Modern fluorescence spectroscopy methods, including excitation–emission matrix (EEM) spectra parsed using parallel factor analysis (PARAFAC) statistical approaches, are widely used to characterize dissolved organic matter (DOM) pools. The effect of soluble reduced iron, Fe(II), on EEM spectra can be significant but is difficult to quantitatively assign. In this study, we examine the effects of Fe(II) on the EEM spectra of groundwater samples from an anaerobic deltaic aquifer containing up to
300 mgL-1 Fe(II), located a few kilometres from the ocean and adjacent to the Fraser River in Richmond, British Columbia, Canada. We added varying quantities of Fe(II) into groundwater samples to evaluate Fe(II)–DOM interactions. Both the overall fluorescence intensity and the intensity of the primary peak, a humic-like substance at excitation and emission wavelengths of 239 and 441–450 nm (peak A), respectively, decreased by approximately 60 % as Fe(II) concentration increased from 1 to 306 mgL-1. Furthermore, the quenching effect was nonlinear and proportionally stronger at Fe(II) concentrations below 100 mgL-1. This nonlinear relationship suggests a static
quenching mechanism. In addition, DOM fluorescence indices are substantially influenced by the Fe(II) concentration. With increasing Fe(II), the fluorescence index (FI) shifts to higher values, the humidification index (HIX) shifts to lower values, and the freshness index (FrI) shifts to higher values. Nevertheless, the 13-component PARAFAC model showed that the component distribution was relatively insensitive to Fe(II) concentration; thus, PARAFAC may be a reliable method for obtaining information about the DOM composition and its redox status in Fe(II)-rich waters. By characterizing the impacts of up to 300 mgL-1 Fe(II) on EEMs using groundwater from an aquifer which contains similar Fe(II) concentrations, we advance previous work which characterized impacts of lower Fe(II) concentrations (less than 2 mgL-1) on EEMs.
Introduction
Fluorescence spectroscopy has been widely used to characterize the properties
of dissolved organic matter as it is highly sensitive to the structures and
functional chemistry of aquatic organic matter (Baker and Spencer, 2004;
Fellman et al., 2010; Helms et al., 2008; Stedmon and Bro, 2008; Weishaar
et al., 2003). In this method, light at a known wavelength (the excitation
wavelength) is passed through a sample, and the absorbance at that frequency
and fluorescence (emission) at other frequencies is measured. Such spectra can
be used to derive commonly utilized fluorescence indices that correlate to
specific forms of organic matter (Aiken, 2014; Coble et al., 2014; Hudson
et al., 2007; Murphy et al., 2013). These indices include the fluorescence
index (FI), which is calculated as the ratio between the emission at
470 nm to that at 530 nm at an excitation wavelength of
370 nm and relates to the concentration of aromatic, microbially
derived lignin-like organic matter (McKnight et al., 2001). An
excitation–emission matrix (EEM) is prepared by systematically repeating the
measurements at a range of different excitation and emission wavelengths.
These measurements are highly sensitive to the structures and functional
chemistry of aquatic organic matter, which determine the unique pattern of
peaks present within the EEM spectra (Aiken, 2014; Coble, 1996; Coble et al.,
2014; Fellman et al., 2010). Due to the complexity of the EEM spectra obtained
from each unique sample, a number of statistical methods have been used to
decompose EEM spectra and relate emission patterns to functional chemistry of
organic matter within a sample. Parallel factor analysis (PARAFAC) is a
commonly utilized statistical means of compartmentalizing EEM spectra into
discrete peaks that may then be compared to broad organic matter classes (Bro,
1997; Chen et al., 2010; Jaffé et al., 2014; Murphy et al., 2013; Stedmon
and Bro, 2008).
It is well accepted that dissolved organic matter (DOM) fluorescence is
quenched or enhanced by interactions with metal ions, including Fe(III) (Ohno
et al., 2008; Poulin et al., 2014; Pullin et al., 2007; Senesi, 1990; Shen et
al., 2020), Fe(II) (Poulin et al., 2014), Al(III) (Ohno et al., 2008), Cu(II)
(Senesi, 1990; Shen et al., 2020), and Hg(II) (Senesi, 1990). Fe(III) is
recognized as an important source of interference for fluorescence
measurements (Ohno et al., 2008; Pullin et al., 2007). Previous studies have
reported a significant quenching effect caused by the binding of Fe(III) to
organic ligands. A possible mechanism that may account for quenching is iron(II)-DOM complexation occurring at the fluorescent sites (Senesi, 1990; Rue
and Bruland, 1995). Such complexes can efficiently decrease the fluorescence
intensity of the fluorophore. Furthermore, the degree of quenching varies
among different organic matter compounds, which increases the complexity and
uncertainty in characterizing and predicting the iron-binding effect across a
range of DOM types (Ohno et al., 2008). However, limited research has focused
on the quenching effect of Fe(II) interference in anoxic groundwater, where
reducing conditions are present. Poulin et al. (2014) first demonstrated that
Fe(II) complexation with DOM decreases fluorescence intensity. Their experiments were only designed to characterize the Fe(II)
quenching effect for surface water with moderately elevated DOM concentrations
(2.3 to 5.0 mgL-1) under low Fe(II) concentrations
(0–1.5 mgL-1). To our knowledge, the extent of fluorescence
quenching in groundwater with higher Fe(II) concentrations is not known.
