Fluorescence characterization of the natural organic matterin deep ground waters from the Canadian Shield, Ontario,Canada

Francois Caron • Karen Sharp-King •

Stefan Siemann • D. Scott Smith

Received: 16 July 2010 / Published online: 19 August 2010

� Akademiai Kiado, Budapest, Hungary 2010

Abstract Deep groundwater samples from a deep bore-

hole in the Canadian Shield, Ontario, Canada, have been

analyzed by fluorometry, to determine the difference in

character of the natural organic matter (NOM) with depth.

This work was done to obtain a set of geochemical char-

acteristics of deep groundwaters at the site. The fluores-

cence signal is a complex signature of excitation and

emission of light from fluorescent molecules which are part

of all natural waters. Fluorescent components have char-

acteristic excitation/emission components, defined as a

humic-like (C1), fulvic-like (C2), and protein-like (C3);

these are found in various proportions in natural samples.

Changes in relative fluorescence intensities of these com-

ponents have been used in the past to determine the origin

and/or processes of the NOM between sampling locations.

In this work, six samples were taken at different depths,

from *108 to 650 m below the surface in the borehole.

The fluorescence signals of the samples showed three main

patterns: (1) the shallower samples (*108, 139 and 285 m)

had a pattern similar to that of surface groundwaters,

dominated by components C1 and C2; (2) the samples in

deep groundwaters (*620 and 650 m) had a weak overall

signal, dominated by component C3; finally (3) the

mid-depth sample (*503 m) had a component pattern

intermediate between the shallower and deeper zones. This

set of data is consistent with other data for the ground-

waters from this borehole, such as chlorinity, suggesting

that the three sampling intervals represent three different

types of groundwaters.

Keywords Fluorescence � Natural organic matter �Deep groundwaters � PARAFAC

Introduction

Natural organic matter (NOM) is a constituent of all types

of natural waters (rain, surface, groundwater, landfills,

marine, etc.). Its role has been linked, among others, to a

change of speciation and transport of metals and radio-

contaminants [1–4], acid–base buffering of natural waters

[5, 6], changes in toxicity and contaminant or nutrient

uptake to biota [7–9], and concerns on the quality of

drinking waters [10–12]. NOM contains molecules of dif-

ferent sizes, with various functional groups and acid–base

properties [5, 13–15]. It is expected that the NOM in

freshwaters and groundwaters contains molecules origi-

nating from the breakdown of carbohydrate residues (cel-

lulose, lignin, etc.), plant residues (chloroplasts, etc.), plus

the breakdown of lipids, fats, and proteins. NOM from

these origins are often called terrestrial (terrigenous) or

allochtonous [11, 13]. The NOM produced from secondary

sources, such as exudates from biological activities,

breakdown of dead biomass, and residues from the build-

up of biomass is often referred to as autochtonous [11, 13].

Most allochtonous NOM in terrestrial waters is refrac-

tory to degradation, as the biodegradable portion contain-

ing sugars is readily assimilated. Because of its plant-based

F. Caron (&) � S. Siemann

Chemistry and Biochemistry Department, Laurentian University,

Sudbury, ON P3E 2C6, Canada

e-mail: fcaron@laurentian.ca

K. Sharp-King

Environmental Technologies Branch, Chalk River Laboratories,

Chalk River, ON K0J 1P0, Canada

D. S. Smith

Department of Chemistry, Wilfrid Laurier University,

75 University Avenue West, Waterloo, ON N2L 3C5, Canada

123

J Radioanal Nucl Chem (2010) 286:699–705

DOI 10.1007/s10967-010-0735-x

origin, the terrigenous material will retain a relatively high

degree of aromaticity from the precursors [13], and hence it

will have a humic-like character. In environments depleted

in sources of organic matter such as in the open sea, almost

all of the NOM is autochtonous [13]. Under these condi-

tions, large molecules making up the NOM are made out of

the build-up of small molecules such as triglycerides,

sugars, amino acids, etc., by condensation, polymerization,

partial oxidation, cross-linking, and other mechanisms.

Historically, the portion of NOM whose chemical

composition is not readily identifiable was referred to as

Humic substances. Humic substances, in turn, have been

operationally defined into 3 groups [13, 14]: (1) Humic

acid, which are soluble in alkaline solutions and can pre-

cipitate upon acidification; (2) Fulvic acid, which is the

aqueous fraction remaining after acidification; and (3)

humin, which cannot be extracted by changes in acid or

base content. Complex schemes, such as column extrac-

tions [16] have been used throughout the years to further

characterize the molecules making up the NOM. This is

useful, but it is time-extensive and large amounts of sam-

ples are required.

