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Depth-dependent molecular composition and photo-reactivity of dissolved organic matter in a boreal lake under winter and summer conditions

Depth-dependent molecular composition and photo-reactivity of dissolved organic matter in a... Open Access Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ Biogeosciences doi:10.5194/bg-10-6945-2013 © Author(s) 2013. CC Attribution 3.0 License. Depth-dependent molecular composition and photo-reactivity of dissolved organic matter in a boreal lake under winter and summer conditions 1, 2 3, 4 2 M. Gonsior , P. Schmitt-Kopplin , and D. Bastviken University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, MD, USA Linköping University, Department of Thematic Studies, Unit Water and Environment, Linköping, Sweden Helmholtz Zentrum Munich, German Research Center for Environmental Health, Neuherberg, Germany Department for Chemical-Technical Analysis, Research Center Weihenstephan for Brewing and Food Quality, Technische Universität München, 85354 Freising-Weihenstephan, Germany Correspondence to: M. Gonsior ([email protected]) Received: 15 April 2013 – Published in Biogeosciences Discuss.: 29 May 2013 Revised: 23 September 2013 – Accepted: 2 October 2013 – Published: 4 November 2013 Abstract. Transformations of dissolved organic matter tion of DOM between surface and deep boreal lake waters. (DOM) in boreal lakes lead to large greenhouse gas emis- The release of DOC from boreal lake sediments also con- sions as well as substantial carbon storage in sediments. Us- tribute to this pattern. Photochemical degradation of DOM ing novel molecular characterization approaches and photo- may be more extensive following ice-out and water column chemical degradation experiments we studied how seasonal turnover when non-light exposed and thereby photosensitive patterns in water column stratification affected the DOM in DOM is photo-mineralized. Hence, the yearly DOM photo- a Swedish lake under early spring and summer conditions. mineralization may be greater than inferred from studies of Dissolved organic carbon (DOC) concentrations were con- recently light-exposed DOM. sistently higher above the sediment when compared to sur- face waters throughout the sampling periods. Photobleach- ing alone could not explain this difference in DOC because the lake was covered by 40 cm-thick ice during late winter 1 Introduction sampling and still showed the same DOC trend. The dif- It is becoming increasingly clear that processes in freshwa- ferences in the molecular diversity between surface DOM ters play a key role for global carbon cycling and in particular in winter and summer were consistent with ongoing pho- for the fate of the organic matter (OM) exported from soils. tobleaching/decarboxylation and a possible bacterial con- Large amounts of OM are decomposed to carbon dioxide and sumption of photo-products. Additional photo-degradation methane in lakes and estimated emissions of these gases cor- experiments using simulated sunlight showed a production of respond to 79 % of the continental uptake of carbon dioxide highly oxidized organic molecules and low molecular weight equivalents (Bastviken et al., 2011). At the same time OM compounds in all late winter samples and also in the deep can also flocculate, sink, and become buried and preserved water sample in summer. In the surface summer DOM sam- in sediments (von Wachenfeldt and Tranvik, 2008; Sobek et ple, few such molecules were produced during the photo- al., 2012). Recent estimates are that sediments harbor 98 % of degradation experiments, confirming that DOM was already the boreal lake OM (Einola et al., 2011), and that the yearly photobleached prior to the experiments. This study suggests sedimentary burial of OM in lakes exceed the burial in the that photobleaching, and therefore also the ice cover dur- oceans (Tranvik et al., 2009). The remainder will be trans- ing winter, plays a central role in surface DOM transforma- ported downstream towards the ocean. The processes behind tion, with important differences in the molecular composi- these different fates are driven by interactions between the Published by Copernicus Publications on behalf of the European Geosciences Union. 6946 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM quantity and quality of the OM, microbial metabolism, and quality and characteristics of OM changed between win- abiotic processes such as photochemistry and the balance be- ter and summer conditions and within the water column, tween flocculation and dissolution of OM. However, the rel- both in situ and after a series of photochemical experi- ative magnitude and the regulation of OM fates are still un- ments, using measurements of optical properties such as ul- clear in most systems. traviolet/visible (UV/Vis) spectroscopy and excitation emis- There have been numerous short-term incubation stud- sion matrix (EEM) fluorescence spectroscopy combined with ies of microbial and photochemical interactions with dis- ultrahigh-resolution electrospray ionization Fourier Trans- solved OM (DOM) degradation in lake waters (Moran and form Ion Cyclotron Resonance Mass Spectrometry (FT-MS). Zepp, 1997). It has been shown that the combination of ini- tial photochemical degradation of the DOM form smaller 2 Methods molecules and that they are more accessible to microbial degradation. This is also important for the total OM miner- 2.1 Sampling alization (Bertilsson and Allard, 1996; Tranvik and Bertils- son, 2001; Anesio et al., 2005). When expanding these find- In the sample collection for the core analyses of this study, ings from short-term incubations over time and space, large one-liter water samples were collected at 0.5 m depth (sur- seasonal differences in lake OM cycling should be expected face), and just above the sediment at 7 m depth in April in stratified and ice-covered systems where different propor- ◦ 0 00 ◦ 0 00 and June 2011 in Lillsjön (58 39 33.05 N, 16 8 34.81 E), tions of the OM are exposed to light during different sea- a lake located in Östergötland, Sweden. During the first sam- sons. Given that 51 % of the global lake area is situated north pling in April, the lake was still covered by 40 cm-thick ice of 48 latitude (Downing and Duarte, 2009), and that these that started to form in November 2010. Dissolved oxygen lakes are chromophoric DOM (CDOM) rich, such seasonal and temperature profiles were measured during sampling us- effects could actually be a major determinant of the fates ing a hand-held probe (Hach HQ40d). of OM in lakes. It is well established that during summer Additional water column profile data on temperatures and stratification in dimictic lakes, the CDOM levels in the sur- DOC was collected as above and the same location (same face layer (epilimnion) is decreased due to photobleaching lake and same sampling area) in August 1999 and March when compared to the deeper waters below the thermocline 2000 (under ice), and analyzed immediately for dissolved or- (hypolimnion) (Bracchini et al., 2006). As a result, photo- ganic carbon (DOC) as described below. These older DOC bleaching is a significant and major player in molecular al- profiles were used as independent supplementary data when teration of DOM (Moran et al., 2000; Stubbins et al., 2012; discussing the potential for DOC release from sediments. Zhang et al., 2006). Modeling attempts stressed that 50 % of All water samples were directly filtered through What- the CDOM light absorption within the entire water column in man GF/F glassfiber filters and acidified after filtration to lakes can be lost in 18–44 days under summer conditions due pH 2 using ultrapure concentrated HCl (Sigma Aldrich 32 %, to photobleaching (Reche et al., 2000). However this model puriss. p.a.). Overall, 800 mL of each water sample was then did not take into account that different CDOM components solid-phase extracted (SPE) as described elsewhere (Dittmar are likely to degrade at different timescales. For example, it is et al., 2008). Briefly, Agilent Bond Elut PPL solid-phase likely that at the onset of sunlight exposure a preferential loss extraction cartridges, filled with 1 g of the PPL resin (a of highly photosensitive components of (C)DOM occurs and styrene-divinylbenzene polymer that has been modified with that slower photo-degrading DOM constituents are relatively a proprietary non-polar surface), were pre-conditioned with increased but continue to photo-degrade at slower rates. methanol, rinsed with acidified (pH 2) Milli-Q water and then Seasonal stratification and the change in redox conditions the water sample was gravity-fed through it. DOC was mea- may also be important for a relatively neglected process in sured on acidified samples before and after the SPE extrac- lake carbon cycling studies – re-dissolution of particulate tion using a Shimadzu 5000 TOC analyzer and carbon ad- OM from lake sediments (Skoog and Arias-Esquivel, 2009; sorption efficiencies ranged between 58 and 64 %, and were Cottrell et al., 2013). During stratification periods, near- very similar to literature values of a variety of DOM samples bottom water layers often become anoxic, which affect all (Dittmar et al., 2008; Gonsior et al., 2011; Shakeri Yekta et redox-sensitive equilibria (Skoog and Arias-Esquivel, 2009) al., 2012). The absorbance of the samples before and after and processes, as well as e.g. pH, and this could affect OM SPE extraction was also measured using an Ultraspec 2100 solubility and availability (Phelps and Zeikus, 1984). The re- Pro (scan over 190–700 nm) and showed an average recovery dissolution can be important for assessments of OM stability, of the CDOM of 63 %. The SPE extracts were eluted with and burial in lake sediments. ◦ methanol and stored at −20 C in a freezer prior to photo- We addressed the hypotheses that seasonal patterns in wa- chemical experiments. Neither the shape of the absorbance ter column stratification are important for lake carbon cy- nor the fluorescence signals of the SPE extract changed no- cling by constraining photo-degradation and by inducing bot- ticeably. tom water conditions that enhance the re-dissolution of sed- imented particulate OM. To do this, we assessed how the Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6947 2.2 Photochemical experiments 2.4 Ultrahigh-resolution mass spectrometric