The fluorescence quenching effect in Fe(II)-rich groundwater is still poorly
understood and warrants further investigation, given the prevalence of high
DOM and Fe(II) in groundwater in deltaic sediments
(Bolton and Beckie, 2011) and sites contaminated
with organics, e.g. from landfills or fuel spills
(van
Breukelen and Griffioen, 2004; Christensen et al., 2001; Heron et al.,
1994). In most instances, these high-Fe(II) groundwaters are found when the
oxidation of organic matter is coupled to solid-phase Fe(III) reduction,
dissolving Fe(II) into groundwater at circumneutral pH. For example, 1.5–10 mgL-1 Fe(II) in groundwater is commonly observed in the organic-rich
groundwaters of the Bengal Basin (Harvey et al., 2002), and up to 90 mgL-1
Fe(II) has been observed in landfill leachate in the Netherlands (van
Breukelen and Griffioen, 2004).
The objective of this study was to assess the influence of high
concentrations of Fe(II) on the fluorescence properties of DOM by titrating
up to 306 mgL-1 (5.4 mM) Fe(II) into groundwater collected from a deltaic
aquifer in Richmond, British Columbia, Canada. This groundwater is
representative of groundwater found in diagenetically immature, organic-rich
deltaic sediments, where Fe(II) concentrations can reach up to 300 mgL-1
(Bolton and Beckie, 2011; Jia, 2015). The biogeochemistry of groundwater at
this site and an analysis of the origin of the extraordinarily high Fe(II)
concentrations are described in Jia (2015). In this study, we identified
the degree of quenching at different Fe(II) concentrations (from 1 to 306 mgL-1) based on the excitation–emission matrix (EEM) regions and peaks. We
fit the EEM spectra to a previously derived 13-component PARAFAC model
(Cory and McKnight, 2005) and calculated commonly used
fluorescence indices to quantify DOM fluorescence properties as a function
of Fe(II) concentration. This study provides a detailed characterization of
the impact of changing Fe(II) concentrations on DOM fluorescence.
The Kidd 2 site is located in the Fraser River delta in southwest
British Columbia.
Plan view of the W3 well location and cross section of the saline
wedge, after Neilson-Welch and Smith (2001).
Deltaic groundwater stock solution
We collected representative deltaic groundwaters from what is known as the
Kidd 2 site, located adjacent to the Fraser River a few kilometres upstream
from its outlet to the ocean, near Vancouver, Canada (49∘11′53.34′′ N, 123∘6′53.25′′ W, Fig. 1), where a near-surface sandy aquifer
is found between 5 and 22 m below ground surface (Jia, 2015;
Bolton and Beckie, 2011). In the anaerobic deltaic aquifer, Fe(II) is
released to groundwater by oxidation of dissolved organic matter (DOM) in a
process that is affected by the circulation of saline ocean water. At the
site, denser, saline ocean water enters the aquifer in the hyporheic zone at
the river bottom, flows inland along the base of the aquifer to a maximum
distance of approximately 500 m inland where it overturns and flows back towards
the river under a regional hydraulic gradient from freshwater recharged
inland (Neilson-Welch and Smith, 2001), forming a wedge of saline water in
the aquifer (Fig. 2). Along the flow path, the saline water mixes with
fresh groundwater. The saline–freshwater mixture eventually discharges to
the river at the top of the saline wedge. Two mixing zones can be identified
along the saline wedge: at the bottom of the wedge, freshwater from the
lower confining silt flows up into the overlying sandy aquifer as the saline
water flows inland (the “lower mixing zone”) and, at the top of the wedge,
terrestrial recharge from inland flows on top of the saline water as it
flows back to the river (the “upper mixing zone”). High concentrations of
Fe(II) are observed along the circulation flow path, especially in the upper
mixing zone, where pore water Fe(II) concentrations peak above 300 mgL-1 (5.4 mM) (Fig. 2) (Jia, 2015).
