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Thesis for the Master’s degree in chemistry Pingchuan Gao
Effect of solar radiation on Dissolved Natural Organic Matter 60 study points DEPARTMENT OF CHEMISTRY Faculty of mathematics and natural sciences UNIVERSITY OF OSLO 05/2016
II
Effect of solar radiation on Dissolved Natural Organic Matter
by
Pingchuan Gao
Department of Chemistry
Faculty of Mathematics and Natural Sciences
University of Oslo
May 2016
III
Abstract Lakes and rivers are the main sources of drinking water in Nordic countries. For aesthetic and
hygienic reasons high concentration levels of dissolved natural organic matter (DNOM) are
not acceptable in drinking water. An increasing amount of DNOM in raw water sources for
drinking water in Fennoscandia is thus raising our concerns. Moreover, the DNOM constitute
an important transport mechanism for the flux of allochthonous nutrients to surface water.
Once in the lake the DNOM photo-degrades due to adsorption of energy from sunlight. This
causes its larger molecular weight (MW) compound to decompose into a variety of smaller
organic photoproducts. Moreover, some of the DNOM is mineralized completely to inorganic
compounds. Mineralization of DNOM causes a release of aluminum (Al) and iron (Fe) ions
along with the release of orthophosphate (PO43-). The metal ions rapidly hydrolyze and form
oxy-hydroxides which co-sorb phosphate and precipitate. Due to the removal of mineralized
PO43- by subsequent precipitation with Fe- and Al-oxy hydroxides it is difficult to detect how
much orthophosphate becomes mineralized by solar radiation. This is needed in order to
assess the amount of this limiting nutrient becoming available for autotrophs. Thus the
essence of this study is to test the option of using algae growth response as an indication of
phosphorus release.
Considerable research has been conducted comparing the changes in characteristics of the
matrix that DNOM is in and structural characterization of DNOM, before and after solar
exposure. A continuous increase in the relative contribution of lower to higher MW DNOM
compounds along with a decrease in dissolved organic carbon (DOC) concentration has been
found with the extension of exposure time. This implies that the photochemical mineralization
of DNOM by the photo-oxidation transforms the higher MW DNOM compounds into lower
MW compounds, and that some of the DNOM is completely mineralized. This is further
corroborated with fluorescence spectroscopy, indicating a decreased ratio of humic to fulvic
acids in the DNOM as a response to sunlight exposure. This would imply that when sunlight
is absorbed by DNOM, the average molecular weight is reduced. Results from assays
showing increased algal growth imply that PO43- has been mineralized from DNOM due to
photochemical reaction. On the other hand the growth response is not always significant. This
may partly be due to concurrent heterotopic mineralization by bacteria. Further work is thus
required in order to exclude the effects of bacteria.
IV
Acknowledgement This study is a part of the project “Drinking water treatment adaptation to increasing levels of
DOM and changing DOM quality under climate change”. This master thesis has been carried
out at the Department of Chemistry, University of Oslo (UIO) in period from January 2015 to
May 2016. Part of the study was carried out at the Department of Biology, Department of
Geosciences (UIO), and Norwegian University of Life Sciences (NMBU).
This thesis would not have been possible without the help of so many people in so many
ways. First and foremost I owe my deepest gratitude to my supervisor Rolf D. Vogt for your
guidance, encouragement and patience over the last two years. I have been amazingly
fortunate to have a supervisor who gave me freedom to explore on my own and at the same
time the instruction to recover when my steps faltered. I would like to thank my co-supervisor
Dag O. Hessen, for introducing me to the biology world, and for expressing values in my
work. You are always there whenever I need your help. Thanks also to co-supervisor Ståle L.
Haaland for picking me up every time I went to Ås, instruction on fluorescence EEMs and for
always sharing your delicious food with me.
I would like to thank Bin Zhou for your guidance, in both scientific and daily life. It is really a
great pleasure sharing an office with you. Thank you to Christian W. Mohr for all your help
throughout this thesis. You were always available for my questions and you were positive and
gave generously of your time and vast knowledge. Thank you to Cathrine B. Gunderson for
all the valuable discussions and assistance in both lab work and writing. Special thanks to
Thrane Jan-Erik for your guidance in algal growth assays and data interpretation. Thank you
to Nita K. Shala sharing the time with me for the preparation of my experiments and
introducing me to Q-SUN. Thank you to Pawel Krzeminski for helping me with the work in
sampling and sharing your data.
A debt of gratitude is also owed to everyone who has been a member of our Environmental
Chemistry group during the last two years: Alexander, Elena, Lena, Yemane, Shafia, Wen
Tan, Liang Zhu and Emelie for your help throughout the course of this thesis.
Thank you to my best friends all over the world for your encouragement and understanding.
Finally, my biggest thanks go beyond all doubt to my parents and families for your countless
support and love!
V
Table of Contents Abstract .................................................................................................................................... III
Acknowledgement .................................................................................................................... IV
Table of Contents ...................................................................................................................... V
1 Introduction ............................................................................................................................. 1
2 Theory ..................................................................................................................................... 3
2.1 Dissolved Natural Organic Matter (DNOM) ................................................................... 3
2.2 Photo degradation ............................................................................................................. 4
2.1.1 Bioavailable photoproducts ....................................................................................... 6
2.1.2 Effect of photo-oxidation of DNOM on algae activity ............................................. 7
2.3 Algae growth assays ......................................................................................................... 8
2.4 Ultraviolet-visible (UV-Vis) absorption spectroscopy .................................................... 9
2.5 UV-Vis Fluorescence ..................................................................................................... 10
3 Material and methods ............................................................................................................ 13
3.1 Raw water samples from DOMQUA project ................................................................. 13
3.2 Reference samples of Reverse Osmosis (RO) isolated DNOM ..................................... 15
3.2.1 Field and Project description ................................................................................... 15
3.3 Sample pre-treatment and flowchart of thesis experiment ............................................. 16
3.3.1 Filtration .................................................................................................................. 16
3.4 Photo-oxidation .............................................................................................................. 18
3.5 Sample matrix characterization ...................................................................................... 19
3.5.1 pH and conductivity ................................................................................................ 19
3.5.2 Alkalinity ................................................................................................................. 19
3.6 Elemental composition and Speciation .......................................................................... 19
3.6.1 Major anions ............................................................................................................ 19
3.6.2 Major cations ........................................................................................................... 20
3.6.3 Dissolved Organic Carbon (DOC) .......................................................................... 20
3.7 DNOM characterization ................................................................................................. 21
3.7.1 UV-Vis Absorbency ................................................................................................ 21
3.7.2 UV-\Vis Fluorescence Spectroscopy ...................................................................... 21
3.8 Algal growth assays ....................................................................................................... 21
VI
4 Results and Discussion .......................................................................................................... 25
4.1 Sample matrix characterization ...................................................................................... 25
4.1.1 pH and conductivity ................................................................................................ 25
4.2 Elemental composition and speciation ........................................................................... 26
4.2.1 DOC ........................................................................................................................ 26
4.2.2 Charge distribution of major anions and cations ..................................................... 27
4.3 Structural Characterization of Dissolved Natural Organic Matter (DNOM) ................. 30
4.3.1 UV-/Vis Absorbency ............................................................................................... 30
4.3.2 Fluorescence excitation-emission matrix (EEM) spectra ........................................ 31
4.3 Changes in characteristics of DNOM due to photo-oxidation ....................................... 35
4.3.1 UV-Vis absorbency spectra ..................................................................................... 35
4.3.2 DOC ........................................................................................................................ 41
4.3.3 Fluorescence excitation and emission matrix spectra (EEMs) ................................ 43
4.4 Algal growth assays ....................................................................................................... 47
4.4.1 General growth response ......................................................................................... 47
4.4.2 Growth rate analysis ................................................................................................ 49
5 Conclusions ........................................................................................................................... 53
6 References ............................................................................................................................. 55
7 Appendix ............................................................................................................................... 67
List of Appendixes ............................................................................................................... 67
7.1.1 Appendix A. Instrumentation and Calibration ........................................................ 67
7.1.2 Appendix B. Results obtained prior and post to solar radiation .............................. 70
1
1 Introduction Dissolved natural organic matter (DNOM) is decomposition products of mainly plants and
microorganisms, which have been oxidized and re-combined into a broad continuum of
organic molecules. Their molecular sizes vary several orders of magnitude from a few
hundred Dalton. DNOM is operationally defined as the fraction of organic matter in solution
not retained by a 0.45µm membrane filter. The DNOM has a profound influence on the
biophysicochemistry of surface waters. It is comprised of aliphatic and aromatic moieties with
a large number of different weak organic acid functional groups, such as carboxylic- and
phenolic acid (Perdue 2009). Its aromatic moieties adsorb radiation and its large number of
weak acid functional groups lower the pH, it complexes and adsorbs pollutants - mediating
their transport to surface waters, thereby increasing their bioavailability, and its content of C,
N, P, S represent commonly the main major resources of nutrients for aquatic organisms.
Over the past few decades the concentrations of DNOM has increased, causing a
brownification of surface waters in the Nordic countries (Hongve et al. 2004; Monteith et al.
2007). This increase of DNOM causes great challenge for waterworks, needing to remove the
organic material from the raw water (Eikebrokk et al. 2004) since waterworks in Nordic
countries rely on lakes and rivers as the main raw water sources for drinking water. Moreover,
it is causing large changes in the habitat conditions in surface waters. Increased flux of
allochthonous nutrients, associated with the DNOM, is expected to affect aquatic flora and
fauna. The radiation adsorbing properties of DNOM decreases the photosynthetically active
radiation (PAR) depth and dampens light for predators. The changes in light penetration
thereby effects primary production as well as the ability of predators to see the pray and
thereby species composition (Watkins et al. 2001). Moreover, the adoption of energy causes
changes in temperature profiles and stratification. Water color has increased more than DOC
concentrations. This indicates a change in DNOM character (Haaland et al. 2010).
This brownification is hypothesized to be due to a combined effect of climate change and
decrease in acid rain loading (Monteith et al. 2007). Reduction in acid deposition (Monteith et
al. 2007) leads to increased solubility of organic matter at higher pH, lower ionic strength (De
Wit et al. 2007) and labile aluminium concentrations (Vogt et al., 2004). Increased
temperature, primarily in winter, and precipitation drive temporal variations in DNOM on a
seasonal and inter-annual time scale by affecting net primary production, soil organic matter
2
mineralization, and catchment flow pathways (Ledesma et al. 2012). Increased amount and
intensity of precipitation in forested regions leads to increased concentrations of DNOM in
runoff due to increased flow through the forest floor directly into the streams (Vogt et al.
1990). On a longer timescale, climate change can allow forest expansion to areas presently
covered with heathland or alpine vegetation, and thereby affecting retention and export of
nutrients, as well as DNOM pools and quality (Vogt 2003). For Norwegian watersheds, even
a moderate increase in temperature with associated increase in vegetation density, could thus
increase DNOM export substantially (Larsen et al. 2011a; Larsen et al. 2011b).
The majority of aqueous DNOM originates from two principal sources: allochthonous and
autochthonous, i.e. terrestrial vascular plants and indigenous phytoplankton, respectively
(Keil & Kirchman 1994). The fluxes of the allochthonous DNOM have increased
significantly over the past 30 years in regions previously suffering heavy loading of acid rain
(Skjelkvåle et al. 2000). Due to this, it is likely that the allochthonous loading of Dissolved
Organic Phosphorus (DOP) to the lakes has also increased, as this is an integral part of the
DNOM. More knowledge is thus needed to reveal the fate and impact of this DOP material
within in the lakes.