Fluorescence spectroscopy has emerged as a leading

technique to characterize NOM [17–19]. A sample is

excited with light, and the signal intensity of the emitted

light is tracked to produce a set of Excitation-Emission

Matrix (EEM). An EEM is analogous to a map of signal

intensity as a function of the excitation and emission of

fluorescing molecules. The interpretation of an EEM can be

simple such as tracking peak maxima [17, 20, 21], to more

sophisticated routines like fluorescence regional integration

(FRI [22]) or multivariate modelling, like parallel factor

analysis (PARAFAC [23]).

The fluorescence technique is fast, non-destructive and

non-invasive, and it can be used to track the origins, changes

and characteristics of NOM in samples from various envi-

ronments [12, 17–22]. Fluorescence has been the method of

choice to isolate and resolve individual and independent

end-member components of the fluorescence EEM [18].

Smith and Kramer [24, 25] have used the technique to

determine metal–NOM interactions in environmental sam-

ples. More recently, the technique has been used in samples

taken in the vicinity of a former radioactive liquid dispersal

area (LDA), to determine the fluorescence behaviour of size-

separated fractions of the NOM [26]. In this work, this

technique is applied in the characterization of deep

groundwaters in the Canadian Shield (CS).

Objectives

The objective of this work is to analyze samples from

a specific borehole in the Canadian Shield using a

fluorescence/EEM technique, to determine the differences

in character of the NOM in deep groundwaters at several

depths. The borehole was selected to transect several

geophysical bedrock features and anomalies, up to a depth

of 704 m. We have also used the PARAFAC routine [26]

to resolve the individual components from the EEMs.

Methodology

Fluorescence

The principles and a review of fluorescence applications in

environmental systems have been known and used for

several decades [17, 27, 28]. Organic molecules containing

aromatic rings normally present in NOM have excitable

electrons in the UV/visible range, and a spontaneous

relaxation rate comparable to non-relative de-excitation.

These conditions for fluorescence are often fulfilled in

aromatic molecules, particularly when NOM contains

quinolines and residues of the amino acids tryptophan and

tyrosine.

Sampling

The deep groundwater samples were taken from the CS

borehole in the fall of 2009. This borehole was originally

drilled in 1979 to intersect several geophysical features and

anomalies in the bedrock of the area. It was left unused

shortly after it was drilled, until it was recovered in 2006. It

was then retrofitted with a Westbay multilevel casing

system, at 12 set intervals between 0 and 704 m from the

surface, for hydrogeologic monitoring and groundwa-

ter sampling. Our samples were taken in early fall of

2009 using pre-cleaned autoclaved stainless steel pipes,

immersed in the borehole at the appropriate depth

(King-Sharp et al. 2010, Unpublished report). It is esti-

mated that the waters in the borehole are well developed at

the time of sampling, with an average of 25 volumes per

interval flushed out.

A list of samples analyzed in this work is given in

Table 1. The depth interval of the samples, organic carbon

(DOC) and the chloride concentrations from this sampling

are also listed in the Table 1.

Analysis and spectral resolution

The samples were analyzed on an Olis RSM 1000 F1

fluorescence spectrophometer with a 150 W Xenon arc

lamp (excitation source) and a DeSa monochromator/

photon counter. The excitation was done at 10 nm scanning

intervals from 250 to 450 nm (10 nm bandpass), and

emission was recorded between 200 and 600 nm (8 nm

700 F. Caron et al.

123

bandpass), using a dual grating monochromator. The

results in the data file were processed using PARAFAC

[23] to isolate user-defined fluorescence components. The

processing was intentionally limited to three components

because: (1) a chemical relevance was meaningless beyond

that point; and (2) the confidence level (given by the per-

cent explained) was high, 96% or better [26].

Standards and benchmarking

Fresh de-ionized water and salicylic acid (10-5 M) were

analyzed with each set of samples. The signal intensities

from the standard were reproducible within ±2% during

the period within which the measurements were made.

Since there are no reference standard materials for NOM,

we used archived shallow groundwater samples (LDA 22)

taken earlier from an adjacent site [26]. These samples

serve as benchmarks to verify the analysis by fluorometry,

the reproducibility between replicates, and the PARAFAC

resolution routine. The replicates from LDA 22, when

resolved, yielded intensities which were reproducible

within ±3.1% for component 1, ±5.0% for component 2,

and ±4.3% for component 3 (5 replicates, 3 components

each).