MethodologySample collection
For the measurements in this study, we collected a single stock solution of
representative natural DOM-containing groundwater, to which we added
(titrated) increasing concentrations of Fe(II). We selected groundwater from
W3-14 (lower mixing zone) at a depth of 20.03 m as the DOM-containing stock solution as it had the lowest Fe(II) concentration (1.3 mgL-1) at the Kidd 2 site (Jia, 2015), allowing us to
explore a large range of Fe(II) concentrations. The multilevel sampling port
consisted of a 0.635 cm inner diameter low-density polyethylene tube with a
5 cm fibreglass-mesh screen (Neilson-Welch and Smith, 2001). Three
tubing volumes of groundwater were purged with a peristaltic pump while pH
was monitored using an OAKTON™ pH/mV/∘C meter in a
sealed flow-through cell to prevent degassing. The pH and temperature
stabilized at 7.44 and 11 ∘C, respectively. The
groundwater was filtered through 0.45 µm cellulose filters and then stored
in a 1 L amber glass bottle with a Teflon-lined plastic cap, without
acidification. The bottle was filled with no headspace and duct tape was
used to further seal the sample and minimize the oxidation of Fe(II). The
collected 1 L stock solution was refrigerated at 4 ∘C until
fluorescence analysis (within ∼14d). Although some
degradation of the DOM may have occurred during the holding period, this
would not significantly affect our conclusions as our intention was to
determine how Fe(II) addition affects the fluorescence properties of DOM rather than to characterize the properties of DOM at the Kidd 2 site. The
stock solution DOM concentration of 10.7 mgL-1 was measured using high-temperature combustion with a HACH™ IL 550 TOC-TN analyser
(detection limit 1 mgL-1) at the Environmental Engineering Laboratory in
The University of British Columbia's
Department of Civil Engineering.
Fe(II) addition experiment and concentration determination
Experimental solutions were prepared from the stock DOM solution in an
anaerobic glovebox (Coy Labs, MI, USA), filled with an N2/H2
mixture (95 % of N2 and 5 % of H2) with a palladium catalyst
inside the chamber, which maintains gaseous O2 levels of less than 5 ppm. The stock DOM solution was de-aerated in the glovebox by purging with
pure N2 for 30 min. The sample addition experiments used glass
cuvettes that were acid-washed with 10 %HNO3 and rinsed with
deionized, distilled water.
As the highest observed Fe(II) concentration in the groundwater at the Kidd 2
site was approximately 300 mgL-1 (Jia, 2015), the Fe(II) addition
experiment was designed for a range of Fe(II) concentrations (from 1 to
300 mgL-1). An Fe spiking solution of 1000 mgL-1
Fe(II) was prepared with FeSO4(H2O)7, following Poulin
et al. (2014), using the DOM stock solution so that spiking with Fe(II) would
not change the overall concentration of DOM. Experiments were performed by
sequentially adding Fe(II) spiking solution to an initial volume of
250 mL of DOM stock solution to reach 10 different concentrations
between 1.3 to 306 mgL-1. Previous analyses of water from W3-14
via ICP-OES (inductively coupled plasma optical emission spectrometry) (Jia, 2015) indicated that the SO42- concentration was
71 mgL-1 and that the Cl- concentration was
1670 mgL-1. Therefore, as the Fe(II) concentration was increased
by a factor of 240 (from 1.3 to 306 mgL-1), the
SO42- concentration only increased by a factor of 8 (from
approximately 71 to 595 mgL-1). We therefore expect that the
dominant effect observed through this addition experiment is the effect of
increasing Fe(II) rather than the effect of increasing SO42- and/or
total anions. The anion and cation concentrations in the experimental spiked
solution were similar to the natural conditions occurring in the aquifer. For
example, for the depths with Fe2+ from 50–435 mgL-1, the
range in SO42- was 13–600 mgL-1, and the range in
Cl- was 50–9600 mgL-1 (Jia, 2015).
If necessary, the pH of the experimental solution was adjusted using 0.1 M
NaOH or 0.1 M HCl (to 7.4 ± 0.3) to match the target pH (7.44) of the
original DOM stock solution. After each Fe(II) addition, 10 mL of the Fe(II)
solution was pipetted into each of two glass cuvettes. One cuvette was
acidified with concentrated HCl to a pH of approximately 2 and used to
determine the total dissolved Fe(II), using a HACH™ DR/2010
spectrophotometer via the colorimetric method (HACH ferrozine method)
(Stookey, 1970). The cuvette of Fe(II) solution used for
fluorescence analysis was capped tightly and transferred out of the glovebox for immediate analysis (Sect. 3.3).
