Dataset title: Chemical characteristics of dissolved organic matter in an oligotrophic subtropical wetland/estuary ecosystem, Everglades National Park (FCE), South Florida from December 2001 to January 2002 Dataset ID: ST_ND_Jaffe_002 Research type: Short-term Dataset Creator Name: Dr. Rudolf Jaffe Position: Lead Principal Investigator Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, Florida 33199 USA Phone: 305-348-2456 Fax: 305-348-4096 Email: jaffer@fiu.edu URL: http://serc.fiu.edu/sercindex/index.htm Metadata Provider Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, FL 33199 USA Phone: 305-348-6054 Email: fcelter@fiu.edu URL: http://fcelter.fiu.edu Dataset Abstract The objective of this study was to investigate in detail the molecular characteristics of DOM in a sub-tropical, oligotrophic aquatic environment, namely the Florida coastal Everglades, along a freshwater wetland, mangrove fringe to estuarine transect. For this purpose, surface waters from five sites along Taylor Slough ranging from freshwater marsh to mangrove ecotone environments, as well as three sites within the Florida Bay estuary were collected for analyses. Fluorescence properties of DOM were measured for a quick assessment of DOM quality. Ultrafiltered DOM samples (UDOM; less than 0.7 um, greater than 1000 Da) were concentrated and freeze dried for the determination of bulk C composition, lignin-phenol concentration, and neutral sugar composition. These molecular characteristics were investigated by 13C-NMR spectroscopy, TMAH thermochemolysis, and hydrolysis with trifluoroacetic acid (TFA), respectively. Furthermore, UDOM samples were classified by cluster analysis based on their pyrolysis products obtained via flash pyrolysis-GC/MS. Geographic Coverage Study Extent Description The Study Extent of this dataset includes water samples collected from the Everglades National Park, South Florida. Bounding Coordinates Geographic description: Water samples were collected in the Everglades National Park, South Florida. West bounding coordinate: -80.938 East bounding coordinate: -80.490 North bounding coordinate: 25.439 South bounding coordinate: 24.913 Geographic description: Florida Coastal Everglades LTER Study Area: South Florida, Everglades National Park, and Florida Bay West bounding coordinate: -81.078 East bounding coordinate: -80.490 North bounding coordinate: 25.761 South bounding coordinate: 24.913 FCE LTER Sites: TS/Ph1a, TS/Ph2, TS/Ph3, TS/Ph6a, TS/Ph7a, TS/Ph9, TS/Ph10, TS/Ph11 All Sites Geographic Description:FCE LTER Site TS/Ph1a Longitude:-80.590 Latitude:25.424 Geographic Description:FCE LTER Site TS/Ph2 Longitude:-80.607 Latitude:25.404 Geographic Description:FCE LTER Site TS/Ph3 Longitude:-80.663 Latitude:25.252 Geographic Description:FCE LTER Site TS/Ph6a Longitude:-80.649 Latitude:25.214 Geographic Description:FCE LTER Site TS/Ph7a Longitude:-80.639 Latitude:25.191 Geographic Description:FCE LTER Site TS/Ph9 Longitude:-80.490 Latitude:25.177 Geographic Description:FCE LTER Site TS/Ph10 Longitude:-80.68 Latitude:25.02 Geographic Description:FCE LTER Site TS/Ph11 Longitude:-80.94 Latitude:24.91 Temporal Coverage Start Date: 2001-12-06 End Date: 2002-01-28 Data Table Entity Name: ST_ND_Jaffe_002 Entity Description: Chemical characteristics of dissolved organic matter in an oligotrophic subtropical wetland/estuarine ecosystem, Everglades National Park, South Florida Object Name: ST_ND_Jaffe_002 Data Format Number of Header Lines: 1 Attribute Orientation: column Field Delimiter: , Number of Records: Attributes Attribute Name: SITENAME Attribute Label: SITENAME Attribute Definition: Name of plant biomass Storage Type: text Measurement Scale: Name of plant biomass Missing Value Code: Attribute Name: Date Attribute Label: Date Attribute Definition: Collection date Storage Type: datetime Measurement Scale: Missing Value Code: Attribute Name: DOC Attribute Label: Dissolved organic carbon Attribute Definition: Total dissolved organic carbon concentration Storage Type: data Measurement Scale: Units: microMolesPerLiter Precision: 1 Number Type: real Missing Value Code: -9999 (Value will never be