analysis After completely drying of 1 mL of each SPE-DOM sam- A Bruker Apex QE 12 Tesla FT-MS with negative mode ple under nitrogen gas and the re-dissolution in 40 mL electrospray ionization located at the Helmholtz Center for high purity water (LC-MS grade water, Chromasolv, Sigma- Environmental Health, Munich, Germany was used to ana- Aldrich), the sample pH was close to the original water sam- lyze the SPE-DOM samples from the lake after a dilution of ple at pH 6.5. This was important to avoid changes in the 1 : 100 and all samples before and after photochemical degra- pH-dependent shift in absorbance and photobleaching as de- dation experiments at similar dilution. Electrospray is a soft scribed elsewhere (Pace et al., 2012; Janot et al., 2010). ionization technique that creates largely intact molecules (no The samples were then divided and half of the sample fragmentation) with a single charge and the ultrahigh resolu- (20 mL) was continuously pumped through a custom-built tion (mass error < 0.2 ppm and mass resolution > 400 000 at photo reactor cell made out of Tefzel tubing for 24 h. A mass 400) allows for the unambiguous assignment of molec- 10 mL gas equilibrator was also attached in-line to allow sat- ular formulae from all m/z peaks (Stenson et al., 2003). The uration of air gases at any given time. The flat-coiled cell accuracy of the used FT-MS is high enough to distinguish was exposed to simulated sunlight (SolSim Luzchem) that masses with a difference of less than an electron and hence closely matched the intensity of one sun at a solar zenith an- multiply charged ions can be distinguished and are rarely ob- gle of z = 48.2 (1.5 AirMass filter) corresponding to tem- served due to their much weaker ionization efficiencies when −2 perate latitudes and an intensity of 870 W m . The Tefzel compared to their singly charged counterparts. The follow- polymer (ethylene tetrafluoroethylene) has excellent light- ing chemical elements were allowed to calculate molecular 12 1 16 14 32 transmitting properties with > 90 % at 300 nm. This system formulae: C , H , O , N and S as 0−∞ 0−∞ 0−∞ 0−5 0−2 has several advantages, including minimizing inner filtering well as the C carbon isotope. Unambiguous molecu- 0−1 effects (due to the very short path length of 1 mm), avoiding lar formula assignments are now possible up to 800 Dalton starvation of air gases (gas equilibrator) and being a closed due to the mass accuracy of less than 0.2 ppm. Further infor- system avoiding contamination during extended sunlight ex- mation on how to accurately assign molecular formulae to posure times. ultrahigh-resolution FT-MS data is given elsewhere (Dittmar Dark controls were stored in the dark at similar tempera- et al., 2007; Schmitt-Kopplin and Hertkorn, 2007). Briefly, ture maintained within the ventilated solar simulator cham- the mass spectra were calibrated across the observed mass ber. All samples (irradiated and dark controls) were diluted range (150–700 Da) using internal standards of high relative with 50 % LC-MS methanol and analyzed by negative mode abundance exact mass peaks with known molecular formulas electrospray FT-MS. The differences between the dark con- that have been previously found in all DOM samples. Exter- trols and the irradiated samples were used to evaluate the nal pre-calibration was undertaken using arginine standards photo-degradation at the molecular level. prior to mass spectrometric analyses. The reproducibility of the SPE method in conjunction with the same mass spec- 2.3 Measurements of optical properties trometer used in this study has been previously described on triplicate samples (Shakeri Yekta et al., 2012). The repro- Absorbance measurements were undertaken on all samples ducibility of the relative abundances of mass peaks was good before and after exposure to simulated sunlight using a Cary and showed on average less than 4 % variability, but maxi- Bio100 Spectrophotometer with 1 cm quartz cuvettes. The mum differences of 10 % of high abundance mass peaks may spectral slopes and slope ratios were calculated using a first- occur; hence we only considered changes in relative abun- order decay function at the wavelengths ranges 275–295 nm dance that exceeded 10 % relative difference. Usually 20– and 350–400 nm and according to a detailed description pre- 30 % of the mass peaks have low enough intensity to be either viously published (Helms et al., 2008). Excitation emission above or below the set signal to noise ratio in replicate sam- matrix (EEM) fluorescence spectra were recorded using a ples. This can cause a large variability between replicates for Horiba Fluoromax 4 Spectrofluorometer at 5 nm excitation such low intensity mass peaks (Shakeri Yekta et al., 2012). intervals between 240–500 nm and an emission range of Correspondingly, an analysis to find unique mass peaks in 290–600 nm at 2 nm intervals. Scatter-correction and normal- each sample revealed that unique mass peaks showed always ization to quinine sulfate equivalency was performed similar very low abundances, indicating that they were likely not to the procedure described elsewhere (Zepp et al., 2004). All unique but just had an intensity above the signal to noise ra- samples were diluted 1 : 10 prior to all optical property anal- tio in some samples. To avoid any artifacts of the data, we yses to avoid inner-filtering effects, and to be able to apply only considered mass peaks that were commonly found in all the linear range of the quinine sulfate calibration of the EEM samples of the same treatment. Van Krevelen diagrams where spectra. the elemental ratios of oxygen to carbon (O / C) are plotted against the hydrogen to carbon (H / C) ratios of each individ- ually assigned molecular formula are particularly useful for visualizing FT-MS data and showing the chemical space of a www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6948 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM DOM sample occupied in this specific elemental plot (Kim et al., 2003) and were used throughout the study. Another useful visualization tool is the Kendrick diagram, where the Kendrick mass defect (KMD) is plotted against the nominal mass (Kendrick, 1963; Wu et al., 2004). The Kendrick mass (KM) is essentially a normalization of the In- ternational Union of Pure and Applied Chemistry (IUPAC) mass to the mass of CH groups (m = 14.01565) and the KMD is the difference between nominal mass (NM) and KM. KM = IUPAC mass × (14.000000/14.01565) (1) KMD = NM − KM (2) All homologues of CH would have the same KMD and therefore it can be used to distinguish CH homologues com- mon in FT-MS data of DOM. A second independent param- eter (z ) was used to assign homologues as Fig. 1. Temperature and dissolved oxygen saturation profiles of Lill- sjön Lake in April and June 2011. z = (mod[NM/14]) − 14 (3) The modulus (mod) function returns the remainder after the high release of DOC at the onset of anoxia (Skoog and Arias- NM is divided by 14 (Stenson et al., 2003). Here, we used Esquivel, 2009). In our case, this may have occurred in the a modified Kendrick plot where the ratio of the KMD is di- month before the April sampling. Another alternative expla- vided by z and then plotted against the mass to show accu- nation could be higher microbial mineralization in the sur- rately unambiguous homologous series in one plot (Shakeri face water, but it is difficult to find good arguments for this, Yekta et al., 2012). given the slightly lower winter temperatures in the surface water and that most parts of the hypolimnion were oxic dur- ing the winter. The strongest support of DOM release from 3 Results and discussion the sediments are derived from DOC distribution under ice During the April sampling, the dissolved oxygen saturation when the temperature profile (and the microbial degradation) was 59 % just underneath the ice and decreased rapidly with and the light influence throughout the water column is more depth to low oxygen levels (1 %) at 0.5 m above the sediment uniform than during summer. The more detailed DOC pro- (Fig. 1). However, the bottom water had probably been oxic files from August 1999 and March 2000 (under ice) support until just before the sampling, as indicated by previous oxic the findings from 2011 and indicate considerable DOC re- profiles taken in the month of March (data not shown). lease from sediments under ice (Fig. 2). In June, the lake was strongly stratified with 100 % oxy- In June the bottom water DOC levels were close to gen saturation at 0.5 m depth, decreasing with depth to about (slightly lower than) surface levels in April. This is log- 18 % at 0.5 m above sediment (Table 1 and Fig. 1). At both ical given the combination of (1) lake circulation mixing sampling periods, the DOC concentrations were consistently the larger surface water volume with a smaller bottom wa- higher 0.5 m above sediment than at 0.5 m below the surface. ter volume, (2) increased temperatures and thereby increased The DOC concentration was highest in the deep sample un- DOC mineralization throughout the water column, and (3) der ice followed by surface sample under ice, deep sample some photo-mineralization during the circulation period af- in summer and surface sample in summer (Table 1). This in- fecting the whole water column. After lake circulation, the dicated either a source of DOM to the bottom water and/or surface water DOC levels was likely reduced further by ad- a DOC dilution/depletion at the surface both under ice and ditional photo-mineralization and enhanced microbial degra- during summer. Under the winter conditions with the exist- dation as temperatures increased further. Additional DOM ing ice cover and frozen soils, it seemed unlikely that the sources that may have influenced the DOC concentrations surface water was diluted (rain, runoff etc.). The most likely between April and June cannot be ruled out but the months reason for the higher DOM concentrations in the bottom wa- of April, May and June 2011 were particularly dry in this ter is release from sediment. A change in the redox potential region and large amounts of leaching from forest soils af- at the sediment surface caused by switching from aerobic to ter spring mixing was unlikely. Photochemical experiments anaerobic conditions has previously been reported to cause a were undertaken to evaluate if differences existed between Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6949 Table 1. Temperature, dissolved oxygen and DOC concentrations during sampling of Lillsjön, a boreal lake in 2011. Sampling 04/01/2011 Sampling 06/28/2011 ◦ −1 ◦ −1 Sample Lillsjön Depth (m) T ( C) O (%) DOC (mg L ) T ( C) O (%) DOC (mg L ) surface 0.5 2.1 59 22.7 ± 0.3 19.4 100 18.7 ± 0.2 bottom 7 4 1 25.0 ± 0.2 4.7 18 21.5 ± 0.2 decrease in molecular weight after irradiation. In contrast, the same photochemical experiments undertaken on water sam- pled in June showed much less photobleaching at the surface when compared to deep water SPE-DOM (Figs. 3 and 4). The photochemically induced changes in S also confirmed this trend with an increase of only 14 % in the June surface sample compared to 22 % in the bottom water sample (Ta- ble 2). This indicates that a significant amount of the sur- face CDOM had already been photobleached by natural sun- light prior to additional laboratory-based photobleaching ex- periments. An alternative explanation would be a microbial degradation of CDOM during spring and the much warmer surface water. However, this was shown to be a rather slow process and a decrease in fluorescent intensity of up to 40 % in 3.5 yr has been demonstrated (Kothawala et al., 2012). An- other study suggested the involvement of lake bacterial com- munities to produce and degrade CDOM, but this observa- tion was associated with increased protein-like fluorescence arising from bacterioplankton biomass (Guillemette and del Fig. 2. Depth profiles of DOC and temperature in Lillsjön Lake in Giorgio, 2012). In this study we did not see any change in 24 August 1999 (summer) and 7 March 2000 (winter). the protein-like fluorescence between winter and summer. Therefore, the involvement of bacteria in the degradation of CDOM in the June surface water sample was probably not the photochemical reactivity (photobleaching) of DOM be- significant when compared to photochemical degradation. tween surface and deep water as a function of season and a It seemed likely that the observed consistent differences comparison between ice-covered and light-protected surface between surface and deep water DOC concentrations are waters in early spring, with strongly exposed surface water in caused by a combination of DOC released from sediments summer after exposure to sunlight during the dry and sunny and photobleaching/microbial degradation of DOM at the spring of 2011. If a release of DOM from sediments occurred surface in summer and mainly driven by DOM fluxes from and the significant differences in DOC concentrations be- sediments in winter. tween surface and deep waters are not primarily related to The ultrahigh-resolution, negative electrospray ionization photobleaching, then there should not be a great difference Fourier transform, ion cyclotron resonance mass spectrom- between photobleaching in surface and deep waters under etry (FT-MS) was used (Fig. 5) to allow for unambiguous winter conditions and during ice-cover. Indeed photochem- assignments of molecular formulae to each exact mass peak ical degradation of solid-phase extracted DOM (SPE-DOM) in each sample. Only masses containing C, H and O atoms and its associated changes in optical properties in April at were used in further analysis because nitrogen- and sulfur- the surface and just above the sediment after the exposure containing molecular formulas were assigned but showed to 24 h simulated sunlight was substantial but revealed only low abundances in negative electrospray mass spectrometry small changes between depths (Figs. 3 and 4). The spectral (Fig. 5). slope ratios (S ), obtained by dividing the slope calculated The comparison between surface and deep SPE-DOM un- between 275–295 nm and 350–400 nm (Helms et al., 2008), der winter conditions collected in April 2011 showed that the showed also significant changes towards higher values after relative abundance of highly oxidized organic compounds irradiation time, but was relatively similar between depths (O / C ratio between 0.50–0.87) was slightly decreased in the (18 % increase in the April surface and 20 % in the April bot- surface compared to deep SPE-DOM, whereas the lower oxi- tom water sample) (Table 2). In general, these results are in dized (O / C ratio < 0.45) organic compounds showed a small agreement with the Helms et al. study and would suggest a www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6950 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM Table 2. UV-Vis spectral slopes at distinct wavelength ranges and the resulting spectral slope ratios (S ) of surface and deep boreal lake water samples collected in April and June 2011. Sampling 04/01/2011 Sampling 06/28/2011 Sample Lillsjön lake S S S S S S S S r r 275−395 350−400 275−395 350−400 surface, before irradiation 0.0144 0.0135 0.0185 0.73 0.0145 0.0139 0.0182 0.76 surface, after irradiation 0.0151 0.0157 0.0176 0.89 0.0151 0.0158 0.0177 0.89 bottom, before irradiation 0.0143 0.0135 0.0183 0.74 0.0141 0.0133 0.018 0.74 bottom, after irradiation 0.0147 0.0159 0.0172 0.92 0.0144 0.0159 0.0167 0.95 Fig. 3. Depth-dependent seasonal changes in the photochemical degradation patterns after 24 h exposure to simulated sunlight of lake SPE- DOM and analyzed by UV/Vis absorption spectroscopy. increase in the surface relative to deep SPE-DOM (Fig. 6). whereby DOC is oxidized and dissolved inorganic carbon is One explanation could be that more oxidized DOM is often formed, seemed to be an effective pathway based on dark in- also more water soluble (because the electronegativity of cubation of water collected in the same lake in a previous oxygen will introduce a polarization of covalent bonds and study and the loss of about 7 % of the initial DOC within increase the overall solubility) and therefore is preferentially 5 months at 15 C (Bastviken et al., 2004). To provide a crude released from sediments. This would increase the relative estimate of relative contribution of microbial versus light- abundance of more oxidized DOM in the bottom water. dependent removal of DOC in surface water between April In June 2011, differences between surface and deep wa- and June in our study we assumed that the total respiration −1 ter were primarily associated with increased abundances of found in this previous study over 5 months (1.7 mg C L ) mass peaks assigned to relatively low H / C and high O / C represented the contribution of the light-independent micro- ratio lower molecular weight compounds in the surface SPE- bial DOC mineralization from April to June (2 months). We DOM sample (Fig. 6). Interestingly, those highly oxidized also assumed negligible input of water and DOC from land molecular formulas appeared in even higher relative abun- given the very dry conditions this spring. Then, given a total −1 dances after light treatment in our photochemical degrada- difference of 2.8 mg DOC L between surface and bottom −1 tion experiments (Figs. 7 and 8). Such highly oxidized for- water in June (Table 1), at least 1.1 mg L or 40 % of this mulas produced after solar simulated irradiation have been relative DOC loss in the surface water should be directly or previously shown and confirmed our observation (Gonsior et indirectly related to photochemical DOC degradation. This al., 2009). This indicates that these molecules were photo- calculation likely overestimates the light-independent micro- chemical products that are labile and depleted by either mi- bial contribution by using a 5-month value for 2 months, crobial degradation or additional photochemical decarboxy- thereby underestimating the influence of photochemistry, and lation (Xie et al., 2004; Xu and Wan, 2000) and the release should not be taken literally. Rather it illustrates similar mag- of carbon mono- and dioxide and other small carboxylic nitudes of light-independent and photochemically induced acids (Bertilsson and Tranvik, 1998). Microbial respiration, DOC mineralization processes in lake surface water. Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6951 Fig. 4. Excitation emission matrix fluorescence of surface and deep SPE-DOM collected in April and June 2011 before and after ex- posure to simulated sunlight including a differential plot of the de- creased fluorescence after irradiation. Note: SPE-DOM was dried and then re-dissolved in pure water at pH 6.5. Fig. 5. Ultrahigh-resolution mass spectrum of SPE-DOM extracted from a boreal lake at the surface in April 2011 in Sweden (Lill- sjön) and the van Krevelen diagram of CHO, CHOS and CHNO formulae. Note: size of bubbles represent relative abundances of as- The absolute decrease in absorption coefficients (Fig. 3) sociated mass peaks. after 24 h simulated sunlight exposure was significant in all samples including the late winter and summer surface and deep samples. The photobleaching under winter condi- tions between surface and deep water CDOM was very sim- sults imply that water that had not been exposed to light for ilar with the only detectable differences below 240 nm. This extended periods (under ice or near the bottom during sum- stands in contrast to the comparison between surface and mer stratification) was much more photo-reactive. deep CDOM in summer, where the surface CDOM is much This reduced photobleaching measured by optical prop- less photobleached in the whole ultraviolet (UV) range (200– erties (Figs. 3 and 4) of the June 2011 SPE-DOM sample 400 nm) (Fig. 3). already indicated that photo-active components in this SPE- The measurements of EEM spectra before and after the DOM were largely degraded prior to photobleaching ex- same irradiation time showed that the photochemically in- periments. As a result, the photobleaching demonstrated by duced changes were reflected in areas in the EEM spectra that changes in optical properties should be also reflected in the are centered around two maxima with excitation, emission ultrahigh-resolution mass spectrometric data and a different couples of 280–380 nm and 340–375 nm (Fig. 4). This pref- photobleaching behavior in the June surface DOM sample erential photo-degradation of FDOM at longer wavelengths was expected given the reduced photobleaching in this sam- indicated a shift towards lower wavelengths of the remain- ple when compared to all others. Indeed, the surface and deep ing fluorescent peaks after irradiation similar to findings re- SPE-DOM sample