Fluorescence data acquisition and analysis
The fluorescence analysis and the PARAFAC modelling were described by
Ishii and Boyer (2012). All fluorescence spectra were obtained
by using a Horiba Aqualog® (Horiba Scientific, Edison, NJ,
USA) spectrofluorometer, equipped with subtractive double excitation
monochromators (Hansen
et al., 2018). A 150 W ozone-free vertically mounted xenon arc lamp was used
as the excitation source. Both excitation and emission were collected at a
bandpass of 5 nm. Fluorescence intensities, as a function of the excitation
and emission wavelengths, were measured across excitation wavelengths
ranging from 240 to 800 nm in 3 nm increments; emission wavelengths, ranging
from 250 to 830 nm, were measured over an integration time of 0.1 s. Water
samples were analysed in 1 cm quartz cuvettes. Between the samples, the
quartz cuvette was rinsed three times with Milli-Q water, followed by three times
with the sample, to reduce possible cross-contamination. If necessary, water
samples were quantitatively diluted with Milli-Q water until the UV
absorbance was lower than 0.2 units (at 254 nm) to minimize inner filter
effects between the Milli-Q water and the water samples. The EEM spectra for
each sample were obtained by subtracting the Milli-Q (blank) spectra to
eliminate the Rayleigh scatter and water Raman peak (Murphy, 2011). Fluorescence intensity within
all EEM data is presented in Raman units (RU) due to the way that raw EEM
spectra are corrected prior to analysis via PARAFAC modelling or calculation
of associated indices. As per standard practice, raw EEMs were instrument
corrected via software provided by the instrument manufacturer. Spectra were
corrected for inner filter effects (Ohno, 2002) and then normalized to the area
under the Raman curve
(Nieke et al., 1997;
Stedmon et al., 2003); second-order Raleigh scatter and Raman bands were
excised at a bandpass of 12 nm
(Bahram et al., 2006; Zepp et al.,
2004), while first-order Raleigh scatter was excised at a bandwidth of 50 nm
to remove all spectral artefacts (Bro, 1997; Stedmon and Bro, 2008).
Specifically, normalization to the area under the Raman curve (which occurs
due to the inelastic scatter of light by water) contributes to instrument
correction that allows for the comparison of spectra between different
instruments and thus different studies. The overall fluorescence intensity
(OFI) was determined for each sample by adding the fluorescence intensities
across all EEMs (Poulin
et al., 2014). The relative fluorescence (OFI/OFI0) can be used to
quantitatively determine the quenching effect of Fe(II)
(Poulin et al., 2014),
where OFI and OFI0 represent the Fe(II) addition samples and the
original groundwater sample, respectively. Similarly, the intensity of the
primary peak (peak A), at an excitation wavelength of 239 nm, and at broad
emission wavelengths ranging from 380 to 460 nm, was determined for each
sample, and the parameter A/A0 was used to quantify the Fe(II)
quenching effect on this diagnostic peak.
The established 13-component PARAFAC model of Cory and McKnight
(2005) was used to fit the EEM spectra within this study. The 13
components consist of seven quinone-like fluorophores, including three
oxidized quinones (Q1, Q2, and Q3), four reduced quinones (SQ1, SQ2, SQ3,
and HQ), two amino acid-like components (tryptophan and tyrosine), and four
remaining unknown fluorophores (Cory and McKnight, 2005). We
chose to use this robust pre-resolved model, which was developed using DOM
from a wide range of aquatic environments and has been subsequently applied
to interpret EEMs from a large variety of aquatic systems
(Jaffé
et al., 2008; Larsen et al., 2010); see Sect. 3.3 for further details. The
use of this 13-component model also facilitates the derivation of the redox
index (RI), calculated by summing the reduced quinone-like inputs over total
quinone-like inputs from components within the model. Finally, derivation of
a unique, site-specific PARAFAC model typically requires a large sample set
composed of samples from a common organic matter context
(Cory and McKnight, 2005; Ishii and Boyer, 2012). As the
aim of this study was to capture how spectral attributes are quenched upon
addition of Fe(II) rather than characterization of the underlying organic
matter properties, the application of a pre-resolved model ensures that
model fitting is not biased by Fe(II) addition.
To ensure that this 13-component model adequately represented the
fluorescent organic matter characteristics within the sample set, the
residual fluorescence remaining after the model was applied was plotted and
analysed. No systematic residuals were found after fitting the EEMs to the
PARAFAC model, suggesting that the model was able to represent the samples
and that Fe(II) additions did not significantly change the structure of
fluorophores in the groundwater stock solution from the Kidd 2 site. The
abundance of each fluorophore was quantified based on its relative
contribution (%) to the total fluorescence. Additionally, commonly used
fluorescence indices, including fluorescence index (FI) (Cory and
McKnight, 2005), humification index (HIX) (Ohno, 2002; Parlanti et al.,
2000), the redox index (RI) (Miller
et al., 2006), and freshness index (FrI, β/α)
(Parlanti et al., 2000; Zsolnay et al.,
1999) were also quantified to provide further DOM characterization (Sect. 4.1.3). The dataset from this study is available on Zenodo
(Jia et al., 2020).
ResultsThe effect of Fe(II) quenching on EEM fluorescenceRelative fluorescence intensity (OFI/<inline-formula><mml:math id="M95" display="inline"><mml:mrow class="chem"><mml:msub><mml:mi mathvariant="normal">OFI</mml:mi><mml:mn mathvariant="normal">0</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>)
Figure 3 shows that the fluorescence intensities of EEMs decrease with
increasing Fe(II), indicating that the DOM fluorescence of the groundwater
stock solution collected from the Kidd 2 site was quenched by the addition
of Fe(II). Figure 4a presents the decrease in the relative fluorescence
intensity (OFI/OFI0) as Fe(II) increases from 1 to 306 mgL-1.