Results

The fluorescence emission intensity is reported as an

excitation-emission matrix (EEM – see Fig. 1). The EEM

resolved with PARAFAC are shown as a set of excitation/

emission contour maps (Fig. 2). The contours, similarly to

the raw EEM, represent fluorescence (emission) intensities,

with the excitation/emission locations. The resolved

components provide two important pieces of information:

(1) the spectral location of the components, which is rep-

resented by the location of the centroid in the Em/Ex

spectrum; and (2) the intensity of each component

(Table 2). Since the Em/Ex location is specific to a mole-

cule, the 3 different regions can be treated as fluorescent

signals from distinct molecular structures. Knowing that

NOM is comprised of a vast array of different molecules, a

fluorescent component does not necessarily represent a

molecule, but rather similar molecules, or similar structures

in larger molecules. In this work, the components are

treated as distinct from one another.

For both the deep groundwater samples (CS) and the

archived samples (LDA), the components had similar fea-

tures (Fig. 2). Component 1 (C1) is characterized by a broad

region in the higher emission wavelength (400–600 nm),

with a low to moderate fluorescence intensity. It is often

referred to as humic-like [17–19, 26, 29]. Component 2 (C2)

occupies a smaller area than C1, its emission region is blue-

shifted compared to C1, i.e., its centroid is of a lower

emission wavelength. In addition, its excitation wavelength

is shorter than for C1. C2 is referred to as fulvic-like (ibid).

Component 3 (C3) occupies a small area in the UV excita-

tion range, with a relatively intense signal, compared to the

other two components. This signal is similar to that of the

amino acids tryptophan and tyrosine. In the current context,

the C3 signal is related to the build-up and breakdown of

proteins; it is referred to as protein-like (ibid). It should be

noted that the humic-, fulvic- or protein-like terminology

may or may not imply the same chemical molecular struc-

ture and behaviour as implied by the names. The terminol-

ogy is based solely on fluorescence activity.

Table 1 List of samples and collection dates for fluorescence

analysis. The relations of these samples with piezometric depths,

chloride ion concentrations and dissolved organic carbon (DOC) are

given for context (from King-Sharp et al. (2010) Unpublished report)

Interval Dates of samplecollection

Piezometric depth Cl- DOCa

(m from surface) mg/L mg/L C

1 – 67.1 22 3.6

2 29-Sep-09 108.3 58 1.7

3 30-Sep-09 138.8 10 5.3

5 1-Oct-09 285.1 28 1.7

6 – 362.9 160 0.88

8 22-Sep-09 503.2 191 1.2

9 – 533.7 987 0.19

10 – 568.8 1,182 1.5

11 23-Sep-09 620.6 989 1.1

12 24-Sep-09 649.7 1,572 0.05

a DOC is the estimator of natural organic matter

Fig. 1 Raw fluorescence spectrum of one archived shallow ground-

water sample (LDA 22). The raw spectrum shows the excitation

(ex, in nm) and emission (em, also in nm). The signal intensity is in

the third dimension, shown by the intensity contour lines. The

Rayleigh–Tyndall scattering lines (where ex = em and 2 ex = em)

are removed in the data processing routine [26]

Fluorescence characterization of the natural organic matter 701

123

Benchmarking using the archived LDA 22 samples gave

reproducible results for signal intensity (see earlier) and for

the location of the resolved components on an Ex/Em map

(Fig. 2a). The location of the centroid of the three com-

ponents was comparable to our previous work [26], and

other investigators [17–19, 29]. We found similar peaks in

the CS samples (Fig. 2b). The locations of the component

centroids from the CS samples were close to those of the

archived LDA 22 sample, although a small blue shift was

observed for fulvic-like C2, and the region for the protein-

like C3 was expanded towards a higher emission

wavelength.

Individually, the three components C1, C2 and C3 were

found in all the deep groundwater samples (Fig. 3a).

A marked decrease in fluorescence intensity for both the

total and the individual components was noted with

increasing interval depth in the borehole. The signal

intensity of the three components was similar among the

shallower samples (intervals 2, 3 and 5). The signal

intensities for C1 and C2 were lower in the interval 8

sample, compared to the shallower samples, but the C3

signal remained approximately the same. In contrast, the

signal intensities for the 3 components were smaller for the

deep samples (intervals 11 and 12). Compared to all the

other samples, the intensity of C1 and C2 was markedly

lower, and it was moderately lower for C3.