recorded ) Attribute Name: FI Attribute Label: Fluorescence Index Attribute Definition: Ratio of emission intensities at 450 and 500 nm at a fixed excitation wavelength of 370 nm. Storage Type: data Measurement Scale: Units: dimensionless Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Max_WL Attribute Label: Maximum Wavelength Attribute Definition: Emission wavelength that gives maximum emission intensity at a fixed excitation of 313 nm Storage Type: data Measurement Scale: Units: nanometer Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Max_I Attribute Label: Maximum Intensity Attribute Definition: Emission intensity of maximum emission wavelength at a fixed excitation of 313 nm. Storage Type: data Measurement Scale: Units: QSUPerMilligramPerLiter Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: %285 Attribute Label: %285 Attribute Definition: Obtained from synchronous fluorescence spectrum at a constant offset of 30 nm (excitation wavelengths = 285, 350, 385, 460 nm). Percentage of the first peak intensity (285 nm). Storage Type: data Measurement Scale: Units: percent Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: C/N Attribute Label: Carbon to Nitrogen ratio Attribute Definition: Carbon to Nitrogen ratio Storage Type: data Measurement Scale: Units: dimensionless Precision: 0.001 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Salinity Attribute Label: Salinity Attribute Definition: Salinity Storage Type: data Measurement Scale: Units: dimensionless Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: %Alkyl_C Attribute Label: % alkyl carbon Attribute Definition: Relative percentage of alkyl carbon defined by 13C NMR Storage Type: data Measurement Scale: Units: percent Precision: 1 Number Type: real Missing Value Code: -9999 (Value will never be recorded ) Attribute Name: %O_alkyl C Attribute Label: % O-alkyl carbon Attribute Definition: Relative percentage of O-alkyl carbon as defined by 13C NMR Storage Type: data Measurement Scale: Units: percent Precision: 1 Number Type: real Missing Value Code: -9999 (Value will never be recorded ) Attribute Name: %Aromatic_C Attribute Label: % aromatic carbon Attribute Definition: Relative percentage of aromatic carbon as defined by 13C NMR Storage Type: data Measurement Scale: Units: percent Precision: .01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Attribute Name: %carbonyl_C Attribute Label: % carbonyl carbon Attribute Definition: Relative percentage of carbonyl carbon as defined by 13C NMR Storage Type: data Measurement Scale: Units: percent Precision: 1 Number Type: real Missing Value Code: -9999 (Value will never be recorded ) Attribute Name: AlkylC/O_alkylC Attribute Label: O-alkyl to alkyl carbon ratio Attribute Definition: Amount of alkyl carbon divided by the amount of O-alkyl carbon Storage Type: data Measurement Scale: Units: dimensionless Precision: 0.01 Number Type: real Missing Value Code: -9999.00 (Value will never be recorded ) Attribute Name: Aromaticity Attribute Label: Aromaticity Attribute Definition: Percentage of aromatic carbon Storage Type: data Measurement Scale: Units: percent Precision: 1 Number Type: real Missing Value Code: -9999 (Value will never be recorded ) Attribute Name: Arabinose Attribute Label: Arabinose Attribute Definition: Percentage of Arabinose Storage Type: data Measurement Scale: Units: percent Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Ribose Attribute Label: Ribose Attribute Definition: Percentage of Ribose Storage Type: data Measurement Scale: Units: percent Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Xylose Attribute Label: Xylose Attribute Definition: Percentage of Xylose Storage Type: data Measurement Scale: Units: percent Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Rhamnose Attribute Label: Rhamnose Attribute Definition: Percentage of Rhamnose Storage Type: data Measurement Scale: Units: percent Precision: 0.1 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Fucose Attribute Label: Fucose Attribute Definition: Percentage of Fucose Storage Type: data Measurement Scale: Units: percent Precision: 0.10 