Approximately 60 % of the fluorescence intensity found in the 1 mgL-1
Fe(II) experimental solution was quenched in the 306 mgL-1 Fe(II)
experimental solution. The fluorescence intensity decreased more rapidly at
lower Fe(II) concentrations: as Fe(II) increased from 1 to 101 mgL-1 the OFI
decreased by ∼40% and as Fe(II) increased from 101 to 306 mgL-1 the OFI decreased by an additional ∼20%. The
magnitude of quenching effect was more pronounced in this study than that
performed by Poulin et al. (2014), who observed nonlinear fluorescence
quenching (7 % to 23 %) in four different surface water samples, by
addition of Fe(II) up to 1.5 mgL-1, significantly lower than the Fe(II)
concentrations used in this study.
Excitation-emission matrices (EEMs) of groundwater from Fraser
River aquifer over a range of Fe(II) concentrations. The Fe(II)
concentration for each sample is indicated in mgL-1 in the top right of each
plot, from (a) 1 mgL-1 to (j) to 306 mgL-1. To distinguish changes in the centre
of the primary peak (peak A), a vertical dashed line at an emission
wavelength of 441 nm is shown (the peak emission wavelength at excitation
239 nm for the 1 mgL-1 solution; see Fig. 4). The fluorescence intensities
are reported in Raman units (RU).
Fe(III) also interacts with DOM and quenches fluorescence intensities
(Ohno
et al., 2008; Poulin et al., 2014). In this experiment, it was expected that
some portion of Fe(II) would have oxidized to Fe(III) since the samples were
directly exposed to the atmosphere when they were transferred to the sample
cuvette for analysis. Nevertheless, the oxidation from Fe(II) to Fe(III) was
limited due to the minimal exposure, and no visible Fe(III) colloids were
observed prior to the analysis. The analysis took about 5–10 min for
each sample; the full analysis was completed within an hour. Therefore, the
quenching effect was unlikely to be caused by dynamic colloid formation.
Furthermore, Poulin et al. (2014) found that almost
no quenching was observed by Fe(III) from the oxidation of Fe(II) to Fe(III)
at pH 6.7. Hence, the quenching effect in this experiment was believed to be
primarily due to Fe(II)–DOM interactions.
Many studies have characterized fluorescence properties of waters based on
the primary peaks in EEM spectra, identified by visual inspection and/or
multivariate data analysis
(Chen
et al., 2003; McKnight et al., 2001; Murphy et al., 2013; Shen et al., 2020;
Stedmon et al., 2003; Stedmon and Bro, 2008). The positions of these peaks
are believed to be linked to the organic matter properties. Coble
(1996; 1990) identified five primary
peaks from a visual inspection of EEMs, including humic-like peaks A, C, and
M and protein-like peaks B and T. We observed only one distinct humic-like
fluorescence peak (peak A) in the EEMs from W3-14. Peak A was in the UV
region at an excitation wavelength of 239 nm and at broad emission
wavelengths ranging from 380 to 460 nm.
Effect of varying Fe(II) concentrations at pH 7.4 on (a) relative
total fluorescence intensity (OFI/OFI0), (c) relative fluorescence
intensity of peak A (A/A0), and (e) peak fluorescence emission wavelength
(nm) at excitation at 239 nm (peak A). Effect of varying Fe(II)
concentration at pH 7.4 on various indices: (b) fluorescence index (FI), (d)
humidification index (HIX), and (f) freshness index (FrI). The results show
that the fluorescence intensities, indices, and peak emission wavelength
change as Fe(II) is increased from 1 to 306 mgL-1.
Similar to the trend observed for relative OFI (Sect. 4.1.1), the relative
intensity of peak A decreased by ∼60% as Fe(II) increased
from 1 to 306 mgL-1, and over 65 % of the quenching occurred below Fe(II)
concentrations of 101 mgL-1 (Fig. 4c). In addition, the position of peak A
continuously migrated towards the shorter (i.e., higher energy) emission
wavelengths with a constant excitation wavelength of 239 nm and increasing
Fe(II) concentration. Figure 4e presents the emission positions of peak A
along with Fe(II) concentrations at excitation 239 nm. Although a linear
relationship was not observed, overall the location of fluorescence response
gradually changed from 441 to 409 nm as Fe(II) increased from 1 to 306 mgL-1.
This result is consistent with quenching experiments conducted with
Everglades F1 water samples, where Poulin et al. (2014) observed a distinct
shift in the quenching locations with increasing ratio of Fe(II) to DOM.
Fluorescence intensities
Iron quenching also affects several indices that are used to quantify DOM
fluorescence properties. The most common indices are the fluorescence index
(FI) (Cory and McKnight, 2005), humification index (HIX)
(Parlanti et al., 2000; Ohno,
2002), the redox index (RI) (Miller
et al., 2006), and freshness index (β/α)
(Zsolnay et al., 1999; Parlanti et al.,
2000). By defining the ratios of fluorescence intensity in different regions
of the EEMs, indices can provide insight into the source of DOM, the degree
of humification, and the relative age of the recently produced DOM.