The load of pollutants and nutrients of DNOM are partly directly bioavailable, while a
significant fraction may become available through photo-oxidation and mineralization.
Surface waters serve as large photo-reaction tanks, photo-bleaching the DNOM into less
aromatic and smaller molecular weight (MW) compounds: In this process some of the metal
ions (Al3+ and Fe3+) and nutrients (PO43-, NO3
-, and NH4+) are mineralized. Moieties of the
low MW organic compounds as well as the orthophosphate and inorganic reactive nitrogen
are important food and nutrient sources for aquatic primary production. The released metal
ions rapidly hydrolyze and form oxy-hydroxides which co-sorb phosphate and precipitate.
This rapid removal of phosphorus from solution by precipitation with Al and Fe makes it
impossible to apply traditional chemical detection method to measure the PO4 (i.e. MBM
method) that is mineralized by solar radiation. Adopting and alternative biological approach,
the essence of this study is to test the option of using algae growth response as an indication
of phosphorus release.
3
2 Theory
2.1 Dissolved Natural Organic Matter (DNOM)
Natural Organic Matter (NOM) is a collective term used for distinguishable Low Molecular
Weight (LMW) organic compounds and Humic Matter (HM) in solution. NOM is formed by
environmental biological decay and inorganic oxidation of organic matter of plant or
microbial origin. The HM does not consist of discrete, well-defined molecules but is a
combination of substances that are produced and reside in soil and water, forming a major
component in aquatic carbon pools. In the hydrosphere, HM typically makes up
approximately 50-75% of DNOM in surface waters (VanLoon & Duffy 2005). HM is a
complex continuous mixture of heterogeneous colored organic macromolecules that occur
ubiquitously in surface waters. Dissolved Natural Organic Matter (DNOM) is defined as the
organic matter fraction in solution that passes through a 0.45um filter. DNOM has a
significant role on freshwater chemistry because of its various weak organic acid functional
groups, strong metal complex capacity and lipophilic sorption abilities (Cronan & Aiken
1985; Liechty et al. 1995; Vance & David 1991). Moreover, DNOM has a major impact on
light attenuation in water reducing primary production, decreasing the predators’ ability to
catch pray, and increases the attenuation of biologically harmful UV-radiation, thereby
changing specie composition. On the other hand, UV photo-oxidation of DNOM may induce
formation of a suite of free radicals and other strong oxidants that both have chemical and
biological impacts. DNOM therefore has effect on chemical and biological processes in the
aquatic environment. Moreover, transport of allochthonous DNOM from the terrestrial to
aquatic compartment has large impact on fluxes of macronutrients as nitrogen and phosphorus
and thereby provides energy and micronutrients for freshwater microorganisms. Given the
current increase in DNOM in many northern waters, a better understanding of its physical,
chemical and biological impacts is thus strongly sought for.
The collective characteristic of HM is largely dependent on its source and environment of
origin, and method of extraction, with more similarities being pronounced than differences
among samples from various sources (Gaffney et al. 1996). HM has a wide range of
molecular weights and sizes, ranging from a few hundred to as much as several thousand
atomic mass units. In general, fulvic acids are of lower molecular weight (1 000-30 000Da)
4
than humic acids (10 000-100 000Da). The range of the elemental mass composition of humic
material is relatively narrow. Approximately 40-60% is carbon, 30-50% oxygen, 4-5%
hydrogen, 1-4% nitrogen, 1-2% sulfur, and 0-0.3% phosphorus (Suffet & MacCarthy 1989).
Generally there is more hydrogen, carbon, nitrogen, and sulfur and less oxygen in humic acids
than fulvic acids (Gaffney et al. 1996). Fulvic acids are therefore characterized by higher O: C
ratio (Stevenson 1985). Although structural similar, humic acids are dominated by conjugated
aromatic compounds while fulvic acids are somewhat more aliphatic and richer in weak acid
functional groups (Gaffney et al. 1996).
The non-HM constituents of DNOM are ascribed to varying amounts of the identifiable LMW
compound classes (Bolan et al. 2011), and this non-humic matter is stabilized to the HM by
hydrogen bonding, covalent bonding, and electrostatic interactions.
2.2 Photo degradation
A range of different reactions do occur when DNOM in surface water is exposed to photons
of different wavelengths and energy. Whipple (1914), interested in the aesthetics of drinking
water, was among the first to demonstrate that the color of DNOM was bleached by sunlight.
There have since then been numerous researches conducted regarding the effect of DNOM
photochemistry in aquatic environments (Moran & Zepp 1997; Richard & Canonica 2005)
DNOM undergoes photochemical oxidation and hydrolysis reactions, leading to its cleavage
to a variety of photoproducts. This causes the loss of visible color (referred to as photo-
bleaching). Though the photo-oxidation occurs mainly due to the absorbance of radiation in
the UV region (Minor et al. 2007; Gao & Zepp 1998; Brinkmann et al. 2003; Vecchio &
Blough 2004) the photochemical processes are mainly hydrolysis reactions breaking of
chemical bonds in the DNOM. This causes an overall reduction in its average molecular mass
(Kieber & Mopper 1987) and partly a total mineralization of DNOM to inorganic compounds,
i.e. carbon monoxide, carbon dioxide, and other forms of dissolved inorganic carbon (DIC)
(Amon & Benner 1996).
By strongly adsorbing sunlight the DNOM act as a photosensitizer and as a reaction substrate,
leading to photoionization (Fischer et al. 1985) and formation of numerous transient
intermediates as well as terminal photoproducts (Blough & Zepp 1995). Among the
intermediates, there are a large number of highly reactive oxygen species such as singlet
5
molecular oxygen and free radicals such as hydroxyl radical ( OH) (Zafiriou 1974; Blough
1998), superoxide anion radical (O2 )(Petasne & Zika 1987; Zafiriou 1990), and peroxy
radicals (ROO )(Faust & Hoigne 1987; Blough 1988). These radical have therefore been
detected by electron paramagnetic resonance (EPR) in solutions of humic and fulvic acids and
in natural waters (Zepp et al. 1985). This formation of reactive oxygen species and free
radicals lead subsequently to chemical transformations of compounds. Radical reactions can
influence chemical and microbial activity in natural waters by causing degradation of
molecules into smaller photoproducts and to the combination of radicals and substrates into
bigger molecular compounds (Sandvik et al. 2000).
The photochemical process does not only change the chemical characteristics of the bulk
DNOM, it also increases the biological reactivity of the DNOM. The formation of DNOM
with smaller molecular mass renders the organic compounds more bioavailable than the un-
irradiated material (Wetzel et al. 1995). Moran et al. (1997) found therefore that DNOM after
photo-degradation was significantly more bioavailable than the parent DNOM.
Aquatic organisms, such as phyto- and bacteria-plankton, play an important role in primary
production and elemental turnover, and constitute the basis of aquatic food webs. Photo-
mineralization of DNOM leads to enhanced activity of the phytoplankton due to increased
availability of CO2 and nutrients (N and P) for autotrophic fixation. Similarly, increased
concentrations of LMW DNOM, and thereby moieties of bioavailable DNOM, promotes
growth of heterotrophic bacteria assimilating the available organic matter (Wetzel et al.
1995). On the other hand, these organisms - and thereby the carbon cycling and transfer of
energy to higher trophic levels – may be harmed by the increased UV radiation (Beardall &
Raven 2004; Leu et al. 2007). Changes in both the quantity and degree of photo-bleaching of
DNOM may thus lead to changes in UV penetration into water bodies with impacts both on
primary production, UV-penetration and formation of oxidants and free radicals. This may
therefore also affect fundamental biological processes and biogeochemical cycles in the
aquatic system.
6
2.1.1 Bioavailable photoproducts
Solar radiation-induced decreases in both absorptivity and fluorescence efficiency of DNOM
are widely reported (Lepane et al. 2003). This may be due to complete mineralization of the
DNOM, but losses of DOC generally do not keep pace with decrease in absorbency and
fluorescence efficiency. This indicates that photo-degradation causes the formation of organic
compounds that, while still a portion of DNOM pool, no longer absorb light to the same
extent as does the parent material (Allard et al. 1994).
Changes in structural characteristics of DNOM due to photo-oxidation are consistent with
degradation of larger, photo-reactive molecules into smaller organic compounds. This
reduction in average molecular size due to light exposure has been confirmed by gel filtration
studies of the molecular weight of bulk DNOM (Allard et al. 1994). It is within the moiety of
photo-chemically modified LMW DNOM that we find the bioavailable photoproducts.
Most of the bioavailable photoproducts identified to date are low-MW organic compounds.
All of the identified low-MW organic substances are carbonyl compounds, with fatty acids
and keto acids being well represented. Normally they contain three or fewer carbon atoms
with a molecular weight less than 100, although their size ranges up to a six-carbon molecule
(Kieber & Mopper 1987; Kieber et al. 1989; Keiber et al. 1990; Wetzel et al. 1995). Except
for low-MW organic compounds, at least three other kinds of photoproducts may also play an
important role in biological activity: I.e.: 1) Inorganic carbon-based gases including CO
(Mopper et al. 1991), CO2 and other forms of DIC (Granéli et al. 1996); 2) Unidentified
photodegraded organic matter; and 3)Nitrogen and phosphorus rich compounds. The
conversion rate of DNOM to identifiable photoproducts was found by Miller and Zepp (1995)
to be less than 20%. This indicates a large unidentified pool of photodegraded organic carbon
that may serve as a source of microbial substratum. In addition, inorganic nitrogen and
phosphorus-rich compounds were also identified to compose a group of bioavailable
photoproducts. Ammonium has been demonstrated to be a photoproduct of the organic
nitrogen component of DNOM from numerous freshwater environments (Bushaw et al.
1996). Phosphate is also believed to be a photoproduct of DNOM, resulting from oxidation of
organic P.
UV radiation has been found to lead to the reduction of iron complexes that bond humic
substances and phosphate together, causing the release of phosphate from humic substances
7
(Francko & Heath, 1982; Francko & Heath, 1979). This photo-chemically driven phosphate
release was observed in situ in the surface waters of a bog lake by Francko & Heath (1982).
They detected a diurnal fluctuation of free phosphate, indicating a reversible reaction; the
hypothesized that in the dark the iron is re-oxidized and phosphate re-adsorbed to the humic
substances. On the other hand mineralized Fe2+ may in addition to be important for algal
growth, would also be oxidized to Fe3+ and thereby bind to, and co-precipitate phosphate,
hence increasing the nutrient scarcity for algae (and potentially also bacteria).
In all, four categories of biologically labile photoproducts are identified, but algae and
bacteria vary in their demand and response to these:
For algae:
carbon gases (carbon monoxide, carbon dioxide, and other forms of inorganic carbon);
inorganic nitrogen and phosphorus-rich compounds (NH4+ and PO4
3-).
For bacteria:
low molecular weight organic compounds (carbonyl compounds with MW of <100);
unidentified photo degraded organic matter.
2.1.2 Effect of photo-oxidation of DNOM on algae activity
Many elements are important for algal growth, such as nitrogen, phosphorus, carbon,
potassium, sulfur, calcium, magnesium and many other trace nutrients. However, among
them, nitrogen and phosphorus are commonly acting as the key limiting factors for autotroph
production in marine and freshwater systems, respectively. This is because of their relatively
low availability in contrast to the high biological demand (Kalff 2002). Soluble reactive
phosphorus (SRP) is an operationally defined parameter and comprises both the inorganic
species and LMW organic form of P believed to be available to pelagic algae and
macrophytes. Orthophosphate species (including H3PO4, H2PO4-, HPO4
2-, and PO43-)
constitute the major component of SRP. The concentration of this fraction is however
maintained at very low concentrations and is thus not easy to detect (Ryding & Rast 1989).
