w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

Available online at www.sciencedirect.com

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journal homepage: www.elsevier.com/locate/watres

Relationships between molecular weight and

fluorescence properties for size-fractionated
dissolved organic matter from fresh and aged
sources
C.W. Cuss a, C. Gueguen b,*
a
b

Environmental and Life Sciences Graduate Program, Trent University, ON, Canada
Chemistry Department, Trent University, ON K9J 7B8, Canada

article info

abstract

Article history:

Relationships between the molecular weight (MW) and fluorescence properties of dissolved

Received 31 July 2014

organic matter (DOM) are important considerations for studies seeking to connect these

Received in revised form

properties to water treatment processes. Relationships between the size and fluorescence

2 October 2014

properties of nine allochthonous DOM sources (i.e. leaf leachates, grass, and headwaters)

Accepted 6 October 2014

were measured using asymmetrical flow field-flow fractionation (AF4) with on-line

Available online 18 October 2014

absorbance and fluorescence detectors. Correlations between optical properties and MW

were readily apparent using parallel factor analysis (PARAFAC) coupled to self-organizing

Keywords:

maps (SOM): protein/polyphenol-like fluorescence (peaks B and T) was highest at lower

PARAFAC-EEM

molecular weights (<0.5 kDa), fulvic/humic-like fluorescence (peaks A, C, and M) was

Principal component analysis (PCA)

highest at mid-weights (0.5e1 kDa), and humic-like fluorescence (Peaks A C) was highest

Molecular weight distribution

at larger molecular weights (>1 kDa). Proportions of peaks B, T, and A C were significantly

(MWD)

correlated with MW (p < 0.001). The first principal component (PC1, 42% of variation in

Self-organizing maps (SOM)

fluorescence properties) was a significant predictor of sample MW (R2 0.63, p < 0.05),

Asymmetrical flow field-flow frac-

while scores on PC2 (27% of total variance) traced a source-based gradient from deciduous

tionation (AF4)

leachates/headwaters through to coniferous leachates/headwaters. PC3 (13% of var.) was

Supramolecular assemblies

also correlated with MW (p < 0.005). A secondary peak in peak T fluorescence was associated with larger size fractions in aged sources, and scores on PC1 also traced a path from
the leachates of fresher leaves, through more humified leaves, to headwaters. Findings are
consistent with the hypothesis that the structure of aged DOM arises through supramolecular assembly.
2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Dissolved organic matter (DOM) is a complex, heterogeneous,

and polymorphous mixture in all natural waters that

* Corresponding author. Tel.: 1 705 748 1011; fax: 1 705 748 1625.
guen).
E-mail address: celinegueguen@trentu.ca (C. Gue
http://dx.doi.org/10.1016/j.watres.2014.10.013
0043-1354/ 2014 Elsevier Ltd. All rights reserved.

functions in several important roles, such as: a primary source

of nutrients and energy for microorganisms (Kirchman, 2003),
binding to heavy metals and pollutants to control their
 guen and Dominik, 2003), and
toxicity, transport, and fate (Gue
generating carcinogenic disinfection by-products (DBP) during