The fluorescence index (FI) is the most widely used index that provides
information about the source of organic matter. FI is defined using
instrument-corrected spectra as the ratio of emission measured at 470 nm to
that measured at 520 nm, both from an excitation of 370 nm (Cory
and McKnight, 2005). In the absence of fluorescence quenching by other
dissolved constituents, high values of FI (approximately 1.80) indicate that
DOM is derived from extracellular microbial activity, whereas low values of
FI (approximately 1.20) suggest that DOM comes from terrestrial plant and
soil organic matter (Cory and McKnight, 2005).
Measured FI values increased from an initial 1.62 to 1.80 (ΔFI=+0.18 FI units) with increased Fe(II) concentrations (Fig. 4b),
indicating the susceptibility of FI to the iron-quenching effect. As FI is a
ratio of emission intensities, nonuniform changes in component emissions
are responsible for the increase in FI values with Fe(II). Nevertheless, the
effects of iron quenching on DOM fluorescence and FI were only observed in
the 1 to 101 mgL-1 Fe(II) concentration range. As the Fe(II) increased from
101 to 306 mgL-1, the measured FI remained stable at ∼1.80.
Poulin et al. (2014) also
observed that FI values increased more rapidly at low Fe(II) concentrations
and began levelling off approaching the maximum Fe(II) concentrations that
they studied (1.5 mgL-1). A stable value of FI was not reached in Poulin et al. (2014) for the Fe(II)
addition experiment, probably because 1.5 mgL-1 of added Fe(II) did not
saturate all available DOM ligands.
The humidification index (HIX) is defined as the peak area under the
emission spectra from 435–480 nm, divided by the peak area from 300–345 nm plus 435–480 nm, at an excitation of 254 nm and typically ranges from 0–1
(Ohno, 2002). Higher values of HIX (closer to 1)
indicate greater humic content and extent of humidification. HIX values
decreased with the addition of Fe(II) (from about 0.93 to 0.84), indicating
that the emission spectra of fluorescence shifted towards shorter wavelengths
(Fig. 4d). Moreover, the decrease in HIX occurred in two phases with
increasing Fe(II), as represented by a change in slope. The steeper decrease
of approximately 4.5 % in HIX was observed as the Fe(II) concentration
changed from 1 to 72 mgL-1. Above 72 mgL-1 Fe(II), HIX only decreased 2.9 %.
The results indicated that changes in emission spectra were therefore more
sensitive to relatively low Fe(II) concentrations.
The freshness index (FrI or β/α) is defined as the ratio of
emission at 380 nm (β) divided by maximum emission between 420 and
435 nm (α), all at an excitation of 310 nm
(Parlanti et al., 2000; Wilson and
Xenopoulos, 2009). It is a measure of the proportion of recently produced
DOM, where β represents freshly produced DOM, and α represents
more decomposed DOM (Parlanti et al., 2000; Wilson and Xenopoulos, 2009). A higher FrI indicates more
recently created DOM, with values >1 indicating freshly released
DOM and lower values (0.6–0.7) corresponding to older DOM with a predominantly
terrestrial source (Parlanti et al., 2000). Overall,
the freshness index ranged between 0.72 to 0.84 and increased with Fe(II),
except for the slight decrease seen at the Fe(II) concentration of 101 mgL-1
(Fig. 4f). Similar to the trend observed for HIX, the more rapid change of
10.4 % occurred as the Fe(II) concentration ranged from 1 to 72 mgL-1 and a
more gradual change of 6.3 % occurred as the Fe(II) concentration ranged
between 72 and 306 mgL-1.
The effect of Fe(II) quenching on PARAFAC modelling and component distribution
Of the 13 components identified by Cory and McKnight (2005),
7 components were identified as quinone-like organic components
(including three oxidized quinones, Q1, Q2, and Q3, and four reduced
quinones, SQ1, SQ2, SQ3, and HQ), based on the similarity of the positions
and relative intensities of the component excitation peaks compared to the
absorbance and excitation peaks of model quinones. Two components were
defined as resembling amino acid fluorophores (C8, tryptophan, and C13,
tyrosine). The remaining four components (C1, C3, C6, and C10) have not been
associated with any class of molecule. The three most abundant components
were Q1, Q2, and Q3; together they contributed 49 %–52 % to the total
fluorescence (Fig. 5). The excitation and emission spectra of the 13
components are included in our dataset published on Zenodo.
(a) Intensity of components at varying Fe quantities including quinones Q1,
Q2, and Q3 (green); reduced quinones (reduced Q, calculated as the sum of
SQ1, SQ2, SQ3, and HQ); amino acid (tryptophan and tyrosine); and total
intensity; (b) redox index (RI) in the presence of varying Fe(II) quantities. In (a)
the Fe concentrations have been slightly offset to better show overlapping
symbols.