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Mannose Attribute Label: Mannose Attribute Definition: Percentage of Mannose Storage Type: data Measurement Scale: Units: percent Precision: 0.10 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Galactose Attribute Label: Galactose Attribute Definition: Percentage of Galactose Storage Type: data Measurement Scale: Units: percent Precision: 0.10 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Glucose Attribute Label: Glucose Attribute Definition: Percentage of Glucose Storage Type: data Measurement Scale: Units: percent Precision: 0.10 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Neutral _Sugars Attribute Label: Neutral sugars Attribute Definition: Neutral sugar concentration Storage Type: data Measurement Scale: Units: milligramsPerGram Precision: 0.10 Number Type: real Missing Value Code: -9999.0 (Value will never be recorded ) Attribute Name: Benzaldehyde, 3,4-dimethoxy- Attribute Label: Benzaldehyde,3,4-dimethoxy- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Benzene, 1,2-dimethoxy-4-(1-propenyl)- Attribute Label: Benzene,1,2-dimethoxy-4-(1-propenyl)- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Ethanone, 1-(3,4-dimethoxyphenyl)- Attribute Label: Ethanone,1-(3,4-dimethoxyphenyl)- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Benzoic acid, 3,4-dimethoxy-, methyl ester Attribute Label: Benzoic Acid,3,4-dimethoxy-methyl ester- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Benzenepropanoic acid, 3,4-dimethoxy-, methyl ester Attribute Label: Benzenepropanoic acid,3,4-dimethoxy-methyl ester- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Ethanone, 1-(3,4,5)-trimethoxyphenyl- Attribute Label: Ethanone,1-(3,4,5)-trimethoxyphenyl- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Benzoic acid, 3,4,5-trimethoxy-, methyl ester Attribute Label: Benzoic Acid,3,4,5-trimethoxy-methyl ester- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Benzaldehyde, 3,4,5-trimethoxy- Attribute Label: Benzaldehyde,3,4,5-trimethoxy- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Phenol 2-methoxy- Attribute Label: Phenol,2-methoxy- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Benzene, 1,2-dimethoxy- Attribute Label: Benzene,1,2-dimethoxy- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: 3,4-Dimethoxytoluene Attribute Label: 3,4-dimethoxytoluene Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: 1,2,3-trimethoxybenzene Attribute Label: 1,2,3-trimethoxybenzene Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Attribute Name: Benzene, 1,2,3-trimethoxy-5-methyl- Attribute Label: Benzene,1,2,3-trimethoxy-5-methyl- Attribute Definition: Compound concentration as determined by thermochemolysis with TMAH Storage Type: data Measurement Scale: Units: milligramsPermilligram Precision: 0.00 Number Type: real Missing Value Code: -9999.000 (Value will never be recorded ) Methods Sampling Description Surface water samples were collected in 25-L white low-density polyethylene Carboy bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted (470 degrees C for 4 hours) 0.7 um GF/F glass fiber filters, followed by concentration using a Pellicon 2 Mini tangential flow ultrafiltration (TFF) system equipped with a nominal 1000 Da molecular weight cut-off regenerated cellulose membrane (Dai et al. 1998). The water samples were concentrated to 100 ml at an inlet pressure of 10 psi, and an outlet pressure of 8 psi. For water samples collected from Florida Bay (sites 9-11), diafiltration was conducted as follows: One liter of Milli-Q water (Millipore) was added to the concentrated sample and then re-concentrated to 100 ml. This process was repeated a total of three times. The concentrated samples were freeze-dried and powdered with an agate mill. Water samples for fluorescence analysis were collected separately in 30 ml brown polyethylene bottles, stored on ice, and transported to the laboratory. The water samples were filtered through pre-combusted (470 degrees C for 4 hours) Whatman GF/F glass fiber filters prior to analysis.Surface water samples were collected in 25-L white low-density polyethylene Carboy bottles during the early