collected in April 2011 together with the ported previously (Helms et al., 2013). The summer surface deep sample from June 2011 showed a similar photochemi- sample was the only sample that showed clear differences cal behavior with a production of highly oxidized and high in photochemically induced degradation of fluorescent DOM O / C ratio molecules (Figs. 7 and 8). Additionally, a shift (FDOM) between surface and bottom water, confirming the to lower molecular weight and a significant increase in rel- same trend shown in the absorbance data (Fig. 3). These re- ative abundance of these low molecular weight mass peaks www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6952 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM Fig. 7. Photochemically induced changes in the molecular composi- tion of a boreal lake SPE-DOM at the surface and bottom in spring (ice covered) and at the surface and bottom in summer shown in van Krevelen diagrams and the observed relative changes in intensity for all mass peaks, respectively. (Note: black bubbles represent molec- ular formulas with an increase in associated relative abundances of the mass peaks after 24 h solar simulated irradiation and open bub- bles with a decrease, respectively.) Fig. 6. Van Krevelen, H / C and O / C versus mass diagrams of rela- tive differences in mass peak abundances between surface and deep been suggested previously (Thomson et al., 2004). The con- SPE-DOM in April and June 2011. Note: the bubbles represent the sistent decrease in all samples of mass peaks associated with relative abundances greater than 10 %. H / C ratios below 0.8 of associated molecular formulae and throughout a broad range of O / C ratios as demonstrated with the June samples (Fig. 8, black circles), is indicative for a was observed in all those samples. This trend is in agree- large pool of presumably aromatic CDOM components that ment with previous studies where ultrafiltered DOM was are photosensitive (Table 3). The high double bond equiv- photo-degraded and analyzed by size exclusion chromatog- alency (DBE, number of double bonds and rings) and the raphy (Lou and Xie, 2006; Thomson et al., 2004). Whole DBE divided by the carbon number (DBE/C) values are in- water samples were also previously analyzed using optical dicative of reduced compounds (Table 3). A set of photo- properties (Helms et al., 2008) and ultrahigh-resolution mass sensitive aromatic compounds in the Cape Fear River estu- spectrometry of Congo River water (Stubbins et al., 2010) ary has been previously shown using FT-ICR-MS (Gonsior with again similar findings. et al., 2009) and long-term exposure to simulated sunlight The photo production of highly oxidized compounds can (57 days) of Congo River water also confirmed the preferen- be explained with reaction of reactive oxygen species, such tial photo-degradation of the aromatic DOM content (Stub- as the hydroxyl radical (OH), super oxide (O ) and hydrogen bins et al., 2010). The loss of aromatics is also in agreement peroxide (H O ). A photochemically induced depolymeriza- with the observed photochemically induced changes in the 2 2 tion and a decrease in molecular weight of DOM have also optical properties and the decrease in conjugated aromatic Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6953 Fig. 8. Photochemically induced changes in the molecular composi- tion of a boreal lake SPE-DOM at the surface and bottom in summer and its associated increase in intensity of mass peaks that showed more than 10 % change and a decrease of more than 10 %, respec- tively. Results are illustrated using van Krevelen diagrams and dia- grams where the relative change in intensity is plotted against mass. Note: the bubble size in the van Krevelen diagrams correspond to the relative intensity change after irradiation, whereas the bubble size in the mass diagram corresponds to relative abundance of a Fig. 9. Conceptual framework of (C)DOM transformation in a bo- mass peak; the red circle highlights the completely photo-degraded, real lake based on FT-MS analysis of SPE-DOM and photochemical more saturated DOM component during spring time sunlight expo- degradation experiments. sure, whereas the black circle indicates slow photo-degrading, un- saturated and presumably aromatic DOM. ically formed compounds in the same sample and after the compounds indicated by the changes in absorbance and fluo- light treatment of this sample strongly support this direct rescence (Figs. 3 and 4). link between low O / C, relatively saturated precursors, and However, the June bottom water SPE-DOM (and all April their highly oxidized and lower mass photo products. Despite samples) showed also another pool of more saturated or the photochemically presumably very labile CDOM pool that aliphatic low oxygen-containing compounds that were pho- was completely absent in the June surface sample (Fig. 8, red tobleached (Fig. 8, red circle and Table 3). These were pre- circle), another CDOM component remained in the summer sumably the precursors for the highly oxidized low molecu- sample that still showed photochemical reactivity (Fig. 8, lar weight compounds photochemically produced in all April black circle). These results suggest the existence of two and the June bottom water samples (Figs. 7 and 8). The DBE distinctly different CDOM components, where one is very and DBE/C values are a lot lower and in conjunction with the photo-reactive and is quickly completely degraded/photo- relatively high H / C ratios, are indicative for the much more transformed even in highly absorbing boreal lake surface saturated nature of this component (Table 3). waters, and one that is more persistent but still undergoes The lack of these precursor DOM components in the June photochemical transformation. Both components are part of surface sample and the lack of highly oxidized photochem- the molecular weight fraction ≥300 Da and are responsible www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6954 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM Table 3. The ten most abundant mass peaks and their associated molecular formulae of each of the two distinctly different pools of (1) aromatic and (2) more saturated/aliphatic photosensitive CDOM components analyzed by FT-MS. Formulae Exact neutral Rel. abundance Rel. abundance Change O / C H / C DBE DBE/C neutral before after (%) ratio ratio mass irradiation irradiation 10 highly photo-degraded members of the aromatic CDOM Component C21H14O5 346.0841 4.7 1.5 −37.5 0.24 0.67 15 0.71 C20H12O6 348.0634 7.5 2.0 −40.4 0.30 0.60 15 0.75 C21H14O6 362.079 6.8 1.9 −37.7 0.29 0.67 15 0.71 C21H12O7 376.0583 8.4 1.9 −41.7 0.33 0.57 16 0.76 C22H12O7 388.0583 7.1 1.1 −46.5 0.32 0.55 17 0.77 C22H10O8 402.0376 5.5 1.0 −44.4 0.36 0.45 18 0.82 C23H14O7 402.074 5.0 1.1 −47.2 0.30 0.61 17 0.74 C23H12O8 416.0532 6.1 1.2 −44.2 0.35 0.52 18 0.78 C24H14O15 542.0333 5.2 2.2 −32.7 0.63 0.58 18 0.75 C24H14O16 558.0282 2.9 1.0 −39.4 0.67 0.58 18 0.75 10 highly photo-degraded members of the more aliphatic/saturated CDOM Component C29H38O8 514.2567 3.3 1.3 −46.5 0.28 1.31 11 0.38 C30H40O9 544.2672 4.2 1.7 −45.7 0.30 1.33 11 0.37 C20H26O4 330.1831 5.5 2.3 −42.2 0.20 1.30 8 0.40 C17H26O4 294.1831 4.7 2.2 −38.3 0.24 1.53 5 0.29 C26H36O7 460.2461 4.3 2.0 −37.7 0.27 1.38 9 0.35 C28H42O9 522.2829 3.5 1.6 −37.3 0.32 1.50 8 0.29 C28H36O8 500.2410 4.8 2.3 −37.1 0.29 1.29 11 0.39 C28H40O8 504.2723 5.3 2.5 −36.4 0.29 1.43 9 0.32 C30H38O10 558.2465 4.0 1.9 −35.8 0.33 1.27 12 0.40 C25H30O7 442.1992 4.5 2.2 −35.8 0.28 1.20 11 0.44 DBE is the double bond equivalency that represents all carbon-carbon and carbon-oxygen double bonds. for the photochemical production of lower molecular weight gest periods with extensive photo-mineralization after ice- compounds (Fig. 8). out and lake turnover when DOM from dark parts of the The FT-MS data is consistent with the observations of water column (under ice or in the hypolimnion) becomes changes in optical properties and demonstrating that the exposed to light (Fig. 9). The increased photo-reactivity of photochemical reactivity of the June surface SPE-DOM sam- deep, aphotic waters has also been demonstrated in other en- ple was significantly reduced to probably natural photo- vironments such as the deep ocean (Mopper et al., 1991). bleaching that had already occurred during the very dry Therefore photo-mineralization may not be uniform over the spring in 2011 and the complete loss of a distinct pool of year but show a distinct seasonal pattern and may be greater photo reactive aliphatic compounds. In general, these find- over the whole year than inferred from experiments using ings are in agreement with previous studies that indicated previously light-exposed DOM. the decrease in DOM photo-reactivity with irradiation time To summarize the presented results, we developed a con- (Zhang et al., 2006; Moran et al., 2000; Stubbins et al., 2012). ceptual framework (Fig. 9) that emphasizes the observed This study suggested that seasonal molecular changes in trends in this study and underlines the importance of seasonal SPE-DOM can be explained by a synergy between photo- photobleaching differences. chemical degradation (including a likely microbial decar- boxylation of highly oxidized photo-products) and a possible Acknowledgements. Many thanks go to H. Reyier for helping release of DOM from sediments. Photobleaching can also be tremendously with the sampling in the lake as well as the technical considered to play a major role in altering the DOM molecu- personnel at the Department of Thematic Studies – Water and lar composition in surface boreal lake waters far beyond the Environmental Studies at Linköping University, Sweden. Without very limited attenuation of light, possibly due to a continuous the highly advanced analytical instrumentation located at the mixing by wind of the upper water column above the thermo- Helmholtz Center for Environmental Health in Munich, Germany, cline. This study further suggests a pool of highly photosen- none of this work would have been possible. This study was sitive relatively saturated CDOM that can be quantitatively financially supported by Linköping University and the Swedish removed through photobleaching in the surface of highly ab- research councils VR and Formas. 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Depth-dependent molecular composition and photo-reactivity of dissolved organic matter in a boreal lake under winter and summer conditions