488

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

water treatment (Reckhow et al., 2004; Beggs and Summers,

2011; Hur et al., 2012).
The application of chemometric methods such as parallel
factor analysis (PARAFAC) (Stedmon et al., 2003) and selforganizing maps (SOM) (Kohonen, 2001; Bieroza et al., 2012a)
to fluorescence excitation-emission matrices (EEM) (Coble,
1996) has allowed the connection of fluorescence properties
to the effectiveness of DOM as: a source of nutrients and energy for microorganisms (Wickland et al., 2007; Fellman et al.,
guen, 2012a), a metal-binding agent (Cuss
2008; Cuss and Gue
 guen, 2012a; Yamashita and Jaffe
, 2008), a fingerand Gue
print for DOM origins and end members (Stedmon and
guen, 2013), a tracer for the
Markager, 2005a; Cuss and Gue
production and degradation of autochthonous DOM (Stedmon
and Markager, 2005b), and a generator of DBP (Beggs and
Summers, 2011; Bieroza et al., 2012a; Pifer and Fairey, 2012).
The size properties of DOM have also been connected to its
source (McElmurry et al., 2013), its effectiveness as a binding
agent for metals (Benedetti et al., 2002; Wu et al., 2004; Chen
et al., 2013), and its DBP formation potential (Hur et al.,
2012). Thus, both the fluorescence and size properties of
DOM are related to its functionality; however, little is known
about relationships between its size and fluorescence properties. The existence of such relationships seems likely given
that the absorbance properties of DOM are frequently used as
a proxy for molecular weight, fluorescent DOM is a subset of
absorbing DOM, and fluorescence is a more sensitive analytical method. Fluorescence also potentially offers a more
finely-grained distinction of DOM constituents via the separation of fluorescence EEMs using PARAFAC; however, the
definitive attribution of underlying chemical species to fluorescence regions remains a challenge (Aiken, 2014). Alternatively, the use of absorbance-based proxies for molecular
weight is based on a greater proportion of underlying chemical species (i.e. all absorbing species) which potentially offers
more information, but less resolving power compared to
EEMs-PARAFAC. However, there are absorbance- and
fluorescence-based proxies that are based on similar chemical
properties that have been associated with molecular weight.
For example, there is a correlation between molecular weight
and absorption at longer wavelengths based on aromaticity
(Chin et al., 1994; Peuravuori and Pihlaja, 1997), which has
been related to molecular weight via the spectral slope (Helms
guen and Cuss, 2011). At the same time,
et al., 2008; Gue
increasing signal in the 'humic-like' fluorescence region has
been associated with increases in relatively aromatic, humic
materials and shifts to longer wavelengths via the humification index (HIX; Zsolnay et al., 1999). Correlations between HIX
and the spectral slope, and between HIX and both molecular
weight and specific UV absorbance (SUVA) have also been
noted, where SUVA is a strong indicator of aromaticity
(Weishaar et al., 2003; Hur and Kim, 2009; Chen et al., 2011).
Tacit indications of relationships between the size and
fluorescence properties of DOM have been suggested by
studies using ultrafiltration (UF) (Liu et al., 2007; Huguet et al.,
guen et al., 2013), size2010; Caron and Smith, 2011; Gue
exclusion chromatography (SEC) (Her et al., 2003; Maie et al.,
 guen and
2007; Romera-Castillo et al., 2014), and AF4 (Gue

 guen, 2012b, 2013). However, efforts

Cuss, 2011; Cuss and Gue
have focussed upon relatively few discrete fractions or samples, single fluorophores and wavelength pairs, or a narrow
range of sizes. Assessments of relationships have also been
primarily descriptive, owing in part to the lack of available
chemometric methods. Consequently, knowledge about relationships between the size and fluorescence properties of
DOM, and how these relationships may change during
biogeochemical cycling, remains limited.
Bioavailability has been positively correlated to the proportion of peak B and peak T fluorescence in DOM (Wickland
guen, 2012a;
et al., 2007; Fellman et al., 2008; Cuss and Gue
Balcarczyk et al., 2009), and it has also been suggested that
the size/structure of larger, more recalcitrant, humified, and
aromatic DOM in part arises from molecular associations that
follow the biodegradation of labile, relatively small constituents (Wershaw, 2004; Sutton and Sposito, 2005; Wickland
et al., 2007; Hur et al., 2009; Cory and Kaplan, 2012). Indeed,
the results of a recent study using size exclusion chromatography and four freshwater samples suggest that the structural
and fluorescence characteristics of DOM may be controlled by
such molecular assemblies, and emphasize the importance of
assessing the optical properties of DOM over the size continuum for understanding its processing and structure (RomeraCastillo et al., 2014). The possibility of such controls on the
structure of biologically processed DOM, with a corresponding
relationship between size and optical properties, is of interest
in drinking water treatment systems that incorporate biological processing. In particular, it has been shown that DOM
leached from different leaf species has different DBP formation potentials that are modified by microbial processing
(Reckhow et al., 2004; Chow et al., 2009; Pellerin et al., 2010;
Beggs and Summers, 2011; Hur et al., 2012).
In the present study, relationships between the size and
fluorescence properties of nine DOM sources that span a range
of humification were investigated using AF4 coupled to
absorbance and fluorescence detectors: three headwaters,
leachates from the senescent leaves of five tree species, and
the partially humified leaves of one species. Results were
effectively visualized by combining PARAFAC and SOM. Principal component analysis (PCA) was used to extract latent
variables from the fluorescence composition of size fractions.
Latent variables were in turn used to predict sample molecular weights using only the fluorescence properties thereof.

2.

Materials and methods

2.1.

