Fig. 1. Sampling sites (GeoB, open circles) at the Galicia-Minho shelf and associated local rivers (distribution of the mid-shelf mudbelt adapted from Dias et al., 2002). Latitude (°N), longitude (°W) and water depth at the sampling sites are listed in Table 1. Fig. 2. Scheme of analytical methods applied in this study. Bulk parameters comprise dissolved organic matter (DOM) and partic- ulate organic matter (POM). At each sampling location, DOM was separated by solid-phase extraction (SPE) from pore or river water; lipid biomarkers and lignin phenols were analyzed in the sedimen- tary OM. Location and water depth of all marine sampling sites in this study. Content of total organic carbon (TOC), total nitrogen (TN) and total sulfur (TS) in %, and their ratios (TOC/TN, TOC/TS) analyzed in the bulk sediment from the Galicia—Minho shelf. Table 1 ‘ig. 3. Gas chromatograms (FID) of the maltene (a and c) and lignin fractions (b and d) of samples from the southern mudbelt (GeoB 11039; , b) and the continental slope (GeoB 11033; c and d). FA — unsaturated and saturated fatty acids, MAGE — mono-O-alkyl glycerol ethers Cy4-C jo), circle — n-alkanes (Cy5—C35), triangle — n-alcohols (C2—C39), black star — cholesterol, cross — brassicasterol, rhomb — dinosterol. IS - nternal standard, | — vanillin, 2 — acetovanillone, 3 — vanillic acid, 4 — syringaldehyd, 5 — acetosyringone, 6 — syringic acid, 7 — p-coumaric cid. Fig. 4. ESI negative FT-ICR mass spectra and expanded sections of mass spectra at nominal mass 407 of: (a and b) northern mudbelt-derived sediment pore water DOM (GeoB 11012) and (c and d) Ave River DOM. Open rhomb — C;3H2g0j0S2, open square — C26H;903N>; following symbols refer to series that are characterized by the replacement of O by CH: filled circles -CHO-series from C,7H)201 to C23H36O¢, cross — nitrogen-bearing series from C;7H16019N2 to CjgH24OgN>, open triangle — sulfur-bearing series from C;gH 1600S to C2;H2g0¢6S. (O/C) wa and (H/C) wa refer to CHO-compounds in the presented mass range and reflect a lower relative O content and a higher relative H content in the pore water DOM. A representative FT-ICR mass spectrum of marine sedi- ment pore water DOM is shown in Fig. 4a. All spectra showed a similar pattern with a slight shift to higher m/z in the river water spectra (Table 3). We observed typical mass spacing patterns such as 14.0156 Da for CH>-groups and an increase of 36.4 mDa for the replacement of O by CHg, A widely used tool for the visualization of compositional variations from FT-ICR-MS data is the van Krevelen dia- gram (e.g., Kim et al., 2003, 2004, 2006; Kujawinski et al., 2004; Wu et al., 2004; Koch et al., 2005) in which individual PrmndiOh Mlbat IE Ret RE nc Alsat BE set Se Md Bela Stone t A Seams Melee ind Characteristic parameters of pore water and river DOM as well as the contents (in tM) of SPE-extracted dissolved organic carbon (DOCgpr) and total dissolved nitrogen (TDNspg). Displayed are carbon to nitrogen ratios in the extract (DOCspp/TDNspg), numbers of identified molecular formulas (75), the intensity weighted average values of the molecular weight ((m/z)wa), double bond equivalents (DBE,,.), molar oxygen to carbon ((O/C),,) and hydrogen to carbon ratios ((H/C)wa), number of carbon (C,,,), nitrogen (Ny) and sulfur atoms (S,,q). TDNspzr close to the limit of quantitation. Fig. 6. Cluster analysis (Bray Curtis Similarity) based on all identified molecular formulas having a relative intensity >2%. Cluster analyses visualize similarities/dissimilarities between sam- ples based on a multidimensional data set. The x-axis represents the degree of similarity; the vertical lines of the branches indicate the similarity value. A similarity value of S = 100 would mean that all molecular formulas in the samples are identical. Fig. 5. van Krevelen diagram of CHO-formulas in DOM from the Galicia—Minho shelf. A shift in direction of the arrow is observed to higher H/C and lower O/C ratios from the Douro River (green rhombs) to the central mudbelt (GeoB 11002, light blue triangles) down to the outer shelf (GeoB 11006, blue circles) and the continental slope (GeoB 11033, black dashes). Black circles correspond to general compound classes (adapted from Kim et al., 2003; Sleighter and