Gonsior, M.; Schmitt-Kopplin, P.; Bastviken, D.
Biogeosciences – Nov 4, 2013
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Open Access Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ Biogeosciences doi:10.5194/bg-10-6945-2013 © Author(s) 2013. CC Attribution 3.0 License. Depth-dependent molecular composition and photo-reactivity of dissolved organic matter in a boreal lake under winter and summer conditions 1, 2 3, 4 2 M. Gonsior , P. Schmitt-Kopplin , and D. Bastviken University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, MD, USA Linköping University, Department of Thematic Studies, Unit Water and Environment, Linköping, Sweden Helmholtz Zentrum Munich, German Research Center for Environmental Health, Neuherberg, Germany Department for Chemical-Technical Analysis, Research Center Weihenstephan for Brewing and Food Quality, Technische Universität München, 85354 Freising-Weihenstephan, Germany Correspondence to: M. Gonsior ([email protected]) Received: 15 April 2013 – Published in Biogeosciences Discuss.: 29 May 2013 Revised: 23 September 2013 – Accepted: 2 October 2013 – Published: 4 November 2013 Abstract. Transformations of dissolved organic matter tion of DOM between surface and deep boreal lake waters. (DOM) in boreal lakes lead to large greenhouse gas emis- The release of DOC from boreal lake sediments also con- sions as well as substantial carbon storage in sediments. Us- tribute to this pattern. Photochemical degradation of DOM ing novel molecular characterization approaches and photo- may be more extensive following ice-out and water column chemical degradation experiments we studied how seasonal turnover when non-light exposed and thereby photosensitive patterns in water column stratification affected the DOM in DOM is photo-mineralized. Hence, the yearly DOM photo- a Swedish lake under early spring and summer conditions. mineralization may be greater than inferred from studies of Dissolved organic carbon (DOC) concentrations were con- recently light-exposed DOM. sistently higher above the sediment when compared to sur- face waters throughout the sampling periods. Photobleach- ing alone could not explain this difference in DOC because the lake was covered by 40 cm-thick ice during late winter 1 Introduction sampling and still showed the same DOC trend. The dif- It is becoming increasingly clear that processes in freshwa- ferences in the molecular diversity between surface DOM ters play a key role for global carbon cycling and in particular in winter and summer were consistent with ongoing pho- for the fate of the organic matter (OM) exported from soils. tobleaching/decarboxylation and a possible bacterial con- Large amounts of OM are decomposed to carbon dioxide and sumption of photo-products. Additional photo-degradation methane in lakes and estimated emissions of these gases cor- experiments using simulated sunlight showed a production of respond to 79 % of the continental uptake of carbon dioxide highly oxidized organic molecules and low molecular weight equivalents (Bastviken et al., 2011). At the same time OM compounds in all late winter samples and also in the deep can also flocculate, sink, and become buried and preserved water sample in summer. In the surface summer DOM sam- in sediments (von Wachenfeldt and Tranvik, 2008; Sobek et ple, few such molecules were produced during the photo- al., 2012). Recent estimates are that sediments harbor 98 % of degradation experiments, confirming that DOM was already the boreal lake OM (Einola et al., 2011), and that the yearly photobleached prior to the experiments. This study suggests sedimentary burial of OM in lakes exceed the burial in the that photobleaching, and therefore also the ice cover dur- oceans (Tranvik et al., 2009). The remainder will be trans- ing winter, plays a central role in surface DOM transforma- ported downstream towards the ocean. The processes behind tion, with important differences in the molecular composi- these different fates are driven by interactions between the Published by Copernicus Publications on behalf of the European Geosciences Union. 6946 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM quantity and quality of the OM, microbial metabolism, and quality and characteristics of OM changed between win- abiotic processes such as photochemistry and the balance be- ter and summer conditions and within the water column, tween flocculation and dissolution of OM. However, the rel- both in situ and after a series of photochemical experi- ative magnitude and the regulation of OM fates are still un- ments, using measurements of optical properties such as ul- clear in most systems. traviolet/visible (UV/Vis) spectroscopy and excitation emis- There have been numerous short-term incubation stud- sion matrix (EEM) fluorescence spectroscopy combined with ies of microbial and photochemical interactions with dis- ultrahigh-resolution electrospray ionization Fourier Trans- solved OM (DOM) degradation in lake waters (Moran and form Ion Cyclotron Resonance Mass Spectrometry (FT-MS). Zepp, 1997). It has been shown that the combination of ini- tial photochemical degradation of the DOM form smaller 2 Methods molecules and that they are more accessible to microbial degradation. This is also important for the total OM miner- 2.1 Sampling alization (Bertilsson and Allard, 1996; Tranvik and Bertils- son, 2001; Anesio et al., 2005). When expanding these find- In the sample collection for the core analyses of this study, ings from short-term incubations over time and space, large one-liter water samples were collected at 0.5 m depth (sur- seasonal differences in lake OM cycling should be expected face), and just above the sediment at 7 m depth in April in stratified and ice-covered systems where different propor- ◦ 0 00 ◦ 0 00 and June 2011 in Lillsjön (58 39 33.05 N, 16 8 34.81 E), tions of the OM are exposed to light during different sea- a lake located in Östergötland, Sweden. During the first sam- sons. Given that 51 % of the global lake area is situated north pling in April, the lake was still covered by 40 cm-thick ice of 48 latitude (Downing and Duarte, 2009), and that these that started to form in November 2010. Dissolved oxygen lakes are chromophoric DOM (CDOM) rich, such seasonal and temperature profiles were measured during sampling us- effects could actually be a major determinant of the fates ing a hand-held probe (Hach HQ40d). of OM in lakes. It is well established that during summer Additional water column profile data on temperatures and stratification in dimictic lakes, the CDOM levels in the sur- DOC was collected as above and the same location (same face layer (epilimnion) is decreased due to photobleaching lake and same sampling area) in August 1999 and March when compared to the deeper waters below the thermocline 2000 (under ice), and analyzed immediately for dissolved or- (hypolimnion) (Bracchini et al., 2006). As a result, photo- ganic carbon (DOC) as described below. These older DOC bleaching is a significant and major player in molecular al- profiles were used as independent supplementary data when teration of DOM (Moran et al., 2000; Stubbins et al., 2012; discussing the potential for DOC release from sediments. Zhang et al., 2006). Modeling attempts stressed that 50 % of All water samples were directly filtered through What- the CDOM light absorption within the entire water column in man GF/F glassfiber filters and acidified after filtration to lakes can be lost in 18–44 days under summer conditions due pH 2 using ultrapure concentrated HCl (Sigma Aldrich 32 %, to photobleaching (Reche et al., 2000). However this model puriss. p.a.). Overall, 800 mL of each water sample was then did not take into account that different CDOM components solid-phase extracted (SPE) as described elsewhere (Dittmar are likely to degrade at different timescales. For example, it is et al., 2008). Briefly, Agilent Bond Elut PPL solid-phase likely that at the onset of sunlight exposure a preferential loss extraction cartridges, filled with 1 g of the PPL resin (a of highly photosensitive components of (C)DOM occurs and styrene-divinylbenzene polymer that has been modified with that slower photo-degrading DOM constituents are relatively a proprietary non-polar surface), were pre-conditioned with increased but continue to photo-degrade at slower rates. methanol, rinsed with acidified (pH 2) Milli-Q water and then Seasonal stratification and the change in redox conditions the water sample was gravity-fed through it. DOC was mea- may also be important for a relatively neglected process in sured on acidified samples before and after the SPE extrac- lake carbon cycling studies – re-dissolution of particulate tion using a Shimadzu 5000 TOC analyzer and carbon ad- OM from lake sediments (Skoog and Arias-Esquivel, 2009; sorption efficiencies ranged between 58 and 64 %, and were Cottrell et al., 2013). During stratification periods, near- very similar to literature values of a variety of DOM samples bottom water layers often become anoxic, which affect all (Dittmar et al., 2008; Gonsior et al., 2011; Shakeri Yekta et redox-sensitive equilibria (Skoog and Arias-Esquivel, 2009) al., 2012). The absorbance of the samples before and after and processes, as well as e.g. pH, and this could affect OM SPE extraction was also measured using an Ultraspec 2100 solubility and availability (Phelps and Zeikus, 1984). The re- Pro (scan over 190–700 nm) and showed an average recovery dissolution can be important for assessments of OM stability, of the CDOM of 63 %. The SPE extracts were eluted with and burial in lake sediments. ◦ methanol and stored at −20 C in a freezer prior to photo- We addressed the hypotheses that seasonal patterns in wa- chemical experiments. Neither the shape of the absorbance ter column stratification are important for lake carbon cy- nor the fluorescence signals of the SPE extract changed no- cling by constraining photo-degradation and by inducing bot- ticeably. tom water conditions that enhance the re-dissolution of sed- imented particulate OM. To do this, we assessed how the Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6947 2.2 Photochemical experiments 2.4 Ultrahigh-resolution mass spectrometric analysis After completely drying of 1 mL of each SPE-DOM sam- A Bruker