chapter 2. Size distribution of absorbing and fluorescing DOM in Beaufort Sea,

Canada Basin ............................................................................................................... 18

Abstract ............................................................................................................................... 19

2.1 Introduction .................................................................................................................. 20

2.2 Methods ........................................................................................................................ 22

2.2.1 Sampling station ..................................................................................................... 22

2.2.2 Asymmetrical Flow field flow fractionation ........................................................... 22

2.2.3 Chromophoric dissolved organic matter (CDOM) and Fluorescent DOM (FDOM) 24

2.3 Results and Discussion .................................................................................................. 26

2.3.1 Hydrographical parameters ................................................................................... 26

2.3.2 Molecular weight depth distribution ..................................................................... 26

2.3.4 Optical Properties .................................................................................................. 27

2.3.5 Relationship between fluorescence properties and MW ...................................... 30

2.4 Conclusions ................................................................................................................... 31

Reference ............................................................................................................................ 33

vi

Figures and tables ............................................................................................................... 41

Chapter 3. Determination of thiols, humic substances and fluorescent dissolved

organic matter during the 2015 Canadian Arctic GEOTRACES cruises .................... 50

Abstract ............................................................................................................................... 51

3.1 Introduction .................................................................................................................. 52

3.2 Methods ........................................................................................................................ 54

3.2.1 Sampling ................................................................................................................. 54

3.2.2 Reagents ................................................................................................................. 54

3.2.3 Instrumentation ..................................................................................................... 55

3.2.4 Fluorescent dissolved organic matter (FDOM) ...................................................... 57

3.3 Results ........................................................................................................................... 57

3.3.1 Water mass definition ............................................................................................ 57

3.3.2 CSV-based DOM characterization .......................................................................... 59

3.3.3 FDOM characterization .......................................................................................... 59

3.3.4 DOM and water masses ......................................................................................... 60

3.3.5 Principal Components Analysis .............................................................................. 62

3.4 Conclusion ..................................................................................................................... 63

Reference ............................................................................................................................ 65

Figures and tables ............................................................................................................... 71

Chapter 4. Conclusion ............................................................................................. 77

4.1 Molecular weight of DOM ............................................................................................. 77

4.2 Composition of DOM .................................................................................................... 78

4.2.1 Fluorescent dissolved organic matter .................................................................... 78

4.2.2 Thiols and humic substances distribution .............................................................. 80

4.3 Conclusions and future directions ................................................................................ 80

Reference ............................................................................................................................ 82

Figures and tables ............................................................................................................... 84

vii

List of figures and tables

Figures

Figure 1.1 Map of the study area ................................................................................ 16

Figure 1.2 Potential temperature as a function of Salinity. A:2014 JOIS Cruise; B: 2015

GEOTRACES Cruise .................................................................................................. 17

Figure 2.1 Sampling locations in Beaufort Sea, Canada Basin .................................. 41

Figure 2.2 AF4 fractograms of CB29 (400m depth; black) and CB28b (1000m depth;

gray) ............................................................................................................................ 42

Figure 2.3 A) Potential temperature and B) FDOM as a function of salinity (Sp) at the

four study sites (Figure 2.1). ASW - Arctic surface waters, PSW – Pacific summer

water, PWW – Pacific winter water, FSB – Fram Strait Branch water, BSB – Barents

Sea Branch water ........................................................................................................ 43

Figure 2.4 MW depth profiles in Beaufort Sea ........................................................... 44

Figure 2.5 Depth distribution of A) a254, (B) a355, and (C) S275-295 ............................. 45

Figure 2.6 Individual components identified by PARAFAC (A- marine humic-like C1,

B- protein-like C2 and C- terrestrial humic-like C3) .................................................. 46

Figure 2.7 Depth distribution of A) Total Fluorescence Intensity (TF), B) marine

humic-like C1, C) protein-like C2 and D) terrestrial humic-like C3 .......................... 47

Figure 2.8 Change in TF vs CDOM MW ................................................................... 48

Figure 2.9 Correlation between MW and fluorescence components (A) C1; (B) C2; (C)

C3; (D) Change in fluorescence abundance with increasing DOM MW ................... 49

Figure 3.1 Sampling locations in Canada Basin and Canadian Arctic Archipelago,

Study transect highlighted in red area ......................................................................... 72

viii

Figure 3.2 (A) Potential temperature as a function of salinity (Sp) at all depths; (B)

Salinity, (C) Potential temperature and (D) Fluo sensor (Chlorophyll-a) distribution

along the transect (Figure 3.1) .................................................................................... 73

Figure 3.3 Distribution of thiol groups (A), humic substances (B), FDOM (C-F) along

the transect (Figure 3.1) .............................................................................................. 74

Figure 3.4 Excitation emission matrices identified by PARAFAC (A-C: humic-like; D:

protein-like) ................................................................................................................. 75

Figure 3.5 PCA models and score plots on all depths (A-B) and samples in top 100m

(C-D); SW: surface waters, OW: Arctic outflow waters, DW: deep waters; Lsd:

Lancaster Sound, CAA: Canadian Arctic Archipelago, CB: Canada Basin ............... 76

Figure 4.1 (A) Vertical distribution of terrestrial humic-like component in Canada

Basin, Canadian Arctic Archipelago and Lancaster Sound; (B) Vertical distribution of

protein-like components and Chl-a signal in all samples from both cruise ................ 85

Tables

Table 3.1 Repeatability, reproducibility, limit of detections and recovery of the CSV

method ......................................................................................................................... 71

Table 4.1 Fluorescent components identified in JOIS cruise and GEOTRACES cruise.

..................................................................................................................................... 84

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List of Abbreviations and Symbols

AF4 - Asymmetrical field flow fractionation

AIW - Arctic intermediate water

ASW - Arctic surface water

BS - Barrow Strait

BSB - Barents Sea Branch water

CAA - Canadian Arctic Archipelago

CB - Canada Basin

CDOM - Chromophoric dissolved organic matter

CSV - Cathodic stripping voltammetry

DOM - Dissolved organic matter

EEMs - Excitation emission matrices

FDOM - Fluorescent dissolved organic matter

FSB - Fram Strait Branch water

HS - humic substances

Lsd - Lancaster Sound

x

LsdSW - Lancaster Sound surface water

MW - Molecular weight

NADW - North Atlantic deep water

OW - Arctic outflow water

PARAFAC - Parallel factor analysis

PSW - Pacific summer water

PWW - Pacific winter water

1

Chapter 1. The chemical nature of dissolved organic

matter and its current situation in the Arctic Ocean

1.1 Introduction

Dissolved organic matter (DOM) is one of the largest reservoirs of organic

molecules (Bushaw et al., 1996) and plays an important role in the global carbon

cycling (Carlson and Hansell, 2014; Mopper and Kieber, 2002). As the most abundant

pool, marine DOM is involved in affecting light penetration (Stedmon et al., 2000),

serving as a food supplement (Cole et al., 2006) and energy transport (Baylor and

Sutcliffe Jr, 1963), as well as regulating trace metal speciation (De Schamphelaere et

al., 2004; Hirose, 2007; Yamashita and Jaffé, 2008).

The difference between DOM and particulate organic matter is only operationally

defined. The definition recommended by the Joint Global Ocean Flux studies is that

DOM is a group of substances passed through a pre-combusted glass fiber (GF) filters

(Knap et al., 1996), while in this study, 0.7 µm GF/F filters were used for the onboard

filtration of marine DOM.

DOM contains a variety of well-defined compounds, including proteins, lipids and

carbohydrates (Leenheer and Croué, 2003; Nebbioso and Piccolo, 2013; Schumacher

et al., 2006). A big part of DOM (13 - 32%, Chanudet et al., 2006) consists of humic

substances (HS), which are heterogeneous in nature and largely refractory in the aquatic

system (Thurman, 2012). While the structure of the DOM complex remains largely

unknown, some organic groups (i.e. thiols and HS) formed strong complexation with

2

trace metals such as Cd, Cu, Fe and Mo (Hunter and Boyd, 2007; Kinniburgh et al.,

1996; Pernet-Coudrier et al., 2013; Yang and Berg, 2009).

It is not easy to understand the fate of DOM due to its various sources and multiple

sinks in aquatic systems. In the marine environment, DOM is mostly formed in-situ

through photosynthesis and metabolism by bacteria and phytoplankton (Aluwihare et

al., 1997; Aslam et al., 2012; Jiao et al., 2010; Teira et al., 2001). Allochthonous source

of DOM was also important. River discharges (i.e Mackenzie River input to Beaufort

Sea, Arctic Ocean) constitute an important source of DOM in coastal areas (Bélanger

et al., 2006; Guéguen et al., 2005) and therefore influence the distribution and

composition of marine DOM (Rachold et al., 2004; Walker et al., 2009; Yang et al.,

2013). Lobbes et al., (2000) reported considerable amounts of DOM released from the

Russian rivers into Arctic Ocean. The major sinks of DOM include photochemical and

microbial mineralization (Brinkmann et al., 2003; Dainard et al., 2015; Helms et al.,

2008; Miller and Moran, 1997; Zhou and Wong, 2000). In this thesis, DOM distribution

and composition would be monitored in the Arctic Ocean, which will help us further

understand the fate of DOM in the area.

