Accepted Manuscript Critical Analysis of Commonly Used Fluorescence Metrics to Characterize Dissolved Organic Matter Julie Ann Korak, Aaron D. Dotson, R. Scott Summers, Fernando L. Rosario-Ortiz PII:

S0043-1354(13)00936-6

DOI:

10.1016/j.watres.2013.11.025

Reference:

WR 10335

To appear in:

Water Research

Received Date: 29 July 2013 Revised Date:

14 October 2013

Accepted Date: 18 November 2013

Please cite this article as: Korak, J.A., Dotson, A.D., Summers, R.S., Rosario-Ortiz, F.L., Critical Analysis of Commonly Used Fluorescence Metrics to Characterize Dissolved Organic Matter, Water Research (2013), doi: 10.1016/j.watres.2013.11.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Peak  Intensity   Specific  Peak   Intensity  

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Which  Metrics?  

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Conserva,ve   Mixing  

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Critical Analysis of Commonly Used Fluorescence Metrics to Characterize

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Dissolved Organic Matter

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Julie Ann Korak1, Aaron D. Dotson2, R. Scott Summers1 and Fernando L. Rosario-Ortiz1*

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Department of Civil, Environmental and Architectural Engineering, 428 UCB,

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University of Colorado-Boulder, Boulder, CO 80309, USA 2

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University of Alaska Anchorage, Anchorage, AK 99515, USA

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Civil Engineering Department, ENGR 201

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*Corresponding author. [email protected]; Phone: 303-492-7607

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Abstract

The use of fluorescence spectroscopy for the analysis and characterization of dissolved organic

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matter (DOM) has gained widespread interest over the past decade, in part because of its ease of use

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and ability to provide insight of bulk DOM chemical characteristics. However, the lack of standard

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approaches for analysis and data evaluation has complicated its use. This study utilized comparative

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statistics to systematically evaluate commonly used fluorescence metrics for DOM characterization to

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provide insight into the implications for data analysis and interpretation such as peak picking methods,

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carbon-normalized metrics and the fluorescence index (FI). The uncertainty associated with peak

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picking methods was evaluated, including the reporting of peak intensity and peak position. The linear

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relationship between fluorescence intensity and dissolved organic carbon (DOC) concentration was

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found to deviate from linearity at environmentally relevant concentrations and simultaneously across

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all peak regions. Comparative analysis suggests that the loss of linearity is composition specific and

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likely due to non-ideal intermolecular interactions of the DOM rather than the inner filter effects. For

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ACCEPTED MANUSCRIPT some DOM sources, Peak A deviated from linearity at optical densities a factor of 2 higher than that of

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Peak C. For carbon-normalized fluorescence intensities, the error associated with DOC measurements

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significantly decreases the ability to distinguish between compositional differences. An in-depth

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analysis of FI determined that the metric is mostly driven by peak emission wavelength and less by

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emission spectra slope. This study also demonstrates that fluorescence intensity follows conservation

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of mass principles, but the fluorescence index does not.

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1. Introduction

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Dissolved organic matter (DOM) is comprised of a wide range of compounds that originate

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from terrestrial or aquatic sources as well as from anthropogenic inputs, such as wastewater effluent

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(Leenheer, 2009). Composition is spatially and temporally dependent and associated with source

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material variation and environmental transformation processes (e.g., redox and photochemical

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processing). DOM is highly heterogeneous and is often characterized by bulk parameters (e.g.,

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dissolved organic carbon (DOC), light absorption/fluorescence, aromaticity, hydrophobicity and

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functional groups) (Leenheer, 2009).

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Fluorescence spectroscopy measures the fraction of DOM that both absorbs and emits light,

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and the fluorescent DOM fraction is often associated with aromaticity (Senesi et al., 1991). Observed

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fluorescence can be a function of individual fluorophores contributing superimposed signals or a

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function of intramolecular charge-transfer mechanisms between interacting fluorophores (Del Vecchio

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and Blough, 2004).