Apex QE 12 Tesla FT-MS with negative mode ple under nitrogen gas and the re-dissolution in 40 mL electrospray ionization located at the Helmholtz Center for high purity water (LC-MS grade water, Chromasolv, Sigma- Environmental Health, Munich, Germany was used to ana- Aldrich), the sample pH was close to the original water sam- lyze the SPE-DOM samples from the lake after a dilution of ple at pH 6.5. This was important to avoid changes in the 1 : 100 and all samples before and after photochemical degra- pH-dependent shift in absorbance and photobleaching as de- dation experiments at similar dilution. Electrospray is a soft scribed elsewhere (Pace et al., 2012; Janot et al., 2010). ionization technique that creates largely intact molecules (no The samples were then divided and half of the sample fragmentation) with a single charge and the ultrahigh resolu- (20 mL) was continuously pumped through a custom-built tion (mass error < 0.2 ppm and mass resolution > 400 000 at photo reactor cell made out of Tefzel tubing for 24 h. A mass 400) allows for the unambiguous assignment of molec- 10 mL gas equilibrator was also attached in-line to allow sat- ular formulae from all m/z peaks (Stenson et al., 2003). The uration of air gases at any given time. The flat-coiled cell accuracy of the used FT-MS is high enough to distinguish was exposed to simulated sunlight (SolSim Luzchem) that masses with a difference of less than an electron and hence closely matched the intensity of one sun at a solar zenith an- multiply charged ions can be distinguished and are rarely ob- gle of z = 48.2 (1.5 AirMass filter) corresponding to tem- served due to their much weaker ionization efficiencies when −2 perate latitudes and an intensity of 870 W m . The Tefzel compared to their singly charged counterparts. The follow- polymer (ethylene tetrafluoroethylene) has excellent light- ing chemical elements were allowed to calculate molecular 12 1 16 14 32 transmitting properties with > 90 % at 300 nm. This system formulae: C , H , O , N and S as 0−∞ 0−∞ 0−∞ 0−5 0−2 has several advantages, including minimizing inner filtering well as the C carbon isotope. Unambiguous molecu- 0−1 effects (due to the very short path length of 1 mm), avoiding lar formula assignments are now possible up to 800 Dalton starvation of air gases (gas equilibrator) and being a closed due to the mass accuracy of less than 0.2 ppm. Further infor- system avoiding contamination during extended sunlight ex- mation on how to accurately assign molecular formulae to posure times. ultrahigh-resolution FT-MS data is given elsewhere (Dittmar Dark controls were stored in the dark at similar tempera- et al., 2007; Schmitt-Kopplin and Hertkorn, 2007). Briefly, ture maintained within the ventilated solar simulator cham- the mass spectra were calibrated across the observed mass ber. All samples (irradiated and dark controls) were diluted range (150–700 Da) using internal standards of high relative with 50 % LC-MS methanol and analyzed by negative mode abundance exact mass peaks with known molecular formulas electrospray FT-MS. The differences between the dark con- that have been previously found in all DOM samples. Exter- trols and the irradiated samples were used to evaluate the nal pre-calibration was undertaken using arginine standards photo-degradation at the molecular level. prior to mass spectrometric analyses. The reproducibility of the SPE method in conjunction with the same mass spec- 2.3 Measurements of optical properties trometer used in this study has been previously described on triplicate samples (Shakeri Yekta et al., 2012). The repro- Absorbance measurements were undertaken on all samples ducibility of the relative abundances of mass peaks was good before and after exposure to simulated sunlight using a Cary and showed on average less than 4 % variability, but maxi- Bio100 Spectrophotometer with 1 cm quartz cuvettes. The mum differences of 10 % of high abundance mass peaks may spectral slopes and slope ratios were calculated using a first- occur; hence we only considered changes in relative abun- order decay function at the wavelengths ranges 275–295 nm dance that exceeded 10 % relative difference. Usually 20– and 350–400 nm and according to a detailed description pre- 30 % of the mass peaks have low enough intensity to be either viously published (Helms et al., 2008). Excitation emission above or below the set signal to noise ratio in replicate sam- matrix (EEM) fluorescence spectra were recorded using a ples. This can cause a large variability between replicates for Horiba Fluoromax 4 Spectrofluorometer at 5 nm excitation such low intensity mass peaks (Shakeri Yekta et al., 2012). intervals between 240–500 nm and an emission range of Correspondingly, an analysis to find unique mass peaks in 290–600 nm at 2 nm intervals. Scatter-correction and normal- each sample revealed that unique mass peaks showed always ization to quinine sulfate equivalency was performed similar very low abundances, indicating that they were likely not to the procedure described elsewhere (Zepp et al., 2004). All unique but just had an intensity above the signal to noise ra- samples were diluted 1 : 10 prior to all optical property anal- tio in some samples. To avoid any artifacts of the data, we yses to avoid inner-filtering effects, and to be able to apply only considered mass peaks that were commonly found in all the linear range of the quinine sulfate calibration of the EEM samples of the same treatment. Van Krevelen diagrams where spectra. the elemental ratios of oxygen to carbon (O / C) are plotted against the hydrogen to carbon (H / C) ratios of each individ- ually assigned molecular formula are particularly useful for visualizing FT-MS data and showing the chemical space of a www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6948 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM DOM sample occupied in this specific elemental plot (Kim et al., 2003) and were used throughout the study. Another useful visualization tool is the Kendrick diagram, where the Kendrick mass defect (KMD) is plotted against the nominal mass (Kendrick, 1963; Wu et al., 2004). The Kendrick mass (KM) is essentially a normalization of the In- ternational Union of Pure and Applied Chemistry (IUPAC) mass to the mass of CH groups (m = 14.01565) and the KMD is the difference between nominal mass (NM) and KM. KM = IUPAC mass × (14.000000/14.01565) (1) KMD = NM − KM (2) All homologues of CH would have the same KMD and therefore it can be used to distinguish CH homologues com- mon in FT-MS data of DOM. A second independent param- eter (z ) was used to assign homologues as Fig. 1. Temperature and dissolved oxygen saturation profiles of Lill- sjön Lake in April and June 2011. z = (mod[NM/14]) − 14 (3) The modulus (mod) function returns the remainder after the high release of DOC at the onset of anoxia (Skoog and Arias- NM is divided by 14 (Stenson et al., 2003). Here, we used Esquivel, 2009). In our case, this may have occurred in the a modified Kendrick plot where the ratio of the KMD is di- month before the April sampling. Another alternative expla- vided by z and then plotted against the mass to show accu- nation could be higher microbial mineralization in the sur- rately unambiguous homologous series in one plot (Shakeri face water, but it is difficult to find good arguments for this, Yekta et al., 2012). given the slightly lower winter temperatures in the surface water and that most parts of the hypolimnion were oxic dur- ing the winter. The strongest support of DOM release from 3 Results and discussion the sediments are derived from DOC distribution under ice During the April sampling, the dissolved oxygen saturation when the temperature profile (and the microbial degradation) was 59 % just underneath the ice and decreased rapidly with and the light influence throughout the water column is more depth to low oxygen levels (1 %) at 0.5 m above the sediment uniform than during summer. The more detailed DOC pro- (Fig. 1). However, the bottom water had probably been oxic files from August 1999 and March 2000 (under ice) support until just before the sampling, as indicated by previous oxic the findings from 2011 and indicate considerable DOC re- profiles taken in the month of March (data not shown). lease from sediments under ice (Fig. 2). In June, the lake was strongly stratified with 100 % oxy- In June the bottom water DOC levels were close to gen saturation at 0.5 m depth, decreasing with depth to about (slightly lower than) surface levels in April. This is log- 18 % at 0.5 m above sediment (Table 1 and Fig. 1). At both ical given the combination of (1) lake circulation mixing sampling periods, the DOC concentrations were consistently the larger surface water volume with a smaller bottom wa- higher 0.5 m above sediment than at 0.5 m below the surface. ter volume, (2) increased temperatures and thereby increased The DOC concentration was highest in the deep sample un- DOC mineralization throughout the water column, and (3) der ice followed by surface sample under ice, deep sample some photo-mineralization during the circulation period af- in summer and surface sample in summer (Table 1). This in- fecting the whole water column. After lake circulation, the dicated either a source of DOM to the bottom water and/or surface water DOC levels was likely reduced further by ad- a DOC dilution/depletion at the surface both under ice and ditional photo-mineralization and enhanced microbial degra- during summer. Under the winter conditions with the exist- dation as temperatures increased further. Additional DOM ing ice cover and frozen soils, it seemed unlikely that the sources that may have influenced the DOC concentrations surface water was diluted (rain, runoff etc.). The most likely between April and June cannot be ruled out but the months reason for the higher DOM concentrations in the bottom wa- of April, May and June 2011 were particularly dry in this ter is release from sediment. A change in the redox potential region and large amounts of leaching from forest soils af- at the sediment surface caused by switching from aerobic to ter spring mixing was unlikely. Photochemical experiments anaerobic conditions has previously been reported to cause a were undertaken to evaluate if differences existed between Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6949 Table 