The fluorescence intensity of each component peaked from 1 to 44 mgL-1 Fe(II)
and then steadily decreased as Fe(II) increased to 306 mgL-1 (Fig. 5a). For
all components except tryptophan (C8), the fluorescence at 306 mgL-1 Fe(II)
was less than the fluorescence at 1 mgL-1 Fe. As Fe(II) increased from 1 to
306 mgL-1, the total fluorescence intensity of the 13 components decreased by
approximately 50 %, from 207 to 101 RU. However, the relative proportions
of the components were relatively stable. The maximum deviation was seen in
C6 (unknown classification), which decreased in relative proportion from
9 % to
5 % as Fe(II) increased from 1 to 306 mgL-1. For the other components, the
changes in proportions were restricted to be within ±3%. We
conclude that the proportions of the 13 components are relatively
insensitive to the Fe(II) concentration for the DOM in the experimental
stock solution from the Kidd 2 site.
The trends in component distribution can be further evaluated using the
redox index (RI), which is calculated as the sum of reduced quinone-like
inputs over the total quinone-like input. RI measures the oxidation state of
the DOM and redox reactivities
(Miller et al., 2006; Mladenov et al., 2008). The RI can be used to determine whether
the quinone-like components within the DOM are more reduced (RI closer to 1)
or more oxidized (RI closer to 0). A shift in the RI usually indicates
changes in the redox status. The total oxidized (Q1, Q2, and Q3) and reduced
(SQ1, SQ2, SQ3, and HQ) quinones changed their contributions to total
fluorescence from 52 % to 50 % and from 21 % to 20 %, respectively. The
small changes for both oxidized and reduced quinones are responsible for the
relatively stable values of RI, which slightly decreased from 0.30 to
∼0.285 (Fig. 5b).
Discussion
Ferrous iron is responsible for the observed quenching of DOM fluorescence.
Both the total fluorescence intensity of EEMs and peak-A intensity decreased
with increasing Fe(II). The likely mechanism accounting for the quenching is
complexation of Fe(II) with the fluorescent sites, efficiently decreasing the fluorescence intensity of fluorophores (Waite and
Morel, 1984; Senesi, 1990). The nonlinear quenching of the fluorescence
intensity with Fe(II) suggests, following Senesi (1990), a static quenching
mechanism. However, our experiment does not allow us to identify quenching
mechanisms. While the maximum ratio of Fe(II) to DOM (mgL-1 per
mgL-1) was approximately 0.4 in Poulin et al. (2014), it is much
larger in our study, with a molar Fe-to-C (as DOC) ratio of approximately
7. While earlier work by Senesi (1990) suggests that quenching primarily
depends upon the fraction of DOM ligands that is complexed to Fe(II), our
study, with a great excess of Fe(II) over C in DOM, could involve other
mechanisms. Poulin et al. (2014) found that Fe(II) quenching of fluorescence
ranged from 7 % to 23 % in four different hydrophobic
acid (HPoA) fractions and surface water samples, with DOC concentrations
ranging from 2.3 to 5.0 mgL-1, and where the degree of quenching was
not related to DOM concentrations. In our study, both total fluorescence
intensity and peak-A intensity decreased by approximately 60 % as
Fe(II) increased from 1 to 306 mgL-1. The differences in the
impact of Fe(II) on fluorescence quenching between the representative
groundwater from Kidd 2 site and those reported by Poulin et al. (2014) could
be related to the much higher Fe(II) background in the organic-rich aquifer
and groundwater at the Kidd 2 site. It should be noted that the DOC
concentration in the Kidd 2 groundwater was 10.7 mgL-1,
which is significantly greater than that in the previous study (Poulin et al., 2014).
Poulin et al. (2014) mainly examined the effect of Fe(II) addition to
terrestrial-derived fresh surface water with undetectable Fe(II) levels. In
contrast, the DOM in the stock solution collected from the Kidd 2 site is
hypothesized to be derived from microbial sources and may respond to high Fe(II) concentrations differently than freshwater terrestrial-derived DOM. The
quenching mechanism for this DOM is not well understood. Similar metal
quenching of humic-like peaks has been observed by other researchers. Ohno
et al. (2008) conducted experiments on the impact of Fe(III) and Al(III)
addition to the deciduous water-soluble organic matter (WSOM) fluorescence
spectra. This result showed that the fluorescence intensity was quenched by
about 30 % in the presence of 25 µM
(1.4 mgL-1) Fe for peak A (Ohno et al., 2008).