part of the dry season (from 05 Dec 2001 to 28 Jan 2002). The bottles were cleaned by soaking in 0.5 mol L-1 HCl followed by 0.1 mol L-1 NaOH for 24 h each. Water samples were filtered through pre-combusted (470 degrees C for 4 hours) 0.7 um GF/F glass fiber filters, followed by concentration using a Pellicon 2 Mini tangential flow ultrafiltration (TFF) system equipped with a nominal 1000 Da molecular weight cut-off regenerated cellulose membrane (Dai et al. 1998). The water samples were concentrated to 100 ml at an inlet pressure of 10 psi, and an outlet pressure of 8 psi. For water samples collected from Florida Bay (sites 9-11), diafiltration was conducted as follows: One liter of Milli-Q water (Millipore) was added to the concentrated sample and then re-concentrated to 100 ml. This process was repeated a total of three times. The concentrated samples were freeze-dried and powdered with an agate mill. Water samples for fluorescence analysis were collected separately in 30 ml brown polyethylene bottles, stored on ice, and transported to the laboratory. The water samples were filtered through pre-combusted (470 degrees C for 4 hours) Whatman GF/F glass fiber filters prior to analysis. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Amelung, W 1996. Determination of neutral and acid sugars in soil by capillary gas-liquid chromatography after trifluoroacetic acid hydrolysis. Soil Biol. Biochem., 28: 1631-1639. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Battin, T J 1998. Dissolved organic materials and its optical properties in a blackwater tributary of the upper Orinoco River, Venezuela. Organic Geochemistry, 28: 561-569. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Dai, M 1998. Evaluation of two cross-flow ultrafiltration membranes for isolating marine organic colloids. Marine Chemistry, 62: 117-136. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation De Souza Sierra, M M 1994. Fluorescence spectroscopy of coastal and marine waters. Marine Chemistry, 47: 127-144. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Donard, O F X 1989. High-sensitivity fluorescence spectroscopy of Mediterranean waters using a conventional or a pulsed laser excitation source. Marine Chemistry, 27: 117-136. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Hatcher, P G 1995. Comparison of two thermochemolytic methods for the analysis of lignin in decomposing gymnosperm wood: the CuO oxidation method and the method of thermochemolysis with tetramethylammonium hydroxide (TMAH). Organic Geochemistry, 23: 881-888. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Hedges, J I 1984. Carbon and nitrogen determinations of carbonate-containing solids. Limnology and Oceanography, 29: 657-663. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Jaffe, R 2004. Source characterization of dissolved organic matter in estuaries of the Florida Everglades by fluorescence analysis . Marine Chemistry, 84: 195-210. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Knicker, H 2000. Nature of organic carbon and nitrogen in physically protected organic matter of some Australian soils as revealed by solid-state 13C and 15N NMR spectroscopy. Australian J. Soil Res., 38: 113-127. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Kogel-Knaber, I 1997. 13C and 15N NMR spectroscopy as a tool in soil organic matter studies. Geoderma, 80: 243-270. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Lu, X Q 2003. Molecular characterization of dissolved organic matter in freshwater wetlands of the Florida Everglades. Water Research, 37: 2599-2606. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Mannino, A 2000. Terrigenous dissolved organic matter along an estuarine gradient and its flux to the coastal ocean. Organic Geochemistry, 31: 1611-1625. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation McKnight, D M 2001. Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnology and Oceanography, 46: 38-48. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Method Step Description DOC concentrations were analyzed using a high temperature catalytic combustion method on a Shimadzu TOC-5000 total organic carbon analyzer. Samples (4 ml) were acidified with 10ul of conc. HCl and sparged for 5 min with nitrogen (150 ml min-1) to remove inorganic carbon. The mean of three to six injections was reported for each sample. Fluorescence spectra were recorded on a Perkin Elmer LS 50B spectrometer equipped with a 150-W Xenon arc lamp as the light source. The emission monochromator was scanned from 250 to 550 nm with excitation at 313 and 370 nm (Donard et al. 1989; De Souza Sierra et al. 1994). Further, synchronous excitation-emission fluorescence spectra at a constant offset value between emission and excitation wavelength of 30 nm were measured from 250 to 550 nm (Lu et al. 2003; Jaffe et al. 2004). Both excitation and emission slits were set at 10 nm. Absorbance of the DOM solution was scanned from 250 to 550 nm for the correction of inner-filter effects on a Shimadzu UV-2101PC UV-visible spectrophotometer. The inner-filter effects were corrected for all the spectra following the procedure described by McKnight et al. (2001). Spectra were not corrected for instrumental response. Milli-Q water was used as a blank to background substract water Raman scatter peaks. The fluorescence intensities were expressed in quinine sulfate units (QSU); 1 QSU = 1 ug L-1 of quinine sulfate monohydrate in a 0.05 mol L-1 H2SO4 solution at excitation/emission (Ex/Em) =350/450 nm (Wu and Tanoue 2001). Four indices were used in this study: (1) maximum intensity (Max I); maximum fluorescence emission intensity with an excitation of 313 nm (Donard et al. 1989); (2) maximum wavelength (Max WL); the wavelength that gives the Intmax (Donard et al. 1989); (3) fluorescence index (FI); the ratio of emission intensities at 450 and 500 nm with an excitation of 370 nm (f450/f500) (Battin 1998; McKnight et al. 2001); (4) %285, calculated from a synchronous spectrum. %285 = Ex285 / (Ex285 + Ex350 + Ex385 + Ex460) x 100, where Ex285, Ex350, Ex385, Ex460 are the emission intensities at the respective excitation wavelengths (nm) noted in subscript (Lu et al. 2003). Total organic C content was measured on a Carlo Erba NA 1500 Nitrogen/Carbon Analyzer at 1050 degrees C, hippuric acid as a standard. To remove carbonate, two to five mg of powered sample was weighed into a silver capsule and exposed to hydrochloric acid vapor for 4 h, followed by drying under vacuum to eliminate any remaining hydrochloric acid (Hedges and Stern 1984). Then, the capsules were closed for analysis. Solid state 13C NMR spectra were obtained at a 13C resonance frequency of 50.3 MHz on a Bruker ASX200 NMR spectrometer equipped with a commercial 7 mm cross polarization magic angle spinning (CPMAS) probe using a standard CPMAS pulse sequence. 13C chemical shifts are expressed with respect to tetramethylsilane by using the carbonyl carbon of glycine (176.48 ppm) as an external reference. Other analytical conditions were as follows: rotation frequency, 4.5 kHz; contact time, 1 ms; recycle delay, 2 s; scans accumulated, 3000-20000; spectral width, 25 kHz; filter frequency, 32 kHz; Lorentzian line-broadening, 120 Hz. NMR spectra were divided into four regions according to chemical shifts as follows: 0-45 ppm (alkyl C), 45-110 ppm (O-alkyl C), 110-160 ppm (aromatic C), 160-210 ppm (carbonyl C) (Kogel-Knabner 1997). The first order spinning sidebands (SSBs) of aromatic and carbonyl signals (220 and 260 ppm, respectively) were corrected if necessary, according to Knicker and Skjemstad (2000). Sugar composition analysis was performed according to Amelung et al. (1996). Briefly, ca. 10 mg of powdered UDOM sample was mixed with an internal standard (50 ug myo-inositol) and 10 ml of 4 mol L-1 trifluoroacetic acid (TFA) and hydrolyzed at 105 degrees C for 4 h. Following filtration with a pre-combusted GF/F glass fiber filter, the solution was rotary-evaporated to remove TFA. The sample was then reconstituted with 2 ml water and passed through Amberlite XAD-4, and Dowex 50 W X 8 columns, successively. The sample was freeze-dried, and derivatized with 400 ul bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) at 75 degrees C for 5 min. Analysis was performed