1.2 Importance of DOM and monitoring methods

The optical properties (i.e. absorbance and fluorescence) of DOM are important

factors affecting its behavior in the marine environment. The light absorbing

chromophoric dissolved organic matter (CDOM; Coble, 1996; Stedmon et al., 2000)

enhances primary production (Arrigo and Brown, 1996) and serves as a protection layer

in the surface water by absorbing harmful radiation such as ultraviolet radiations (Hill,

2008). However, high levels of light absorption may result in competition with

3

phytoplankton for the amount and quality of available light resource, which may

impede primary production (Bidigare et al., 1993; Kowalczuk et al., 2003; Thrane et

al., 2014). Based on the absorption spectrum, information on relative CDOM

concentration (absorption coefficient at a specific wavelength at 254 or 355 nm, Chen

et al., 2004; Dainard and Guéguen, 2013; Granskog et al., 2007), as well as molecular

size and aromaticity (spectral slope S, Helms et al., 2008; Stedmon et al., 2011), could

be determined.

In addition to absorbing properties, DOM can be characterized by its fluorescing

properties (FDOM, Coble, 1996; Mopper and Schultz, 1993). The fluorescence

excitation emission matrices (EEMs, Coble, 1996) had been proved to be capable of

differentiating humic-like DOM of terrestrial or marine origin (Coble, 1996). The

coupling with parallel factor analysis (PARAFAC) allows to decompose EEMs into

more detailed fluorescent components (Dainard and Guéguen, 2013; Guo et al., 2011;

Murphy et al., 2008). For example, a PARAFAC model consisted of marine (M peak,

Coble, 1996) and terrestrial (A and C peak, Coble, 1996) humic-like, protein-like (T

peak, Coble, 1996) fluorescent components was validated for DOM samples in Arctic

Ocean (Dainard and Guéguen, 2013; Guéguen et al., 2014; Walker et al., 2009).

DOM – metal complexation is of vital importance in the marine environment,

which had been demonstrated to be related with aromaticity and DOM molecular size

(Benoit et al., 2001; Guo and Santschi, 2007; Laglera and van den Berg, 2003; Louis

et al., 2014; Nierop et al., 2002; Sun et al., 1997; Wu et al., 2004). Thus, assessment

on the molecular weight (MW) could help us better understand the function of DOM

4

in the ocean system. There were multiple ways of determining DOM MW, like

ultrafiltration (Guo and Santschi, 2007; Pokrovsky et al., 2012), high pressure size

exclusion chromatography (HPSEC, Conte and Piccolo, 1999; Zhou et al., 2000) and

asymmetrical flow field flow fractionation (AF4, Guéguen and Cuss, 2011; Lin et al.,

2016; Stolpe et al., 2014, 2010). However, ultrafiltration only gives a single MW cutoff

separation while AF4 could provide a continuous and detailed MW distribution.

HPSEC and AF4 were both proved to be capable of detecting DOM MW with rapid

throughput and high accuracy. But HPSEC is limited in the size range and choice of

buffers (Liu et al., 2006; Narhi, 2013), while AF4 displays broad dynamic range and is

capable of running under high salinity condition (Cao et al., 2009; Narhi, 2013), which

is more applicable for marine DOM. Besides, AF4 is capable of maintaining the

original DOM composition without the high pressure applied in HPSEC system.

Moreover, only a few researchers applied AF4 on the marine DOM (Hassellöv, 2005;

Lin et al., 2016; Stolpe et al., 2014, 2010) and for the first time, AF4 was selected as

the detector for marine DOM MW in the Arctic Ocean in this thesis.

Besides DOM MW, available organic ligands are another important factors

affecting DOM-metal complexation. Some metal-binding agents are fluorescent and

can be determined by fluorimeter (McIntyre and Guéguen, 2013; Tani et al., 2003; Wu

et al., 2001; Yamashita and Jaffé, 2008). For example, HS was involved with iron

solubility and measured as humic-like type fluorescent components in north Pacific

Ocean (Tani et al., 2003). But not all the organic ligands are fluorescent, like thiols,

however, thiol groups (i.e. GSH) showed great binding affinity with metals in the

aquatic system (Hunter and Boyd, 2007; Kinniburgh et al., 1996; Yang and Berg, 2009).

5

Thus, methods other than EEMs are needed for the non-fluorescing organic ligands (i.e.

thiols). High performance liquid chromatography (HPLC) and cathodic stripping

voltammetry (CSV) have been applied in detecting thiols in ocean studies (Kading,

2013; Laglera and van den Berg, 2003; Pernet-Coudrier et al., 2013; Swarr et al., 2016).

Although HPLC analysis features high resolution in thiol determination, time

consuming thiol derivatization is required (Kading, 2013; Swarr et al., 2016; Tang et

al., 2003). On the other hand, CSV technique allows the determination of GSH

(Kawakami et al., 2006) without extensive sample preparation (Marie et al., 2015;

Pernet-Coudrier et al., 2013). Regarding refractory organic matter in the aquatic

systems, HS could be characterized as humic-like components based on its fluorescing

property (Tani et al., 2003) and detected by CSV (Whitby and Van den Berg, 2014).

But the comparison between humic-like fluorescent components and CSV-based HS

remains unknown, and this comparison will provide novel insights on marine DOM

composition in this thesis. For the simultaneous detection purpose of thiols and HS in

seawater samples (Marie et al., 2015; Pernet-Coudrier et al., 2013), CSV is used in this

thesis.

1.3 The hydrology in the Arctic Ocean

The Arctic Ocean (Fig 1.1) serves as a channel between the Pacific Ocean and the

Atlantic Ocean. Pacific-origin water flows into Beaufort Sea via Bering Strait while

Atlantic-origin deep water enters through Fram Strait Branch and Barents Sea Branch.

Canadian Arctic Archipelago (CAA) and other passages like Smith Sound serve as

exiting pathways for Arctic waters into the North Atlantic Ocean.

6

Surface waters in the Canada Basin (CB) are controlled by a strong wind-driven

circular current, the Beaufort Gyre (Woodgate, 2013), accumulating freshwater from

sea ice melting and Eurasian and American river inputs. With the contribution of

summer ice melting inputs, Pacific summer water (PSW) is formed, which is on the top

of a saline and cold Pacific winter water (PWW). The great difference in salinity and

temperature (Fig 1.2A) impeded the vertical exchange between PSW and PWW.

Underlying Pacific waters, saline and warm Atlantic waters was found below 400 m.

For the spatial distribution, sub-surface Pacific waters flowed eastwards through CAA

and encountered warm and saline Atlantic waters at Lancaster Sound where they join

the thermocline circulation at Baffin Bay.

Four main water masses (Carmack et al., 2015; McLaughlin et al., 2011, 2004;

Melling et al., 2008; Michel et al., 2006; Rudels et al., 2012; Woodgate, 2013) are

identified based on the temperature/salinity (T/S) diagram (Fig 1.2):

Surface waters (SW) occupy the top 30 m and include the Arctic surface

waters (ASW) in CB and western and Lancaster Sound surface waters

(LsdSW).

Arctic Outflow waters (OW) are found in the top 300 m (excluding SW).

OW encompasses the PSW and PWW in CB.

In CB, Atlantic-derived Arctic intermediate water (AIW) are found

underlying Pacific waters, a temperature maximum near 400 m

characterizes Fram Strait Branch (FSB) waters while lower temperature

characterizes Barents Sea Branch (BSB) water.

7

Deep layers near Lancaster Sound are occupied by North Atlantic Deep

waters (NADW).

The Arctic Ocean is undergoing rapid changes due to global warming (Purkey and

Johnson, 2010; Shimada et al., 2006). Increased river discharges in Arctic Ocean

(Peterson et al., 2002) may bring more terrestrial influence to the Arctic Ocean, while

change in sea ice cover (Comiso et al., 2008) may make a difference in DOM

composition and distribution. Although, remote sensing techniques have been applied

in DOM monitoring in Arctic Ocean (Matsuoka et al., 2013), in-situ sampling and

assessment of DOM in Arctic Ocean are still required and will provide better insights

into the Arctic Ocean under global warming issues.

1.4 Objectives of my study

For the purpose of monitoring DOM distribution in the Arctic Ocean, DOM MW,

optical properties and metal-binding ligands (i.e. thiols and HS) concentration would

be determined in samples collected in CB and CAA during the Joint Ocean Ice Studies

(JOIS) cruise (September 2014) and the Canadian Arctic GEOTRACES cruises (July-

September 2015).

Two major research questions will be solved in this thesis:

Is the distribution of DOM characteristics associated with water masses of

different origin?

Is there a connection between voltammetry-based HS and humic-like

fluorescent component?

8

For the first time, AF4 system will be applied to assess marine DOM MW in the

Arctic Ocean, as well as the first monitoring of thiols and humic substances. Besides,

a novel comparison between voltammetry-based HS and humic-like fluorescent

components will be conducted.

Since the Arctic Ocean is highly stratified and consisted of waters from Pacific and

Atlantic origin, the DOM characteristics (i.e. DOM MW, CDOM, thiol and HS) are not

homogeneously distributed throughout the Arctic Ocean. Tracing water masses by

DOM characteristics will improve the understanding of water circulation, fate and

transport of DOM in the Arctic. The DOM monitoring conducted in this thesis will

provide unique insights on the DOM distribution and composition in the Arctic Ocean.

At the same time, DOM MW, thiols and HS concentrations, CDOM (including FDOM)

will be studied as fingerprints to distinguish water masses of different origin.