DOM samples

A variety of leachates and freshwater samples were chosen in

order to span the ranges of molecular weights, optical properties, and degree of humification typically encountered in
headwaters (Table 1). Fresh senescent leaves or needles were
gathered from the ground beneath, or shaken from the
branches of three different tree species: A. rubrum (senescent
red maple gathered from ground, source SRM), A. saccharinium
(silver maple, shaken from branches; SSM), and P. glauca

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

(white spruce, shaken from branches; SWS). Senescent and

browned reed canarygrass (P. arundinacea; SRC) was also
leached, as were overwintered leaves collected from beneath
an A. saccharinium after being exposed to freezeethaw cycles
over winter, and leached by spring melt (source OSM). Finally,
a leaf sample (STL) was collected by stripping senescent needles from a Tamarack (L. laricina). The procedures used for
storing and leaching leaves followed those outlined in earlier
 guen, 2012a, 2013).
studies (Cuss and Gue
Headwater samples were collected from three locations
just after the peak of spring freshet (May/June 2013): seasonal
meltwater at the perimeter of a cattail marsh dominated by
cattails, tussock sedge, and reed canarygrass (source WW;
Peterborough, ON., 44.34 N, 78.36 W), and two vernal headwaters with sub-catchments dominated by deciduous (DHW)
and coniferous (CHW) vegetation (Dorset, ON., 45.14 N,
79.092 W and 45.14 N, 79.090 W, respectively) (Figure S1). The
coniferous site was heavily influenced by grass immediately
upstream of the sampling point (site photographs are shown
in Figure S1). Headwater samples were taken just after the
peak of spring freshet, so that the primary source was snowmelt running across the surface of the soil.
The DOM content of leachates and headwater samples was
isolated by vacuum filtration using combusted 0.45-mm glassfibre filters, and filter-sterilized using rinsed, 0.2-mm polycarbonate filters (Millipore). The resulting DOM samples were
refrigerated at 4  C in combusted (450  C for 5 h) amber-glass
vials prior to analysis, which was completed within seven
days of leaching/collection.

2.2.

AF4-DAD-EEM

The methods and apparatus used for on-line size fractionation with UVeVisible and fluorescence detection are outlined
guen and Cuss, 2011). Two
in detail in an earlier study (Gue
millilitres of each sample was injected into the 0.3-mL sample
loop of an asymmetrical flow field-flow fractionator (AF2000
Focus; Postnova Analytics), equipped with a polyethersulfone
membrane (300-Da molecular weight cut-off; Postnova), and

Table 1 e Peak-maximum molecular weights (Mp) and

origins of DOM sources.
Source

Origin

Senescent white spruce (SWS) Tree branches

Senescent red maple (SRM)
Ground under
tree (in autumn)
Senescent silver maple (SSM) Shaken from tree
Senescent
Tree branches
tamarack/larch (STL)
Over-wintered silver
Ground under tree
maple (OSM)
(in spring)
Wetland (WET)
Spring headwater
Deciduous-dominated
Spring headwater
headwater (DHW)
Coniferous-dominated
Spring headwater
headwater (CHW)
Senescent reed
Senescent/dying
canarygrass (SRC)
grass (in autumn)

Mean Mp 95%
CI (kDa)
0.42 0.03
0.50 0.04
0.48 0.03
0.43 0.03
1.05 0.04
1.12 0.04
1.48 0.13
1.71 0.06
2.06 0.12

489

coupled to on-line diode array and fluorescence detectors

(Shimadzu SPD-M20A and Agilent 1200 series FLD model
G1321A, respectively). In this study a lower cross flow rate was
applied to achieve higher resolution in the low molecular
weight range, necessitating changes in other flow rates to
optimize focussing position and maintain channel pressure at
<15 bar. Instrument settings were as follows: 7-min. focussing
stage with a tip flow of 0.15 mL min1, cross flow of
2.2 mL min1, and detector flow of 0.4 mL min1; 13-min.
elution stage with a tip flow of 2.6 mL min1 and a cross flow of
2.2 mL min1 throughout. Longer focussing times were also
tested to ensure that samples were fully focussed. Similar
separation conditions have shown improved resolution in the
guen, 2012b, 2013;
low molecular weight range (Cuss and Gue
Neubauer et al., 2013; Stolpe et al., 2014).
To facilitate the direct comparison of DOM properties for
all samples at constant pH and ionic strength, and to replicate
the conditions of local headwaters, the carrier fluid was
adjusted to a pH of 6 using NaOH (SigmaeAldrich), and an
ionic strength of 120 mS cm1 using NaCl (Caledon Laboratories). A blank sample of carrier fluid was run before each
sample to ensure the absence of memory effects. Calibration
was conducted at the beginning and end of each analysis day
by injecting a mixture of macromolecules with molecular
guen and
weights spanning a range of 0.48e14.3 kDa (Gue
Cuss, 2011; 2012b; 2013). A sample calibration curve is provided in the supplementary information (Figure S2). The mean
R2 value for the calibration curves in this study was
0.993 0.006 (n 36). All fractograms (i.e. the absorbance
profile measured at a wavelength of 254 nm over a complete
size fractionation) were converted to molecular weight distributions (MWD) using the averaged slope and intercept from
the calibration curves measured on the same day. The molecular weight at peak maximum (Mp) of each sample was
estimated by applying both calibration curves to the retention
time of the fractogram peak maximum (Fig. 1). The reported
MWD and Mp of each source were each measured twice (n 2).
The fluorescence properties of each DOM sample were
measured at 8 to 11 points along its MWD, at which EEMs were
subsequently measured (Fig. 1). To measure each EEM, an
additional 2-mL aliquot was fractionated, and the on-line flow
was stopped. The retention times of EEMs were chosen to
adequately span each MWD, which differed by source. Thus,
the molecular weight of the size fractions (SFMW) at which
EEMs were measured was not the same for different sources.
The complete list of SFMWs for all EEMs is shown in Table S1.
EEMs were generated in S/R mode over excitation/emission
(Ex/Em) wavelength ranges of 200e450/280e600 nm in increments of 1 and 5 nm, respectively. The wavelengths of the
fluorescence
detector
were
calibrated
using
the
manufacturer-recommended procedure.
To demonstrate the resolving power achieved using the
current fractionation method, a single leaf leachate (S. bebbiana) was fractionated in quadruplicate, and its fluorescence
properties were measured over the complete size spectrum
using multi-emission scanning (Figure S3). For each multiemission scan the excitation wavelength was held constant
(230, 275, 310, and 370 nm), while emission was scanned from
Ex50e550 nm. EEMs were also measured at both Mp for
comparison. Animations demonstrating MWDs and stopping