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1.1. Application of Fluorescence Spectroscopy for DOM Characterization

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Fluorescence spectroscopy has been used extensively to characterize differences in DOM

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signatures in natural systems (Hudson et al., 2007; Fellman et al., 2010). Recently, fluorescence has

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been applied to engineered treatment systems to characterize DOM changes (Win et al., 2000; Cheng 2

ACCEPTED MANUSCRIPT et al., 2004; Swietlik and Sikorska, 2004; Allpike et al., 2005; Beggs et al., 2009; Dwyer et al., 2009;

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Kraus et al., 2010; Baghoth et al., 2011; Beggs and Summers, 2011; Bieroza et al., 2011; Beggs et al.,

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2013). Early applications of fluorescence included two-dimensional emission, excitation and

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synchronous scans and focused on spectral peak intensity and positions. Analysis of two-dimensional

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scans also led to the development of indices such as the fluorescence index (FI) and humification index

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(HIX) (Zsolnay et al., 1999; McKnight et al., 2001; Ohno, 2002). Recent analysis of DOM

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fluorescence has focused on the use of three dimensional excitation-emission matrices (EEMs), as

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illustrated in Figure 1. Peak picking methods have driven a significant portion of quantitative

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fluorescence analysis (Coble, 1996). Furthermore, parallel factor analysis (PARAFAC) has been used

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to deconvolute EEMs into components, but relies on collecting large datasets of EEMs (Stedmon et al.,

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2003; Stedmon and Bro, 2008; Murphy et al., 2013).

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A wide range of metrics has been used to analyze EEMs. The simplest EEM characterization

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method is qualitative observation of the presence or absence of regional intensity (Bu et al., 2010). A

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fluorescence regional integration (FRI) method was developed that sums all intensities within a region

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(Chen et al., 2003; Zhou et al., 2013). Peak picking is a quantitative method that records the peak

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intensities within pre-defined regions of interest. Common peak regions relevant to freshwater include

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two humic-like (A, C) and two heterocyclic nitrogen/polyphenolic/protein-like peaks (B, T) and are

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outlined in Table S-1 (Coble, 1996; Leenheer, 2009). Peak picking has been executed using two

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distinct methods. The first method reports the intensity of a stationary point (fixed wavelength) within

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each region of interest (Leenheer, 2009; Lønborg et al., 2010; Murphy et al., 2010; Romera-Castillo et

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al., 2011). The second approach is to define a region of interest and systematically extract the

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maximum intensity within the region even though the location of maximum may vary between samples

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and replicates (Senesi et al., 1991; Wang et al., 2009; Beggs and Summers, 2011; Bieroza et al., 2011;

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Esparza-Soto et al., 2011; Guéguen and Cuss, 2011; Yang et al., 2013). This method will be referred to 3

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ACCEPTED MANUSCRIPT as an “algorithm-based” approach herein. Many studies that employ this method also record the

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excitation and emission wavelength at which the maximum is located and use this information to infer

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changes in DOM composition (Del Vecchio and Blough, 2004; Wang et al., 2009; Bieroza et al., 2011;

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Esparza-Soto et al., 2011; Yang et al., 2013). Furthermore, much in the same way as specific UV

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absorbance (SUVA) has been used to characterize DOM (Weishaar et al., 2003), carbon-normalized

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peak intensities (intensity divided by DOC), have been used to characterize fluorescence per unit DOC,

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often termed specific intensities (Alberts et al., 2002; Alberts and Takács, 2004; Cheng et al., 2004;

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Jaffe et al., 2004; Allpike et al., 2005; Cumberland and Baker, 2007; Hudson et al., 2007; Wu et al.,

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2007; Dwyer et al., 2009; Fellman et al., 2010; Beggs and Summers, 2011; Fleck et al., 2013).

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FI has been correlated to DOM aromaticity and is often used as a surrogate for DOM origin,

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i.e., allochthonous or autochthonous (Win et al., 2000; McKnight et al., 2001; Cheng et al., 2004;

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Swietlik and Sikorska, 2004; Allpike et al., 2005; Beggs et al., 2009; Dwyer et al., 2009; Kraus et al.,

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2010; Baghoth et al., 2011; Beggs and Summers, 2011; Bieroza et al., 2011; Beggs et al., 2013). FI

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was originally defined for fulvic acids isolated from a range of DOM sources, but has since been

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applied to whole water analyses (McKnight et al., 2001; Beggs et al., 2009; Kraus et al., 2010; Beggs

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and Summers, 2011). FI was initially defined as a ratio of emission intensities at 450 nm and 500 nm

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when excited at a wavelength of 370 nm (Coble, 1996; McKnight et al., 2001). The 450 nm point was

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chosen because it was between the peak emission for Pony Lake and Suwannee River end members.