9

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Figures and tables

Figure 1.1 Map of the study area

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Figure 1.2 Potential temperature as a function of Salinity. A:2014 JOIS Cruise; B: 2015 GEOTRACES Cruise

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Chapter 2. Size distribution of absorbing and fluorescing

DOM in Beaufort Sea, Canada Basin

Zhiyuan Gao1, Céline Guéguen2,*

1 Environmental and Life Sciences Graduate program, Trent University, ON, Canada

2 Chemistry Department, Trent University, ON, Canada

*Corresponding author: Tel: +1 (705) 748 1011; Fax: +1 (705) 748 1625; email:

celinegueguen@trentu.ca

19

Abstract

The molecular weight (MW) of dissolved organic matter (DOM) is considered as an

important factor affecting the bioavailability of organic matter and associated chemical

species. Chromophoric DOM (CDOM) MW distribution was determined, for the first

time, in the Beaufort Sea (Canada Basin) by asymmetrical flow field-flow fractionation

(AF4) coupled with online diode array ultra violet-visible photometer and offline

fluorescence detectors. The apparent MW ranged from 1.07 kDa to 1.45 kDa, congruent

with previous studies using high performance size exclusion chromatography and

tangential flow filtration. Interestingly, a minimum in MW was associated with the

Pacific Summer Waters, while higher MW was associated with the Pacific Winter

Waters. The Arctic Intermediate Waters did not show any significant change in MW

and fluorescence intensities distribution between stations, suggesting homogeneous

DOM composition in deep waters. Three fluorescence components including two

humic-like components and one protein-like component were PARAFAC-validated.

With the increase of MW, protein-like fluorescence component became more dominant

while the majority remained as marine/microbially derived humic-like components.

Overall, it is concluded that water mass origin influenced DOM MW distribution in the

Arctic Ocean.

Keywords

DOM, Asymmetrical flow field-flow fractionation (AF4), Molecular weight,

PARAFAC-EEMs

20

2.1 Introduction

Dissolved organic matter (DOM), usually measured as dissolved organic carbon

(DOC), is a major carbon pool in aquatic systems (Amon and Benner, 1994; Benner et

al., 1992; Jiao et al., 2010). DOM serves as a source of food for microorganisms (Cole

et al., 2006), contributing to the food web and energy transport (Baylor and Sutcliffe

Jr, 1963). DOM also plays an important role in regulating the speciation and

distribution of trace metals such as Cu, Hg, Fe and Zn (Hirose, 2007; De Schamphelaere

et al., 2004; Yamashita and Jaffé, 2008). Previous studies showed that increased Cu-

binding affinity was observed with increasing DOM molecular weight (MW) in natural

waters (Midorikawa and Tanoue, 1998; Wu and Tanoue, 2001). High MW DOM

complexation was favoured by metals with high binding strength while low MW DOM

was preferentially bound by weak binding strength metals (Wu et al., 2004). Besides

affecting metal binding affinity, DOM MW could influence its uptake by

microorganisms (Guo and Santschi, 2007; Sun et al., 1997). For example, high MW

oceanic DOM was favoured by bacterial utilization (Amon and Benner, 1996), boosting

growth rates of some bacterial group and algae (Tulonen et al., 1992).

The heterogeneous nature of DOM requires the use of complementary analytical

techniques. The absorbance and fluorescence properties of DOM have been

increasingly used in differentiating allochthonous and autochthonous DOM sources

(Stedmon et al., 2003) and tracing riverine supply into the ocean system (Fichot et al.,

2013; Fichot and Benner, 2012; Guéguen et al., 2011, 2012a; Nelson et al., 2010;

Stedmon et al., 2011). The ultraviolet-visible absorbance properties of chromophoric

dissolved organic matter (CDOM) have been used as molecular size and aromaticity

21

proxies (Guéguen and Cuss, 2011; Helms et al., 2008). Excitation emission matrix

(EEM; Coble, 1996) technique coupled to parallel factor analysis (PARAFAC; Murphy

et al., 2008) have been used to facilitate the identification and quantification of

independent fluorescent classes such as protein-like, microbially derived and terrestrial

humic-like in marine studies (Dainard and Guéguen, 2013; Guo et al., 2011; Murphy

et al., 2008). Asymmetrical flow field-flow fractionation (AF4) is a chromatographic-

like method that has been recently applied to investigate the sources and dynamics of

DOM in aquatic systems (Boehme and Wells, 2006; Guéguen and Cuss, 2011; Lin et

al., 2016; Stolpe et al., 2010, 2014). For example, Lin et al., (2016) showed that the 1-

10 kDa CDOM fraction was influenced by terrestrial input. Stolpe et al., (2014)

concluded that the small MW DOM (2-3 nm) in surface northern Gulf of Mexico

originated from terrestrial sources whereas the larger MW DOM (>6 nm) was protein-

rich and freshly produced.

The Arctic regions are undergoing rapid changes in air temperatures, river

discharge, sea ice extent and permafrost integrity (Frey and McClelland, 2009;

Overland and Wang, 2013; Peterson et al., 2002; Schuur et al., 2009; Shimada et al.,

2006). Continued climate change will likely have profound effect on carbon cycle.

Documenting the composition and distribution of DOM (i.e. MW and optical properties)

could provide valuable insights into the biogeochemical carbon cycle.

In this study, DOM samples were collected at four sites and five water masses in

Beaufort Sea: Arctic Surface Water (ASW), Pacific Summer Water (PSW), Pacific

Winter Water (PWW) and Arctic Intermediate Waters (AIW) including Fram Strait

Branch (FSB) waters and Barents Sea Branch (BSB) water (Carmack et al., 2015;

22

McLaughlin et al., 2011; 2004; Rudels et al., 2012; Woodgate et al., 2005). The DOM

MW distribution in the Arctic Ocean was assessed, for the first time, using

asymmetrical flow field-flow fractionation (AF4) coupled with online diode array ultra-

violet/visible photometer and offline fluorescence detectors.

2.2 Methods

2.2.1 Sampling station

A total of 88 samples, including 20 samples for MW analysis, were collected at 4

sites (CB28b, CB29, CB50 and CB51; Fig. 2.1) in Beaufort Sea in September 2014 as

part of the Joint Ocean Ice Studies cruise. Water samples were collected at depths

ranging from 5 m to 1000 m at each site using Niskin bottles mounted on a rosette

together with a conductivity – temperature – depth profiler and immediately filtered

using pre-combusted (450 ℃ for 4 h) 0.7 µm glass fiber filters (GF/F, Whatman) and

stored in the dark at 4 ℃ in pre-combusted amber glass vials until measurement. The

AF4 based MW distribution was carried out on samples collected at 10 m, sub-

chlorophyll maximum (i.e. 50 - 68 m), salinity 33.1 (i.e. 148 - 161 m) and temperature

maximum (i.e. 414 – 433 m) and 1000 m at each site. High-resolution vertical profiles

of humic-like fluorescent DOM (FDOM), primarily derived from terrestrial sources,

were also acquired using a WET Labs probe (WETStar Ex/Em 370/460 nm) at each

site.

2.2.2 Asymmetrical Flow field flow fractionation

MW distribution was assessed using an asymmetrical flow field-flow fractionation

(AF4) system within 10 days of collection to avoid MW alteration (Guéguen and Cuss,

23

2011). AF4 is carried out using an AF2000 Focus fractionation system (Postnova

Analytics, Landsberg, Germany) which includes two PN1130 HPLC pumps to control

the axial and focus flows, a PN1610 syringe pump to control the crossflow rate, and a

PN7505 degasser to remove gas from the carrier solution prior to introduction to the

pumps (Guéguen and Cuss, 2011). Absorbance scan was recorded from 270 nm to 700

nm, using an on-line diode array ultra-violet/visible photometer (Shimadzu SPD-

M20A). The fractionation system is equipped with a 300 Da polyethersulfone

membrane (Guéguen and Cuss, 2011; Guéguen et al., 2013; Zhou and Guo, 2015). It

should be noted that DOM smaller than 300 Da will not retained on the AF4 membrane

and thus not measured. The pH and conductivity of the NaCl carrier solution matched

that of the Arctic samples (20 mS/cm, pH 7.9) to preserve the native chemical

environment of DOM.

The AF4 system was operated under the following conditions: detector flow rate at

0.35 mL/min, focusing at 0.15 mL/min focus flow rate for 7 min, elution at 2.20

mL/min cross flow rate for 13 min. MQ blank were performed under the same operating

conditions between DOM samples to avoid any cross contamination. In order to get

accurate MW distribution, the AF4 system was calibrated at 254 nm using a log-log

calibration based on four macromolecules laser grade rhodamine B (479 Da),

bromophenol blue (692 Da), cytochrome C (12,400 Da) and lysozyme (14,300 Da)

twice a day, at the beginning and the end of each day (Guéguen and Cuss, 2011). The

simultaneous multi wavelength measurements of the online absorbance detector allow

us to accurately determine the retention of each of the four macromolecules in a single

injection. The retention time at 254 nm was used to estimate the CDOM MW at peak

24

maximum intensity (Guéguen and Cuss, 2011; Lin et al., 2016; Stolpe et al., 2010,

2014). For example, a typical calibration recorded at 254 nm was log (Time) = (0.1634

± 0.0086) * Log (MW) + (0.4831 ± 0.0228) (r2 = 0.999). The smallest and largest

macromolecules (rhodamine-B and lysozyme, respectively) were eluted at 8.680 ±

0.165 (n = 10) and 14.759 ± 0.503 min (n = 10), respectively. It can be noted that the

retention time (i.e. MW) of the Beaufort samples was 4.8% higher (n = 20) at 350 nm

than at 254 nm, confirming the higher MW of aromatic CDOM (Cuss and Guéguen,

2013). A typical chromatogram (Fig. 2.2) showed a very sharp peak representing the

void peak with unfocused and small MW materials and a smooth peak containing the

focused and eluted material.