This is partly due to that bioavailable P is rapidly assimilated by autotroph and partly due to
low solubility in the environment. Only within a small pH region (4.5-7) the amount of
8
phosphorus in the form of orthophosphate may be found in significant amounts in most
freshwaters. This is not because pH plays an important role in the solubility of orthophosphate
but rather the case that pH affects or reflects the solubility of other compounds that adsorb or
precipitate out orthophosphate. When pH is lower than 4.5 the solubility of iron and
aluminum ions are significant. These ions form insoluble salts and precipitate with
orthophosphate (FePO4, Ksp = 4×10-27, AlPO4, Ksp = 6.3×10-19). While where pH is higher
than 7 the water is typically buffered by carbonate solubility rendering high concentrations of
calcium. Similar to Al3+ and Fe3+ the Ca2+ causes orthophosphate to precipitate in the form of
Ca3 (PO4)2 (Ksp=1.2x10-29) or Ca5 (PO4)3OH (Ksp=6.8x10-37) (Lide 2004).
Practically most of the phosphorus found in freshwater systems is thus not readily
bioavailable as it is bound up in the biomass, to refractory DNOM or adsorbed/ bond
inorganically to silt and clay particles. Mineralization of DNOM causes a release of aluminum
and iron ions along with the release of orthophosphate. The metal ions rapidly hydrolyze and
form oxy-hydroxides which co-sorb phosphate and precipitate. Due to the removal of
mineralized PO43- by subsequent precipitation with Fe- and Al-oxy hydroxides it is difficult to
detect how much orthophosphate becomes mineralized by solar photo-oxidation.
The release of SRP (soluble reactive phosphorus) by photo-oxidation of DOP (Dissolved
Organic Phosphorus) is believed to be important process in the biogeochemical cycling of P
in freshwater lakes.
2.3 Algae growth assays
Experiments with algal cultures have been instrumental in providing basic knowledge on the
functioning and role of photosynthesis (Govindjee & Krogmann 2004). Moreover, ecological
aspects on aquatic microorganisms like carbon assimilation, nutrients demand, temperature
dependent growth, photosynthetic rates (Eppley 1972; Geider & Geider 1987; Goldman &
Carpenter 1974; Turpin 1991), as well as assessments of influence of future environmental
conditions (Fu et al. 2007), are to a large extent obtained from algal bioassays.
In previous algal assays it has been common to use culture flask. Nowadays the use of
microplate assays as an appropriate alternative to flasks is increasing due to several
advantages such as small sample volume requirement, incubation space economy, increased
yield in processing samples, elimination of post-experimental washing of glassware
9
(microplates are disposable), and elimination of potential contamination and/ or toxicity
problems resulting from re-use of glassware (Blaise et al. 1986).
All autotrophs contain the primary photosynthetic pigment chlorophyll a. The quantitation
through extracted analysis, or estimation through in vivo (meaning “within a living
organism”) measurements, of chlorophyll a concentration supplies information on the
abundance of phytoplankton present in all aquatic environments. Even though chlorophyll a
concentration shows great differences among lakes, correlations between phytoplankton
biomass and chlorophyll a concentration are expected to be significant (Vörös & Padisák
1991).
In vivo measurements of the fluorescence (IVF) by chlorophyll a has for a long time been
used for non-destructive estimation of in-situ phytoplankton biomass and thereby growth rates
in microalgal cultures (Brand & Guillard 1981; Huot & Babin 2011). In combination with
microplates the IVF technique has been used for estimating growth in filamentous algae
(Karsten et al. 1996). It has also been suggested suitable for planktonic species (Fu et al.
2007). Today the measurement of in vivo chlorophyll a fluorescence is considered as one of
the most versatile, sensitive and easy ways to measure the concentrations of phytoplankton in
water (Juneau & Popovic 1999).
2.4 Ultraviolet-visible (UV-Vis) absorption spectroscopy
Absorption of light within the UV (190-400nm) and visible (400-800nm) region of the
electromagnetic spectrum involves excitation of outer electrons from ground state to higher
energy levels. In molecules the absorbance is due to specific segments or functional groups
that contain σ-, π-, or η-electrons in the bonds between atoms. These specific moieties are
termed chromophores.
Chromophores in charge of the absorbance by DNOM are composed of conjugated double
bonds and unbounded electrons. Long conjugated chains of double (π) bonds are responsible
for the characteristic color of DNOM (Stevenson 1994). The UV-Vis spectra of DNOM are
usually poorly resolved with a characteristic strong increase of the absorbance of lower
wavelengths. This is typical for complex mixtures of substances that have strong
intermolecular interactions and a significant amount of unsaturated bonds and lone-pair
10
electrons (Stevenson 1994; Frimmel & Abbt-Braun 2009). Nevertheless, based on what
moiety of DNOM contributes to the absorbance at a particular wavelength, the UV-visible
spectra of DNOM do provide many useful applications. Around 254nm a weak shoulder is
usually found in the spectra of DNOM. Absorbency at this wavelength is assigned to
chromophores of C=C and C=O double bonds that can be conjugated (Frimmel & Abbt-Braun
2009). Absorbance by DNOM obtained at 254nm has thus been found to be strongly
correlated with the aromatic content found by NMR spectroscopy (Weishaar et al. 2003).
Scientists have developed a number of simple proxies according to radiation adsorption, in the
UV, Visible and IR range. Among them, several are commonly used: Specific UV absorbance
(sUVa or sUVa254) is defined as the samples’ UV absorbance at 254nm divided by the DOC
concentration of the solution. As described above the absorbency at ʎ254nm is mainly due to
double bonds. This proxy is thus indicative of the relative amount of aromatic C in the
DNOM. Longer chained conjugated systems are conceptually perceived to be responsible for
the absorbency in the visible region, in accordance with bathochromic shift. Specific VIS
absorbance (sVISa or sVISa400) is the DOC normalized specific absorbance within the visible
region. This is used as a proxy for the amount of higher molecular weight chromophores.
Specific Absorbance Ratio (SAR), which is defined as the ratio of absorbency at 254nm to
400nm, serves as a proxy for the relative of lower to higher molecular weight chromophores.
This proxy is frequently used for DNOM characterization. It is expected to increase (blue
shift) with decreasing degree of aromaticity and molecular weight (Peuravuori & Pihlaja
1997).
2.5 UV-Vis Fluorescence
Fluorescence occurs when a molecule absorbs energy resulting in an electron to be excited to
a higher energy level, and as the electron returns to the original ground state, energy is lost as
light or fluorescence. Organic compounds that absorb and re-emit light are referred to as
fluorophores (Mopper et al. 1996).
Fluorescence spectroscopy involves measurement of radiation caused by the relaxation
through emission of photons. This radiation will always have lower energy than the incident
light (Harris 2007). The absorption and emission wavelengths at which fluorescence occurs
are characteristic to specific molecular structure (Fellman et al. 2010). Fluorescence
11
spectroscopy opens thus a new window for characterization of DNOM. The fluorescence of
DNOM is attributed to C=O bonds, as well as phenolic, aromatic, and quionine moieties. The
fluorescence spectra provide thereby reliable information about the source, redox state, and
biological reactivity of DNOM (Miller et al. 2009).
A 3-D fluorescence excitation-emission matrix (EEM) (Cabaniss 1987; Matthews et al. 1996;
Senesi et al. 1989) spectra is obtained by recording several emission spectra at different
excitation wavelengths. The results of these spectra can be presented in a 3-dimensional
topographical manner with excitation and emission wavelength on the x-axis and y-axis, and
fluorescence intensity on the z-axis.
In a review paper on EEM spectra of DNOM Chen et al. (2003) presented a graph describing
location of five different EEM peaks, with operationally defined excitation and emission
boundaries (Figure 1). These were based on EEM spectra obtained from marine, aquatic and
terrestrial derived DNOM. A sum-up of the information in Figure 1 is presented in Table 1.
Table 1: Association of different moieties in the DNOM to different regions of the EEM spectra.
Region Exitation ʎex Emisson ʎem Association
1 <250 <330 Aromatic proteins
2 <250 330nm<ʎ < 380nm Aromatic proteins
3 250nm<ʎ< 340nm <380nm Soluble microbial by-product
like material.
4 250nm<ʎ< 400nm >380nm Humic acid-like materials
5 <250nm >380nm Fulvic acid-like materials
12
Figure 1: Location of EEM peaks (symbols) based on literature reports and operationally defined excitation and emission wavelength boundaries (dashed lines) for five EEM regions (Chen et al. 2003).
13
3 Material and methods Laboratory experiments were performed on two sets of samples as follows in order to capture
a span in factors that explains differences in photo-oxidation response:
a. Fresh water samples from waterworks of project “Drinking water treatment adaptation on
increasing levels of DOM and changing DOM quality under climate change” (DOMQUA).
b. A set of thoroughly characterized reverse osmosis and freeze-dried DNOM material from
the project entitled “Natural Organic Matter in the Nordic Countries” (NOMiNiC) (Vogt et al.
2004).
3.1 Raw water samples from DOMQUA project
DNOM quality in raw water affects choice for optimal drinking water treatment, and affects
microbial growth and formation of biofilms in pipelines and other surfaces (Torvinen et al.
2004). These properties will vary both between localities (Weyhenmeyer et al. 2012)
depending on hydrology and catchment properties as well as water residence time, but also
seasonally within localities depending on microbial activity, solar exposure, lake-water
mixing regimes (Wachenfeldt et al. 2009). Therefore the main drinking water sources for
Helsinki (Lake Päijänne), Stockholm (Lake Mälaren) and Oslo (Lake Maridalsvannet),
hereafter referred to as PMM, were selected in DOMQUA project. The raw water samples
from PMM were collected from two different seasons: spring and fall 2015, respectively.
Sampling campaigns:
#1: 2-6 March 2015 (2nd in Oslo, 2nd in Helsinki, 6th in Stockholm)
#2: 17-24 August 2015 (17th in Oslo, 20th in Helsinki, 24th in Stockholm)
The sampling campaigns were conducted by the project members adhering to the same
protocol and distributed to individual research group (Figure 2). All the samples were
collected directly from the raw water taps in water works (Figure 3). Samples from March
represent spring samples that have been exposed to a minimum of solar radiation due to ice
cover during the preceding winter, while fall samples collected in August had been exposed to
more solar sunlight.
14
Figure 2: Sampling campaigns within DOMQUA project
Figure 3: Sampling of raw water samples at Oset water works at Maridalsvannet.
15
3.2 Reference samples of Reverse Osmosis (RO) isolated DNOM
The freeze-dried RO isolates from the NOMiNiC project are stored in a freezer with a
temperature below 4°C. The goal of the NOMiNiC project was to conduct a thorough
characterization on a common set of DNOM through an international collaboration. All of the
samples were distributed to more than 20 research groups within both Europe and North-
America. A large data matrix with DNOM characteristics has thereby been generated (pH,
conductivity, alkalinity, major anions and cations, UV-Vis absorption, UV-Vis fluorescence
etc) (Vogt et al. 2004) and thus available for this study for data quality control as well as a
reference material.
3.2.1 Field and Project description
Sampling sites within the NOMiNiC project are presented in Figure 4. Totally 5 sites from
Norway, Sweden and Finland were included in the project: Hietajärvi, Valkea-Kotinen,
Svartberget, Birkenes and Skjervatjern. These sites were carefully chosen to represent
difference in properties such as climate, vegetation, retention time, etc. The NOMiNiC project
aimed at investigating the effect of anthropogenic loading on DNOM. Seasonal variations
(spring/fall) were further included in the NOMiNiC project resulting in 10 samples in total.