490

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their optimization. The SOM algorithm was applied to proportional PARAFAC loadings and log-transformed SFMW
using the SOM toolbox in Matlab R2010a by adapting the
tutorial of Bieroza et al. (2012b). To eliminate, source-specific
clustering caused by differences in the fluorescence compoguen, 2013), component
sition of leaf leachates (Cuss and Gue
proportions and log-SFMWs were standardized on [0e1] by
source according to the following formula:
N

Fig. 1 e Molecular weight distribution of sample WW

normalized to absorbance at Mp, showing EEM stopping
points on days 1 (d, ;) and 2 (- - -, 6).

points with corresponding EEMs modelled using PARAFAC

components are provided as visualization aids (Figure S4).

2.3.

Data analysis

The number, identities, and relative concentrations of independent fluorophores in EEMs were assessed using parallel
factor analysis (PARAFAC) in Matlab R2010a (MathWorks) according to the procedure outlined in the DOMFluor toolbox
(Stedmon and Bro, 2008). A total of 81 EEMs were preprocessed by subtracting the EEM of Milli-Q water measured
on the same day, and normalizing the fluorescence intensities
to Raman units (r.u.) using the area under the Raman peak of
the Milli-Q EEM at an excitation wavelength of 350 nm
(Lawaetz and Stedmon, 2009). Excitation wavelengths
<210 nm were removed due to a low signal/noise ratio. Blank
subtraction, Raman normalization, and the removal of scatter
and wavelengths <210 nm were conducted using an in-house
modified, instrument-optimized version of the EEMCut algo guen, 2012a). To assess the relationship
rithm (Cuss and Gue
between molecular weight and total fluorescence composition, the loadings of PARAFAC components were transformed
into proportions of total fluorescence (i.e. loading of each
component divided by the sum of loadings for all components
in the same EEM).
Kohonen's self-organizing maps (SOM) (Kohonen, 2001) are
a type of artificial neural network originally developed for
pattern recognition in artificial intelligence research. Due to
their excellent visualization capabilities, SOM are increasingly
employed in situations involving complex data distributions,
structures, and interrelationships. For example, SOM have
been used to assess the optical properties of DOM removed in
water treatment (Bieroza et al., 2012a), to detect diesel
contamination by distinguishing DOM fluorescence (Carstea
et al., 2010), and in several diverse water resource applications (Kalteh et al., 2008, and references therein). Please refer
to the supplementary information for details about SOM and

X  minX
maxX  minX

(1)

where N is the normalized value of the variable X (either

SFMW or the proportional fluorescence of PARAFAC components). Hence, the patterns displayed by SOM were limited to
correlations between molecular weight and fluorescence
composition, as projected onto a two-dimensional surface.
The strength of the relationships between PARAFAC component proportions and SFMW was also formally assessed using
Spearman's rank-order correlation coefficient (Statistica 8,
StatSoft).
Principal component analysis (PCA) was applied to SFMW
fluorescence compositions using the 'princomp' function in R
version 2.15.0 (R Foundation for Statistical Computing). In
preparation for PCA, the centred-log-ratio transformation was
used to eliminate spurious correlations that could be caused
by the constant-sum constraint (Pawlowsky-Glahn and
Egozcue, 2006). The scores produced by applying the loadings of the first principal component to fluorescence compositions at Mp were assessed as linear predictors for molecular
weight by regressing them against molecular weights at Mp.
Where EEMs were not measured exactly at Mp, distanceweighted averages of loadings from adjacent EEMs were
used (e.g. Fig. 1).