The fluorescence indices FI, HIX, and β/α provided evidence of the
susceptibility to Fe(II) quenching, while RI was relatively insensitive to
Fe(II) concentration. FI values increased by approximately 0.18 units from the
addition of 300 mgL-1 of Fe(II) and shifted towards a greater
microbial-derived origin. This relatively small change may not affect the
utility of FI towards inferring DOM origin. Moreover, FI values are more
sensitive at low Fe(II) concentrations, and they gradually reach a plateau once
Fe(II) is above 101 mgL-1, as all available DOM ligand sites are
likely fully occupied by Fe(II)–DOM interactions. This result also supports a
static quenching mechanism, since quenching does not depend on the Fe(II)
concentration (Senesi, 1990). Similar to FI, both HIX and β/α
show more pronounced changes at low Fe(II) concentrations (Fe(II) <72mgL-1). The decrease in HIX and increase of the β/α ratio are consistent, as they both indicate that the DOM shifted to more
freshly produced, with a higher H:C ratio and less polycondensation (Ohno,
2002). Nevertheless, the HIX and the β/α ratio are not likely to reach
a constant value with Fe(II) concentration. Therefore, conclusions about
organic matter origin based on humidification and freshness indices must
consider the DOM sensitivity to Fe(II) concentration when reporting values.
In the 13-component PARAFAC model (Cory and McKnight, 2005), all components
except C8 were quenched in terms of their fluorescence
intensities. Nevertheless, changes in their “relative” component
distribution were relatively small (within ±4% contribution
to total fluorescence). This result was consistent with the observation by Poulin et al. (2014), where changes in the component distributions were within ±3% of the total fluorescence in their 13-component PARAFAC
model. The small or negligible change in component distribution results in the
relatively constant RI. Although few fluctuations were observed in the Fe(II)
addition experiment, the overall trend of the RI is stable; thus, it can be
used as a reliable index to infer the redox condition of the aquatic
environment. Still, the component distribution may vary in different PARAFAC
models. There were more pronounced distribution changes in a 7-component
model than in a 13-component model (Poulin et al., 2014). This suggests that
the degree of quenching should always be associated with fluorophore
classification. Besides fluorophore classification, DOM composition is another
important factor that controls the quenching characterization. Ohno et al. (2008)
found that quenching of fluorescence intensity varies depending on the DOM
composition. In their three-component PARAFAC model, Fe(III) quenching was
observed in all three components for the deciduous WSOM sample. For the
coniferous WSOM sample, however, only components 1 and 2 were quenched, and
component 3 increased slightly with the initial addition of Fe(III) and
decreased with further Fe(III) additions.
While our study shows that high Fe(II) concentrations can influence
fluorescence properties of a representative deltaic groundwater, it is
difficult to generalize our results to other terrestrial waters with different
DOM compositions without further analyses. We observed nonuniform quenching
as a function of Fe concentration in this study, with smaller increases in
quenching at concentrations above 100 mgL-1. We note that both
the DOM and Fe(II) concentrations that we examined were higher than those
examined by Poulin et al. (2014).
Summary
This study demonstrates the quenching effect of Fe(II) in organic-rich anoxic
groundwater from the Kidd 2 aquifer in the Fraser River Delta, Richmond, BC,
where Fe(II) concentrations range from 1 to 300 mgL-1 and the DOM
concentration is ∼10mgL-1. In our experiments, total
fluorescence intensity decreased by approximately 60 % as Fe(II)
increased from 1 to 300 mgL-1. While our results are likely
applicable to similar deltaic groundwaters, further analyses are required to
quantify the quenching effect on fluorescence indices in other terrestrial
waters (for example, surface waters with sufficiently high Fe concentrations
such that quenching is likely). In this study, FI values tended to shift to be
more autochthonous in origin with increasing Fe(II), but the small changes are
unlikely to invalidate conclusions about the source of DOM. Changes in the
humidification index and freshness index both indicated that the addition of
Fe(II) would shift these two indices towards more recently produced DOM.
Therefore, the sensitivity of these indices should be evaluated when water
samples contain Fe(II). The nonlinear relationship between the indices and
Fe(II) can be seen in all of the indices, especially the FI. Although the
intensities of all 13 components varied as a function of Fe(II), the
relatively stable component distribution suggests that the Fe(II) quenching
effect has a negligible effect on the 13-component PARAFAC model. As a result,
the PARAFAC model can be a reliable method for obtaining information about DOM
composition in their relative distributions and redox status via the RI.
Code and data availability
The data presented in this article have been published on Zenodo at 10.5281/zenodo.3737108 (Jia et al., 2020).
Author contributions
KJ, AJ, and RDB designed experiments and KJ conducted experiments. KJ and AJ performed data analysis. KJ and CCMM prepared figures. CM compiled and archived the data. KJ and CCMM prepared the article with contributions from AJ and RDB.
Competing interests
The authors declare that they have no conflict of interest.
Disclaimer
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We thank Mark Bolton for assistance with sample analysis and three anonymous reviewers for their helpful feedback that improved the article.
Financial support
This research has been supported by the NSERC (grant no. 121436-13).
Review statement
This paper was edited by Marnik Vanclooster and reviewed by three anonymous referees.
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