1. Temperature, dissolved oxygen and DOC concentrations during sampling of Lillsjön, a boreal lake in 2011. Sampling 04/01/2011 Sampling 06/28/2011 ◦ −1 ◦ −1 Sample Lillsjön Depth (m) T ( C) O (%) DOC (mg L ) T ( C) O (%) DOC (mg L ) surface 0.5 2.1 59 22.7 ± 0.3 19.4 100 18.7 ± 0.2 bottom 7 4 1 25.0 ± 0.2 4.7 18 21.5 ± 0.2 decrease in molecular weight after irradiation. In contrast, the same photochemical experiments undertaken on water sam- pled in June showed much less photobleaching at the surface when compared to deep water SPE-DOM (Figs. 3 and 4). The photochemically induced changes in S also confirmed this trend with an increase of only 14 % in the June surface sample compared to 22 % in the bottom water sample (Ta- ble 2). This indicates that a significant amount of the sur- face CDOM had already been photobleached by natural sun- light prior to additional laboratory-based photobleaching ex- periments. An alternative explanation would be a microbial degradation of CDOM during spring and the much warmer surface water. However, this was shown to be a rather slow process and a decrease in fluorescent intensity of up to 40 % in 3.5 yr has been demonstrated (Kothawala et al., 2012). An- other study suggested the involvement of lake bacterial com- munities to produce and degrade CDOM, but this observa- tion was associated with increased protein-like fluorescence arising from bacterioplankton biomass (Guillemette and del Fig. 2. Depth profiles of DOC and temperature in Lillsjön Lake in Giorgio, 2012). In this study we did not see any change in 24 August 1999 (summer) and 7 March 2000 (winter). the protein-like fluorescence between winter and summer. Therefore, the involvement of bacteria in the degradation of CDOM in the June surface water sample was probably not the photochemical reactivity (photobleaching) of DOM be- significant when compared to photochemical degradation. tween surface and deep water as a function of season and a It seemed likely that the observed consistent differences comparison between ice-covered and light-protected surface between surface and deep water DOC concentrations are waters in early spring, with strongly exposed surface water in caused by a combination of DOC released from sediments summer after exposure to sunlight during the dry and sunny and photobleaching/microbial degradation of DOM at the spring of 2011. If a release of DOM from sediments occurred surface in summer and mainly driven by DOM fluxes from and the significant differences in DOC concentrations be- sediments in winter. tween surface and deep waters are not primarily related to The ultrahigh-resolution, negative electrospray ionization photobleaching, then there should not be a great difference Fourier transform, ion cyclotron resonance mass spectrom- between photobleaching in surface and deep waters under etry (FT-MS) was used (Fig. 5) to allow for unambiguous winter conditions and during ice-cover. Indeed photochem- assignments of molecular formulae to each exact mass peak ical degradation of solid-phase extracted DOM (SPE-DOM) in each sample. Only masses containing C, H and O atoms and its associated changes in optical properties in April at were used in further analysis because nitrogen- and sulfur- the surface and just above the sediment after the exposure containing molecular formulas were assigned but showed to 24 h simulated sunlight was substantial but revealed only low abundances in negative electrospray mass spectrometry small changes between depths (Figs. 3 and 4). The spectral (Fig. 5). slope ratios (S ), obtained by dividing the slope calculated The comparison between surface and deep SPE-DOM un- between 275–295 nm and 350–400 nm (Helms et al., 2008), der winter conditions collected in April 2011 showed that the showed also significant changes towards higher values after relative abundance of highly oxidized organic compounds irradiation time, but was relatively similar between depths (O / C ratio between 0.50–0.87) was slightly decreased in the (18 % increase in the April surface and 20 % in the April bot- surface compared to deep SPE-DOM, whereas the lower oxi- tom water sample) (Table 2). In general, these results are in dized (O / C ratio < 0.45) organic compounds showed a small agreement with the Helms et al. study and would suggest a www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6950 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM Table 2. UV-Vis spectral slopes at distinct wavelength ranges and the resulting spectral slope ratios (S ) of surface and deep boreal lake water samples collected in April and June 2011. Sampling 04/01/2011 Sampling 06/28/2011 Sample Lillsjön lake S S S S S S S S r r 275−395 350−400 275−395 350−400 surface, before irradiation 0.0144 0.0135 0.0185 0.73 0.0145 0.0139 0.0182 0.76 surface, after irradiation 0.0151 0.0157 0.0176 0.89 0.0151 0.0158 0.0177 0.89 bottom, before irradiation 0.0143 0.0135 0.0183 0.74 0.0141 0.0133 0.018 0.74 bottom, after irradiation 0.0147 0.0159 0.0172 0.92 0.0144 0.0159 0.0167 0.95 Fig. 3. Depth-dependent seasonal changes in the photochemical degradation patterns after 24 h exposure to simulated sunlight of lake SPE- DOM and analyzed by UV/Vis absorption spectroscopy. increase in the surface relative to deep SPE-DOM (Fig. 6). whereby DOC is oxidized and dissolved inorganic carbon is One explanation could be that more oxidized DOM is often formed, seemed to be an effective pathway based on dark in- also more water soluble (because the electronegativity of cubation of water collected in the same lake in a previous oxygen will introduce a polarization of covalent bonds and study and the loss of about 7 % of the initial DOC within increase the overall solubility) and therefore is preferentially 5 months at 15 C (Bastviken et al., 2004). To provide a crude released from sediments. This would increase the relative estimate of relative contribution of microbial versus light- abundance of more oxidized DOM in the bottom water. dependent removal of DOC in surface water between April In June 2011, differences between surface and deep wa- and June in our study we assumed that the total respiration −1 ter were primarily associated with increased abundances of found in this previous study over 5 months (1.7 mg C L ) mass peaks assigned to relatively low H / C and high O / C represented the contribution of the light-independent micro- ratio lower molecular weight compounds in the surface SPE- bial DOC mineralization from April to June (2 months). We DOM sample (Fig. 6). Interestingly, those highly oxidized also assumed negligible input of water and DOC from land molecular formulas appeared in even higher relative abun- given the very dry conditions this spring. Then, given a total −1 dances after light treatment in our photochemical degrada- difference of 2.8 mg DOC L between surface and bottom −1 tion experiments (Figs. 7 and 8). Such highly oxidized for- water in June (Table 1), at least 1.1 mg L or 40 % of this mulas produced after solar simulated irradiation have been relative DOC loss in the surface water should be directly or previously shown and confirmed our observation (Gonsior et indirectly related to photochemical DOC degradation. This al., 2009). This indicates that these molecules were photo- calculation likely overestimates the light-independent micro- chemical products that are labile and depleted by either mi- bial contribution by using a 5-month value for 2 months, crobial degradation or additional photochemical decarboxy- thereby underestimating the influence of photochemistry, and lation (Xie et al., 2004; Xu and Wan, 2000) and the release should not be taken literally. Rather it illustrates similar mag- of carbon mono- and dioxide and other small carboxylic nitudes of light-independent and photochemically induced acids (Bertilsson and Tranvik, 1998). Microbial respiration, DOC mineralization processes in lake surface water. Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6951 Fig. 4. Excitation emission matrix fluorescence of surface and deep SPE-DOM collected in April and June 2011 before and after ex- posure to simulated sunlight including a differential plot of the de- creased fluorescence after irradiation. Note: SPE-DOM was dried and then re-dissolved in pure water at pH 6.5. Fig. 5. Ultrahigh-resolution mass spectrum of SPE-DOM extracted from a boreal lake at the surface in April 2011 in Sweden (Lill- sjön) and the van Krevelen diagram of CHO, CHOS and CHNO formulae. Note: size of bubbles represent relative abundances of as- The absolute decrease in absorption coefficients (Fig. 3) sociated mass peaks. after 24 h simulated sunlight exposure was significant in all samples including the late winter and summer surface and deep samples. The photobleaching under winter condi- tions between surface and deep water CDOM was very sim- sults imply that water that had not been exposed to light for ilar with the only detectable differences below 240 nm. This extended periods (under ice or near the bottom during sum- stands in contrast to the comparison between surface and mer stratification) was much more photo-reactive. deep CDOM in summer, where the surface CDOM is much This reduced photobleaching measured by optical prop- less photobleached in the whole ultraviolet (UV) range (200– erties (Figs. 3 and 4) of the June 2011 SPE-DOM sample 400 nm) (Fig. 3). already indicated that photo-active components in this SPE- The measurements of EEM spectra before and after the DOM were largely degraded prior to photobleaching ex- same irradiation time showed that the photochemically in- periments. As a result, the photobleaching demonstrated by duced changes were reflected in areas in the EEM spectra that changes in optical properties should be also reflected in the are centered around two maxima with excitation, emission ultrahigh-resolution mass spectrometric data and a different couples of 280–380 nm and 340–375 nm (Fig. 4). This pref- photobleaching behavior in the June surface DOM sample erential photo-degradation of FDOM at longer wavelengths was expected given the reduced photobleaching in this sam- indicated a shift towards lower wavelengths of the remain- ple when compared to all others. Indeed, the surface and deep ing fluorescent peaks after irradiation similar to findings re- SPE-DOM sample collected in April 2011 together with the ported previously (Helms et al., 