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The 500 nm index represented the point where emission intensity for microbial end members was half

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of its maximum intensity. Due to instrument specific corrections that shift the emission maxima to

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longer wavelengths by about 15 nm (red-shifted), the indices for FI were modified to the ratio of 470

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nm to 520 nm (Cory et al., 2010).

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Past work has made great advances in standardizing the procedures by which EEMs are

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collected, corrected and normalized (Lawaetz and Stedmon, 2009; Murphy et al., 2010). Murphy et. 4

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effects and a comparison of Raman and quinine sulfate normalization methods. The study also

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compared the reproducibility of single intensities and intensity ratios through an inter-laboratory

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comparison. These, however, are only a few of the commonly used metrics that have been employed,

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and the literature lacks a thorough discussion of metric limitations and a validation of underlying

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assumptions.

Many metrics rely on an underlying assumption that fluorescence intensity and DOC

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concentration are linearly related. Using this assumption, fluorescence can be used as a surrogate for

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DOC concentration in monitoring applications by developing site-specific correlations (Bergamaschi et

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al., 2012; Carpenter et al., 2012; Goldman et al., 2012). Online monitoring is one application where

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there are limitations inhibiting the use of inner filter corrections (IFCs), which in turn affects linearity

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assumptions. Identifying metrics that do not require IFCs would benefit such applications.

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illustrate another example, for the ratio of two intensities (i.e., peak C/ peak T, FI) to be an intrinsic,

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compositional metric over a range of DOC concentrations, the peak intensities must vary

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proportionally across the range of concentrations. Fundamental theory supports a linear response

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between fluorescence intensity and DOC concentration given a number of constraints described herein

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(Parker and Rees, 1962; Kubista et al., 1994). According to the Beer-Lambert law, at low

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concentrations absorption is directly proportional to concentration when the composition (decadic

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molar extinction coefficient) and cell path length are constant (Lakowicz, 2006). Non-linear absorption

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behavior often occurs in the presence of scattered light from turbidity, fluorescing species, and

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molecular aggregation (Lakowicz, 2006). Once organic molecules are in the excited singlet state, the

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fluorescence quantum yield dictates the fraction of absorbed energy that is released through a radiative

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fluorescing mechanism versus non-radiative or phosphorescing energy dissipation mechanisms. The

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quantum yield is dependent on the fluorophore composition and surrounding environment. The 5

To

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presence of quenchers, enhancers, or the formation of excimers can all impact the quantum yield

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(Lakowicz, 2006). Past research has demonstrated that DOM behaves as a complex mixture where

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charge transfer interactions play an important role (Del Vecchio and Blough, 2004). For given application, a variety of metrics have been used to investigate the same process. For

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example, across a range of DOM coagulation studies, some report changes in peak intensities

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(Afcharian et al., 1997; Cheng et al., 2004; Swietlik and Sikorska, 2004; Bieroza et al., 2009; Dwyer et

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al., 2009; Gone et al., 2009; Kraus et al., 2010; Baghoth et al., 2011; Bieroza et al., 2011), some report

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specific intensities (Cheng et al., 2004; Allpike et al., 2005; Dwyer et al., 2009; Beggs and Summers,

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2011), and others use indices such as the FI (Beggs et al., 2009; Kraus et al., 2010; Beggs and

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Summers, 2011). With the increasing use of EEMs to study DOM, the literature lacks a comprehensive

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study on metric sensitivity and applicability. This study utilized comparative statistics to systematically

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evaluate the commonly used fluorescence metrics to provide insight into the implications for data

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analysis and interpretation such as peak picking methods, carbon-normalized metrics and the

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fluorescence index (FI). The intent is to aid researchers in understanding limitations and pitfalls of

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different DOM fluorescence data analysis tools. Application of the concepts described here will

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provide a strong foundation for future experimental design and data analysis to allow for more

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consistent practices and better comparison across literature. While this study relied on well-

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characterized DOM standards and some surface waters, the results highlight important considerations

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for any DOM analysis by evaluating spectral features common to many DOM samples. Metrics were

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evaluated both with and without IFCs. The article will highlight metrics where inner filter effects can

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be considered negligible with the remaining analysis documented in the Supplemental Information.