3.

Results and discussion

3.1.

Molecular weight and PARAFAC

The Mp of DOM samples spanned a broad range: from

0.42 0.03 kDa for SWS, to 1.48 0.13 kDa for DHW, to
2.06 0.12 kDa for SRC (Table 1). These Mp were similar to those
previously measured for leaf leachates (0.31e3.9 kDa) (Cuss and
guen, 2013), and for DOM from fluvial and lacustrine enviGue
ronments (0.68e1.95 kDa; Zanardi-Lamardo et al., 2004;
 guen and Cuss, 2011; Pifer et al., 2011; Gue
guen et al.,
Gue
2013). In agreement with the current study it has been previously found that the molecular weight of the DOM leached
from senescent grass was greater than that leached from leaves
using size-exclusion chromatography (Scully et al., 2004), and
more than 70% of the DOM found in the leachates of each of
three wetland grasses had a molecular mass > 1 kDa, as
assessed by ultrafiltration (Osborne et al., 2007). The MWD of
leachates with higher Mp were also skewed further towards the
higher MW range, indicating that a greater proportion of
absorbing DOM was in larger size fractions.
Specific excitation/emission wavelength maxima and full
EEMs of the six components detected by PARAFAC are shown
in Table 2 and Figure S5, respectively. Six EEMs measured at

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

Table 2 e Excitation and emission wavelengths (in nm) of

primary (and secondary) maxima of PARAFAC
components.
Component
C1
C2
C3
C4
C5
C6
C7

Excitation

Emission

Peak

<250 (279)
<250 (326)
<250 (276)
265 (382)
<250 (315)
258 (346)
(<250) 329

315
420
340
470
430
395
460

B
A
T
AC
M
N*
C

the tails of MWDs (i.e. SWS and SFMW 7.72 kDa; SRM, 3.11 kDa;
SSM, 2.87 kDa; STL, 2.33 kDa; SRC, 16.2 and 28.5 kDa) had
loadings of zero on all PARAFAC components, indicating that
fluorescence was below the detection limit. These EEMs were
removed prior to subsequent analysis. The spectra of PARAFAC components were similar to those widely observed in
water samples (Coble et al., 1998; Stedmon and Markager,
2005a,b; Fellman et al., 2008), leaf leachates (Wickland et al.,
2007; Fellman et al., 2010; Beggs and Summers, 2011; Cuss
 guen, 2012a, 2012b; 2013), and products of microbial
and Gue
activity (Li et al., 2008; Andrade-Eiroa et al., 2013). Component
C6 (Ex/Em 258(346)/395 nm) was similar to peak N (Ex/
Em 280/370 nm; Coble et al., 1998), but was blue-shifted in
excitation and red-shifted in emission wavelengths. Hence,
C6 has been named peak N* herein to emphasize these
differences.
Fluorescence compositions at Mp were distinct by litter species and age (Fig. 2), in agreement with prior studies of leaf
guen, 2013; Wong
leachates (Wickland et al., 2007; Cuss and Gue
and Williams, 2010). Senescent leachates were dominated by
components C1 and C3 excepting SRM, which was dominated by
C5. The DOM samples isolated from headwaters (CHW, DHW,
WET) were dominated by C2 and C4. Component C1 was absent
from WET and C6 was absent from CHW, while DHW contained
small amounts of both components. Leachates SRC and OSM
were also dominated by C2 and C4, but were distinguished from
headwaters by greater amounts of C1, C3, and C6.

3.2.

Self-organizing map

Relationships between fluorescence composition and molecular weight were clearly and vibrantly demonstrated by the