2013). The summer surface deep sample from June 2011 showed a similar photochemi- sample was the only sample that showed clear differences cal behavior with a production of highly oxidized and high in photochemically induced degradation of fluorescent DOM O / C ratio molecules (Figs. 7 and 8). Additionally, a shift (FDOM) between surface and bottom water, confirming the to lower molecular weight and a significant increase in rel- same trend shown in the absorbance data (Fig. 3). These re- ative abundance of these low molecular weight mass peaks www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6952 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM Fig. 7. Photochemically induced changes in the molecular composi- tion of a boreal lake SPE-DOM at the surface and bottom in spring (ice covered) and at the surface and bottom in summer shown in van Krevelen diagrams and the observed relative changes in intensity for all mass peaks, respectively. (Note: black bubbles represent molec- ular formulas with an increase in associated relative abundances of the mass peaks after 24 h solar simulated irradiation and open bub- bles with a decrease, respectively.) Fig. 6. Van Krevelen, H / C and O / C versus mass diagrams of rela- tive differences in mass peak abundances between surface and deep been suggested previously (Thomson et al., 2004). The con- SPE-DOM in April and June 2011. Note: the bubbles represent the sistent decrease in all samples of mass peaks associated with relative abundances greater than 10 %. H / C ratios below 0.8 of associated molecular formulae and throughout a broad range of O / C ratios as demonstrated with the June samples (Fig. 8, black circles), is indicative for a was observed in all those samples. This trend is in agree- large pool of presumably aromatic CDOM components that ment with previous studies where ultrafiltered DOM was are photosensitive (Table 3). The high double bond equiv- photo-degraded and analyzed by size exclusion chromatog- alency (DBE, number of double bonds and rings) and the raphy (Lou and Xie, 2006; Thomson et al., 2004). Whole DBE divided by the carbon number (DBE/C) values are in- water samples were also previously analyzed using optical dicative of reduced compounds (Table 3). A set of photo- properties (Helms et al., 2008) and ultrahigh-resolution mass sensitive aromatic compounds in the Cape Fear River estu- spectrometry of Congo River water (Stubbins et al., 2010) ary has been previously shown using FT-ICR-MS (Gonsior with again similar findings. et al., 2009) and long-term exposure to simulated sunlight The photo production of highly oxidized compounds can (57 days) of Congo River water also confirmed the preferen- be explained with reaction of reactive oxygen species, such tial photo-degradation of the aromatic DOM content (Stub- as the hydroxyl radical (OH), super oxide (O ) and hydrogen bins et al., 2010). The loss of aromatics is also in agreement peroxide (H O ). A photochemically induced depolymeriza- with the observed photochemically induced changes in the 2 2 tion and a decrease in molecular weight of DOM have also optical properties and the decrease in conjugated aromatic Biogeosciences, 10, 6945–6956, 2013 www.biogeosciences.net/10/6945/2013/ M. Gonsior et al.: Molecular composition and photo-reactivity of DOM 6953 Fig. 8. Photochemically induced changes in the molecular composi- tion of a boreal lake SPE-DOM at the surface and bottom in summer and its associated increase in intensity of mass peaks that showed more than 10 % change and a decrease of more than 10 %, respec- tively. Results are illustrated using van Krevelen diagrams and dia- grams where the relative change in intensity is plotted against mass. Note: the bubble size in the van Krevelen diagrams correspond to the relative intensity change after irradiation, whereas the bubble size in the mass diagram corresponds to relative abundance of a Fig. 9. Conceptual framework of (C)DOM transformation in a bo- mass peak; the red circle highlights the completely photo-degraded, real lake based on FT-MS analysis of SPE-DOM and photochemical more saturated DOM component during spring time sunlight expo- degradation experiments. sure, whereas the black circle indicates slow photo-degrading, un- saturated and presumably aromatic DOM. ically formed compounds in the same sample and after the compounds indicated by the changes in absorbance and fluo- light treatment of this sample strongly support this direct rescence (Figs. 3 and 4). link between low O / C, relatively saturated precursors, and However, the June bottom water SPE-DOM (and all April their highly oxidized and lower mass photo products. Despite samples) showed also another pool of more saturated or the photochemically presumably very labile CDOM pool that aliphatic low oxygen-containing compounds that were pho- was completely absent in the June surface sample (Fig. 8, red tobleached (Fig. 8, red circle and Table 3). These were pre- circle), another CDOM component remained in the summer sumably the precursors for the highly oxidized low molecu- sample that still showed photochemical reactivity (Fig. 8, lar weight compounds photochemically produced in all April black circle). These results suggest the existence of two and the June bottom water samples (Figs. 7 and 8). The DBE distinctly different CDOM components, where one is very and DBE/C values are a lot lower and in conjunction with the photo-reactive and is quickly completely degraded/photo- relatively high H / C ratios, are indicative for the much more transformed even in highly absorbing boreal lake surface saturated nature of this component (Table 3). waters, and one that is more persistent but still undergoes The lack of these precursor DOM components in the June photochemical transformation. Both components are part of surface sample and the lack of highly oxidized photochem- the molecular weight fraction ≥300 Da and are responsible www.biogeosciences.net/10/6945/2013/ Biogeosciences, 10, 6945–6956, 2013 6954 M. Gonsior et al.: Molecular composition and photo-reactivity of DOM Table 3. The ten most abundant mass peaks and their associated molecular formulae of each of the two distinctly different pools of (1) aromatic and (2) more saturated/aliphatic photosensitive CDOM components analyzed by FT-MS. Formulae Exact neutral Rel. abundance Rel. abundance Change O / C H / C DBE DBE/C neutral before after (%) ratio ratio mass irradiation irradiation 10 highly photo-degraded members of the aromatic CDOM Component C21H14O5 346.0841 4.7 1.5 −37.5 0.24 0.67 15 0.71 C20H12O6 348.0634 7.5 2.0 −40.4 0.30 0.60 15 0.75 C21H14O6 362.079 6.8 1.9 −37.7 0.29 0.67 15 0.71 C21H12O7 376.0583 8.4 1.9 −41.7 0.33 0.57 16 0.76 C22H12O7 388.0583 7.1 1.1 −46.5 0.32 0.55 17 0.77 C22H10O8 402.0376 5.5 1.0 −44.4 0.36 0.45 18 0.82 C23H14O7 402.074 5.0 1.1 −47.2 0.30 0.61 17 0.74 C23H12O8 416.0532 6.1 1.2 −44.2 0.35 0.52 18 0.78 C24H14O15 542.0333 5.2 2.2 −32.7 0.63 0.58 18 0.75 C24H14O16 558.0282 2.9 1.0 −39.4 0.67 0.58 18 0.75 10 highly photo-degraded members of the more aliphatic/saturated CDOM Component C29H38O8 514.2567 3.3 1.3 −46.5 0.28 1.31 11 0.38 C30H40O9 544.2672 4.2 1.7 −45.7 0.30 1.33 11 0.37 C20H26O4 330.1831 5.5 2.3 −42.2 0.20 1.30 8 0.40 C17H26O4 294.1831 4.7 2.2 −38.3 0.24 1.53 5 0.29 C26H36O7 460.2461 4.3 2.0 −37.7 0.27 1.38 9 0.35 C28H42O9 522.2829 3.5 1.6 −37.3 0.32 1.50 8 0.29 C28H36O8 500.2410 4.8 2.3 −37.1 0.29 1.29 11 0.39 C28H40O8 504.2723 5.3 2.5 −36.4 0.29 1.43 9 0.32 C30H38O10 558.2465 4.0 1.9 −35.8 0.33 1.27 12 0.40 C25H30O7 442.1992 4.5 2.2 −35.8 0.28 1.20 11 0.44 DBE is the double bond equivalency that represents all carbon-carbon and carbon-oxygen double bonds. for the photochemical production of lower molecular weight gest periods with extensive photo-mineralization after ice- compounds (Fig. 8). out and lake turnover when DOM from dark parts of the The FT-MS data is consistent with the observations of water column (under ice or in the hypolimnion) becomes changes in optical properties and demonstrating that the exposed to light (Fig. 9). The increased photo-reactivity of photochemical reactivity of the June surface SPE-DOM sam- deep, aphotic waters has also been demonstrated in other en- ple was significantly reduced to probably natural photo- vironments such as the deep ocean (Mopper et al., 1991). bleaching that had already occurred during the very dry Therefore photo-mineralization may not be uniform over the spring in 2011 and the complete loss of a distinct pool of year but show a distinct seasonal pattern and may be greater photo reactive aliphatic compounds. In general, these find- over the whole year than inferred from experiments using ings are in agreement with previous studies that indicated previously light-exposed DOM. the decrease in DOM photo-reactivity with irradiation time To summarize the presented results, we developed a con- (Zhang et al., 2006; Moran et al., 2000; Stubbins et al., 2012). ceptual framework (Fig. 9) that emphasizes the observed This study suggested that seasonal molecular changes in trends in this study and underlines the importance of seasonal SPE-DOM can be explained by a synergy between photo- photobleaching differences. chemical degradation (including a likely microbial decar- boxylation of highly oxidized photo-products) and a possible Acknowledgements. Many thanks go to H. Reyier for helping release of DOM from sediments. Photobleaching can also be tremendously with the sampling in the lake as well as the technical considered to play a major role in altering the DOM molecu- personnel at the Department of Thematic Studies – Water and lar composition in surface boreal lake waters far beyond the Environmental Studies at Linköping University, Sweden. Without very limited attenuation of light, possibly due to a continuous the highly advanced analytical instrumentation located at the mixing by wind of the upper water column above the thermo- Helmholtz Center for Environmental Health in Munich, Germany, cline. This study further suggests a pool of highly photosen- none of this work would have been possible. This study was sitive relatively saturated CDOM that can be quantitatively financially supported by Linköping University and the Swedish removed through photobleaching in the surface of highly ab- research councils VR and Formas. 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