For more information regarding the site description and the process of sampling, see Vogt et
al. (2004).
16
Figure 4: Sampling sites for Reverse Osmosis (RO) isolates marked with numbers according to Table 1 (Vogt et
al. 2004). ①Hietajärvi, ②Valkea-Kotinen, ③Svartberget, ④Birkenes and ⑤Skjervatjern.
3.3 Sample pre-treatment and flowchart of thesis experiment
The raw water samples from DOMQUA project were received on 10th March and31st August.
The samples collected in spring (March) had been kept in a dark room for six months while
fall (August) samples were stored one month at 4 before analysis. RO reference material
were attempted to be dissolved to an estimated of the same level of DOC concentration as raw
water samples prior to filtration. But a big deviation between the measured and estimated
DOC has been found (Chapter 4.2.1) in the samples collected in Svartberget. This may be
ascribed to wrong operation in dissolving procedure. Before dissolution, RO reference
material was thawed carefully by placing them at 7 °C for 3h and subsequently put in a room
temperature circumstance. Thawed RO reference material was re-dissolved using 3L of Milli-
Q (ultrapure, de-ionized) water. RO reference material was magnetically stirred for 48 hours
in the dark to pursue a more complete dissolution.
3.3.1 Filtration
Flowchart of the thesis experiments is presented in Figure 5. Both raw water samples and
dissolved RO reference material were filtered through a 0.45µm filter cellulose nitrate filter
17
(Sartorius) to obtain the DNOM fraction (Thurman 1985). The filters were rinsed with 150mL
Type 1 water before filtration. Filtration was conducted with a water vacuum pump. After
filtration, all samples were stored in amber bottles to avoid photo-oxidation, and put back into
cold room for 2 days before analysis.
Characterization of sample matrix and the DNOM were conducted both before and after
exposure to artificial sunlight in Q-SUN (See Chapter 3.4). Due to sample volume constraints
only three different exposure times were included. 0h represents the non-exposed samples. 4h
was chosen to represent a relative shorter exposure time. 20h was chosen as maximum
exposure as this is the longest working duration for Q-SUN without experiencing problem of
shutdown due to heating.
Figure 5: Flowchart of thesis experiments
18
3.4 Photo-oxidation
The photo-oxidation was conducted on a Q-Sun Xenon test chamber (Figure 6) (Q-Panel Lab
Products, Cleveland, OH) at the Department of Biosciencs, UiO. This is a laboratory
simulator of the damaging effects of sunlight. Irradiance was set at 0.65Wm-2 at 340nm which
is commonly used as a simulation of sunlight (Kragh et al. 2008)(Figure 7) and controlled
with a CR 20 calibration radiometer (Q-panel Lab Products), as described by Scully et al.
(2003).
Figure 6: Q-SUN photo-oxidation instrument. The quartz bottles are the containers for samples which are stored in a tank. Tap water is always switched on during the experiments for cooling with a drain system.
Figure 7: Comparison of irradiance spectrum from artificial sunlight produced from the Q-Sun Xenon Test Chamber using “daylight” filters and direct sunlight.
19
3.5 Sample matrix characterization
Analysis of pH, conductivity and alkalinity was conducted on samples both prior and post to
artificial sunlight exposure. Each measurement was conducted with three replicates. Water
characterization is conducted to provide information regarding the chemical environment that
the DNOM is in and what changes the photo-oxidation has made.
3.5.1 pH and conductivity
Measurements of pH and conductivity were conducted according to ISO-10523 (2008) and
ISO-7888 (1985), respectively. The measurement of conductivity was first analysed with a
Mettler-Toledo AG FiveGOTM electrode, calibrated with a standard solution of 85 µS cm-1,
followed by pH analysis using an Orion pH-meter equipped with a combined Ross electrode,
calibrated with buffer solution with pH4.00 and pH7.00. Samples aliquots of approximately
20mL were consumed.
3.5.2 Alkalinity
Measurement of total Alkalinity was conducted according to ISO-9963-1 (1994). This is a
method for the titrimetric determination of alkalinity using 0.02M HCl, and with
potentiometric endpoint detection using a pH-meter. Analysis was performed using a Ω
Methrom Swissmade 702 SM Titrimo instrument with approximately 50mL of samples and
25mL of titrand used.
3.6 Elemental composition and Speciation
Major anions and cations as well as DOC concentrations were measured in order to acquire
changes in elemental composition of DNOM caused by sunlight.
3.6.1 Major anions
Determination of major anions, i.e. chloride (Cl-), fluoride (F-), sulphate (SO42-) and nitrate
(NO3-) were measured by the Department of Geosciences, UiO, according to international
standard ISO-10304-1 (2007) in compliance with the instrument manual. The measurements
of major anions were performed both prior and post to photo-oxidation with approximately
20
10mL of samples consumed. The Dionex ICS-2000 Ion Chromatography System (ICS-2000)
performs ion analyses using suppressed conductivity detection. In an ion chromatograph the
anions are separated in a chromatographic column based on size and charge. Cations
associated with the anions are exchanges for H+ in a ion suppressor before the conductivity is
measured.
3.6.2 Major cations
The concentration of major cations (Ca2+, Mg2+, Na+, K+) along with total Fe and Al were
determined by a Varian VISTA Inductively Coupled Plasma Optical Emission Spectrometer
(ICP-OES) according to ISO-11885 (2007). The instrument is equipped with a Charge
Coupled Device (CCD) Echelle polychromator, and the emission was measured axially for an
increase in detection limit compared to radically view. It is a type of emission spectroscopy
using the inductively coupled plasma to produce exited atoms and ions that emit
electromagnetic line spectrum with radiation at specific wavelengths characteristic of a
particular element. The intensity of this emission is the indication of the concentration of
specific element within the sample. Before measuring, all standard solutions and samples
were conserved by acidification by adding 1% (v/v) of a 32.5% (m/v) nitric acid with 50000
ppm Cesium (Cs). The purpose of adding acids is to mobilize the ions in the samples. Cs is
used as an ionization buffer in order to increase atomic emission intensity of Na and K.
3.6.3 Dissolved Organic Carbon (DOC)
Dissolved Organic Carbon (DOC) was measured using a Shimadzu TOC-5000A total organic
carbon analyser, according to ISO 8245 (1999) and the TOC-5000A instruction manual. A
Non-Purgeable Organic Carbon (NPOC) was applied as the instrument setting, which include
the preliminary step of removing inorganic carbon by purging acidified (pH≤2) samples with
high purity air. With the aid of an oxidation catalyst, organic carbon is decomposed to carbon
dioxide (CO2). The final determination of CO2 is carried out by a nondispersive infra-red
(NDIR) gas analyser.
21
3.7 DNOM characterization
Structural characterization of DNOM in raw water samples and dissolved RO reference
material were performed both prior and post to photo-oxidation. Structure, composition and
size distribution of both humic and fulvic substances in DNOM (Lepane et al. 2003) would be
influenced by exposure to sunlight.
3.7.1 UV-Vis Absorbency
Two specific wavelengths, 254nm (UV) and 400nm (colour), were used to measure the
absorbency on a Varian Cary 100 Bio UV-Vis spectrophotometer using 1cm quartz cuvettes.
Data were processed according to Chapter 2.4.
3.7.2 UV-\Vis Fluorescence Spectroscopy
Fluorescence Excitation-Emission matrix (EEM) spectroscopy is an established tool of
organic matter fingerprinting in aqueous systems. This was measured at Norwegian Institute
of Life Sciences (NMBU) with a clear faced quartz cuvette on Varian Cary Eclipse
Fluorescence Spectrophotometer. The EEM spectra were generated by subsequently scanning
the emission from 300-600nm by incrementing the excitation wavelength by 10nm from 240
to 450nm. Excitation and emission slit width were set to 10 and 2nm, respectively. Scan speed
was set to 600nm min-1. Data was processed using Varian Cary Eclipse software.
3.8 Algal growth assays
As described in the introduction (Chapter 1) the exposure of DNOM to solar radiation causes
photo-bleaching. This is, as described in the theory chapter (Chapter 2.2), due to photo-
oxidation of high molecular weight DNOM molecules which are broken down into lower
molecular weight organic compounds. It is within the moiety of photo-chemically modified
LMW DNOM that we find the bioavailable photoproducts, i.e. lower MW organic
compounds, unidentified photo degraded organic matter, carbon gases and inorganic nitrogen
and phosphorus-rich compounds Algae, as a species of autotrophs, rely on inorganic
compounds to grow. Thus algal growth assays were conducted in order to indicate the release
22
of PO43- as it is the limiting nutrients for algae. Three different durations of artificial sunlight
0h, 4h and 20h were chosen as described in Chapter 3.3.
At start of the experiment each well on the microplates (Figure 8) were inoculated with 5μL
(ca. 1000 cells) of Chlamydomonas reinhardii (strain CC-1690 21 gr mt+) stock culture
prepared by colleagues at the Dept. of Biosciences, UiO, in a WC medium rich in other
nutrients than phosphorus to ensure that algae are phosphorus limites(Guillard & Lorenzen
1972). Chlamydomonas reinhardii is a single-cell green alga which is commonly used in
laboratory experiments as it has a robust growth response. Before adding into the well the
stock culture bottle was shaken in order to make it homogenous. Transparent sealing-tape
(BarSeal, Thermo scientific Nunc, Waltham, USA) was used to reduce contamination of
bacteria from the air. Temperature was kept stable at 19°C (in a climate room) and a 12/12
hour light/dark cycle was applied for algal growth. The measurement started from the second
day after inoculation. Estimates of the algal growth rates were made based on the fluorescent
properties of algae, as described in Chapter 2.3. This was conducted by daily measuring in
vivo fluorescence (IVF) using a microplate reader fitted with a fluorometer. IVF represents a
non-destructive proxy for algae population size. In addition, IVF was measured as a proxy of
the biomass of the algae in the plates, adhering to the theory outlined in Chapter 2. IVF
readings were made with an FLx 800 microplate reader (BioTek, Inc. USA) with
excitation/emission at 460/ 680 nm and filter bandwidths of 20nm. Fluorescence is the
phenomena of some compounds to absorb specific wavelengths of light and almost
instantaneously emit longer wavelengths of light. Chlorophyll a naturally absorbs blue light
and emit, or fluoreces, red light. Fluoremeters detect chlorophyll a by transmitting an
excitation beam of light in the blue range (440nm for extracted analysis and 460nm for in
vivo analysis) and by detecting the light fluoresced by cells or chlorophyll in a sample at
680nm (red). Generally, this fluorescence is directly proportional to the biomass of algae.
Each treatment combination was performed in three replicates including blank samples as
control. The specific growth rate (µ) for each sample was calculated with Equation 1. It is a
measure of how fast the algal population increases, independent of population size. It is
calculated as the logarithm of the relative rate of increase from one time t1 to a new time t2,
i.e.
μ log 2 1⁄ / 2 1 (Eqn.1)
23
The following equation describes the exponential growth of the population:
0 (Eqn.2)
where Nt denotes population at time t, N0 denotes the initial population and µ is growth rate.
Figure 8: Sealing 96-cell culture microplates
24
25
4 Results and Discussion This chapter starts with presenting the water chemistry of the studied samples in order to
characterize the matrix in which the DNOM is studied. Following this, the structural character
of the studied DNOM is described. The effect of photo-oxidation on water chemistry and
DNOM characteristics is studied by comparing the results obtained both before and after
photo-oxidation. Finally, a comparison of the results from the algae growth assays on the
samples both prior (t=0) and post to photo-oxidation for 4 and 20 hours is discussed. By
combining these findings the effects of sunlight may have on DNOM will be revealed.