The fluorescence intensity signals for the components

were normalized relative of the total signal, e.g., C1/total,

C2/total, and C3/total (Fig. 3b), to determine if the signal

changes preferentially affect some components. Similarly,

signal ratios were also calculated for the ratio of C1 to C2,

and for allochtonous to autochtonous signal ratios (C3 to

the total of C1 and C2).

This analysis reveals that the shallower samples (inter-

vals 2, 3 and 5) have quasi-identical patterns (Fig. 3b),

suggesting similar components of the NOM. Both deep

Fig. 2 PARAFAC-resolved

spectra for the samples for the

shallow groundwaters from

the adjacent liquid dispersal

area site (a) and the Canadian

Shield borehole samples (b).

The concentric shapes in the

graph represent contour lines, in

arbitrary fluorescence units (the

centre of the shape corresponds

to the highest intensity)

Table 2 Fluorescence

intensity of the PARAFAC-

resolved components of the

Canadian Shield samples, and

replicates from archived

shallow groundwaters (liquid

dispersal area [LDA] 22)

a Replicates measured at the

same time as the deep

groundwater samples

Sample ID Fluorescence intensitya (arbitrary units)

C1 C2 C3

Interval 2 (Sept 29) 27.0 21.0 9.7

Interval 3 (Sept 30) 25.4 20.3 8.7

Interval 5 (Oct 1) 23.4 17.8 8.3

Interval 8 (Sept 22) 14.8 8.8 7.3

Interval 11 (Sept 23) 3.6 1.5 5.6

Interval 12 (Sept 24) 3.5 1.2 5.3

LDA 22 archived – replicate 6a 27.1 18.2 12.2

LDA 22 archived – replicate 7a 26.9 18.5 12.0

702 F. Caron et al.

123

samples (intervals 11 and 12) have similar signal patterns,

whereas the sample from interval 8 at intermediate depth is

different from the two other sets. It is noted that the relative

proportion of component C3 becomes higher with depth. It

is also apparent that the shallower samples (intervals

2, 3 and 5) are similar to one another, as the ratio of humic-

like to fulvic-like components (C1/C2) is slightly higher

than 1.0 for all three shallower samples (Fig. 3c). The same

observations can be made for the archived shallow samples

(Table 2). The deeper samples are distinct from these by

having a high C1/C2 ratio (high proportion of fulvic-like

components), and a high proportion of the protein-like

autochtonous (C3) material, compared to allochtonous

(C1 ? C2) material. The sample in interval 8, at interme-

diate depth, has a distinct character, which is intermediate

from the samples in other intervals.

The overall fluorescence signal intensity decrease with

depth was plotted along with the dissolved organic carbon

(DOC, Fig. 4). The DOC generally decreased with depth,

but the trend is less obvious than for the fluorescence

signal. The decrease in signal with depth was mostly due to

the C1 and C2 components, whereas the C3 intensity was

almost invariant. The fluorescence intensity per unit of

DOC was inconsistent, when this was plotted against depth

(not shown).

Discussion

Even though this set of data from the CS borehole is

fragmented because data is not available for all intervals,

there was a discernable pattern of fluorescence intensity

decrease with depth: the fluorescence character in the

shallower samples (intervals 2, 3 and 5,\285 m) was fairly

similar among the samples, resembling the shallow

groundwaters samples (LDA). The sample from interval

8 had a distinct character from the shallower samples, and

the deep samples (intervals 11 and 12) were also of a

different character from the others. In other words, the

fluorescence signal suggests three distinct types of NOM,

in three different layers of deep groundwaters. The rest of

the geochemical data corroborates with this, for example,

the chloride ion concentrations (Table 1; King-Sharp et al.

(2010) Unpublished report). The increase in salinity is

generally consistent with the geochemical development of

groundwaters at great depths.

The contrasting character of the NOM in the deeper

samples, especially for intervals 11 and 12, might be

related to two factors: (1) the character of the NOM is

different from that of the shallower samples; or (2) the

different salinity and ionic content impacts the signal either

by a change in conformation of the NOM, or by quenching

due to the high salinity.