In terms of repeatability, MW of bromophenol blue sodium salt (Sigma-Aldrich®)

was measured daily (n = 10) and was within 6% of the recommended MW (669 Da).

The relative standard deviation of MW of Suwannee River natural organic matter (n =

5) was 3% (or 85 Da). Similar precision was reported in earlier work (Guéguen and

Cuss, 2011).

2.2.3 Chromophoric dissolved organic matter (CDOM) and Fluorescent DOM (FDOM)

Chromophoric dissolved organic matter (CDOM) samples were allowed to warm

to room temperature before analyzing on a Shimadzu UV 2550 spectrophotometer.

Milli-Q water was measured between each samples to avoid carry-over. A slit width

of 0.5 nm was applied, with an absorbance acquisition interval of 1 nm. The measured

absorbance at wavelength λ was converted to absorption coefficient a (m-1) as

following Eq (1):

25

𝑎𝜆 = 2.303𝐴𝜆/ 𝑙 (1)

where 𝑎𝜆 is the absorption coefficient at wavelength λ , 𝐴𝜆 is the instrumental

absorbance signal, l is the path-length of the optical cell in meters (i.e. 10 cm).

Absorbance coefficient at 254 nm (a254) and 355 nm (a355) were used as indicator for

CDOM concentration in natural samples (Chen et al., 2004; Dainard and Guéguen,

2013; Granskog et al., 2007; Penru et al., 2013).

Three dimensional excitation-emission fluorescence matrices (EEMs) were

generated by measuring fluorescence intensity across excitation wavelengths ranging

from 250 to 500 nm and emission wavelengths ranging from 300 to 600 nm through

successive scans with a Fluoromax-4 Jobin Yvon spectrofluorometer. Excitation and

emission slit widths were set to 5 nm. Blank Milli-Q EEMs were measured daily to

correct background and scatter peak. Before loading samples, a cuvette was rinsed to

avoid cross contamination and scanned at excitation 270 nm and emission 280 – 500

nm for confirmation. All EEMs were normalized to Raman units (R.U.) by dividing the

signal at all Ex/Em pairs by the integrated area under the Raman water peak at

excitation 350 nm using a daily acquired Milli-Q blank EEM (Lawaetz and Stedmon,

2009). Absorption spectra were obtained for all natural seawater samples to make sure

the intensity were within the linear range (A250 < 0.05).

Parallel factor analysis (PARAFAC) was carried out on 88 EEMs (20 AF4 fractions

and 68 bulk) in Matlab using the DOMFluor (Stedmon and Bro, 2008) and DrEEM

toolboxes (Murphy et al., 2013). The model was constrained to nonnegative values and

run for three to ten components. The appropriate number of components was

26

determined by split-half and Tucker congruence coefficient analyses. Percent

fluorescence was calculated as follows:

C1% =𝐶1

(𝐶1+𝐶2+𝐶3) (2)

2.3 Results and Discussion

2.3.1 Hydrographical parameters

Five main water masses were found based on the temperature/salinity (T/Sp)

diagram (Fig. 2.3A): Arctic surface waters (ASW) in the top 50 m (Sp < 30 and -1.5 ℃

< T < -0.5 ℃) were influenced by river runoff (Cory et al., 2007; Pegau, 2002; Walker

et al., 2009), primary production (Fouilland et al., 2007; Hill and Cota, 2005), sea ice

formation and melt (Logvinova et al., 2016; Walker et al., 2009), and photobleaching

(Bélanger et al., 2006); Pacific summer waters (PSW, 30 < Sp < 32) were characterized

by a temperature maximum (T ~ -0.8 ℃; Steele et al., 2004); Pacific winter water

(PWW, Sp ~ 33.1) was characterized by a temperature minimum (T ~ -1.4°C;

McLaughlin et al., 2004) and a terrestrial humic-like maximum (Fig. 2.3B) (Guéguen

et al., 2012b); Deeper layers (> 400 m) were dominated by Atlantic-derived Arctic

intermediate water (AIW). A temperature maximum near 400 m characterized Fram

Strait Branch waters (FSB; Sp ~ 34.8, T ~ 0.6 °C) while lower temperature

characterizes the Barents Sea Branch waters (BSB; Sp ~ 34.8, T ~ 0 °C) (McLaughlin

et al., 2004; Woodgate et al., 2005).

2.3.2 Molecular weight depth distribution

Natural marine CDOM MW ranged from 1.07 kDa to 1.45 kDa (Fig. 2.4),

congruent with previous studies on DOM MW using AF4, high performance size

27

exclusion chromatography and tangential flow filtration (Beckett et al., 1992; Everett

et al., 1999; Guéguen and Cuss, 2011; Huguet et al., 2010; Landry and Tremblay, 2012;

Penru et al., 2013; Stolpe et al., 2010). For example, colloidal DOM MW averaged 1.4

± 0.4 kDa in Northern Gulf of Mexico (Stolpe et al., 2010). Higher MWs (1.62-2.52

kDa) were reported for colloidal DOM in Chukchi Sea (Lin et al., 2016). The average

MW reported in Beaufort Sea agreed with values reported for fulvic acids (Beckett et

al., 1987; Guéguen and Cuss, 2011), confirming that fulvic acids are good proxy for

marine DOM (Tipping et al., 2015).

Average CDOM MW (1.37 ± 0.01 kDa) in ASW was remarkably consistent across

the study area. This contrasts with the halocline complex and deeper layers where

significant MW differences were found (Fig. 2.4). Compared to ASW, the CDOM MW

in PSW was lower, likely due to the influence of sea ice melt waters rich in low MW

DOM (Logvinova et al., 2016). CDOM MW increased again in PWW, which resulted

from the interactions with C-rich shelf bottom sediment (Moore et al., 1983)

remobilizing high MW DOM (Skoog et al., 1996). Below the halocline complex, lower

MW CDOM (~1.21 kDa) was found with the saline Atlantic waters. The BSB MW was

relatively consistent (1.27 ± 0.06 kDa), confirming the CDOM homogeneity in deeper

layer.

2.3.4 Optical Properties

The a254 and a355 values (Fig. 2.5A-B) compared well with previous Arctic studies

(Dainard and Guéguen, 2013; Spencer et al., 2009). Relatively low a254 and a355 values

(2.444 and 0.2014 m-1, respectively) were found in ASW likely due to photobleaching

(Guéguen et al., 2016; Logvinova et al., 2015) and/or sea ice melt (Logvinova et al.,

28

2015) in surface waters. Similar trends were previously observed (Dainard and

Guéguen, 2013; Stedmon et al., 2011). The PSW-PWW waters were characterized by

high CDOM values (Fig. 2.5A-B), congruent with previous studies. As proxy for

molecular size and aromaticity (Guéguen and Cuss, 2011; Helms et al., 2008), S275-295

ranged from 0.018 to 0.033 nm-1 (Fig. 2.5C), congruent with previous Arctic DOM

studies (Fichot et al., 2013). Although S275-295 was not significantly correlated with

AF4-based MW in this study, the increase in S275-295 (i.e. decrease in MW) from PSW

to deeper waters was consistent with the overall vertical decrease in AF4-based MW

(Fig. 2.4).

Three fluorescent components (Fig. 2.6) were PARAFAC-validated and reported

in previous studies (Dainard and Guéguen, 2013; Kowalczuk et al., 2009; McIntyre and

Guéguen, 2013; Singh et al., 2010; Stedmon and Markager, 2005; Walker et al., 2009).

Component C1 displayed a maximum excitation wavelength at 305 nm and emission

wavelength at 410 nm, similar as to peak M (Coble, 1996), representing marine humic-

like component or biological and microbial origin DOM (Shimotori et al., 2012;

Yamashita et al., 2008). This component was widely found in both freshwater and

marine systems (Dainard and Guéguen, 2013; Kowalczuk et al., 2009; McIntyre and

Guéguen, 2013; Singh et al., 2010; Walker et al., 2009). Component C2 showed a

maximum excitation wavelength at < 280 nm and emission wavelength at 330 nm,

which was similar to peak T, traditionally considered as protein-tryptophan-like

component (Coble, 1996). Tryptophan-like component was linked to biological

production (Coble et al., 1998) and commonly found among different aquatic systems

(Dainard and Guéguen, 2013; McIntyre and Guéguen, 2013; Stedmon and Markager,

29

2005). Component C3 had maximum excitation wavelengths at < 270 nm and 365 nm

and a maximum emission wavelength at 465 nm. This component was comparable to

peaks A and C (Coble, 1996). Despite of its terrestrial origin, similar components have

been reported along a large salinity scale (Dainard and Guéguen, 2013; Kowalczuk et

al., 2009; Stedmon and Markager, 2005; Walker et al., 2013).

