C.W. Cuss

Characterization of aquatic dissolved organic matter by asymmetrical flow field-flow fractionation coupled to UV–Visible diode array and excitation emission matrix fluorescence

Flow field-flow fractionation (FlFFF) with on-line UV/Visible diode array detector (DAD) and excitation emission matrix (EEM) fluorescence detector has been developed for the characterization of optical properties of aquatic dissolved organic matter (DOM) collected in the Otonabee River (Ontario, Canada) and Athabasca River (Alberta, Canada). The molecular weight (MW) distribution of DOM was estimated using a series of organic macromolecules ranging from 479 to 66,000 Da. Both the number-average (M(n)) and weight-average (M(w)) molecular weights of Suwannee River fulvic acid (SRFA) and Suwannee River humic acid (SRHA) determined using these macromolecular standards were comparable to those obtained using polystyrenesulfonate (PSS) standards, suggesting that organic macromolecules can be used to estimate MW of natural organic colloids. The MW of eight river DOM samples determined by this method was found to have an M(n) range of 0.8-1.1 kDa, which agrees with available literature estimates. The FlFFF-DAD-EEM system provided insight into the MW components of river DOM including the optical properties by on-line absorbance and fluorescence measurement. A red-shift in emission and excitation wavelength maxima associated with lower spectral slope ratios (S(R)=S₂₇₅₋₂₉₅:S₃₅₀₋₄₀₀) was related to higher MW DOM. However, DOM of different origins at similar MW also showed significant difference in optical properties. A difference of 47 and 40 nm in excitation and emission peak C maxima was found. This supports the hypothesis that river DOM is not uniform in size and optical composition.

Determination of relative molecular weights of fluorescent components in dissolved organic matter using asymmetrical flow field-flow fractionation and parallel factor analysis.

Dissolved organic matter in aquatic systems is of variable structure and composition. Asymmetrical flow field-flow fractionation coupled to UV/vis diode array and fluorescence detectors (AF4-DAD-EEM) was used to assess the size and optical properties of dissolved organic matter. The results were analyzed using parallel factor analysis (PARAFAC) and statistical fractogram deconvolution to correlate fluorescing components with molecular weight fractions. This coupling, which is shown for the first time in this work, is a powerful method capable of revealing novel information about the size properties of PARAFAC components. Tyrosine/polyphenol-like fluorescence (peak B) was significantly correlated (p<0.05) with the smallest size group (relative molecular weight=310±10 Da), microbial humic-like and terrestrial visible humic-like fluorescence (peaks M, C, A) with the intermediate size group (1600±150 Da), and terrestrial fulvic-like and tryptophan/polyphenol-like fluorescence (peaks A and T) with the largest size group (4300±660 Da).

Distinguishing dissolved organic matter at its origin: size and optical properties of leaf-litter leachates.

Dissolved organic matter (DOM) was leached from eight distinct samples of leaves taken from six distinct trees (red maple, bur oak at three times of the year, two sugar maple and two white spruce trees from disparate soil types). Multiple samples were taken over 72-96h of leaching. The size and optical properties of leachates were assessed using asymmetrical flow field-flow fractionation (AF4) coupled to diode-array ultraviolet/visible absorbance and excitation-emission matrix fluorescence detectors (EEM). The fluorescence of unfractionated samples was also analyzed. EEMs were analyzed using parallel factor analysis (PARAFAC) and principal component analysis (PCA) of proportional component loadings. Both the unfractionated and AF4-fractionated leachates had distinct size and optical properties. The 95% confidence ranges for molecular weight distributions were determined as: 210-440Da for spruce, 540-920Da for sugar maple, 630-800Da for spring oak leaves, 930-950Da for senescent oak, 1490-1670 for senescent red maple, and 3430-4270Da for oak leaves that were collected from the ground after spring thaw. In most cases the fluorescence properties of leachates were different for individuals from different soil types and across seasons; however, PCA of PARAFAC loadings revealed that the observed distinctiveness was chiefly species-based. Strong correlations were found between the molecular weight distribution of both unfractionated and fractionated leachates and their principal component loadings (R(2)=0.85 and 0.95, respectively). It is concluded that results support a species-based origin for differences in optical properties.