491

SOM (Fig. 3). Each hexagon is labelled with the sources and
size fractions that were the most similar to the corresponding
map vector (Fig. 3A), alongside the resulting distributions of
source-normalized PARAFAC component proportions and
size-fraction molecular weight (SFMW; Fig. 3B). The lowest
proportion of each component and molecular weight for each
DOM source are coloured blue, and the highest values are red.
Thus, source EEMs measured at the lowest MW are clustered
in the bottom left corner of the map (i.e. '-2'), while those
measured at the highest MW are in the upper right corner (i.e.
'-10'). The relative amount of each PARAFAC component for
each source and size fraction can be assessed by visually
overlaying the hexagon containing the EEM of interest in
Fig. 3A and the SOM components of interest in Fig. 3B. For
example, EEMs SSM-2 and SSM-9, (lower left corner and upper
right corner of Fig. 3A), respectively contained the highest and
lowest amounts of C3 in sample SSM because the lower left
corner of the map for C3 is dark red, and the upper right corner
is dark blue. In kind, visually overlaying the variable distribution plots for SFMW, C1, C3, and C4 in Fig. 3B reveals that
PARAFAC components C1 and C3 were negatively correlated
with molecular weight, while C4 was positively correlated
(p < 0.001 for all).
A shoulder in the middle of the distribution map for
component C3 was associated with distributional differences
for two groups of leachates (Fig. 3B, circled). Despite consistent decrease with increasing MW, the proportion of C3
remained relatively high at all MW in the first group (SSM, STL,
SWS; Mp < 0.50 kDa for all), so that its normalized value
remained high even at higher MW (e.g. at MW 1.1 kDa, SSM7 had normalized MW and C3 0.53 and 0.84, respectively). In
the second group of larger and more aged DOM (headwaters,
OSM, SRC; Mp > 1.0 kDa for all), the central shoulder corresponded with a secondary maximum in the proportion of C3
in the range of 1.3e2.5 kDa (e.g. at MW 2.4 kDa, SRC-7 had
normalized MW and C3 0.32 and 0.50, respectively). The
EEMs of the second group were distributed around the edges
of the map as C3 initially decreased with increasing MW,
rather than running through the centre like group 1. The
second group then re-converged towards the centre of the
map for 1e2 EEMs that were associated with the secondary
maximum of C3 (see also animations in Figure S4). Peak T
fluorescence similar to C3 has been previously associated with
aged DOM of different molecular weights in wetland,

Fig. 2 e FDOM compositions at sample Mp, described in terms of PARAFAC components C1-7 (darkest e lightest). Source
abbreviations are shown in Table 1.

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w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

Fig. 3 e Self-organizing map showing distributions of (A) size fractions, and (B) corresponding molecular weights (SFMW)
and proportions of PARAFAC components C1 e C7 normalized by source: red denotes the largest proportion in each source,
and blue the lowest. Molecular weight increases by number (i.e. lowest SFMW for each source is -2 and highest is -10).
Source abbreviations are shown in Table 1. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article).

estuarine, and marine systems using size exclusion chromatography (Maie et al., 2007; Romera-Castillo et al., 2014), cross
flow ultrafiltration (Liu et al., 2007), and AF4 (Stolpe et al.,
2014). It has been suggested that this secondary maximum
in peak T fluorescence may arise as a consequence of protein
encapsulation by humic substances in the molecular aggregation that follows the diagenetic processing of fresh DOM
(Wershaw, 2004; Sutton and Sposito, 2005; Tomaszewski et al.,
2011; Romera-Castillo et al., 2014). The differences observed in
the distribution of C3 between fresh leachates and larger,
DOM sources dominated by peaks A, C, and A C also supports this claim.
The distributions of PARAFAC components C2 and C6 were
bimodal, with maxima in the map corners (Fig. 3B). Accordingly, the SFMWs of sources dominated by C2 (CHW, DHW,
SRC, WET) increased around the map edges from the bottomleft to the upper-right corner, distinguishing them from the
peak-T-dominated sources in the middle (Fig. 3A, B). The
maxima of C2 were thus associated with size fractions of intermediate molecular weights (~1 kDa). One maximum of
component C6 (i.e. in the upper right corner of the map)
exhibited significant positive correlation with MW for SSM,
STL, and SRC (p < 0.001), and exhibited an increasing trend
with OSM, SRM, and SWS. The other maximum of C6 was
negatively correlated with MW for the wetland sample (WET;
p < 0.001), but composed only  5% of the fluorescence
signature in all WET EEMs.
Components C5 and C7 also exhibited source-dependent
trends, as suggested by their partitioned distributions
(Fig. 3B). C5 was negatively correlated with the MWs of SWS
(p < 0.05) and SRC (p < 0.005), and exhibited maxima at medial

MWs for SRM, STL, SSM, and CHW. Component C7 was positively correlated with MW for SSM and SRC, and negatively
correlated for WET (p < 0.001 for all). Component C5 has been
previously associated with the mid-ranged size fraction cenguen, 2012b).
tred at 1.6 kDa in leaf leachates (Cuss and Gue
However, this fluorophore has also been associated with a size
fraction <0.50 kDa in fresh and marine waters using ultrafiltration (Huguet et al., 2010).
In summary, the relationship between MW and the proportion of PARAFAC components was consistent across all
DOM sources for four of seven components: C1 and C3 were
significantly negatively correlated with MW and C4 was
significantly positively correlated, while C2 exhibited maxima
at medial MW. However, the significant correlation between
C3 and MW did not detect the important small secondary
maximum associated with more aged, larger DOM sources. In
addition to visualizing significant correlations and consistent
relationships with MW, the secondary maximum of C3 was
revealed when the SOM was applied to component loadings.
This illustrates the advanced pattern detection capabilities of
SOM, and its ability to visualize non-linear relationships between variables. SOM visualization also illustrated the
inconsistent relationships between MW and proportions of
C5, C6, and C7, which were significant for some DOM sources.