Total fluorescence intensity (TF=C1+C2+C3) as well as the fluorescent component

(C1, C2 and C3) intensities (Fig. 2.7) were within the ranges reported earlier (Dainard

and Guéguen, 2013; Guéguen et al., 2014; Walker et al., 2009). TF was relatively stable

within each water mass except in ASW likely due to the influence of biological

activities and sea ice melt. Maximum TF and humic-like intensities (C1 and C3) were

associated with PWW (150 m; Fig. 2.7A-B, D), consistent with CDOM distribution

(Fig. 2.5). Similar distributions were found in previous studies (Dainard et al., 2015;

Guéguen et al., 2007, 2012b; Nakayama et al., 2011). Low TF in ASW is likely due to

enhanced photobleaching (Dainard et al., 2015; Guéguen et al., 2016; Logvinova et al.,

2016) associated with a greater influence of DOM-poor sea ice melt waters (Belzile et

al., 2002) in surface waters. The prominent FDOM (and CDOM; Fig. 2.5) maximum

at PWW was due to humic-rich materials brought by injection of cold saline shelf

waters (Guéguen et al., 2007; Moore et al., 1983; Woodgate et al., 2005). No significant

difference in humic-like fluorescence intensities was found in FSB and BSB between

sites (0.0137 ± 0.0014 r.u. for C1; 0.0090 ± 0.0010 r.u. for C3), suggesting

homogeneous humic-like intensity in deeper layers. Protein-like C2 intensity maximum

(Fig. 2.7C) was associated with the highly productive (Arrigo et al., 2008; Fouilland et

al., 2007; Hill and Cota, 2005) ASW, confirming the biological origin of protein-like

30

component. Lower C2 intensities were associated with the PSW-PWW complex and

Atlantic-derived waters. Homogeneous distribution (p > 0.05) was observed in AIW.

2.3.5 Relationship between fluorescence properties and MW

Despite a 40% difference in TF (0.042 vs 0.060 r.u., p < 0.05; Fig. 2.8), ASW and

PWW showed similar average CDOM MW values (1.37 vs 1.38 kDa) (Fig. 2.8). PWW

was largely associated with the increase in aromatic CDOM (low S275-295; Fig. 2.5C)

and humic-like FDOM (Fig. 2.7) due to sediment interactions (Guéguen et al., 2007;

Moore et al., 1983). Although FDOM concentration and composition did not

significantly influence MW in ASW and PWW, the significant decrease in MW from

PWW and AIW was associated with a dramatic decrease in humic-like intensities (p <

0.05). Similarly, PSW and BSB displayed comparable mean MW values (1.27 kDa in

both water masses) but a 39 % reduction (p < 0.05) in TF in BSB. Together these results

showed that a change in MW does not necessarily mean a change in TF. This contrasts

with previous studies where higher TF, and particularly higher humic-like intensities,

was generally associated with higher MW material (Cuss and Guéguen, 2013; Huguet

et al., 2010). Because the PARAFAC-validated components cannot be attributed to

pure compounds but to a group of compounds sharing similar fluorescence properties,

it is possible that a change in the concentration and/or nature of fluorescing compounds

affects the fluorescence efficiency and therefore FDOM signal without changing MW.

Furthermore, Stolpe et al. (2014) showed that protein-like fluorescence (275/340 nm)

in marine waters had a size distribution that was very different from the size distribution

of CDOM measured at 254 nm. Further studies should be carried out to assess the

influence of absorbing and fluorescing DOM composition on MW.

31

In terms of fluorescence composition in bulk samples (Fig. 2.9A-C), humic-like C1%

and C3% decreased with increasing MW (r2 = 0.38 and 0.40, respectively). This

contrasts with protein-like C2% which was more abundant at higher MW (r2 = 0.41;

Fig. 2.9D). The change in size distribution between protein- and humic-like FDOM

was also found in the northern Gulf of Mexico (Stolpe et al., 2014) where humic-like

components dominated small size marine DOM while protein-like DOM was enriched

in larger MW DOM. Similarly, increase in protein-like component was observed with

decreasing S275-295 (i.e. increase in MW) in North Pacific seawater studies (Helms et al.,

2013). Despite of variation in S275-295, humic-like components were found to be

dominant (> 50%) throughout the study area (Dainard and Guéguen, 2013).

2.4 Conclusions

For the first time, asymmetrical flow field-flow fractionation system was applied in

the Arctic Ocean to assess CDOM MW distribution in relation with optical properties

and water masses. The CDOM MW ranged from 1.07 to 1.45 kDa, consistent with

earlier works. The vertical distribution of CDOM MW and optical properties showed

some water-mass related distribution pattern. DOM in Arctic surface waters displayed

similarity in MW and fluorescence intensity across the study area. PWW formed in

from ice formation and freshwater injection, showed higher MW CDOM associated

with higher fluorescence intensity. Protein-like fluorescence became more dominant as

MW increased, but humic-like components remained dominant throughout the water

column.

32

Acknowledgments

This work was supported by the Northern Scientific Training Program, Canada

Research Chair Program (CG) and National Sciences and Engineering Research

Council of Canada (CG). The field program on the Louis S. St-Laurent was funded by

the U.S. National Science Foundation’s Beaufort Gyre Observation System and the

Department of Fisheries and Oceans Canada. We gratefully acknowledge the support

of C. Wylie, S. Zimmerman and W. Williams for field assistance in the JOIS cruise.

We thank the captain and crew of the CCGS Louis S. Saint-Laurent and the other

participants of the cruise.

33

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41

Figures and tables

Figure 2.1 Sampling locations in Beaufort Sea, Canada Basin

42

Figure 2.2 AF4 fractograms of CB29 (400m depth; black) and CB28b (1000m depth; gray)

43

Figure 2.3 A) Potential temperature and B) FDOM as a function of salinity (Sp) at the four study sites (Figure 2.1). ASW -

Arctic surface waters, PSW – Pacific summer water, PWW – Pacific winter water, FSB – Fram Strait Branch water, BSB –

Barents Sea Branch water

44

Figure 2.4 MW depth profiles in Beaufort Sea

45

Figure 2.5 Depth distribution of A) a254, (B) a355, and (C) S275-295

46

Figure 2.6 Individual components identified by PARAFAC (A- marine humic-like C1, B- protein-like C2 and C- terrestrial

humic-like C3)

47

Figure 2.7 Depth distribution of A) Total Fluorescence Intensity (TF), B) marine humic-like C1, C) protein-like C2 and D)

terrestrial humic-like C3

48

Figure 2.8 Change in TF vs CDOM MW

49

Figure 2.9 Correlation between MW and fluorescence components (A) C1; (B) C2; (C) C3; (D) Change in fluorescence

abundance with increasing DOM MW

50

Chapter 3. Determination of thiols, humic substances and

fluorescent dissolved organic matter during the 2015

Canadian Arctic GEOTRACES cruises

Zhiyuan Gao1, Céline Guéguen2,*

1 Environmental and Life Sciences Graduate program, Trent University, ON, Canada

2 Chemistry Department and Trent School of the Environment, Trent University, ON,

Canada

*Corresponding author: Tel: +1 (705) 748 1011; Fax: +1 (705) 748 1625; email:

celinegueguen@trentu.ca

51

Abstract

Distribution of thiols, humic substances (HS) and fluorescent dissolved organic matter

(FDOM) were determined in seawater samples from Canada Basin and Canadian Arctic

Archipelago (CAA) by differential pulse cathodic stripping voltammetry (DP-CSV)

and excitation-emission matrix (EEM). The simultaneous determination of thiols and

HS by DP-CSV featured high repeatability (RSD, 0.64 % for GSH, 0.60 % for HS),

high recovery (~ 100% for both) and low LOD (1.12 nM for GSH, 21.19 µg C/L for

HS). The thiol concentration ranged from 8 to 78 nM (glutathione equivalent) and HS

ranged from 31 to 222 µg C/L, congruent with ranges reported in CSV-based seawater

studies. Three humic-like (C1-C3) and one protein-like (C4) components identified and

validated using parallel factor analysis (PARAFAC) have been reported in previous

ocean studies. The distributions of voltammetry-based HS and humic-like components

(C1 and C2) along CAA were similar, whereas thiol and protein-like component C4

were closely related in the top 100 m depth. Maximum thiol concentrations were

associated with chlorophyll-a maximum, confirming the biogenic origin of thiols. The

distribution and concentrations of thiols, HS and FDOM varied between four distinct

water masses (i.e. surface waters, Arctic outflow waters, Arctic intermediate waters and

North Atlantic deep waters) with the lowest levels associated with the surface waters,

likely due to photobleaching and sea ice melt dilution. The concentrations in HS and

FDOM in the Arctic outflow waters decreased from western to eastern CAA, reflecting

the influence of DOM-rich Pacific derived waters.

Keywords

DOM, Cathodic stripping voltammetry, Thiols, Humic substances, PARAFAC-EEMs

52

3.1 Introduction

Dissolved organic matter (DOM) is one of the major carbon groups in the ocean

system, contributing to energy transport and food supplement. DOM plays a significant

role in regulating the speciation and distribution of trace metals, such as Cu, Fe and Hg,

affecting their bioavailability and toxicity (Hirose, 2007; Stockdale et al., 2011; Wen

et al., 1999). For example, the photolysis of iron-siderophore complexes increases the

availability of Fe uptake by planktonic assemblages (Barbeau et al., 2001). DOM was

reported to be an important complexing agent for Hg (Benoit et al., 2001) whereas

terrestrial humic substances (HS) were responsible for reduction of copper toxicity in

the seawater (Kogut and Voelker, 2001).

Low molecular weight sulphur-containing DOM (i.e. thiols) is one of the most

important organic ligands for metal binding (Benoit et al., 2001; Laglera and van den

Berg, 2003), with thiourea, cysteine and glutathione (GSH) being the most common

thiol groups in marine environment (Ahner et al., 2002; al-Farawati and Van Den Berg,

2001; Dupont et al., 2006; Kading, 2013; Tang et al., 2000). Algal culture studies have

shown that thiols are biologically produced as a response to the increase of free metal

ion concentrations in aquatic systems (Ahner et al., 2002; Dupont and Ahner, 2005;

Leal et al., 1999). High performance liquid chromatography (HPLC) and cathodic

stripping voltammetry (CSV) have been applied in detecting thiol abundance in ocean

studies (Kading, 2013; Laglera and van den Berg, 2003; Pernet-Coudrier et al., 2013).