Impacts of Microbial Activity on the Optical and Copper-Binding Properties of Leaf-Litter Leachate

Dissolved organic matter (DOM) is a universal part of all aquatic systems that largely originates with the decay of plant and animal tissue. Its polyelectrolytic and heterogeneous characters make it an effective metal-complexing agent with highly diverse characteristics. Microbes utilize DOM as a source of nutrients and energy and their enzymatic activity may change its composition, thereby altering the bioavailability and toxicity of metals. This study investigated the impacts of microbial inoculation upon the optical and copper-binding properties of freshly produced leaf-litter leachate over 168 h. Copper speciation was measured using voltammetry, and using fluorescence quenching analysis of independent fluorophores determined using parallel factor analysis (PARAFAC). Two protein/polyphenol-like and two fulvic/humic-like components were detected. Thirty-five percent of total protein/polyphenol-like fluorescence was removed after 168-h of exposure to riverine microbes. The microbial humic-like and tryptophan-like PARAFAC components retained significantly different log K values after 168 h of incubation (p < 0.05), while their complexing capacities were similar. Using voltammetry, a sixfold increase in copper-complexing capacity (CC, from 130 to 770 μmol Cu g C−1) was observed over the exposure period, while the conditional binding constant (log K) decreased from 7.2 to 5.8. Overall binding parameters determined using voltammetry and fluorescence quenching were in agreement. However, the electrochemically based binding strength was significantly greater than that exhibited by any of the PARAFAC components, which may be due to the impact of non-fluorescent DOM, or differences in the concentration ranges of metals analyzed (i.e., different analytical windows). It was concluded that the microbial metabolization of maple leaf leachate has a significant impact upon DOM composition and its copper-binding characteristics.

Absorption and fluorescence of dissolved organic matter in the waters of the Canadian Arctic Archipelago, Baffin Bay, and the Labrador Sea

The optical properties of dissolved organic matter (DOM) were investigated for the first time in the Canadian Arctic Archipelago (CAA), Baffin Bay (BB), and Labrador Sea (LS) as part of the International Polar Year Canadas Three Oceans project (C3O). The dynamics and composition of absorbing DOM (i.e., colored DOM, or CDOM) and fluorescent DOM (FDOM) were evaluated in several distinct water masses occupying the three regions: surface waters, Arctic outflow waters, West Greenland Intermediate waters (WGIW), upper Labrador Sea waters (uLSW), and Bottom Baffin Deep Water (BBDW). Four fluorescent components were identified by applying parallel factor analysis (PARAFAC) to 522 excitation emission matrix (EEM) spectra: three humic-like and one protein-like. The FDOM in surface waters of the CAA and BB differed considerably in character from those of the LS, with higher fluorescence intensity in the former. The fluorescence intensities of the two terrestrial humic-like components (C1 and C3) were linearly correlated with apparent oxygen utilization (AOU) in the CAA Arctic outflow and the WGIW whereas only humic-like C3 was significantly correlated with AOU in uLSW. These findings suggest that the humic-like components were produced in situ as organic matter was bio-oxidized. The slopes of the linear relationship between humic-like intensity and AOU were significantly greater in the WGIW relative to the Arctic outflow waters, which implies that FDOM in the Arctic-derived deeper layer was less prone to mineralization.

Changes in the fluorescence composition of multiple DOM sources over pH gradients assessed by combining parallel factor analysis and self‐organizing maps

Dissolved organic matter is a ubiquitous constituent of natural waters that plays key roles in several important processes. The fluorescence properties of DOM have been linked to its functionality, but these properties may vary with pH. In this study Kohonens self-organizing maps (SOMs) were applied to excitation-emission matrices (EEMs) of fresh dissolved organic matter (DOM) from three sources: senescent sugar-maple leaves and white spruce needles, and humified white spruce needles, over a pH range of ~4.5 – 12.5. SOMs were applied to raw EEMs, EEMs reduced in dimensionality by pre-processing using parallel factor analysis (PARAFAC), and PARAFAC loading proportions normalized to values at initial pH. Some separation of EEMs into source-based clusters was achieved in the SOM of raw EEMs, but commingling was apparent and evidence of changes over pH gradients was overshadowed. SOMs of PARAFAC component proportions demonstrated clear source-based clustering, and pH-based gradients were visible for DOM from senescent and humified spruce needles. Changes in optical properties were obvious over pH gradients in the SOM of components normalized to starting condition. Component proportions decreased to values as low as 5% of the initial values for microbial humic-like peak M and increased to as high as 278% for a humic-like component. Tyrosine-like fluorescence increased to 112% of initial over increasing pH in humified spruce leachates but decreased to as low as 45% in the other leachates. The combination of PARAFAC and SOM drastically enhanced visualization and interpretability of pH-induced changes in DOM compared to either method alone.

Snowpack deposition of trace elements in the Athabasca oil sands region, Canada

The total recoverable and dissolved concentrations of 29 metals and metalloids were analyzed in snowpack collected at 91 sites in the Athabasca oil sands region, Canada in winter 2011. Based on deposition pattern from geographical centre, three groups were found: Type-1 metals (i.e. dissolved and total recoverable V; Mo) showed a significant exponential decrease with distance, suggesting oil sands development sources; Type-2 elements (e.g. Al, Sb, As, Ba, Fe, Ni, Tl, and Ti and Zn) showed exponentially decline patterns but with some local point sources; Type-3 elements (e.g. Cd, Cl, Cr, Mn, Sr and Th) deposition pattern represented local sources. A self-organizing map showed that sites with the highest elemental concentrations (Cluster I) were mainly located in the vicinity of upgrading facilities and along the north-south transects. The lowest elemental concentration sites (Cluster III) were the most distal sites or located in the western region of the study area.