3.3.

Principal component analysis

The first three principal components (PC1e3) were sufficient

to explain the behaviour of latent variables (82% of total
variance), as determined using a scree plot. Major
(magnitude  0.20) variable loadings on PC1 (42% of variance)

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

493

Fig. 4 e Biplot and component loadings from principal component analysis of size-fraction fluorescence compositions: A
senescent red maple (SRM); - senescent tamarack leachate (STL); : senescent silver maple (SSM); deciduous headwater
(DHW); over-wintered silver maple (OSM); , wetland (WW); senescent reed canarygrass (SRC); C senescent white
spruce (SWS); B coniferous headwater (CHW). Major loadings (magnitude 0.20) are shown in bold type.
were positive for C1, C3, and C6, and negative for C2 and C4
(Fig. 4). These loadings reflect the structure and distributions
of variables and samples discussed for the SOM. In PC2 (27% of
variance), C5 and C6 had negative loadings, while those of C2,
C3, and C7 were positive. In PC3 (13% of variance), C4, C6, and
C7 were positively loaded, and were balanced by C1, C2, and
C5. Considered over all EEMs, scores on PC1 and PC3 were
negatively (p < 0.001) and positively (p < 0.005) correlated with
SFMW, respectively, while no such relationship was apparent
for PC2 (p > 0.05). Scores were clustered by source in the biplot
of PC1 and PC2, and also showed trends in MW and source,
respectively (Fig. 4).
Size-dependent trends were investigated further by producing scores for the fluorescence properties of samples at Mp

using the loadings on PC1. Scores thus produced were a significant predictor of Mp (R2 0.63, p < 0.05; Fig. 5A). A very
similar relationship has been previously observed for a
different collection of whole samples and fractionated leaf
 guen, 2013), suggesting that fluoresleachates (Cuss and Gue
cence composition may be a useful estimator of DOM molecular weight. Scores on PC1 also separated aged headwater
sources from fresh leachates owing to the relatively low proportions of C1 and C3 (peaks B and T) in the former, and the
relatively low proportions of C2 and C4 (peaks A and A C) in
the latter (Fig. 5B). These sources were therefore also separated along a continuum of age/humification, since SRM was
collected from the ground beneath the tree, SRC was highly
browned when it was collected, and the other leaves/needles

Fig. 5 e Relationships for latent variables from PCA of PARAFAC loadings from EEMs of size fractions: (A) PC1 scores as
predictors of Mp for different DOM types, and (B) PC2 scores at Mp by DOM origin. A senescent red maple (SRM); - senescent
tamarack leachate (STL); : senescent silver maple (SSM); deciduous headwater (DHW); over-wintered silver maple
(OSM); , wetland (WW); senescent reed canarygrass (SRC); C senescent white spruce (SWS); B coniferous headwater
(CHW).

494

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

were either collected directly from the tree or shaken from the
branches (Table 1). Thus, as DOM aged and increased in MW,
C1 and C3 decreased while C2 and C4 increased. Similar patterns of change have been previously associated with very
similar PARAFAC components during the photo- and biotransformation of tannins and plant biomass leachates
, 2014). Additionally, Maie
(Maie et al., 2008; Chen and Jaffe
et al. (2008) found that leaf tannins exhibited progressively
less fluorescence in regions associated with proteins and
polyphenols (peaks B and T), and more fluorescence in regions
associated with humic material (peaks A and C) during photoand bio-transformation, suggesting that polyphenols were
converted into humic material as DOM ages. Although the use
of distinct fresh and aged sources in this study prevented the
tracking of fluorescence conversions, the common relationships between PARAFAC components in PC1 and the negative
correlation between PC1 scores and DOM age suggest that
these changes in fluorescence may be universally related to
DOM aging and concomitant molecular assembly. Scores on
PC2 traced a gradient from freshly leached deciduous DOM
through to the coniferous headwater (Fig. 5B). Interestingly,
scores for coniferous leachate (SWS) were the most similar to
those of coniferous headwater (CHW, and the loadings for
deciduous leachates (SRM, SSM, STL) were the most similar to
that of deciduous headwater DHW. The wetland water sample
(WW) was central in this gradient, bracketed by the grass and
overwintered leachate (SRC, OSM, respectively). Despite significant correlation between MW and scores on PC3, PC3 was
not linearly related to MW and no relationship was evident
between scores and source. Thus, the latent variables that
were primarily MW/age-related (PC1 and PC3) explained 55%
of the observed variation in optical properties, while variation
that was primarily attributable to source (PC2) accounted for
27%.