Although HPLC analysis featured high resolution in thiol determination, time-

consuming thiol derivatization is required (Kading, 2013; Swarr et al., 2016; Tang et

al., 2003). On the other hand, the CSV technique allows the determination of GSH

53

(Kawakami et al., 2006) without extensive sample preparation (Marie et al., 2015;

Pernet-Coudrier et al., 2013). The limit of detection (LOD) of CSV-based thiol

determination (1 nM; Pernet-Coudrier et al., 2013) is compatible with the natural range

of seawater thiol compounds (10 to 410 nM; Le Gall and van den Berg, 1993; Marie et

al., 2015; Pernet-Coudrier et al., 2013).

Humic substances (HS), consisting of fulvic and humic acids (Coble, 1996; Harvey

et al., 1983; Rashid, 2012), are considered as the refractory fraction of DOM and

comprise 10-50 % of total organic carbon (Harvey et al., 1983). The two main sources

of marine HS in the marine domain are: allochthonous part of terrestrial origin

(Guéguen et al., 2005; Meyers-Schulte and Hedges, 1986; Opsahl et al., 1999; Opsahl

and Benner, 1997) and autochthonous part produced in-situ through photo-oxidation

and biological processes (Harvey et al., 1984; Kieber et al., 1997; Shimotori et al., 2012;

Yamashita and Tanoue, 2008). HS can be characterized by its fluorescence property as

humic-like components (Coble, 1996) and detected through electrochemical techniques

like CSV (Pernet-Coudrier et al., 2013; Whitby and Van den Berg, 2014). However,

the comparison between humic-like components and CSV-based HS remained to be

shown.

Regarding metal binding capability, documenting the concentration and

distribution of their main ligands (i.e. thiols and HS) will provide valuable insights into

distribution of DOM and associated trace metals. For the first time, differential pulse

cathodic stripping voltammetry (DP-CSV) was applied on samples collected in the

Canada Basin and Canadian Arctic Archipelago as part of the 2015 Canada Arctic

54

GEOTRACES cruises. Comparison between CSV-based HS and humic-like

components will provide novel insights into DOM distribution in the Arctic Ocean.

3.2 Methods

3.2.1 Sampling

Eleven sites (Fig 3.1) in the Canadian Arctic Archipelago (9 sites) and Canada

Basin (2 sites) were sampled in July and August, 2015 (leg 2) and September, 2015

(leg 3b) as part of the 2015 Canadian Arctic GEOTRACES cruise program. Water

samples were collected using Niskin bottles mounted on a trace metal rosette together

with a conductivity – temperature – depth profiler (Fig 3.2A). Samples were

immediately filtered using pre-combusted (450 ℃ for 4 h) glass fiber filters (GF/F,

Whatman) and stored in the dark at 4 ℃ in pre-combusted amber glass vials for ~2

months. Duplicate samples for voltammetry analysis were acidified to pH 1.95 with 50

µL HCl immediately after filtration and analyzed within the recommended preservation

time (i.e. 2 months; Pernet-Coudrier et al., 2013).

3.2.2 Reagents

Milli-Q water (Millipore, Fisher scientific) was used for rinsing glass cells and

fluorescence cuvette, and preparation of stock and working solutions. HCl (CALEDON)

and NaOH (J.T.Baker) were used to acidify and adjust pH during voltammetric analysis.

The choice of standards is an important factor affecting the CSV-based thiol

concentration. Several thiol groups have been previously used as standards, resulting

in huge difference in the reported concentrations. For example, 50 nM of GSH-

equivalent thiol compounds was found in ocean studies (Le Gall and van den Berg,

55

1993), while a range of 0.7 – 3.6 nM of thiourea-equivalents thiol compounds was

found in English Channel and North Sea studies (al-Farawati and Van Den Berg, 2001).

In this study, glutathione (GSH) was used as a standard for thiol groups (Pernet-

Coudrier et al., 2013). GSH stock solution (oxidized, Sigma-Aldrich) was prepared in

Milli-Q water and stored in dark at 4 ℃ and diluted daily to 20 µM; the pH was adjusted

to 1.95 by adding 2 % HCl. Suwannee river fulvic acid (SRFA) (1S101F; International

Humic Substances Society) was selected as a standard to quantify HS in voltammetry

analysis (Pernet-Coudrier et al., 2013). SRFA stock solution (251 mg C/L) was

prepared in NaOH (~ 0.01 M) for better dissolution and diluted to 150 mg C/L, adjusted

to pH 1.95 before using as the working solution.

3.2.3 Instrumentation

Voltammetry analysis was carried out using a Metrohm uAutolab Type III

potentiostat/galvanostat coupled with a three electrode basis 663 VA stand (Metrohm)

controlled by Autolab GPES software version 4.9. A static mercury drop electrode

(SMDE) was applied in the electrochemical cell as working electrode with the mercury

drop at size one (r0 = 1.41 × 10-4 m). A Ag/AgCl (3 mol/L, Fluka) electrode and a glass

carbon rod (Metrohm) were used as reference and auxiliary electrodes, respectively. A

PTFE stirrer was used with a rotation rate of 1500 rpm for the purpose of better purging

and deposition. A daily calibrated dual channel pH meter (accumet) was used during

the measurement. The DP-CSV method was based on Pernet-Coudrier et al. (2013).

Briefly, 25 mL of seawater samples was loaded into a voltammetric glass cell and 100

µL of 100 ppm Mo solution (EMD) was added to amplify HS signal. A 600-s N2

purging period was applied, followed by a 120-s deposition time with deposition

56

potential at 0.00 V with stirring. After deposition, a 5-s rest time was applied and

stripping scan using differential pulse mode started from 0.0 V to -0.6 V, where the

step potential was 2 mV and amplitude was 60 mV. The range of current was

determined automatically by the GPES software.

Repeatability, reproducibility, LOD and recovery (Table 3.1) experiments were

conducted on freshly made artificial seawater (Stein, 1979) without EDTA and trace

metal solution. The artificial seawater was adjusted in conductivity and pH to match

that of natural seawater samples. Repeatability calculated as 10 consecutive

measurements of artificial seawater (2.50 nA and 32.11 nA) was 0.64 %, 0.60 % for

GSH and HS, respectively. The CSV reproducibility obtained after 10 consecutive

measurements of one sample was 40.75 nM and 672.05 µg C/L for GSH and HS,

respectively. LODs were calculated as 3 times the standard deviation of 10 consecutive

measurements, which were 1.12 nM and 21.19 µg C/L, respectively for GSH and HS

(Table 3.1). Repeatability, reproducibility and LODs are comparable to previous study

(Marie et al., 2015; Pernet-Coudrier et al., 2013). Recovery was assessed by measuring

artificial seawater with known concentration of GSH and HS (10.3 nM and 168 µg C/L,

respectively). The recovery was ~ 100% for both GSH and HS.

Though the presence of HS was reported to overlap the GSH signal in CSV (Le

Gall and van den Berg, 1993), seawater contains low DOM concentration (i.e. < 1 ppm;

Carlson and Hansell, 2014) which is not enough to obscure GSH signal.

57

3.2.4 Fluorescent dissolved organic matter (FDOM)

Three dimensional excitation-emission fluorescence matrices (EEMs) were

generated by measuring fluorescence intensity across excitation wavelengths (Ex)

ranging from 250 to 500 nm and emission wavelengths (Em) ranging from 300 to 600

nm through successive scans with a Fluoromax-4 Jobin Yvon spectrofluorometer.

Excitation and emission slit widths were set to 5 nm. Blank Milli-Q EEMs were

measured daily to correct background and scatter peaks. Between each sample, the

quartz cuvettes were thoroughly rinsed with Milli-Q and scanned at Ex/Em 270/280 –

500 nm. The absence of significant fluorescent intensity at 300-500 nm confirmed the

cleanliness of the cuvette. All EEMs were normalized to Raman units (R.U.) by

dividing the signal at all Ex/Em pairs by the integrated area under the Raman water

peak at Ex 350 nm using a daily acquired Milli-Q blank EEM (Lawaetz and Stedmon,

2009).

Parallel factor analysis (PARAFAC) was carried out in Matlab using the

DOMFluor (Stedmon and Bro, 2008) for the decomposition of EEMs dataset. The

model was constrained to nonnegative values and run for three to ten components. The

appropriate number of components was determined by split-half and Tucker

congruence coefficient analyses.

3.3 Results

3.3.1 Water mass definition

Arctic Ocean serves as a conduit between the Pacific Ocean and the Atlantic Ocean.

Pacific-derived water exits the Canada Basin through Canadian Arctic Archipelago

58

(CAA), Barrow Strait (BS) and other passages like Jones Sound and Smith Sound into

North Atlantic Ocean (Woodgate, 2013). The current description of water masses is

based on previous works in CB (Carmack et al., 2015; McLaughlin et al., 2011, 2004;

Rudels et al., 2012; Woodgate, 2013) and CAA (Melling et al., 2008; Michel et al.,

2006; Woodgate, 2013). Three main water masses were found (Fig 3.2A):

Surface waters (SW; top 30 m). Surface waters (ASW) in CB and western CAA

were highly affected by runoff and ice melt (Cory et al., 2007; Logvinova et

al., 2016; Walker et al., 2009) and characterized by a low salinity and cold

temperature (Tp < 2 ℃ and Sp < 32; Fig 3.2B-C), while Lancaster Sound

surface waters (LsdSW) were affected by warmer and saline Atlantic waters

(Tp > 2 ℃ and Sp > 32). Chlorophyll showed relative low level in ASW while

it is relatively higher with LsdSW (Fig 3.2D).