4.1 Sample matrix characterization
The water chemistry of the raw water samples, which were collected during the spring (S) and
fall (F) seasons from the waterworks in Oslo, Helsinki and Stockholm, and the dissolved RO
reference materials are presented here. Spring samples are considered to have DNOM that has
been exposed to less sunlight than the fall samples. Water chemistry of the samples provides
us information regarding the chemical environment that the DNOM is in.
4.1.1 pH and conductivity
pH and conductivity in the raw water samples and RO isolates are given in Table 2 and 3,
respectively. The pH values of the raw water samples are circumneutral, ranging from 6.60 to
7.91, while the RO reference samples are more acidic with pH values from 4.90 to 6.75.
These differences are mainly due to that the water from the waterworks are from high order
lakes while the RO reference materials are generated from head water lakes and streams with
generally higher concentrations of DNOM. Overall, samples collected in fall (F) have a
relatively lower pH than the one collected in early spring (S) for raw water samples and
majority of the RO isolates. There is a large span in conductivity of the raw water samples.
The water samples from Stockholm have a high conductivity. The cause for these differences
will be addressed in the following chapters. Measured conductivity is found to be strongly
correlated (R2=0.993) with the calculated conductivity implying good data quality.
26
Table 2: pH and conductivity of the raw water samples collected in Oslo (Osl), Helsinki (Hel), and Stockholm (Sto). (S) and (F) denotes to spring and fall respectively.
Sample pH Conductivity
(µS/cm)
Osl(S) 6.79 0.03 23.2 0.16
Osl(F) 6.60 0.02 23.5 0.16
Hel(S) 7.32 0.02 62.5 0.49
Hel(F) 7.28 0.02 64.8 0.37
Sto(S) 7.91 0.01 203 1.25
Sto(F) 7.73 0.01 200 0.82
Table 3: pH and conductivity of the RO isolates collected in Birkenes (Bir), Hietajärvi (Hie), Skjervatjern (Skj), Svartberget (Sva), and Valkea-Kotinen (Vk). (S) and (F) denotes to spring and fall, respectively.
Sample pH Conductivity
(µS/cm)
Bir(S) 5.120.02 58.60.25
Bir(F) 4.900.03 37.70.51
Hie(S) 6.750.22 18.70.11
Hie(F) 5.950.18 17.60.03
Skj(S) 5.110.02 27.50.09
Skj(F) 5.280.01 15.70.29
Sva(S) 5.040.04 21.80.12
Sva(F) 6.040.11 14.40.03
Vk(S) 5.700.13 26.00.36
Vk(F) 5.570.04 24.70.08
4.2 Elemental composition and speciation
4.2.1 DOC
DOC is short for dissolved organic carbon, which is the main component in DNOM. DOC is
used as a proxy for the concentration of DNOM in the samples. Concentrations of DOC are
listed in Table 4. Raw water samples from Oslo have lower DOC values compared to the ones
27
from Helsinki and Stockholm. The RO isolate solutions were prepared by dissolving
estimated amounts of freeze dried material aiming to reconstruct the same level in DOC as in
the original raw water of which it was isolated. Large deviations in the resulting DOC
compared to the original sample may partly be due to problems encountered during the
dissolution operation. The DOC concentration in the Sva (F) sample deviates the most
compared to the targeted value (Table 4b). UV absorbency (λ=254nm) is commonly found to
correlate with DOC. Strong correlation is also found for the raw water samples from water
works (r2=0.99) while a relatively weaker but still strong correlation is found for the RO
reference samples (r2=0.91).
Table4a: DOC of the raw water samples collected in Oslo (Osl), Helsinki (Hel), and Stockholm (Sto). (S) and (F) denotes to spring and fall respectively.
Sample DOC(mg/L) Sample DOC(mg/L)
Osl(S) 3.88 Osl(F) 3.63
Hel(S) 6.55 Hel(F) 6.25
Sto(S) 7.35 Sto(F) 7.40
Table4b: DOC of the RO isolates collected in Hietajärvi (Hie), Valkea-Kotinen (Vk), Svartberget (Sva), Birkenes (Bir), and Skjervatjern (Skj). (S) and (F) denotes to spring and fall, respectively. Data for the original raw water are from Vogt et al. (2004).
Sample DOC(mg/L) Sample DOC(mg/L)
Original Re-dissolved Original Re-dissolved
Hie(S) 4.69 7.26 Hie(F) 6.35 8.64
Vk(S) 11.1 8.32 Vk(F) 9.44 8.09
Sva(S) 18.6 11.8 Sva(F) 10.9 4.07
Bir(S) 3.60 7.56 Bir(F) 5.00 8.55
Skj(S) 5.92 7.59 Skj(F) 10.2 7.91
4.2.2 Charge distribution of major anions and cations
Charge distribution of major anions (HCO3-, Cl-, SO4
2-, NO3-, F-) and cations (Ca2+, Na+, K+,
Mg2+, H+) is presented in Figure 9a. HCO3- was calculated based on alkalinity data assuming
that other inorganic buffering species in the sample are insignificant. HCO3- which is
represented by DIC (Dissolved Inorganic Carbon) in the figure can then be calculated by
28
subtracting the amount of acid needed to change the pH from the sample pH to pH 4.5 as well
as the equivalents of organic charge that is protonated from sample pH to pH 4.5. The latter
were calculated by the method of Oliver et al. (1983). The anion deficiency observed in all
samples, except for Sto(S), is constituted by organic anions. The high conductivity in the Sto
samples, described in Chapt 4.1.1., is here explained by the much higher ionic strength in
these samples.
Figure 9a: Charge distribution of major anions and cations for both raw water samples and RO isolates.
The relative charge distribution (%) of the major species in raw water samples (left side) and
RO isolates (right side) are presented in Figure 9b. Overall, Ca2+ and HCO3- are the dominant
cation and anion in raw water samples, respectively. The high concentrations of these species
are the result of dissolution of carbonate minerals such as limestone in the watershed. Na+ and
Cl- are also accounting for a big part of the cation and anion charge, respectively, in raw water
samples. This is mainly due to strong sea salt influence. Compared to raw water samples, the
dominant cation in RO isolates is Na+. This is due to the procedure for preparing RO isolates
in which the water was pumped through a cation exchanger replacing other cations with Na+
to prevent precipitation of insoluble salts, such as CaSO4(s) and CaCO3(s) (Gjessing et al.
0
500
1000
1500
2000
2500
HIE(S)
HIE(F)
VK(S)
VK(F)
SVA(S)
SVA(F)
BIR(S)
BIR(F)
SKJ(S)
SKJ(F)
Osl(S)
Osl(F)
Hel(S)
Hel(F)
Sto(S)
Sto(F)
eq DIC Org. charge HPO42‐
Tot‐F Cl‐ NO3‐
SO42‐ K+ Na+
Mg2+ Ca2+ H+
29
1999). Nearly no inorganic labile Al can be found in the raw water samples and RO isolates
as the pH of majority of these samples are higher than 5.5 where the solubility of Al3+ is very
low.
The relative composition of the water work samples do not differ significantly. The large
differences in absolute amounts seen in Fig. 9a are therefore mainly due to dilution/up-
concentration effects caused by differences in precipitation amount and evapotranspiration.
Correlation coefficients between the dominant ions Na+, Cl- and SO42- determined on the
reference material by Gundersen (2012) and in this study varies from 0.945 to 0.987,
confirming good data quality.
Figure 9b: Distribution (%) of charge contribution species for both raw water samples and RO isolates.
The electrical balance of these samples is listed in Table 5 in which we can see the accuracy
of chemical analysis for raw water samples is of good standard according to Appelo &
Postma (2005) with a percentage less than 10%. Some of the RO reference material
(Hietajärvi and Birkenes) show a relatively higher electrical balance than raw water samples
illustrating lower accuracy but still acceptable.
0 %
10 %
20 %
30 %
40 %
50 %
60 %
70 %
80 %
90 %
100 %
HIE(S)
HIE(F)
VK(S)
VK(F)
SVA(S)
SVA(F)
BIR(S)
BIR(F)
SKJ(S)
SKJ(F)
Osl(S)
Osl(F)
Hel(S)
Hel(F)
Sto(S)
Sto(F)
eq DIC
Org. charge
HPO42‐
Tot‐F
Cl‐
NO3‐
SO42‐
K+
Na+
Mg2+
Ca2+
H+
30
Table 5: Electrical balance of both raw water samples collected in Oslo (Osl), Helsinki (Hel), and Stockholm (Sto), and RO isolates collected in Hietajärvi (Hie), Valkea-Kotinen (Vk), Svartberget (Sva), Birkenes (Bir), and Skjervatjern (Skj). (S) and (F) denotes to spring and fall respectively.
Sample E.B. (%) Sample E.B. (%)
Osl(S) 0 Osl(F) 6
Hel(S) -4 Hel(F) 0
Sto(S) -7 Sto(F) -7
Hie(S) 21 Hie(F) 16
Vk(S) 1 Vk(F) -4
Sva(S) -14 Sva(F) -1
Bir(S) -8 Bir(F) -15
Skj(S) -11 Skj(F) 3
4.3 Structural Characterization of Dissolved Natural Organic Matter (DNOM)
UV radiation may influence the structure, composition and size distribution of both humic and
fulvic substances in DNOM (Lepane et al. 2003) constituting the most important moieties of
chromophores. Absorption of sunlight induces photo-bleaching by photo-oxidation producing
LMW organic compound and inorganic species. These processes may result in the loss of
specific functional moieties as well as a loss of DNOM aromaticity causing changes in optical
properties of DNOM.
4.3.1 UV-/Vis Absorbency
Results for DNOM absorbency of light within UV (λ=254nm) and visible region (λ=400nm)
of the electromagnetic spectrum were presented in Appendix B-1. According to that, two
indices of Specific Absorbency, sUVa ((Abs254nm/ [DOC])*100) and sVISa ((Abs400nm/
[DOC])*1000), as well as Specific Absorbency Ratio, SAR (Abs254nm/ Abs400nm) are
calculated and presented in Table 6 and Table 7. sUVa is a proxy for the aromaticity and
sVISa is the specific color of the DNOM. The SAR denotes the relative size of the DNOM.
31
The sUVa, sVISa and SAR values of the RO reference material are found to be strongly
correlated (R2= 0.799, 0.921 & 0.871, respectively) to values previously reported by
Gundersen (2012) implying good data quality.
Table 6: Proxy values of the characteristics of DNOM for RO reference material
Hie(S) Hie(F) Vk(S) Vk(F) Sva(S) Sva(F) Bir(S) Bir(F) Skj(S) Skj(F)
sUVa 4,31 4,00 4,57 4,73 5,68 5,25 3,91 4,27 5,18 5,00
sVISa 5,09 5,32 5,41 5,93 7,47 6,89 3,75 4,49 7,11 6,96
SAR 8,46 7,51 8,46 7,97 7,60 7,62 10,42 9,51 7,28 7,18
Table 7: Proxy values of the characteristics of DNOM for raw water samples
Osl(S) Osl(F) Hel(S) Hel(F) Sto(S) Sto(F)
sUVa 4,01 4,24 3,26 3,30 3,09 3,04
sVISa 4,04 4,23 2,54 2,61 2,13 2,07
SAR 9,91 10,0 12,8 12,6 14,5 14,7
The sUVa and sVISa values indicate that the raw water samples have generally low
aromaticity and color compared to the RO reference material. Especially the specific color of
DNOM from Hel and Sto is low. The SAR on the other hand is relatively high, especially in
the sample from Osl. This indicates that the DNOM has relatively low molecular weight.