Hatcher, 2008) and are indicated for reference, not necessarily implying the presence of these com- pounds in the DOM samples. Distributional pattern and source assignments of identified compound classes in river and sediment pore water DOM. Table 4 Fig. 7. Detailed van Krevelen diagram of aromatic compounds (modified aromaticity index Al,,oq > 0.67), including N and S for: (a) the Douro River, (b) the southern (GeoB 11039) and (c) the central mudbelt (GeoB 11002). The non-modified aromaticity index >0.67 (red line) assigns all condensed aromatics with low oxygen content. Circles mark highly oxygenated aromatics of terrestrial origin. Fig. 8. van Krevelen diagram of nitrogen-bearing molecular formulas in DOM of: (a) the Douro River (rectangle marks compounds of terrestrial origin) and (b) the continental slope (GeoB 11033). Circles correspond to O/C and H/C ratios of proteins and amino sugars (Kim et al., 2003). ‘sussitimientnaniiaialinnemememies ihe van WKrevelen diagram in Fig. 6 retlects the high variety in nitrogen-bearing compounds in the pore water DOM from the continental slope in comparison to Douro River DOM. Striking features are the contributions from compounds with two N-atoms, plotting mainly in regions of the van Krevelen diagram where proteins, oligopeptides, amino sugars and highly aromatic compounds are expected. These compounds were exclusively present in the continen- tal slope DOM. A high relative abundance of nitrogen- bearing compounds, which were concentrated in the protein area of the van Krevelen diagram, was also detected by Kujawinski et al. (2004) who analyzed DOM produced by bacteria and protozoan cultures. Although proteins are not in our analytical window (200 < m/z < 600), the occur- rence of degradation products and intermediates with lower molecular weight are expected constituents in pore water DOM. The lack of those molecules in the other pore water samples may be related to variations in the exchange be- tween pore water and bottom water at the different sam- pling locations. Sediment deposits at the Galicia—Minho shelf are subjected to sediment mixing due to strong cur- rents and storm induced bottom waves in winter (Jouan- neau et al., 2002; Vitorino et al., 2002b) and therefore, pore water and bottom water are regularly exchanged on a seasonal basis. In contrast, calm sedimentation conditions at the continental slope result in a continuously accumula- tion of clay-rich sediment. Additionally, diffusion rates of pore water are reduced in clay-rich densely packed sedi- ments (e.g., Tryon et al., 2001; Janssen et al., 2005), enhanc- transported in the POM fraction via rivers or as aerosols via the atmosphere, can be oxidized by microbes in mar- ine sediments, where the products dissolve and accumu- late in the pore water DOM. Fig. 9. FT-ICR mass spectra at 353 m/z for: (a) the outer shelf (GeoB 11006), (c) the southern mudbelt (GeoB 11039) and (e) the Ave River and van Krevelen diagram of all characterized S-bearing compounds for: (b) the outer shelf (GeoB 11006), (d) the southern mudbelt (GeoB 11039) and (f) the Ave River. Molecular formulas and exact masses refer to filled circles (a), to open circles (b) and to open triangles (c). Cross — nitrogen series, where CH, replaces O from C,4H)4O9N2 to Cj6H2207Ng, filled squares — Cj2H)gO12, C13H22011. All DOM samples contained several compounds with one or two sulfur atoms in the formulas. Compounds with higher sulfur (and also nitrogen) content have been omit- ted, since it was necessary to restrict the number and type of isotopes for the molecular formula calculation in an a priori assumption (Koch et al., 2007). A specific compound group with O/C ratios of 0.4-0.7 and relatively low H/C ratios of 0.6 and 0.8 was observed in the river DOM (rectangle in Fig. 8a). Molecular formu- las related to these ratios (e.g., Cj9H,;0,,;N) were also present in the southern mudbelt but missing in all other pore water samples, thus pointing to a terrestrial source of this compound group. Nevertheless, at this point, the Identified sum formulas in pore water DOM from the southern mudbelt (GeoB 11039) shown for the mass range from 350 to 400 Da. Appendix A (continued) Appendix A (continued) Appendix A (continued) Appendix A (continued)