Analysis of dissolved organic matter fluorescence using self-organizing maps: mini-review and tutorial

The analysis of dissolved organic matter (DOM) fluorescence has facilitated tracing throughout both natural and engineered aquatic systems, and the connection of DOM quality to its effectiveness as a binding agent, nutrient source, and generator of disinfection byproducts. While methods that deconvolute fluorescence into distinct underlying components have been instrumental in this endeavour, the vast majority of subsequent analyses has been limited to isolated parts/components, whereas DOM is a complex and dynamic system of interacting molecules. Tools for pattern recognition such as Kohonens self-organizing maps (SOMs) are capable of analyzing DOM fluorescence holistically, either by recomposing the parts isolated via PARAFAC, or by analyzing fluorescence in its entirety; however, the SOM has been underutilized in this endeavour, largely due to the difficulty of implementation and optimization. This report briefly summarizes the prior application of the SOM to DOM fluorescence, followed by a brief introduction to its theory and utility. An instructional tutorial and user-friendly ‘Fluor_SOmap’ Matlab package have been included to assist researchers in implementing SOMs. Four modes of fluorescence data analysis are facilitated by the package, including: whole EEMs, excitation–emission wavelength pairs, component scores from PARAFAC, and overall fluorescence composition. It is hoped that this tutorial and the associated package will help researchers implement and interpret SOMs to extend the scope and utility of fluorescence analysis.

Asymmetrical flow field-flow fractionation and excitation-emission matrix spectroscopy combined with parallel factor analyses of riverine dissolved organic matter isolated by tangential flow ultrafiltration

Asymmetrical flow field-flow fractionation (AF4) with sequential on-line UV/visible and fluorescence detectors was used to investigate the composition of dissolved organic matter (DOM) in permeate and retentate fractions isolated by tangential flow ultrafiltration (TFF) at various concentration factors (i.e. ratio of initial volume to the retentate volume; CF). The permeation coefficient model, which defines the log-log relationship between DOM in the permeate fractions and CFs, described the permeation behaviour of DOM with regression coefficients r 2 > 0.99. The dominance of higher-molecular weight retentate chromophoric DOM (CDOM) observed in TFF was consistent with the results of AF4. The weight-averaged molecular weights (M w) of the integral permeate and retentate at CF = 20 were determined to be 1160 and 2320 by AF4, respectively, while their molecular weight distributions (MWD) were centered at 1120 and 1600 Da. M w, MWD, and aromaticity (i.e. ratio of absorbance at 250 and 365 nm; E2/E3) in permeate fractions were altered significantly during the early stages of TFF (CF < 9). These changes, however, were not evident in excitation-emission matrix fluorescence properties as determined using the parallel factor analysis model. The application of AF4 to TFF fractions suggests that the choice of CF may have an important impact on the size distribution and aromaticity of permeate fractions, whereas fluorescence properties appear insensitive to concentration factor. These results suggest that the choice of CF is crucial only in the study of the permeate fraction where similar CF (i.e. > 9) should be used to obtain meaningful comparison among samples.

Advanced Residuals Analysis for Determining the Number of PARAFAC Components in Dissolved Organic Matter

Parallel factor analysis (PARAFAC) has facilitated an explosion in research connecting the fluorescence properties of dissolved organic matter (DOM) to its functions and biogeochemical cycling in natural and engineered systems. However, the validation of robust PARAFAC models using split-half analysis requires an oft unrealistically large number (hundreds to thousands) of excitation–emission matrices (EEMs), and models with too few components may not adequately describe differences between DOM. This study used self-organizing maps (SOM) and comparing changes in residuals with the effects of adding components to estimate the number of PARAFAC components in DOM from two data sets: MS (110 EEMs from nine leaf leachates and headwaters) and LR (64 EEMs from the Lena River). Clustering by SOM demonstrated that peaks clearly persisted in model residuals after validation by split-half analysis. Plotting the changes to residuals was an effective method for visualizing the removal of fluorophore-like fluorescence caused by increasing the number of PARAFAC components. Extracting additional PARAFAC components via residuals analysis increased the proportion of correctly identified size-fractionated leaf leachates from 56.0 ± 0.8 to 75.2 ± 0.9%, and from 51.7 ± 1.4 to 92.9 ± 0.0% for whole leachates. Model overfitting was assessed by considering the correlations between components, and their distributions amongst samples. Advanced residuals analysis improved the ability of PARAFAC to resolve the variation in DOM fluorescence, and presents an enhanced validation approach for assessing the number of components that can be used to supplement the potentially misleading results of split-half analysis.