using a Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector. One ul of the solution was injected into a JandW DB1MS capillary column. Quantification was based on the comparison of the area on total ion chromatogram with known concentration of standard materials (arabinose, ribose, xylose, rhamnose, fucose, mannose, galactose, and glucose) that were processed in the same way as the samples. Initial oven temperature was set at 160 degrees C, held for 0.5 min, ramped at 8 degrees C min-1 to 185 degrees C, followed by 3 degrees C min-1 to 191 degrees C, by 0.5 degrees C min-1 to 195 degrees C, and thereafter by 10 degrees C min-1 to 250 degrees C and held for 5 min. Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. Detection limit was around 100 ppm in UDOM. The TMAH thermochemolysis was performed according to Hatcher et al (1995). Briefly, 4 to 10 mg C powdered UDOM sample was placed in a 5-ml glass ampoule and 200 ul of solution consisting of 25% TMAH in methanol and 200 ul of an internal standard, n-eicosane (50 ug ml-1 in methanol) were added. The methanol was evaporated under vacuum, and the ampoule was flame sealed and placed in a gas chromatographic oven at 250 degrees C for 30 min. After cooling, the ampoule was cracked open, and the inner glass surface was washed with 1 ml of methylene chloride three times, and concentrated to approximately 200 ul under a gentle stream of nitrogen. Analysis of this extract was performed on a Hewlett Packard 6890 GC-MS series gas chromatograph (GC) coupled to a 5973 mass selective detector. One ul of the solution was injected into a DB5MS (5% phenyl, 95% methyl silicone; 30 m length,0.25 mm i.d.,0.25 um film thickness) capillary column. Helium served as the carrier gas. The column temperature was programmed as follows: initial temperature at 40 degrees C, ramped at 10 degrees C min-1 to 120 degrees C, followed by 3 degrees C min-1 to 200 degrees C, and thereafter by 4 degrees C min-1 to 300 degrees C (Mannino and Harvey 2000). Mass spectra were recorded under electron impact ionisation conditions (70 eV) at 1 scan s-1 in the m/z = 50-500 mass range. The detection limit was 1 ng for the eicosane standard. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. A response factor for phenolic compounds to eicosane was calculated by averaging the relative response of methylation products of vanillin, vanillic acid, and acetovanilone to that of the internal standard. The concentration of phenolic compounds was estimated by comparing the area with that of the eicosane standard. Py-GC/MS analyses were on UDOM samples. Briefly, UDOM samples (ca. 5 mg) were pyrolyzed at 650 degrees C for 20 s in a helium atmosphere using a pyroprobe 1500 pyrolyzer. Separation of pyrolysis products was carried out on a DB5MS fused-silica column (30 m length, 0.25 mm i.d., 0.25 mm film thickness) at a split ratio of 1:75 under helium atmosphere. The oven was connected to the split/splitless injection port of a Hewlett Packard 6890 GC coupled to a HP 5973 mass spectrometer. The oven temperature program was as follows: initial temperature was held at 40 degrees C for 2 min, ramped at 7 degrees C min-1 to 300 degrees C where it was held for 15 min. The assignment of peaks was based on the comparison of mass spectra with the spectral library (NIST 98, Gaithersburg, MD, USA) and/or mass spectral interpretation. Other analytical conditions were identical with those of TMAH thermochemolysis described above. Based on the relative abundance of individually identified pyrolysis products (approximately 100 compounds; peak area of individual compound to total peak area of identified compounds in pyrogram) a hierarchical cluster analysis (HCA) was performed using an agglomerative method with SPSS version 11.0.1 software for the interpretation of the multivariate pyrolysis data set. Citation Wu, F 2001. Molecular mass distribution and fluorescence characteristics of dissolved organic ligands for copper (II) in Lake Biwa, Japan. Organic Geochemistry, 32: 11-20. Instrumentation Whatman GF/F glass fiber filters (Whatman International Ltd. Maidstone, England) Nalgene polyethylene