4.1. Peak Excitation and Emission Maxima, Peak Intensities, and Peak Ratios
 Humic-type peaks within permeate samples, representing LMW terrigenous organic matter, appeared almost universally to bleach more at lower salinities (Figures 1 and 2), the principal exception was freshwater permeates from DB Mar03. There was often (especially with estuarine permeate) a drastic increase in fluorescence photobleaching below 8 salinity (Figures 1 and 2). This fluorescence photobleaching effect appears to overlap with an anomalous increase in fluorescence within this region due to mixing effects (assumed to be ionic strength-related) alone [Boyd et al., 2010]. One could speculate that either processed end-member (freshwater perhaps) or in situ produced LMW organic matter is impacted by modest salinity changes (ionic strength) which may induce either conformational changes or alter complexation which in turn impacts photodegradation potential. The LMW organic matter would presumably consist of smaller biomolecules, amino acids, and small peptides [Yamashita and Tanoue, 2003], but the fluorescence signal might be dominated by fulvic acids [Leenheer et al., 1995a, 1995b]. B and T peak fluorescence (discussed further in this section) has been presumed to be derived from tyrosine and tryptophan residues within proteinaceous components; however, recent evidence suggests a LMW DOM source for this fluorescence [Yamashita and Tanoue, 2003]. While largely confined to the cell biochemistry community, reports are available which describe considerable fluorescence shifts in small peptides under different divalent ionic strength regimes such as KI [Weljie and Vogel, 2000]. We hypothesize that components of the mid-estuarine LMW organic matter pool are closely associated with divalent cations, which in turn modulate their fluorescence properties and photoreactivity. There is evidence that divalent metals can significantly impact the fluorescence properties of LMW organic matter during sea-ward mixing off the West Florida shelf [Zanardi-Lamardo et al., 2004]. In this study, the authors also noted a higher relative fluorescence for lower molecular masses. While perhaps more relevant to UDOM, decreases in photoreactivity have been noted with divalent metal-binding ligands [Witt et al., 2007]. With humic-type material, photochemical “protection” was noted when complexed with divalent copper [Brinkmann et al., 2003], although these elements are not likely in high abundance in the sampled estuaries. Dissolved element measurements during DOM photodegradation also argues for tightly coupled organic matter–trace element behavior in estuaries [Shiller et al., 2006].
 For freshwater and marine humic-type UDOM, there was slight increase in fluorescence photobleaching at mid-salinities and a decrease toward the freshwater and ocean end-members (Figures 1 and 2). The exception to this general trend was freshwater UDOM from DB Mar03 (Figures 1 and 2). Humic-type components were separated by type in a study from Isa Bay in Japan [Yamashita et al., 2008]. Type II humic materials were described as having non-conservative mixing behavior when plotted against salinity. While not separated into LMW and HMW fractions, type II humics were thought to be produced mid-estuary, possibly as a product of type I (C and A peak) photo- or bio-degradation. Another possible suggestion for type II humic creation was POM dissolution [Yamashita et al., 2008]. As we are aware of no other photobleaching experiments on LMW and UDOM from the same sample matrix, it is not possible to directly relate this data set to other analyses. It is likely that at very low salinities, LMW fluorophores, perhaps dominated by fulvic acids, are highly photoreactive. Their photoreactivity is diminished upon mixing with a small amount of seawater, possibly due to divalent ion concentrations increasingly complexing them. Lower salinity waters appear to have a smaller fluorescence photobleaching effect on UDOM but there appears perhaps to be a photolabile component of UDOM at mid-salinities. Based on peak and PARAFAC score changes with salinity, photolabile material may consist of HMW humic acids [Osburn et al., 2009].
 Quite possibly, a bi-phasic estuarine mixing scenario may exist in estuaries wherein LMW organic matter becomes increasingly complexed and more photo-resistant toward mid salinities while higher molecular weight organic matter (relatively photo-resistant at lower salinities) becomes more photo-reactive as it mixes into mid-estuary. This might be consistent with the mid-estuarine humic source explanation offered by Yamashita et al. , particularly if UDOM represented a more refractory DOM component derived from riverine upper reaches. This scenario is consistent with earlier reports for the Chesapeake Bay system [Osburn et al., 2009].
 EEMs resulting from photoexposure had B and T peaks which differed considerably in character, both in terms of LMW and UDOM fractions. While these peaks have traditionally been thought to represent proteinacous material with tyrosine and tryptophan residues, respectively, their behaviors are beginning to be seen as different in natural waters [Yamashita et al., 2008]. Certainly in this series of experiments, both in terms of mixing alone [Boyd et al., 2010], and mixing with photoexposure, the T peak most closely resembled the N peak, which has been classified as non-humic and biologically labile [Coble et al., 1998; Cory and McKnight, 2005]. The tracking of these two peaks with salinity caused Yamashita et al.  to classify them together as “type III” components. Photobleached fluorescence in both T and N peaks showed no trend in low salinity regions (<16), sometimes increasing and sometimes decreasing with decreasing salinity. Because the T (and N) peak showed different behavior based on size (LMW and UDOM), it is likely that they may represent proteinaceous material (HMW), fulvic acids, or phenolic precursors of humic material [Maie et al., 2007], each, of course, with different reactivity.