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2. Materials and Methods

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2.1. DOM sources 6

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waters were used in this study. The DOM isolates include: Suwannee River NOM (SRNOM,

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1R101N), Suwannee River Fulvic Acid (SRFA, 1S101F), Suwannee River Humic Acid (SRHA,

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2S101H), and Pony Lake Fulvic Acid (PLFA, 1R109F). One surface water, Big Elk Meadows Lake

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(BEM), was used throughout the study and is from a mountain lake in Colorado located at about 7500

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feet above sea level that naturally has a high DOC concentration, low conductivity and low alkalinity.

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The other four waters, Boulder Reservoir, Betasso Water Treatment Plant influent, Barr Lake and

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Boulder Wastewater (WW) effluent, were analyzed to augment the FI discussion. All samples were

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filtered with GF/F 0.7µm filters (Whatman) that were muffled at 550°C for 4 hours and rinsed with

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250 mL of 18MΩ-cm lab-grade water (LGW). The DOM isolates and BEM sources were

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dissolved/diluted with 10 mM, pH 7.5 phosphate buffer using LGW to 8 different concentrations

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targeting the linear absorbance range according to the Beer-Lambert law (Lakowicz, 2006). The final

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pH of the dilutions was 7.5±0.15.

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After dilution, the solutions had DOC concentrations that ranged from 0.83-40.5 mgC L-1, as

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measured by high-temperature combustion in the presence of platinum catalyst and non-dispersive

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infrared detection using a non-purgeable organic carbon method (TOC-VCSH, Shimadzu, MD).

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Samples were analyzed with repeated injections with the criteria that the peak area coefficient of

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variation was less than 2% or the standard deviation is less that 0.2, which ever was less. The

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instrument was calibrated with potassium hydrogen phthalate and the study-wide accuracy and

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precision of 5 mgC L-1 standards were both within 5%. The minimum reporting limit was 0.16 mgc L-1

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following EPA method 415.3.

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2.2. Fluorescence Spectroscopy Analysis.

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Fluorescence EEMs were collected using a spectrofluorometer (John Yvon Horiba FluoroMax-

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4, NJ). All samples were measured in triplicate with a new sample aliquot for each EEM. Fluorescence 7

ACCEPTED MANUSCRIPT intensity was measured during emission scans (300 nm to 550 nm every 2 nm) at set excitation

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wavelengths in 10 nm increments from 240 nm to 500 nm. A 5 nm bandpass for excitation and

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emission wavelengths and 0.25 s integration time were used. Fluorescence data were corrected

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following published methods (Murphy et al., 2010). The EEMs were collected in signal divided by

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reference mode and incorporated instrument-specific correction factors. Unless specifically stated

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otherwise for the parallel metrics analysis, all EEMs were corrected for primary and secondary inner

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filter effects using a UV-Vis absorbance spectrum collected in a quartz cell with a 1 cm path length

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(Cary-100, Agilent Technologies, CA) (Lakowicz, 2006). EEMs were blank subtracted and Raman

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normalized based on the Raman peak area for LGW collected at an excitation wavelength of 350 nm

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(Lawaetz and Stedmon, 2009). Corrected EEMs are presented in Raman Units (RU). Lamp scans to

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verify monochromator calibration and cuvette contamination checks with LGW were performed daily.

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EEMs were corrected and analyzed using MATLAB (Mathworks, MA) software. Specific

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fluorescence intensities were calculated by dividing the fluorescence intensity by the DOC (RU L mgc-

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1

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nm (Cory et al., 2010). Local total optical density (ODTotal) was defined as the sum of absorbance

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measurements at a fluorescence peak excitation and emission wavelengths, because this quantity

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determines the magnitude of inner filter corrections applied to the measurements.

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). FI was calculated as the ratio of emission intensities at 470 nm and 520 nm at an excitation of 370

2.3. Statistical Methods

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Coefficient of variation (CV) was used as a comparative metric when triplicate reproducibility

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was evaluated. For carbon-normalized fluorescence metrics, error propagation using the root mean

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square approach for the quotient of two measurements was carried out to account for the uncertainty of

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both measurements (Taylor, 1997). Fluorescence in the Peak B region for the three Suwannee River

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isolates was not evaluated due to a lack of local intensity; fluorescence intensity was less than 0.02 RU

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and did not increase with concentration. Regression and ANOVA analyses were performed at a 95% 8

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residuals analysis. The coefficient of determination, R2, was not used to judge model adequacy because

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it cannot identify systematic residuals or curvature.