Changes in fluorescence behaviour with salinity have

been reported before, in which the authors have proposed

empirical relationships [17, 30]. In these two studies, the

0

5

10

15

20

25

30

Inte

nsi

ty (

)

C1 C2 C3

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Rat

io (

)

C1/Total C2/Total C3/Total

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Interv. 2 Interv. 3 Interv. 5 Interv. 8 Interv. 11 Interv. 12

Rat

io (

)

C1/C2 C3/(C1+C2)

(a)

(b)

(c)

Fig. 3 PARAFAC-resolved peak intensities of the Canadian Shield

borehole samples. All samples share the same x-axis, where ‘‘Interv.’’is an abbreviation for interval; (a) absolute values (arbitrary

fluorescence units); (b) ratios of components C1, C2 and C3 to the

total area (unitless); and (c) ratio of components (C1/C2), and

composite C3/(C1 ? C2)

0

10

20

30

40

50

60

70

Interv. 2 Interv. 3 Interv. 5 Interv. 8 Interv. 11 Interv. 12

Interval No.

Flu

ore

scen

ce in

ten

sity

( )

0

1

2

3

4

5

6

7

DO

C (

mg

/L)

Total intensityC1C2C3DOC (right axis)

Fig. 4 Total fluorescence signal intensity and dissolved organic

carbon changes with interval depth

Fluorescence characterization of the natural organic matter 703

123

salinity gradient was compounded with NOM from

different sources (marine and terrestrial origins). The pro-

tein-like fraction decreased to a lesser extent than the

humic-like or fulvic-like component. The authors men-

tioned that these components were cycled at a faster rate

than protein-like components. This is consistent with our

observations between the shallower intervals (2, 3 and 5)

and the deeper intervals (11 and 12); see Fig. 3b, c. There

was a suggestion [30] that bacterial breakdown of terrestrial

fluorophores contribute to the production of protein-like

fluorophores in marine waters, making these fluorophores

recalcitrant. This would decrease the relative intensities of

humic-like and fulvic-like components (C1 and C2), com-

pared to the protein-like component (C3), which is what we

are observing in the deep samples.

The changes in signal intensity with a salinity gradient

may not be a significant variable in our work. Coble [17]

and Kowalczuk et al. [30] have suggested empirical rela-

tionships that predicted a decrease of *4–8% in humic-

like fluorescence intensity per salinity unit. In our work, the

chloride data corresponds to a salinity of *0.05–0.13% for

the shallower samples, to *1.8–2.9% for the deeper

samples. Based on this, one would predict a *8–24%

decrease in the C1 signal due to salinity only. This is much

lower than the six-fold decrease in total intensity observed

in our work (Fig. 4): we are reporting a *eight-fold

decrease for C1 and a *17-fold decrease for C2. The

decrease in C3 was less than two-fold. No decrease of the

protein-like component was reported with salinity [30],

further suggesting that salinity is not a factor in our case.

It was mentioned earlier that the C3 component occu-

pied slightly different spectral regions (Fig. 2): in the

archived LDA sample, the spectral region was typical of

tyrosine, whereas the signal was typical of tryptophan

fluorescence in the deeper CS samples. This difference

could be caused by the degree of denaturation of the

tryptophan-like fluorophores. For example, Burstein et al.

[31] have reported that tryptophan exhibited an emission

maximum at *330–332 nm when it is buried in non-polar

regions of a protein or cell residues. When proteins are

denatured, a portion of the tryptophan is exposed to a polar

solvent (water), resulting in an emission red shift, to

*350–353 nm. One can speculate that this shift towards a

higher emission wavelength in the deep samples (Fig. 2b),

could be due to highly degraded tryptophan-like molecules.

This is also consistent with a local origin and recycling of

NOM.

Conclusions

Deep groundwater samples in the borehole have been

analyzed by fluorometry. Three fluorophores were

observed in the NOM: a humic-like component (C1),

fulvic-like component (C2), and a tyrosine-like component

(C3). These components were ubiquitous but in various

proportions in the samples taken at different depths. The

shallower samples (intervals 2, 3 and 5) had a similar

character to those previously sampled in near-surface

groundwater at an adjacent site, whereas the deeper sam-

ples (intervals 11 and 12) exhibited a marked decrease of

the C1 and C2 components, with a lesser decrease in the C3

component. One sample at intermediate depth (interval 8)

showed a mixed fluorescence character, which is somewhat

transitional and distinct, compared to the shallower and

deeper groundwaters.

The fluorescence spectroscopic approach indicated that

the NOM in shallow groundwater had a character similar to

that of a surface-based source. The NOM in deep

groundwaters has a different character from the other

samples, suggesting that the NOM at deep locations could

be autochtonous, which is the result of local recycling of

NOM.

Acknowledgments The main author wishes to thank the contribu-

tors (who wish to remain anonymous) for funding this project.

Administration of the contract by MIRARCo (Sudbury, ON),

specifically Sherry Greasly (administrative support) and Stephen Hall

(signing authority) is appreciated.

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