Arctic outflow waters (OW; T < 0 ℃ and Sp < 33.7) (Azetsu-Scott et al., 2010;

Tang et al., 2004). OW occupied the top 300 m (excluding SW) and included

chlorophyll maximum along the section (Fig 3.2D). OW also encompassed

Pacific summer waters (T ~ - 0.8℃; Fig 3.2C) and Pacific winter waters (Sp ~

33.1) in CB. OW is affected by Canadian rivers input and ice melting in CAA

(Michel et al., 2006; Myers, 2005).

Deep waters (DW; T > 0 ℃ and Sp > 34). Warm and saline Atlantic origin

waters dominated the deep layers (> 300 m) with low level bioactivity (i.e. low

chlorophyll, Fig 3.2D). Arctic intermediate waters (AIW; > 400 m, Sp > 34.5)

were observed in CB and western CAA, whereas North Atlantic deep waters

(NADW; T ~ 2 ℃) was found in Lancaster Sound (Lsd).

59

3.3.2 CSV-based DOM characterization

The CSV-based thiol concentration of all samples ranged from 8 to 78 nM (Fig

3.3A) with an average of 21.9 nM (GSH equivalent), congruent with CSV-based

studies (Le Gall and van den Berg, 1993; Marie et al., 2015; Pernet-Coudrier et al.,

2013). HS of all samples ranged from 31 to 222 µg C/L (Fig 3.3B) with an average of

94 µg C/L and fell into the range reported earlier (Pernet-Coudrier et al., 2013; Whitby

and Van den Berg, 2014). Assuming that the dissolved organic carbon (DOC)

concentration was 90 µM in surface Western Arctic Ocean (Cai et al., 2011), the

abundance of HS represented between 2.8 % and 20.5 % of DOC, which was

comparable to previous studies (Chanudet et al., 2006; Weishaar et al., 2003).

3.3.3 FDOM characterization

Four components were identified and validated in PARAFAC model (Fig 3.4). The

cross validated PARAFAC components were compared with the online repository of

published PARAFAC components (Tucker congruence > 0.95; Murphy et al., 2014).

All components were previously found.

Components C1 to C3 were reported previously and considered as humic-like as

their maximum emission wavelengths were above 400 nm. C1 showed an excitation

wavelength at < 250 nm and 370 nm, an emission wavelength at 460 nm, which is close

to traditionally considered UV/visible terrestrial humic-like components (Coble, 1996).

The longest maximum emission wavelength of C1 suggested its elevated degree of

aromaticity (Stedmon et al., 2003). Despite of its terrestrial origin, similar components

had been found in open ocean studies (Dainard and Guéguen, 2013; Guéguen et al.,

2014; Walker et al., 2009). C2 and C3 showed a primary excitation wavelength

60

maximum at < 250 nm, a secondary excitation maximum at 310 nm and a maximum

emission at ~ 400 nm. These two components were identified as a combination of UV

humic-like and marine humic-like components (Coble, 1996), which were observed in

both terrestrial and marine systems (Dainard and Guéguen, 2013; McIntyre and

Guéguen, 2013; Stedmon et al., 2007; Walker et al., 2009). C4 featured a peak at Ex/Em

275/325 nm, which is similar to tryptophan-like fluorescence component (Coble, 1996)

associated with biological production (Coble et al., 1998). This component had been

reported widely in aquatic studies (Dainard and Guéguen, 2013; McIntyre and Guéguen,

2013; Stedmon et al., 2007; Stedmon and Markager, 2005).

The intensity ranges of four fluorescence components (C1: 0.014 to 0.039 r.u.; C2:

0.007 to 0.031 r.u.; C3: 0.005 to 0.032 r.u.; C4: 0.001 to 0.079 r.u.; Fig. 3.3C-F) agreed

with reported studies in the Arctic Ocean (Dainard and Guéguen, 2013; Guéguen et al.,

2014; Stedmon et al., 2007; Stedmon and Markager, 2005; Walker et al., 2009).

3.3.4 DOM and water masses

Thiol concentration (Fig 3.3A) increased from SW (16.87 ± 5.29 nM) to the

subsurface chlorophyll-a maximum depth (27.18 ± 5.59 nM). Maximum thiol

concentrations were coincident with chlorophyll-a maximum, but not universal along

the transect. The similar biological origin of thiol compounds was also reported in the

English Channel (al-Farawati and Van Den Berg, 2001) and North Atlantic Ocean

(Swarr et al., 2016). High HS concentrations (Fig 3.3B) were associated with the top

300 m through the study area and the highest HS concentrations were found in Viscount

Melville Sound (~150m) and in BS bottom waters. For deep layers, low level thiol

compounds and HS was found from 500 m to 1,000 m in AIW, except a DOM-rich

61

source was observed over 1,000 m, likely due to shelf break sediment interactions

(Hioki et al., 2014; Nebbioso and Piccolo, 2013; Shi et al., 2016). In NADW,

homogeneous distribution of thiol compounds and HS was observed, but the average

thiol concentration from 300 m to 1,000 m was higher than that of AIW (22.59 ± 3.63

nM vs. 15.90 ± 3.74 nM, p < 0.05) while HS (< 50 µg C/L) remained similar.

In terms of FDOM intensities (Fig 3.3C-F), humic-like C1 and C2 showed similar

distribution throughout the study area, suggesting similar processes affecting their

distribution as previously reported in CAA (Guéguen et al., 2014). High C1 and C2

intensities were found to be associated with the subsurface Pacific haloclines in CB,

occupying the top 300 m excluding ASW, the similar distribution was reported

previously in the Arctic Ocean (Dainard et al., 2015; Guéguen et al., 2012, 2007; Hioki

et al., 2014; Nakayama et al., 2011). A significant lateral reduction in C1-C2 intensities

in top 300 m near Lsd is caused by mixing with less humified Atlantic-origin waters

(Guéguen et al., 2014). Low C1 and C2 intensities (0.019 and 0.012 r.u. respectively)

in SW may be caused by enhanced photobleaching (Bélanger et al., 2006; Dainard et

al., 2015) and FDOM-poor sea ice contribution (Logvinova et al., 2016). Almost

identical vertical distribution (C1 and C2) in top 300 m from CB to BS suggested that

photo degradation is limited due to extensive sea ice cover in western CAA (Fransson

et al., 2009). However, the UV humic-like C3 (peak A; Coble, 1996) displayed different

distribution in western CAA. For protein like C4, higher intensities in SW and OW

were found in western CAA than in eastern CAA (0.023 vs 0.017 r.u., p < 0.05). Since

similar protein-like component had been reported to be linked to biological production

62

(Coble et al., 1998), the eastward decrease confirmed Pacific waters in western CAA

were more nutrients-rich (Michel et al., 2006) than eastern CAA waters.

In DW, a decreasing trend was observed for C1-C4 from 300 m to 1,000 m in

BAW, this contrasted with the distribution of FDOM in NADW, where identical

distribution was observed at relatively low level.

3.3.5 Principal Components Analysis

Principal components analysis (PCA) was carried out using samples from all

depths (Fig 3.5A) and using samples collected in the top 100 m (Fig 3.5C).

Five variables (HS, FDOM C1- C4) were selected in PCA on all depths. Principal

components 1 (PC1) and 2 (PC2) explained 39% and 25% of total variance (Fig 3.5A).

Humic-like C2 (A/M peak) and protein-like C4 were closely related (negative PC2

values), suggesting similar processes affecting both PARAFAC components. Both

components C2 and C4 were previously reported to be linked to microbial or bioactivity

production (Coble et al., 1998; Shimotori et al., 2012). On the other hand, voltammetry-

measured HS and humic-like components (C1 and C3) were in close proximity

(positive PC2 values), confirming the humic character of C1 and C3. The PCA revealed

differences in DOM characteristics between three main water masses (i.e. SW, OW and

DW) (Fig 3.5B). The DW and SW samples were clustered together in the left quadrants

whereas the OW samples were further on the right side of the score plot. SW took over

the top left quadrant while the DW samples dominated the bottom left quadrant, except

those affected by shelf sediments leaching in deep CB sites (> 1,000 m).

63

When considering all variables (i.e. GSH, HS and C1-C4) in the top 100 m (Fig

3.5C), the influence from intense biological activity (i.e. chlorophyll maximum), sea

ice contribution and river discharge could be revealed. The first and second principal

components accounted for 36% and 20% of the total variance explained, respectively.

Humic-like fluorescent components (C1 – C3) displayed negative PC2 loadings

whereas protein-like C4 and GSH concentration showed positive PC2 loadings. The

closer proximity of protein-like C4 and voltammetry-measured GSH concentration in

the top 100 m suggested similar biological origin. Interestingly, a trend moving from

CB to Lsd was observed in the score plots (Fig 3.5D), supporting the observation that

Pacific waters in CB were more nutrients-rich and productively (Michel et al., 2006)

than Atlantic waters in Lsd.