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3. Results and Discussion

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3.1. Method Comparison for Peak Intensity Reproducibility

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An intra-laboratory comparison was performed to compare the reproducibility of both peak

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picking methods to determine if the algorithm-based approach introduced greater uncertainty

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compared to the fixed wavelength approach. The fixed wavelength method evaluated the CV between

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triplicates at a fixed point at the center of each peak region (Table S-1, S-2). Intensities at the center of

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Peak A and C regions (Table 1) were the most reproducible with CVs less than 1% for most samples.

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As concentration and fluorescence intensity decreased (0.13

Peak C ODTotal 0.09-0.13 0.18-0.26 0.14-0.20 0.20-0.25 >0.07

Peak T ODTotal 0.20-0.28 0.39-0.58 0.31-0.43 >1.90 >0.17

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DOC mg L-1 4.3-6.5 13.2-20.7 7.1-9.6 4.7-6.0 >8.2

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DOM SRNOM PLFA SRFA SRHA BEM

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Figure 1. Excitation emission matrix for BEM. Intensity is presented in Raman units (RU). Boxes outline the four peak regions (A, B, C and T) with labels above. Hollow circles indicate the location of the peak intensity in each region. The two points used in the I470/I520 Fluorescence Index (FI) metric are represented by filled circles connected by a line at an excitation of 370 nm. 440

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Figure 2. Peaks C (a) and T (b) intensity as a function of DOC for five DOM sources. Data is IFC corrected and error bars representing the standard deviation are smaller than marker size.

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Figure 3. Peak A and C intensity as a function of DOC concentration for SRNOM. All data are inner filter corrected and the lines represent a linear model fit to the lowest 5 DOC concentrations. Error bars representing the standard deviation between triplicate measurements may be smaller than the marker.

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Figure 4. Specific Peak C intensity for 5 DOM sources within linear concentration range with IFCs applied. The inner (shorter) error bar represents the uncertainty associated with only fluorescence. The outer (wider) error bar is the uncertainty associated with both fluorescence and DOC measurements.

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Figure 5. Local curvature as a function of location relative to the peak emission. FI is the ratio of two intensities (I1/I2) spaced 50 nm apart with I1 occurring at the lower wavelength. The x-axis refers to the position of I1 relative to the emission peak. A distance of 0 nm means that the I1 is located at the peak and the second is located 50nm higher. The markers indicate where FI is calculated using the I470/I520 ratio according to Cory et. al. (46). All FIs are measured at an excitation wavelength of 370 nm.

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Figure 6. Normalized emission spectra at an excitation of 370nm plotted according to a) emission wavelength and b) relative to peak emission wavelength. The lines in plot a) indicate the location of the I470 and I520 and in b) the range of emission wavelengths where the slopes calculated 50nm apart are statistically the same. b)

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Figure 7. Fluorescence index and Peak C intensity as a function of composite ratio between SRHA and PLFA. Error bars representing the standard deviation may be smaller than the marker.

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Highlights  

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Fluorescence  data  analysis  methods  for  characterizing  DOM  are  evaluated.   Fluorescence-­‐DOC  linear  relationship  deviates  due  to  quantum  yield  change.   Fluorescence  Index  largely  driven  by  emission  peak  location  not  curvature.   Peak  intensity  follows  conservative  mixing  but  fluorescence  index  does  not.  

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Critical Analysis of Commonly Used Fluorescence Metrics to Characterize Dissolved Organic Matter 1

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Julie Ann Korak1, Aaron D. Dotson2, R. Scott Summers1 and Fernando L. Rosario-Ortiz1* Department of Civil, Environmental and Architectural Engineering, 428 UCB, University of Colorado-Boulder, Boulder, CO 80309, USA 2

Civil Engineering Department, ENGR 201

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University of Alaska Anchorage, Anchorage, AK 99515, USA

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*Corresponding author. [email protected]; Phone: 303-492-7607

Table S-1. Defined peak regions based on the values in literature (22). Peak

Range Ex: 240-270 nm Em: 380-470 nm Ex: 260-290 nm Em: 300-320 nm Ex: 300-340 nm Em: 400-480 nm Ex: 260-290 nm Em: 326-350 nm

A B

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Type

Humic-like

Tyrosine-like Humic-like

Tryptophan-like

Table S-2. Excitation and emission wavelength coordinates for the center of each peak region.