Seasonal differences in the DNOM proxies are insignificant.
Summing up, the absorbancy proxies indicate that the DNOM in the raw water samples are of
relatively low aromaticity and of low molecular size.
4.3.2 Fluorescence excitation-emission matrix (EEM) spectra
Fluorescence excitation-emission spectra (EEMs) for the raw water samples and RO reference
materials are presented in Figure 10. Two major peaks (peak A and peak B) appear in the
spectra of the samples. Peak A, observed at λex/ λem= 320-330/420-458nm, is due to
fluorophores in the phenolic acids contributing to fluorescence. This peak is thus assigned to
humic acids (Chen et al. 2003). The wavelength region at λex/ λem= 240/420-440nm, where
peak B is observed, is generally attributed to fulvic acids (Chen et al. 2003). In the sample
collected in fall at Stockholm a shoulder C (at about λex/ λem= 420/495) appears in the spectra
32
in another view of the plot (see appendix B-2). This is an indication of more highly
conjugated aromatic compounds (Blaser et al. 1999).
There is unfortunately no agreement between peak intensities and ratios of the reference
materials determined in this study compared to previous studies (Gunderson 2012; Vogt et al.
2004).
Fluorescence EEM spectra of the samples from water works generally displays the same kind
of shapes as the RO isolates. The absolute intensity of peak A is low in samples collected in
Oslo and high in samples from Stockholm. This is mainly due to a higher concentration of
DNOM in Stockholm samples. This is reflected in a correlation of R2 = 0.700 between DOC
and peak A intensity measured in the raw water and reference material. The same is not
found for the peak B. That the intensity of peak A is strongly governed by DOC follows from
that the main constituent in DNOM is the humic acid fraction. The two diagonal lines
(Rayleigh scatter lines) which can be clearly seen in the plots originate from the interaction
between molecules in the solution and the incident light that do not contain information on the
chemical properties of the sample (Rinnan & Andersen 2005).
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(S)90‐10575‐9060‐7545‐6030‐4515‐300‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
33
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(S) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(S) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(F)90‐10575‐9060‐7545‐6030‐4515‐300‐15
34
Figure 10: Fluorescence Excitation-Emission Matrix spectra (EEMs) for the water samples originating from waterworks and RO reference materials. Two different kinds of plots were made for each sample but only one of them was chosen here to be presented (Another one see appendix).
Wavelength locations (λex & λem), absolute (I) and relative (I/DOC) intensities of peak A
and peak B, which is attributed to humic acids and fulvic acids respectively, as well as peak
ratio (A: B) for both the samples from the waterworks and RO reference materials were
presented in Appendix B-3. Wavelength locations (λex & λem) were determined by tracing
back to the raw data following by checking the highest intensity after peaks were found from
the plots. No distinct differences in EEM based indexes between water samples and RO
isolates were found. Wavelength locations of the maximum of peaks A and B are
approximately found in the same region in each sample. Peak ratio is the ratio between the
highest intensities of peak A and peak B. This ratio is considered to reflect the degree of
aromaticity. There is thus a positive correlation between the A: B ratio and sUVa as well as
sVISa (R2=0.588 and 0.581, respectively) as all proxies are reflecting the relative content of
aromatic moieties. The changes in peak ratios for all the samples prior and post to sunlight
will be discussed in Chapter 4.3.3.
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(S) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
35
A rather constant intensity ratio of the peaks A and B (i.e. A: B) among the water work
samples implies that the raw water samples have the same relative content of humic and
fulvic substances. The low ratio (0.6) suggests that the raw water samples are relatively richer
in fulvic acids than in humic acids compared to the NOMiNiC reference material.
4.3 Changes in characteristics of DNOM due to photo-oxidation
The chemical and structural characteristics of DNOM affects its photo-reactivity and
bioavailability (Bertilsson & Tranvik 2000; Granéli et al. 1996; Keiber et al. 1990; Miller &
Zepp 1995; Mopper et al. 1991). Photo-mineralization and photo-transformations remove
DOC and structurally alter DNOM, respectively, causing the DNOM to loose color (i.e.
photo-bleaching). This is subsequently influencing its biological utilization and fate
(Obernosterer & Benner 2004). Photo-oxidation has thus a significant impact on the cycling
of DNOM in aquatic environments and thereby affects ecosystem structure and function. The
reduction in color can be followed by monitoring the sUVa and sVISa. The transformations
are from the more aromatic and large molecular weight humic acids to the less aromatic and
smaller fulvic acids (Moran & Zepp 1997). Such changes can be detected from Fluorescence
EEM spectra. Results are given according to different parameters below.
4.3.1 UV-Vis absorbency spectra
Percentage reduction in absorbency within the UV (λ=254nm) and visible region (λ=400nm)
for raw water samples and RO reference material are presented in Figure 11a and 11b,
respectively. A clear trend of decrease in absorbance at both wavelengths can be found as the
percentage reduction is always rising with increasing irradiation time. This indicates that both
aromatic moieties and longer chained conjugated molecules are decreasing due to UV
radiation.
The average percentage reduction in absorbency at two specific wavelength-254nm and
400nm for both raw water samples and RO reference material are presented in Table 8. It can
be clearly summarized that percentage reduction in absorbency at 400nm is relatively higher
than the one at 254nm. This blue shift indicates that higher molecular weight molecules have
36
a more significant reduction caused by the photo-bleaching. Similar changes associated with
different sample sources have been found. No seasonal differences have been found.
Figure 11a: Percentage reduction in UV-Vis absorbency (@254nm and 400nm) for raw water samples from water works with 4h and 20h of exposure time.
37
Figure 11b: Percentage reduction in UV-Vis absorbance (@254nm and 400nm) for RO reference material with 4h and 20h of exposure time to artificial sunlight.
Table 8: Average percentage reduction in absorbency at two specific wavelength-254nm and 400nm for both raw water samples and RO reference material.
Raw water samples RO isolates
Exposure time(h) 254nm 400nm 254nm 400nm
4h 7.2% 8.0% 10.4% 14.5%
20h 16.9% 25.7% 35.7% 45.9%
Results for two indices of specific absorbency, sUVa and sVISa, prior and post to photo-
oxidation are presented in Figure 12 and Figure 13, respectively. The sUVa reflects the
relative amount of aromatic moieties contained in the DNOM. DNOM samples with high
sUVa are therefore generally more hydrophobic and humic. sUVa values decrease due to the
photo-oxidation. This implies a decrease in the aromaticity of the DNOM upon exposure to
UV radiation which is in accordance with Weishaar (2003). No seasonal differences are
found.
The percentage reduction in sUVa of the RO reference samples is found to be strongly
correlated to the relative intensity of the humic (A; R2=0.746) and fulvic (B; R2=0.617) acid
38
peaks in the Fluorescence EEM. This is likely due to that it is mainly these moieties that
absorbs radiation and becomes photo-bleached or completely photo-mineralized.
sVISa also decreases with exposure time. Nearly all the samples see the same trend except the
RO isolates collected in Skj(S). The sVISa in this reference sample increase from 0h to 4h
and then decrease from 4h to 20h, ending with an overall reduction from 0h to 20h. sVISa
serves as a proxy for the relative amount of higher molecular weight chromophores. This
reduction in sVISa therefore means a relative decrease in higher molecular weight DNOM
compounds. Similar to the percent reduction in sUVa, the amount of reduction in sVISa
caused by the photo-oxidation is also in the reference samples governed by the relative
intensity of the A and B fluorescence EEM peaks (R2=0.710 and 0.560, respectively). The
percent reduction in sVISa is also correlated to the SAR (R2=0.702).
These findings are also reflected by the effect of photo-oxidation on the Specific Absorbency
Ratio, SAR, presented in Figure 14. A clear increase in SAR indicates that the relative
contribution of lower to higher molecular weight DNOM compounds (Peuravuori & Pihlaja
1997) are increasing. A weaker visible color (i.e. increased SAR) is due to reduced length of
the long conjugated double bonds and is as such also conceptually related to the molecular
weight.
Figure 12: Values for Specific UV Absorbency, ((Abs254nm/ [DOC])*100), obtained for the samples from the water works and RO isolates after 0h, 4h and 20h of exposure time to artificial sunlight.
39
.
Figure 13: Values for the Specific Visible Absorbency, ((Abs400nm/ [DOC])*100), obtained for the samples from the water works and RO isolates after 0h, 4h and 20h of exposure time to artificial sunlight.
Figure 14: Values for the Specific Absorbency Ratio, SAR, (Abs254nm /Abs400nm) for the samples from the water works and RO isolates after 0h, 4h and 20h of exposure time to artificial sunlight.
40
Average percentage reduction in three specific absorbency indexes (sUVa, sVISa and SAR)
with 4h and 20h of exposure time compared to no exposure are presented in Table 9. A higher
average percentage reduction in sVISa compared to sUVa has been found which implies that
higher molecular weight compounds have a slightly higher decrease than lower molecular
weight compounds. Correlation coefficients for percentage reduction/increase in specific
absorbency indexes, ‘sUVa (PR) and SAR (PI)’, ‘sVISa (PR) and SAR (PI)’ and ‘sUVa (PR)
and SAR (PI)’ for both raw water samples and RO isolates have been calculated and listed in
Table 10. ‘PR’ and ‘PI’ equals to percentage reduction and percentage increase respectively.
It can be clearly seen from the table that the percentage reduction in sVISa and increase in
SAR in raw water samples are correlated to each other. This is indicating that the decrease in
higher molecular weight compounds has a more significant effect on the increase in
contribution of lower to higher molecular weight compounds compared to the decrease of
lower molecular weight compounds in DNOM after photo-oxidation. For RO isolates a strong
relationship between the reduction in sUVa and sVISa is found while there is no clear
relationship between sUVa and SAR or between sVISa and SAR. This is also reflecting that
both the lower and higher molecular weight compounds in DNOM are decreasing due to UV
radiation, but that the lower molecular weight compounds have a slightly less decrease than
higher molecular weight compounds.
Table 9: Average percentage reduction in specific absorbency indexes (sUVa, sVISa, and SAR) with different exposure time-4h and 20h.
Raw water samples RO isolates
Exposure
time(h) sUVa sVISa SAR sUVa sVISa SAR
4h 5.1% 6.0% 1.0% 4.0% 8.4% 5.0%
20h 10.2% 19.4% 10.8% 17.2% 33.4% 21.0%
Table 10: Correlation coefficients between specific absorbency indexes (sUVa, sVISa and SAR) pairs of both raw water samples and RO isolates with different exposure time-4h and 20h.
Raw water samples RO isolates
Exposur
e time
sUVa/SA
R
sVISa/SA
R
sUVa/sVIS
a
sUVa/SA
R
sVISa/SA
R
sUVa/sVIS
a
4h -0.34 0.77 0.34 0.07 0.03 0.84
20h 0.51 0.95 0.76 0.26 0.28 0.91
41
4.3.2 DOC
Percentage reduction of concentrations in DOC of both raw water samples and RO reference
materials after 4h and 20h of natural sunlight are presented in Figure 15a and 15b,
respectively. The percentage reductions after 4h and 20h exposure time are relative to
reference samples that were not exposed to artificial solar radiation (t=0). Nearly all samples,
including both raw water samples and RO reference material, show a reduction in DOC
concentrations after photo-oxidation. The exception is for the raw water sample collected in
fall from Helsinki which has a slightly higher DOC after 4h of exposure compared to no
exposure. This is due to measurement error.