3.4 Conclusion

The distribution of FDOM, thiols and HS showed significant spatial changes

between eastern Canada Basin to Lancaster Sound and great stratification throughout

the entire depth. The ranges of GSH (8 to 78 nM) and HS (31 to 222 µg C/L) determined

by CSV were consistent with previous studies, suggesting that CSV-based DOM

characterization is suitable for marine studies. Four fluorescent components including

three humic-like (C1- C3) and one protein-like components (C4) were identified and

previously reported. Maximum thiol concentration was associated with the subsurface

chlorophyll-a maximum, but not universal along CAA. HS and humic-like fluorescent

components showed similar distribution, whereas thiols groups (i.e. GSH) was

observed to be closely related to protein-like C4. For the first time, thiols and HS were

measured in the Arctic Ocean by CSV and the method was proved to be capable in

64

marine studies, enabling the future monitoring of these important metal-binding ligands,

which will help better understand the distribution of trace metals in the Arctic Ocean.

Acknowledgments

This work was supported by the Canada Research Chair Program (CG) and National

Sciences and Engineering Research Council of Canada (CG). The field program on the

CCGS Amundsen was funded by Geotraces and ArcticNet. We thank Roger François,

Philippe Tortell, Jay Cullen, Richard Nixon and, the captain and crew of the CCGS

Amundsen.

65

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Figures and tables

Table 3.1 Repeatability, reproducibility, limit of detections and recovery of the CSV method

GSH HS

Repeatability Average 2.50 nA 32.11 nA

n = 10 Stdev 0.016 nA 0.192 nA

RSD % 0.64 0.60

Reproducibility Average 40.75 nM 672.05 µg C/L

n = 10 Stdev 0.375 nM 7.062 µg C/L

RSD % 0.92 1.05

Recovery % Average 100.81 101.29

n = 3 Stdev 0.74 1.78

Limit of detection

deposition time 120 s

1.12 nM 21.19 µg C/L

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Figure 3.1 Sampling locations in Canada Basin and Canadian Arctic Archipelago, Study transect highlighted in red area

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Figure 3.2 (A) Potential temperature as a function of salinity (Sp) at all depths; (B) Salinity, (C) Potential temperature and

(D) Fluo sensor (Chlorophyll-a) distribution along the transect (Figure 3.1)

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Figure 3.3 Distribution of thiol groups (A), humic substances (B), FDOM (C-F) along the transect (Figure 3.1)

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Figure 3.4 Excitation emission matrices identified by PARAFAC (A-C: humic-like; D: protein-like)

76

Figure 3.5 PCA models and score plots on all depths (A-B) and samples in top 100m (C-D); SW: surface waters, OW:

Arctic outflow waters, DW: deep waters; Lsd: Lancaster Sound, CAA: Canadian Arctic Archipelago, CB: Canada Basin

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Chapter 4. Conclusion

Dissolved organic matter (DOM) was monitored over two consecutive years (i.e.

2014 and 2015) in the Arctic Ocean. Samples were collected aboard the CCGS Louis

S. Saint-Laurent (Joint Ocean Ice Studies JOIS cruise, September 2014) and the CCGS

Amundsen (Canadian Arctic GEOTRACES cruises, July-September 2015). DOM

Molecular weight (MW) was assessed using asymmetrical flow field-flow fractionation

(AF4), DOM characterization was carried out using Parallel factor analysis

(PARAFAC) – Excitation-emission matrices (EEMs) and concentrations of thiols and

humic substances was monitored using cathodic stripping voltammetry (CSV).

Two research questions were solved in this thesis:

Is the distribution of DOM characteristics associated with water masses of

different origin?

Is there a connection between voltammetry-based HS and humic-like

fluorescent component?

4.1 Molecular weight of DOM

For the first time, AF4 system was used in detecting DOM MW (JOIS cruise) in

Arctic waters. The DOM MW ranged from 1.07 to 1.45 kDa, consistent with earlier

studies using field-flow fractionation, high performance size exclusion

chromatography and tangential flow filtration (Beckett et al., 1992; Everett et al., 1999;

Guéguen and Cuss, 2011; Huguet et al., 2010; Landry and Tremblay, 2012; Penru et

al., 2013; Stolpe et al., 2010). The vertical distribution of DOM MW showed water-

78

mass related distribution pattern. DOM in Arctic surface waters displayed similarity in

MW. A minimum in MW was associated with the Pacific summer waters, while higher

MW was associated with Pacific winter waters. Arctic intermediate waters did not show

any significant change in MW in the area.

It is noted that there were some limitations in applying AF4 system to determine

DOM MW in seawater samples. For example, the 300 Da membrane used in the system

prevented molecules less than 300 Da to be focused and thus analysed. As fulvic acids

are good proxy for marine DOM (Tipping et al., 2015), the small molecules (< 300 Da)

are not signficant compared to the natural range of marine DOM. The molecules used

in the calibration solution did not exactly resemble natural DOM, thus the application

of more representative standards is needed for future work. The presence of salts in

samples and AF4 eluent was one of the issues affecting AF4 system. Although

extensive cleaning between samples allowed us to minimize salt built-up in the AF4

system, running seawater samples still resulted in a short life of membrane. A future

focus on pre-treatment (e.g. solid phase extraction) for seawater desalt process may

provide novel information. Furthermore, the preservation time (i.e. 10 days) was too

short to allow a large scale monitoring using in-lab measurements. An onboard AF4

using miniature AF4 channel should be developed in future oceanographic studies.

4.2 Composition of DOM

4.2.1 Fluorescent dissolved organic matter

The PARAFAC-validated fluorescent components in this study were found in

previous Arctic studies (Dainard and Guéguen, 2013; Guéguen et al., 2014; Stedmon

79

and Markager, 2005; Walker et al., 2009). Table 4.1 summarized the fluorescent

components identified in both cruises. C1-JOIS and C2-Geotraces both contained

marine humic-like component M peak (Coble, 1996). Protein-like (C2-JOIS and C4-

Geotraces) and terrestrial humic-like (C3-JOIS and C1-Geotraces) were identified in

both PARAFAC models. Humic-like C3 was only found in Geotraces dataset.

With the influence of sea ice contribution and river inputs, Pacific waters occupied

the top 400 m while saline and warm Atlantic waters stayed underneath. The great

difference in salinity and temperature impeded the vertical exchange between different

water masses, resulting in the Pacific haloclines in top 400 m. As Arctic waters flowed

through CAA, the haloclines became weak while the Pacific waters merged with

Atlantic waters. Humic-like component (A and C peak) was widely used to track the

movement of water mass due to its terrestrial origin (Walker et al., 2009), since similar

component had been observed in both two cruises, a comparison of vertical distribution

pattern of this terrestrial component in different sites was shown in Figure 4.1. In order

to discriminate the difference between sites, the signal had been normalized to the

surface ones. A strong halocline was observed at CB site and it weakened as water

flowing eastwards to Lancaster Sound. For protein-like component, similar vertical

distribution patterns on all samples collected in two years were observed with

fluorescence probe signal, which was associated with the chlorophyll-a concentration

(Fig. 4.1B). This observation supported the previous findings on the biological origin

of protein-like components (Coble et al., 1998).

80

4.2.2 Thiols and humic substances distribution

Thiols and humic substances (HS) concentrations were monitored the first time in

the Arctic Ocean, the ranges of thiols (8 - 78 nM GSH-equivalent) and HS (31 - 222

µg C/L) determined by CSV were consistent with previous marine studies (Le Gall and

van den Berg, 1993; Marie et al., 2015; Pernet-Coudrier et al., 2013; Whitby and Van

den Berg, 2014). Interestingly, maximum thiol concentration was associated with the

subsurface chlorophyll-a maximum for most sites, but not universal along Canadian

Arctic Archipelago. Highest HS concentration was associated with Arctic outflow

waters in top 300 m. Comparable distributions of CSV-based HS and humic-like

fluorescent components suggest the close proximity between each other.

Filella, (2014) showed that the concentrations of CSV-based thiols are greatly

affected by the choice of standards, a consistency in standards would be very helpful.

Alternatively, calibrating each CSV standard (GSH, cysteine, thioacetamide, thiourea)

against each other would be recommended for future study. Besides, novel information

would be revealed if CSV-based metal ligands concentrations were analyzed together

with the trace metal distribution in the area.

4.3 Conclusions and future directions

Overall, water mass related distribution of DOM fingerprints (i.e. DOM MW,

FDOM and CSV-based DOM) was observed in the study. The range of DOM MW

determined by AF4 system was similar to those done by high performance size

exclusion chromatography and tangential flow filtration. CSV-based thiols and HS

concentration was consistent with other marine studies and provided comparable trends

81

to previous work by HPLC. The results (i.e. DOM MW, thiol and HS distribution)

supported AF4 and CSV-based DOM analysis are suitable for marine studies.

For the future project, in-situ measurement and field work focus are required for a

larger scale monitoring. Miniature AF4 channel and onboard CSV measurement could

make it possible for future in-situ monitoring. A large scale future ocean monitoring on

DOM MW and CSV-based DOM would provide valuable insights on the fate and

transport of DOM, thus help us better understand global carbon cycle.

82

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Figures and tables

Table 4.1 Fluorescent components identified in JOIS cruise and GEOTRACES cruise.

JOIS cruise (ex/em) GEOTRACES cruise (ex/em) Traditionally (Coble, 1996) (ex/em)

Component 1 C1 (305/410) M peak

Component 2 C2(<280/330) C4 (275/325) T peak

Component 3 C3(<270 and 365/465) C1 (250 and 370/460) A and C peak

Component 4 C2 (<250 and 310/400) A and M peak

Component 5 C3 (<250/400) A peak

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Figure 4.1 (A) Vertical distribution of terrestrial humic-like component in Canada Basin, Canadian Arctic Archipelago and

Lancaster Sound; (B) Vertical distribution of protein-like components and Chl-a signal in all samples from both cruise