3.4.

Implications for water treatment

The finding of similar relationships between molecular weight

and fluorescence for leaf leachates and headwaters in an inguen, 2013) suggests that a
dependent study (Cuss and Gue
consistent relationship between size and fluorescence properties may exist. Such a relationship could arise as a consequence of more or less consistent biogeochemical processing
(e.g. humification causing supramolecular assembly), or simply as a consequence of underlying molecular properties (e.g.
stronger absorption of high-molecular-weight humic-like
DOM at higher wavelengths; Peuravuori and Pihlaja, 1997;
guen and Cuss, 2011). Given that the size and optical
Gue
properties of DOM are related to its potential for forming
carcinogenic disinfection byproducts and its removal efficiency (Beggs and Summers, 2011; Bieroza et al., 2012a; Hur
et al., 2012; Pifer and Fairey, 2012; Lyon et al., 2014), the processes that govern the relationship between size and optical
properties are of particular importance in water treatment
applications that seek to incorporate biological processing. In
particular, it has been found that the disinfection byproduct
formation potential (DBPFP) of DOM with higher levels of
protein-like fluorescence (i.e. peaks B and T) is lower than that
of DOM dominated by humic-like fluorescence (Johnstone
et al., 2009; Beggs and Summers, 2011; Bieroza et al., 2012a;

Hur et al., 2012), and the DBPFP of larger DOM is higher than
that of smaller DOM (Hur et al., 2012). However, peak B and T
fluorescence that is present in relatively large DOM and
encapsulated in humic substances (i.e. following biological
processing) may not impact DBPFP in the same fashion as
exposed proteins, so that protein-like content alone may not
reflect DBPFP. Additionally, the source of protein-like fluorescence is not entirely clear because the Ex/Em region associated with proteins is also associated with smaller
polyphenolics such as tannins that are abundant in freshly
leached material (Aiken, 2014). Indeed, it has been demonstrated that the leachates of fresh litter and older duff from
the same species possess different DBPFPs (Chow et al., 2009;
Beggs and Summers, 2011; Hur et al., 2012); however, in these
studies the DBPFP also differed by species and leachate pretreatment (i.e. exposure to ambient levels of microbes or
sunlight vs. freshly leached). Given the uncertain source and
contribution of protein-like fluorescence, the measurement of
both size and optical properties may be necessary for an accurate assessment of DBPFP.

4.

Conclusions

Significant relationships between the molecular weight and

fluorescence properties of size-fractionated DOM from nine
headwaters and leaf leachates were detected using asymmetrical flow field-flow fractionation coupled to on-line
absorbance and fluorescence detectors. These relationships
were effectively visualized by applying SOM to PARAFACbased fluorescence composition. Principal component analysis of the fluorescence compositions of multiple size fractions revealed latent variables related to molecular weight,
DOM age, and source. The latent variable most associated
with the molecular weight of size fractions was also a significant predictor of overall sample molecular weight given only
the fluorescence properties at Mp (R2 0.63); however, this
linear relationship also described a continuum of DOM
freshness.
Though relationships between optical properties and
source/age were not entirely extricated from sizeefluorescence relationships, the proportion of variation
explained by latent variables that were primarily size-related
(55%) was twice that explained by source -related variables
(27%). Thus, the relationship between the MW of allochthonous DOM and fluorescence composition may be similar to
the strength of the relationship between fluorescence
composition and source. Since fluorescence composition has
been shown to be an effective tracer of DOM source, it may
therefore also be useful as an indicator of molecular weight.
The tight coupling of DOM size and age/freshness apparent in
PC1 suggests that DOM increases in size as it ages. The singular maximum in peak T fluorescence in the leachates of
fresh leaves and secondary maximum in peak T fluorescence
in older, more humic-like DOM suggests that proteins may be
encapsulated as DOM ages, in agreement with the molecular
assembly hypothesis. The possibility of such consistent relationships and processes should be investigated further,
particularly for other DOM sources (e.g. soil-derived, autochthonous, marine, waste-derived).

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 8 7 e4 9 7

Acknowledgements
The authors thank S. Watmough and K. Pinder for assistance
with field sampling at Dorset, and A. McDonough for assistance with the classification of plant species. This work was
funded in part by the Canada Research Chairs program and
the Canadian National Science and Engineering Research
Council (CG). CWC gratefully acknowledges the financial
support accorded by the Canadian Graduate Scholarship
(NSERC). Finally, we thank two reviewers who provided
constructive comments that helped improve our manuscript.

Appendix A. Supplementary data

Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.10.013.

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