For raw water samples, an average of 2% and 7% reduction in DOC was found after 4h and
20 h exposure to sunlight, respectively. RO reference materials have a relatively larger
response, with 7% and 21% reduction after 4h and 20h of exposure, respectively. These
results confirm previous studies that DOC concentrations are decreased due to photo-
oxidation. This is probably mainly attributed to photochemical mineralization, as well as
possibly some biological assimilation of bioavailable moieties formed by the photo-oxidation
(Obernosterer & Benner 2004). Fall raw water samples are found to have a less decrease in
DOC (avg. 5.8% after 20 h) compared to spring samples (7.8%). This may reflect that the fall
samples have already been exposed to sunlight while the spring samples have been protected
by the ice cover. The percent mineralization after 20h was strongest correlated to the Fe (R2=
0.448) and H+ concentrations (=0.440). The effect of iron is likely due to that it acts as a
chromophore adsorbing radiation energy (Bertilsson & Tranvik 2000). The connection to
acidity may be linked to the content of aluminum. Studying only the RO reference materials
we find a strong correlation between the % mineralization found in this study and the total Al
concentration taken from Vogt et al (2004).
42
Figure 15a: Percentage reduction of concentrations in DOC of raw water samples from water works with photo-oxidation exposure time 4h and 20h.
Figure 15b: Percentage reduction of concentrations in DOC of RO reference materials with photo-oxidation exposure time 4h and 20h.
43
4.3.3 Fluorescence excitation and emission matrix spectra (EEMs)
Fluorescence excitation-emission matrix (EEM) spectra for one of the raw water samples and
RO isolates prior and post to photo-oxidation are presented in Figure 16a and 16b,
respectively. The spectra appear quite similar to each other for different sampling sites so the
rest plots are given in Appendix.
It has been reported that higher molecular weight moieties of the DNOM in natural water
samples are more easily photo-degraded than the lower molecular weight fraction (Lepane et
al. 2003). This is in accordance with the results from fluorescence spectra in that the peak
ratio, A: B, which is presented in Figure 17, illustrating humic/ fulvic ratio in the samples,
decreases with extension of UV radiation time. Details of the wavelength locations (λex/λem)
for the highest fluoresecence intensities of these two peaks and relative intensities which
equals to the ratio between the highest intensities and DOC concentrations as well as peak
ratio were in Appendix. The wavelength locations for the highest fluorescence intensities of
these two peaks were presented in order to illustrate that peak A refers to humic acids and
peak B refers to fulvic acids according to Chen (2003). The correlation coefficients between
relative intensity (both peak A and peak B) and peak ratio prior and post to photo-oxidation
were calculated (Table 11). A strong relationship between reduction in relative peak A
intensity and peak ratio has been found. This indicates that humic/ fulvic reduction is mainly
due to the decrease in humic acids. RO reference materials exhibit a more significant
decrease in peak ratios (Figure 17). This may be attributed to a higher initial relative amount
of humic acids (i.e. higher A/B), allowing the less refractory parts of the humic acids fraction
to be photo-oxidized. Results from changes in fluorescence EEMs are in accordance with the
changes in indices of specific absorbency in the UV and Visible region of electromagnetic
spectrum shown in Chapter 4.3.2. Both of these are corroborated with the view that larger
molecular weight compounds of the DNOM have been transformed into smaller ones due to
UV radiation. The percentage increase in SAR and decrease in Peak A/Peak B ratio for both
raw water samples and RO reference material was presented in Table 12. The SAR increase is
smaller than the decrease in peak A/peak B ratio indicating that the relative conversion from
humic to fulvic acids is greater than the conversion from HMW to LMW compounds. Then
the relative production of LMW compounds relative to fulvic acids, and thereby indirectly to
the relative production of LMW aliphatic compounds.
44
Figure 16a: Fluorescence Excitation-Emission Matrix (EEM) spectra obtained for the samples collected from Stockholm with different exposure time: 0h, 4h and 20h. Locations of the two peaks A and B.
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(S)90‐10575‐9060‐7545‐6030‐4515‐300‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(F)90‐10575‐9060‐7545‐6030‐4515‐300‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(S)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(F)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(S)‐20h90‐10575‐9060‐7545‐6030‐4515‐300‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(F)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
45
Figure 16b: Fluorescence Excitation-Emission Matrix (EEM) spectra obtained for one RO isolates collected from Hietajärvi with different exposure time: 0h, 4h and 20h.
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(S) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(S)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(F)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(S)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(F)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
46
Figure 17: Peak ratio variations for both raw water samples and RO isolates with 0h, 4h and 20h of exposure time.
Table 11: Correlation coefficients between relative intensity (both peak A and peak B) and peak ratio prior and post to photo-oxidation
Sample A/ Peak ratio B/ peak ratio Sample A/ Peak ratio B/ Peak ratio
Osl(S) 0,96 0,85 Osl(F) 0,88 0,83
Hel(S) 0,91 0,90 Hel(F) 0,56 0,50
Sto(S) 0,99 0,98 Sto(F) 0,97 0,96
Hie(S) 0,99 0,98 Hie(F) -0,72 -0,95
Vk(S) 0,98 -1 Vk(F) 0,82 -0,98
Sva(S) -0,99 -0,99 Sva(F) -0,33 -0,79
Bir(S) 0,87 0,04 Bir(F) -0,35 -0,93
Skj(S) 0,14 -0,99 Skj(F) -0,85 -0,99
47
Table 12: The percentage increase in SAR and decrease in Peak A/Peak B ratio with 4h and 20 of exposure time
for both raw water samples and RO isolates.
Raw water samples RO isolates
Exposure time SAR Peak A/peak B SAR Peak A/Peak B
4h 1.0% 3.4% 5.0% 18.6%
20h 10.8% 12.2% 21.0% 37.2%
4.4 Algal growth assays
4.4.1 General growth response
Algae growth assays were conducted to test if nutrients (PO43-) were released from DNOM
due to photo-oxidation. A 10-day average percentage increase in growth response of algae
(determined by the value of ¨chlorophyll a¨) for raw water samples and RO reference material
with different exposure times is presented in Figure 18 and 19. Three replicate experiments
for each sample were conducted. In general, a clear growth response can be figured out
compared to blanks. This demonstrates that algae in the water samples exposed to sunlight
increased in numbers. The algae rely on available nutrients to grow. The increase in algae
growth thus implies an increased access to nutrients. As phosphate is the limiting nutrient for
algae growth in freshwaters this increased algae growth is related to the amount of phosphate
released through photo-mineralization of DOP. The algae growth rate increases for
approximately 3 days until the nutrients were used up. At this time, the biomass of algae
reaches to the top, before being reduced when nutrients are depleted.
48
Figure 18: Average percentage increase in growth response of algae within 10 days with different exposure time of all the raw water samples. ¨Chlorophyll a¨ was measured as an indication of the biomass of algae. Y-axis denotes to percentage increase, X-axis denotes to time (d).
Figure 19: Average percentage increase in growth response of algae within 10 days with different exposure time
of all the RO reference material. ¨Chlorophyll a¨ was measured as an indication of the biomass of algae. Y-axis
denotes to percentage increase, X-axis denotes to time (d).
Overall, there is not always an increase of ‘chlorophyll a’ found with the extension of
exposure time in the plots with absolute biomass of algae (See appendix B-4). Several reasons
can be ascribed to this phenomenon. First of all, since the samples were not sterilized both
algae and bacteria may be competing for the food released from DNOM. The variation in
growth response may thus be partly due to that not only algae but also bacteria in the water
‐30,00%
‐20,00%
‐10,00%
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
2 3 4 5 6 7 8 9 10
0h
4h
20h
blank
‐30,00%
‐20,00%
‐10,00%
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
60,00%
2 3 4 5 6 7 8 9 10
0h
4h
20h
blank
49
samples were consuming some of the inorganic phosphate released by DNOM, making the
growth response slightly erratic. In addition, phytoplankton cells exposed to sunlight undergo
a series of physiological changes that influence cell volume and intracellular morphology, as
well as biochemical pathways and products, eventually affecting nutrient uptake as well
4.4.2 Growth rate analysis
The inoculation of algae culture was conducted with a micropipette, but still the initial
population of algae may have varied somewhat between samples. Avoiding such potential
differences in inoculum size, the average maximum growth rate of algae (µ) achieved after 3-
5 days for raw water samples and RO reference material was calculated according to Equation
1 and presented in Figure 20a and 20b, respectively. It can be clearly seen that all the raw
water samples and majority of RO reference samples have an increase in growth rate from 0h
to 4h of exposure time, while only half of raw water samples and 70% of RO reference
samples continue increasing from 4h to 20h. The initial increase may be ascribed to photo-
mineralization of DNOM releasing PO43- previously bond to DOP to the solution, promoting
growth of algae. The lack of continued growth with longer exposure time may be due to
competition from bacteria that also take up PO43- with high efficiency due their high surface-
to-volume ratios(Løvdal et al. 2007). In addition, the inhibitory effects of UV radiation may
have on the growth of algae (Gao & Zheng 2010; Xenopoulos et al. 2002) could offset the
stimulus caused by P-mineralization over extended exposure times.
50
Figure 20a: Average growth rate for raw water samples from waterworks with three different exposure time: 0h, 4h and 20h. Three different sampling places: Oslo, Helsinki and Stockholm with two sampling dates: Spring and Fall are included.
Figure 20b: Average growth rate for RO reference samples with three different exposure time: 0h, 4h and 20h. Five different sampling places with two sampling dates: spring and fall are included.
51
The empirical correlation between average growth rate vs. % DOC loss and SAR decrease
for RO reference material is found to be relatively strong (R2=0.66, 0.60, respectively). That
the reduction of DOC in RO reference materials is related to the algal growth provides strong
empirical support for that bioavailable PO43- is released by the mineralization of DNOM. The
correlation with decrease in SAR, reflecting a transformation of bigger molecular weight
DNOM compounds into smaller ones, suggests that the mineralized P may have been bound
to the larger more humic acid moieties of DNOM,
On the other hand, in terms of the raw water samples only samples collected in Stockholm
region gave a similar relationship (R2=0.85 and 0.51 for DOC and SAR, respectively). This
may be due to the rather small differences in DNOM characteristics among these samples.
52
53
5 Conclusions Exposing DNOM to artificial sunlight caused a loss in DOC concentration as well as specific
color (sVISa) and UV absorbency (sUVa) in the water matrix. The decrease in DOC implies
that some of the DNOM has been completely mineralized. The decrease in color was greater
than UV absorbance causing an increase in the specific adsorption ratio (SAR). The decrease
in sVISa and sUVa reflects a loss of especially the aromatic moieties of the DNOM.
Increasing SAR implies that the photo-oxidation generates a relative larger contribution of
lower to higher MW compounds of DNOM. The changes were also reflected in fluorescence
EEM spectra which indicate a transformation of more aromatic and larger MW humic acids to
the less aromatic and smaller fulvic acids. These changes are in accordance with previous
findings and are attributed to the absorption of sunlight energy by DNOM inducing photo-
oxidation and hydrolyzation reactions causing photo-bleaching and photo-production of
LMW organic compounds and inorganic species.
The mineralization of DNOM is hypothesized to lead to release of bioavailable inorganic
phosphate. Preceding studies have found this difficult to detect using traditional chemical
methods due to concurrent mineralization and release of iron and aluminum ions, rapidly
hydrolyzing and precipitating and thereby co-adsorbing the phosphate. Algal growth assays
were conducted as an alternative ‘in-situ’ method to verity the release of inorganic
orthophosphate based on that the assimilation of phosphate by algae is more rapid than the
concurrent precipitation.