 The B peak showed no trend with salinity mixing alone [Boyd et al., 2010] and little to no photobleaching in either the LMW or UDOM samples. Yamashita et al.  classify the B peak fluorescene as a type IV component and argue it represents relatively ubiquitous, highly degraded (thus refractory) LMW organic matter [Yamashita et al., 2008; Yamashita and Tanoue, 2003]. The B peak may represent the recalcitrant LMW fraction of DOM reported elsewhere [Amon and Benner, 1996].
 As fluorescence peak ratios are often used to track organic matter from various sources in coastal systems, it is interesting to note that for surveyed ratios (with UDOM), there were significant changes based on the salinity gradient. With freshwater UDOM, the McKnight aromaticity index varied by at least 0.02 units for all sites/seasons with the lowest variation coming from DB Mar03 (Figure 3a). There appeared, as with other parameters, significant shifts at the end of salinity ranges (0–12, 28–36). For estuarine UDOM, “dry season” samples from CB (Aug04, Sep03) showed extreme variation (up to 0.5 units) depending on salinity. Mechanistically, it is not possible without further chemical analysis to determine if fluorescence photobleaching changed the amount of aromaticity within the UDOM, but the presence of positive and negative fluctuations, especially in estuarine UDOM (Figure 5a), argues that small changes in spectral regions used for ratio calculations can have a significant impact on their values. The increase in this ratio at the ocean end-member for freshwater UDOM seems consistent within sites/seasons (with the common exception of DB Mar03). The 265F375/400 ratio has been used to infer a terrigenous vs ocean origin for natural organic matter. As with peak intensities, this ratio was extremely variable at low salinities, in the case of estuarine UDOM, well within the “marine” range of greater than 1.0 (Figures 4b and 5b). The 305F430/540 ratio also demonstrated considerable variability at freshwater end-member salinities and tended to be lower at the low salinity range and remain relatively constant at mid salinities toward the ocean-member. Without mixing and photoexposure history, it might be difficult to absolutely apportion organic matter fluorescence to a given “origin” solely using fluorescence ratios. The less specific CDOM fluorescence signal 355F450 also showed variation due to photobleaching at different salinities (Figures 4c and 5c), often mimicking humic-type peak variations (with higher fluorescence photobleaching at mid salinities). Again, without molecular-level knowledge of the mechanism altering UDOM during estuarine mixing and fluorescence photobleaching, it may be difficult to apply fluorescence ratios as a static tracer of CDOM origin.
4.2. PARAFAC Analyses
 PARAFAC analysis on bleached EEM stacks was able to identify several factors which corresponded to known fluorescence peak regions. However, PARAFAC on bleached EEM stacks produced scores which in many cases failed to overlap known peak areas (e.g., Table 2). In many PARAFAC runs, peaks corresponding to a recently reported region (component 3 [Yamashita et al., 2008], SQ1 [Cory and McKnight, 2005], or peak 2 [Stedmon and Markager, 2005]) were evident but were not universal. In general, peaks identifiable in PARAFAC scores followed peak intensity characteristics: linear or exponential decreae across the salinity gradient for humic-type permeate peaks (Figures 6c and 6d), and extremely variable scores in low or high salinities with UDOM (Figure 7b).
 The most enlightening PARAFAC analysis came from stacking the mixing alone and photobleached EEMs into a 4-dimensional model wherein changes across the fourth dimension (bleached vs non-bleached) could be evaluated. In this PARAFAC model, four components were modeled: One, overlapping with the A peak region; two, overlapping the A, M and Intermediate C-M region; three, overlapping the A and C region; and four, overlapping (bisecting) the T and to a lesser extent, B peak region (Figures 9a–Figures 9d). The major finding in this analysis is that while the humic-type peaks all decrease in abundance from pre- to post-exposure, the T-B peak component actually increases due to photobleaching (Figure 9e). Interpretation of a 4D model takes one more level of complexity to understand. The loadings underlying those scores show a profile of behavior, certain EEM components decreasing with photoexposure for components 1 through 3, and increasing with exposure for component 4. A positive slope in the loading for the T-B factor in the exposure mode could mean that the T-B peak in the exposed EEMs is decreasing less quickly with salinity than in the unexposed EEMs. Alternatively there could be real photochemical conversion of material to this florescent region. Clearly, as indicated by other studies, there is a different dynamic affecting the T-B peak with respect to salinity and photobleaching than with the humic regions of the EEM. Considering the biological importance of amino acids for instance, this should not be surprising.