bottles (25-L and 30-mL) (Nalge Nunc International, Rochester, NY) Pellicon 2 Mini tangential ultrafiltration system Shimadzu TOC-5000 total organic carbon analyzer (Shimadzu, Kyoto, Japan) Perkin Elmer LS 50B Spectrometer (Perkin Elmer, Wellesly, MA, USA) Shimadzu UV-2101PC UV-visible Spectrophotometer (Shimadzu, Kyoto, Japan) Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy) Bruker ASX200 NMR Spectrometer (Bruker, Rheinstetten, Germany) Hewlett Packard 6890 GC-MS series gas chromatograph coupled to a 5973 quadrupole mass selective detector (Hewlett-Packard, Avondale, PA, USA) Pyroprobe 1500 pyrolizer (Chemical Data Systems, Oxford, PA, USA) Hewlett-Packard 6890 GC coupled to a HP5973 mass spectrometer. Quality Control Standards, data was graphed Distribution Online distribution: http://fcelter.fiu.edu/perl/public_data_download.pl?datasetid=ST_ND_Jaffe_002.txt Intellectual Rights These data are classified as 'Type II' whereby original FCE LTER experimental data collected by individual FCE researchers to be released to restricted audiences according to terms specified by the owners of the data. Type II data are considered to be exceptional and should be rare in occurrence. The justification for exceptions must be well documented and approved by the lead PI and Site Data Manager. Some examples of Type II data restrictions may include: locations of rare or endangered species, data that are covered under prior licensing or copyright (e.g., SPOT satellite data), or covered by the Human Subjects Act, Student Dissertation data and those data related to the FCE LTER Program but not funded by the National Science Foundation (NSF) under LTER grants #DEB-9910514, and # DBI-0620409. Researchers that make use of Type II Data may be subject to additional restrictions to protect any applicable commercial or confidentiality interests. All publications based on this dataset must cite the data Contributor, the Florida Coastal Everglades Long-Term Ecological Research (LTER) Program and that this material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DEB-1237517, #DBI-0620409, and #DEB-9910514. Additionally, two copies of the manuscript must be submitted to the Florida Coastal Everglades LTER Program Office, LTER Program Manager, Florida International University, Southeast Environmental Research Center, OE 148, University Park, Miami, Florida 33199. For a complete description of the FCE LTER Data Access Policy and Data User Agreement, please go to FCE Data Management Policy at http://fcelter.fiu.edu/data/DataMgmt.pdf and LTER Network Data Access Policy at http://fcelter.fiu.edu/data/core/data_user_agreement/distribution_policy.html. Dataset Keywords FCE Florida Coastal Everglades LTER ecological research long-term monitoring Everglades National Park DOM Characteristics Taylor Slough Shark River Slough Florida Bay Ultrafiltered DOM lignin-phenol concentration neutral sugar composition 13C-NMR spectroscopy TMAH thermochemolysis flash pyrolysis-gas chromatography/mass spectroscopy dissolved organic carbon emissions aromaticity acidic carbon organic matter nitrogen freshwater fluorescence organisms Data Submission Date: 2005-09-08 Maintenance This is a short-term DOM dataset. This dataset replaces the original version named ST_ND_Jaffe_002. The FCE program is discontinuing its practice of versioning data as of March 2013. Dataset Contact Position: Information Manager Organization: LTER Network Office Address: UNM Biology Department, MSC03-2020 1 University of New Mexico Albuquerque, NM 87131-0001 USA Phone: 505 277-2535 Fax: 505 277-2541 Email: tech-support@lternet.edu URL: http://www.lternet.edu Position: Information Manager Organization: Florida Coastal Everglades LTER Program Address: Florida International University University Park OE 148 Miami, FL 33199 USA Phone: 305-348-6054 Fax: 305-348-4096 Email: fcelter@fiu.edu URL: http://fcelter.fiu.edu Dataset Submission Date 2005-09-08 Information Management Notes This is a short-term DOM dataset. This dataset replaces the original version named ST_ND_Jaffe_002. The FCE program is discontinuing its practice of versioning data as of March 2013.