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Peak Ex (nm) Em (nm) A 260 426 B 280 310 C 320 440 T 280 338

Sp. A

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DOC

RU L mgc-1 RU L mgc-1 0.12 ± 0.006 0.06 ± 0.003 0.10 ± 0.005 0.02 ± 0.0017 0.14 ± 0.007 0.03 ± 0.002 0.10 ± 0.005 0.02 ± 0.004 0.08 ± 0.004 0.05 ± 0.003 0.11 0.03 0.05 0.04 0.1 0.09 ---

FI 1.46 ± 0.007 1.29 ± 0.007 1.31 ± 0.011 1.08 ± 0.008 1.46 ± 0.007 1.34 1.4 1.83 1.95

EP

UV 254

RU L mgc-1 RU L mgc-1 0.30 ± 0.015 0.04 ± 0.002 0.28 ± 0.014 -0.41 ± 0.02 -0.27 ± 0.013 -0.25 ± 0.012 0.05 ± 0.005 0.31 0.02 0.15 0.04 0.32 0.09 ---

IHSS Peak wavelength Aromatic at Ex=370 Carbon nm % 461 ± 1.4 12 474 ± 1.4 23 474 ± 0.6 24 484 ± 1.9 31 463 ± 2.4 -468 -468 -462 -444 --

Table S- 3. Summary of compositional metrics for IHSS isolates and individual waters used for FI analysis. For the IHSS isolates and BEM, an average value across with linear range is reported with its standard deviation.

DOM Source (cm-1) mgc L-1 L mgc-1 m-1 PLFA --2.56 ± 0.03 SRNOM --3.74 ± 0.16 SRFA --3.97 ± 0.12 SRHA --6.20 ± 0.27 BEM --2.18 ± 0.05 Betasso WTP* 0.3 7.7 4 Boulder Reservoir 0.07 3.5 2 Barr Lake 0.11 6.2 1.8 Boulder WW 0.11 n/a n/a *diluted before analysis Aromatic Carbon values from (Thorn et al., 1989)"

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Figure S-1. Coefficient of variance between triplicate measurements as a function of mean signal intensity for each peak center with inner filter corrections applied.

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Deviations from the Fluorescence-DOC Linearity Assumption 5 Points

9 Points Peak A % Difference

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−4

−10

−30

10

10

10

0

−5

−5

1

Peak A

% Difference

0.00

Peak T

−0.20 0.04

1

TE D

0.01

8

14 1

11

22 0

21

2

Peak T

3 1

3

4 1

DOC Concentration (mgc/L)

30

5

9

8 Points

0

−5

−10

−4

−20

−30

4

10

20

0

−5

−10

−4

−20

−40

6

5

10

2

−3

−5

1

−10 14 1

8

3 Points

% Difference

EP 0.00

1

15

11

−20 22 0

21

42

DOC Concentration (mgc/L)

Peak A

Peak A Peak C

Residual Intensity (RU)

0.00

−0.00 0.01

Peak T

7 Points

10

5 Points

−0.01 0.00

AC C

BEM

0.00

4 Points

−20 7 0

10

−2

42

Peak C

3 Points 0.01

4

4

DOC Concentration (mgc/L)

−0.01

−20 4 1

3

6 Points

Peak C

Peak A Peak C

Residual Intensity (RU)

−0.20 0.20

Peak T

PLFA

0.20

−0.02

0

M AN U

6 Points

10

DOC Concentration (mgc/L)

DOC Concentration (mgc/L) 0.60

9 Points

10

−10

30

6 Points

4

RI PT

6 Points

SC

5 Points 0.20

4 Points

5 Points

6

4

5

2

0

0

−2

−4

−5

4

2

4

1

−1

1

−2

−4

−2

15

5

15

5

0

5

−5

1

2

3

−5

1

3

4

−5

1

5

9

DOC Concentration (mgc/L)

Figure S-2. Residual analysis for SRNOM, PLFA and BEM showing the upper and lower limits of the linearity threshold and full range of DOC concentrations. The left column presents the residuals in Raman units and the right column presents the residuals as a percentage of the measured intensity.