A relative robust algae growth response was found as a response to the photo-oxidation
reflecting that nutrients (PO4) have been mineralized from DNOM due to sunlight exposure.
On the other hand, higher growth rate were not always detected with the extension of
exposure time. This can be attributed to three possible reasons: First of all, bacteria are
competing with algae for food as biological reactivity of some bacteria can also be enhanced
by making use of inorganic orthophosphate. Secondly, there is not enough DNOM in the
water samples of which provide enough orthophosphate for algal reactivity. Last but not the
least, UV radiation may be an inhibitory effects on the growth of algae that could offset the
stimulus caused by P-mineralization over extended exposure times.
54
Suggestion for future work
1. Further studies are needed to provide more conclusive evidence on the growth response of
algae. Extra work on exclusion of the bacteria can be conducted to make sure the only species
consuming orthophosphate in the inoculum is algae which may render a more robust growth
response.
2. Quantify the photo-degradation of DNOM.
3. Use 31P-NMR techniques to determine which phosphorus are broken as a result of photo
degradation.
55
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67
7 Appendix
List of Appendixes
Appendix A. Instrumentation and Calibration
A-1 ICP-OES
A-2 DOC
Appendix B. Results obtained prior and post to solar radiation
7.1.1 Appendix A. Instrumentation and Calibration
A-1 ICP-OES
Table 1: Concentration (mg/L) range of the six cations (calcium, potassium, magnesium, sodium, aluminium and
iron) in the calibration solution prepared for the analysis with the ICP-OES.
Analyte Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7
Ca 1.0 2.0 6.0 12.0 20.0 40.0 64.0
K 1.0 2.0 6.0 12.0 20.0 40.0 64.0
Mg 1.0 2.0 6.0 12.0 20.0 40.0 64.0
Na 1.0 2.0 6.0 12.0 20.0 40.0 64.0
Al 0.1 0.2 0.6 1.2 2.0 4.0 6.4
Fe 0.1 0.2 0.6 1.2 2.0 4.0 6.4
68
Table 2: Instrumental settings applied for the ICP-OES
Parameter Setting
RF Power 1.05kW
Plasma Ar Flow 15.0 L min-1
Auxillary Ar Flow 1.50 L min-1
Nebulizer Ar Flow 0.90 L min-1
Read time 1.00s
Rinse time 20s
Sample update delay 30s
Pump rate 20rpm
Table 3: Wavelength selections for the determination of the major cations (calcium, potassium, magnesium,
sodium, aluminium and iron) using ICP-OES.
Analyte Wavelength
Ca 396.847
K 766.491
Ma 285.213
Na 589.592
Al 237.312
Fe 238.204
69
A-2 DOC
Calibration solutions prepared from potassium hydrogen phthalate (HOOCC6H4COOK), dried
for 1h at 110 °C, and dissolved in Type I water. The calibration solutions were within the
following range in concentrations; 0, 2, 5, 10, 15, 20 mg C L-1. All glass equipment used in
this experiment was baked at 500 °C.
The limit of detection (LOD) of the DOC measurement was determined to 0.75 mg C L-1.
This was calculated from 3x st.dev. of 10 repeated measurements of blank samples (Type 1
water).
Table 4: Instrument settings applied for DOC
Parameter Setting
Pressure 5 bar
Flow rate 150 mL min-1
No. Of injections 3
Max. No. Injections 5
Min. No. Injections 3
No. Of washes 4
Sparge time 1min
70
7.1.2 Appendix B. Results obtained prior and post to solar radiation
B-1 Results for DNOM absorbency of light within UV (λ=254nm) and visible region
(λ=400nm) of the electromagnetic spectrum.
Table 5: Values for absorbency within UV (λ=254nm) and visible region (λ=400nm) for raw water samples
Osl(S) Osl(F) Hel(S) Hel(F) Sto(S) Sto(F)
λ (nm) Absorbency
254 0.155 0.154 0.213 0.206 0.227 0.225
400 0.016 0.015 0.017 0.016 0.016 0.015
Table 6b: Values for absorbency within UV (λ=254nm) and visible region (λ=400nm) for RO isolates.
Hie(S) Hie(F) Vk(S) Vk(F) Sva(S) Sva(F) Bir(S) Bir(F) Skj(S) Skj(F)
λ (nm) Absorbency
254 0.313 0.345 0.381 0.382 0.671 0.213 0.294 0.365 0.393 0.395
400 0.037 0.046 0.045 0.048 0.088 0.028 0.028 0.038 0.054 0.055
71
B-2 Results obtained from Fluorescence Excitation and Emission matrix spectra
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Osl(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Osl(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Osl(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
72
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Osl(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Osl(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Osl(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Osl(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
73
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hel(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hel(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hel(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
74
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hel(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hel(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hel(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hel(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
75
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sto(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sto(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sto(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
76
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450260
290320350380
410440
0153045607590105
300330360390420450480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sto(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sto(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sto(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sto(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
77
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hie(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hie(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hie(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
78
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hie(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hie(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Hie(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Hie(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
79
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450480510540570600
Excitation (nm)
Intensity
Emission (nm)
Vk(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Vk(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Vk(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
80
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450480510540570600
Excitation (nm)
Intensity
Emission (nm)
Vk(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(F)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450480510540570600
Excitation (nm)
Intensity
Emission (nm)
Vk(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Vk(F)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450480510540570600
Excitation (nm)
Intensity
Emission (nm)
Vk(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
81
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(S) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sva(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(S)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sva(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(S)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sva(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
82
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sva(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(F)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sva(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Sva(F)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Sva(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
83
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(S) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Bir(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(S)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Bir(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(S)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Bir(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
84
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Bir(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(F)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Bir(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Bir(F)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Bir(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
85
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(S) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Skj(S)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(S)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Skj(S)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(S)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Skj(S)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
86
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(F) 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Skj(F)90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(F)‐4h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Skj(F)‐4h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240
270
300
330
360
390
420
450
300 330 360 390 420 450 480 510 540 570 600
Excitation (nm)
Emission (nm)
Skj(F)‐20h 90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
240270
300330
360390
420450
0
15
30
45
60
75
90
105
300330
360390
420450
480510540570600
Excitation (nm)
Intensity
Emission (nm)
Skj(F)‐20h90‐105
75‐90
60‐75
45‐60
30‐45
15‐30
0‐15
87
B-3 Wavelength locations (λex & λem), absolute (I) and relative (I/DOC) intensities of peak
A and peak B
Table 9: Locations (λex/λem), intensity (I), relative intensity (I/DOC) and peak ratio (A: B) for the two peaks, A and B, evident fluoresecence EEM spectra obtained for raw water samples and RO isolates with 0h, 4h, and 20h of exposure.
Peak A Peak B Peak
ratio
Sample λex λem Intensity
(I)
Rel. I
(I/DOC)
λex λem Intensity
(I)
Rel. I
(I/DOC)
A:B
Osl(S) 320 440 29.2 7.5 240 430 46.5 12.0 0.63
Osl(S)4h 330 434 22.4 6.2 240 426 40.9 11.3 0.55
Osl(S)20h 310 432 16.3 4.7 240 430 31.5 9.1 0.52
Osl(F) 330 450 28.6 7.9 240 428 48.1 13.3 0.59
Osl(F)4h 330 440 23.5 6.6 240 432 39.5 11.1 0.59
Osl(F)20h 320 424 17.7 5.2 240 438 31.7 9.4 0.56
Hel(S) 330 432 38.0 5.8 240 438 65.3 10.0 0.58
Hel(S)4h 320 426 29.7 4.6 240 428 52.4 8.2 0.57
Hel(S)20h 310 438 23.4 3.9 240 430 43.1 7.2 0.54
Hel(F) 320 442 35.0 5.6 240 428 60.7 9.7 0.58
Hel(F)4h 320 434 28.1 4.5 240 446 51.1 8.1 0.55
Hel(F)20h 310 430 24.8 4.1 240 420 43.7 7.3 0.57
Sto(S) 310 420 49.1 6.7 240 424 82.1 11.2 0.60
Sto(S)4h 320 424 36.2 5.0 240 428 64.4 8.8 0.56
Sto(S)20h 310 428 24.3 3.5 240 432 44.7 6.3 0.54
Sto(F) 320 428 45.7 6.2 240 430 77.1 10.4 0.59
Sto(F)4h 310 424 34.6 4.8 240 432 60.9 8.4 0.57
Sto(F)20h 310 428 24.1 3.5 240 434 46.2 6.7 0.52
Hie(S) 320 424 43.8 6.0 240 432 67.4 9.3 0.65
Hie(S)4h 320 444 37.7 5.3 240 436 63.3 8.9 0.60
Hie(S)20h 320 448 31.1 4.6 240 444 55.6 8.3 0.56
Hie(F) 320 436 43.9 5.1 240 436 56.2 6.5 0.78
Hie(F)4h 320 434 50.2 6.3 240 434 69.7 8.8 0.72
Hie(F)20h 320 450 43.6 6.2 240 426 71.2 10.1 0.61
88
Vk(S) 330 450 53.9 6.5 240 438 71.6 8.6 0.75
Vk(S)4h 330 448 50.3 6.3 240 436 76.9 9.6 0.65
Vk(S)20h 320 432 45.1 6.1 240 434 74.6 10.1 0.60
Vk(F) 330 450 46.4 5.7 240 440 63.4 7.8 0.73
Vk(F)4h 320 430 45.1 5.7 240 442 69.6 8.9 0.65
Vk(F)20h 320 430 38.8 5.5 240 442 65.4 9.3 0.59
Sva(S) 330 450 58.9 5.0 240 434 51.9 4.4 1.13
Sva(S)4h 330 446 73.3 6.2 240 442 81.5 6.9 0.90
Sva(S)20h 330 450 71.3 7.3 240 436 97.2 9.9 0.73
Sva(F) 330 448 29.3 7.2 240 436 43.0 10.6 0.68
Sva(F)4h 320 442 35.3 10.2 240 444 54.9 15.8 0.64
Sva(F)20h 310 434 24.1 8.9 240 430 45.2 16.6 0.53
Bir(S) 330 446 59.0 7.8 240 436 82.9 11.0 0.71
Bir(S)4h 330 442 57.8 7.8 240 446 95.3 12.9 0.61
Bir(S)20h 330 430 35.2 5.6 240 432 68.8 10.9 0.51
Bir(F) 330 446 63.9 7.5 240 436 81.0 9.5 0.79
Bir(F)4h 330 446 67.9 9.4 240 438 104.7 14.5 0.65
Bir(F)20h 330 430 43.7 8.1 240 446 81.9 15.2 0.53
Skj(S) 330 458 47.2 6.2 240 438 58.6 7.7 0.81
Skj(S)4h 320 446 42.6 6.0 240 442 65.0 9.2 0.66
Skj(S)20h 320 440 38.1 6.2 240 444 66.8 10.8 0.57
Skj(F) 330 458 43.9 5.5 240 434 51.7 6.5 0.86
Skj(F)4h 330 442 48.4 6.7 240 440 69.5 9.7 0.70
Skj(F)20h 320 446 40.3 6.6 240 442 71.0 11.5 0.57
B-4 Algal growth response within 10 days for each sampling site (both raw water samples and
RO reference materila): three replicates (one dot), three different exposure time (0h, 4h, and
20h), two differnt season spring (March) and fall (August) were included.
89
Oslo
Helsinki
90
Stockholm
Hietajärvi
91
Valkea-Kotinen
Svartberget
92
Birkenes
Skjervatjern