ACCEPTED MANUSCRIPT

9 Points

6 Points Peak A % Difference

0.00

Peak T

−0.04 0.02 0.00 −0.01

Peak C

Peak A Peak C

Residual Intensity (RU)

−0.10 0.04

Peak T

SRHA

0.00

1

3

5 1

3

6 0

12

DOC Concentration (mgc/L) 6 Points

7 Points

9 Points

−0.01

Peak A

Peak C

Peak T

1

4

7 1

5

−5

−10

−10

−20

−40

5

10

10

0

0

−10

−5

−10

−30

1

40

40

0

10

10

1

−20 5 1

3

6 Points

% Difference

0.00

0

10−1

19

38

TE D

DOC Concentration (mgc/L)

3

−20 6 0

7 Points

12

25

9 Points

5

5

10

−3

−3

−10

M AN U

Peak A Peak C

Residual Intensity (RU)

0.00 −0.10 0.01

Peak T

SRFA

−0.20 0.10

20

DOC Concentration (mgc/L)

0.60 0.20

9 Points

10

−1

25

7 Points

10

RI PT

7 Points

SC

6 Points 0.10

−10

−10

−30

10

10

10

3

0

−10

−5

−10

−30

10

10

10

0

0

−5

−10

1

4

−10 7 1

5

−20 10 −1

19

38

DOC Concentration (mgc/L)

AC C

EP

Figure S-3. Residual analysis for SRHA and SRFA showing the upper and lower limits of the linearity threshold and full range of DOC concentrations. The left column presents the residuals in Raman units and the right column presents the residuals as a percentage of the measured intensity.

ACCEPTED MANUSCRIPT

8

1.6

7

1.4

6

1.2

4 3 2 Peak A Peak C

0.6

10

20 DOC (mgc/L)

30

0.2 0

40

0

2

M AN U

0

SRHA 2.5

4 DOC (mgc/L)

6

8

SRFA

TE D

1

0.5

Peak Intensity (RU)

5

1.5

4

3

2

1

0

2

EP

Peak A Peak C

0

Peak A Peak C

6

2

Peak Intensity (RU)

0.8

0.4

1 0

1

SC

5

RI PT

BEM 1.8

Peak Intensity (RU)

Peak Intensity (RU)

PLFA 9

4

6 8 DOC (mgc/L)

10

Peak A Peak C 0

0

5

10 DOC (mgc/L)

15

AC C

Figure S-4. Peak A and C intensity as a function of DOC concentration for PLFA, BEM, SRHA and SRFA. The lines represent a linear model fit to the lowest 5 DOC concentrations. Error bars representing the standard deviation between triplicate measurements may be smaller than the marker.

ACCEPTED MANUSCRIPT

1.6

0.4

1.4

0.35

1.2

0.3

0.8 0.6 0.4

0.2 0.15 0.1

Peak B Peak T 0

10

20 DOC (mgc/L)

30

0.05

40

0

0

2

M AN U

0.2 0

0.25

SC

1

RI PT

BEM 0.45

Peak Intensity (RU)

Peak Intensity (RU)

PLFA 1.8

4 DOC (mgc/L)

Peak B Peak T 6

8

AC C

EP

TE D

Figure S-5. Peak B and T intensity as a function of DOC concentration for PLFA and BEM. The lines represent a linear model fit to the lowest 5 DOC concentrations. Error bars representing the standard deviation between triplicate measurements may be smaller than the marker.

ACCEPTED MANUSCRIPT

SRNOM

SRHA

0.3

0.14

0.15

0.1

0.05

0

2

4

6 8 DOC (mgc/L)

10

12

SRFA

0.4 0.35

0.04

0

0

2

4

0.3 0.25

6 8 DOC (mgc/L)

10

TE D

Peak T Intensity (RU)

0.06

0.02

0.45

0.2 0.15 0.1

0

EP

0.05 0

0.08

M AN U

0

0.1

SC

Peak T Intensity (RU)

Peak T Intensity (RU)

0.2

RI PT

0.12

0.25

5

10 DOC (mgc/L)

15

AC C

Figure S-6. Peak T intensity as a function of DOC concentration for SRNOM, SRHA and SRFA. The lines represent a linear model fit to the lowest 5 DOC concentrations. Error bars representing the standard deviation between triplicate measurements may be smaller than the marker.

ACCEPTED MANUSCRIPT

BEM SRHA SRFA

DOM Source SRNOM PLFA BEM SRHA

Max. DOC mg L-1 4.3 13.9 9.3 40.4 8.2 3.02 12.0 4.6 18.7

Peak C Slope Regression p y=0.107x-0.021

Critical analysis of commonly used fluorescence metrics to characterize dissolved organic matter.

The use of fluorescence spectroscopy for the analysis and characterization of dissolved organic matter (DOM) has gained widespread interest over the p...
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