Environ. Sci. Technol. 2007, 41, 7864-7869

investigate floc/aggregate structures in freshwater systems Spectromicroscopy Mapping of (8, 9) in particular, as well as to obtain morphological Colloidal/Particulate Organic Matter information on the colloidal fraction (1-1000 nm) in freshwater (10, 11) and groundwater systems (12, 13). in Lake Brienz, Switzerland Low nutrient or oligotrophic alpine lakes have attracted increased interest over the past few years as they are sensitive THORSTEN SCHA¨ FER,† indicators of climate change. Recent publications have shown VINCENT CHANUDET,‡,§ that the properties of the catchment have a clear impact on FRANCIS CLARET,| , ⊥ AND the macroinvertebrate communities in these lakes, while the MONTSERRAT FILELLA*,‡ influence of geographical patterns is minor (14). Large inputs Institut fu¨r Nukleare Entsorgung (INE), Forschungszentrum of suspended sediments and glacial silt/colloids may also Karlsruhe, P.O. Box 3640, D-76021 Karlsruhe, Germany, limit the spring phytoplankton peak in alpine lakes, which Department of Inorganic, Analytical, and Applied Chemistry, occurs in a number of other temperate lakes (15, 16). As part University of Geneva, Quai Ernest-Ansermet 30, CH-1211 of a large project focused on understanding the severe Geneva 4, Switzerland, Institut F.-A. Forel, University of productivity limitation conditions existing in a peri-alpine Geneva, Route de Suisse, 10, CH-1290 Versoix, Switzerland, lake (17)sLake Brienz, Switzerlands,submicrometer natural and CEA Saclay, CEA/DPC/SECR/LSRM, Gif sur Yvette, France organic matter (NOM), in particular, refractory organic matter (ROM) (18), and inorganic colloids (19, 20) have been studied for over a year in the lake and its two main tributaries, the Aare and Lu¨tschine Rivers. Transmission electron microscopy (TEM), soft X-ray The focus of this study is on (i) the characterization of the scanning transmission X-ray microscopy (STXM), and NOM found in the lake and in its tributary rivers, both alone µ-FTIR spectromicroscopy were used to map colloidal/ and in association with inorganic colloids; (ii) inter- particulate material in an ultra-oligotrophic lake, Lake Brienz, comparison of the data obtained by the three different Switzerland, with a special focus on organic functionality. methods used, namely, TEM, STXM, and µ-FTIR microscopy; Within the statistical margin of error and the uncertainties and (iii) comparison of organic functional group spectral arising from the representativeness of the results, the research signatures found in the lake with those of the two rivers. reveals that organic material was associated with potassium- Materials and Methods rich inorganic colloids present in surface and deep water (depths of 1 and 100 m, respectively), which indicates Sampling Sites. Samples were collected from the Lu¨tschine River (46°38′52′′N, 7°52′29′′E) and the Aare River (46°44′37′′N, a vertical transfer of aggregates by sedimentation. Pure ° ′ ′′ ° ′ ′′ organic colloids could only be detected in surface waters. 8 3 2 E) and from the middle of Lake Brienz (46 43 2 N, 7°56′59′′E) in July 2005. Lake water samples were collected In addition, correlation map analysis of synchrotron- at depths of 1 and 100 m using a membrane pump, with the based µ-FTIR and carbon K-edge STXM spectromicroscopic end of the tube directly attached to a multi-parameter Zu¨llig data using spectra from the Lu¨tschine and Aare Rivers HPT-D probe. River water samples were collected directly as target spectra revealed spectral similarities with organic into bottles at a depth of about 10 cm. Samples for dissolved components from both tributary rivers in deeper regions organic carbon (DOC) determination were collected in (100 m) of the lake. The results prove that STXM and µ-FTIR precombusted (3 h at 550 °C) glass bottles. Samples for ROM can characterize colloidal and particulate organic material and carbohydrate analysis were collected in clean polyeth- in low organic carbon systems. ylene bottles. Immediately after collection, all samples were acidified to pH 2 with Suprapur grade HCl and filtered through precombusted (3 h at 550 °C) 1.2 µm glass filters (Whatman Introduction GF/C filters) by vacuum filtration. All samples were stored in a cooler in double plastic bags and kept in a refrigerator The application of chemical microscopy to biological systems until measured. All standard and sample solutions were has benefited greatly from synchrotron light, particularly prepared with 18 MΩ cm Milli-Q water. through scanning transmission X-ray microscopy (STXM) Methods. DOC was determined by a high-temperature µ and synchrotron-based infrared microscopy ( -FTIR) (1). combustion method using a TOC 5000-A Shimadzu analyzer. STXM, which uses X-ray absorption spectroscopy (NEXAFS) Milli-Q water was used as the blank (0.00 mg C L-1 with a as the contrast medium, is a very powerful tool for analyzing SD less than 0.005 mg C L-1). fully hydrated samples such as colloids (2-4). Dynes and co-workers have recently applied this method to investigating Total dissolved carbohydrates were analyzed using a microbial biofilms and the metal/chlorhexadine speciation modified MBTH (3-methyl-2-benzothiazolinone hydrochlo- ride) method (21, 22). Calibration was performed with in them (5, 6). The combination with transmission electron -1 microscopy (TEM) gives additional structural information at glucose. Results are expressed as mg C L . Polysaccharide the highest resolution (7). TEM has been extensively used to morphology was assessed by TEM with specifically stained (0.1 mmol of Ruthenium Red) TEM grids (18). * Corresponding author phone: (+41 22) 379 6046; fax: (+4122) ROM was measured by following the adsorptive stripping 3796069; e-mail: [email protected]. voltammetry response of the complex formed by these † Institut fu¨r Nukleare Entsorgung. compounds in the presence of trace amounts of Mo(VI) (23). ‡ Department of Inorganic, Analytical, and Applied Chemistry, This method is particularly well-suited to the quantitative University of Geneva. determination of low concentrations of humic-type com- § Institut F.-A. Forel, University of Geneva. | CEA Saclay. pounds in fresh water. The same concentrations were ⊥ Present address: BRGM, Environment and Process Division, 3 obtained when using standard river fulvic (IHSS Suwannee Avenue Claude Guillemin, F-45060 Orleans Cedex 2, France. River fulvic acid standard (1S101F)) or humic (IHSS Suwannee

7864 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 22, 2007 10.1021/es071323z CCC: $37.00  2007 American Chemical Society Published on Web 10/09/2007 Laboratories (BNL) in New York, undulator beamline X1A1, operated by the State University of New York at Stony Brook. The principle of this method is described in detail elsewhere (27, 28). Carbon K-edge spectra were recorded in a constant helium atmosphere using an undulator gap of 36.8 mm. The Fresnel zone plate utilized for carbon-edge measurements had a diameter of 160 µm and an outermost zone width (δ) of 45 nm. Energy calibration of the spherical grating monochromator was performed using the photon energy of the CO2 gas adsorption band at 290.74 eV (29). Infrared measurements were performed at the U10B beamline (NSLS, BNL) using a Spectra-Tech Continuum IR microscope coupled with a Nicolet Magna 860 FTIR. The microscope utilizes a dual remote masking aperture and FIGURE 1. Mineralogical composition of inorganic colloids in Lake × Brienz and its tributaries as determined by TEM-EDS-SAED analysis. matching 32 Schwatzchild objectives. Spectra were collected using a 10 × 10 aperture and by averaging 512 scans in the mid-IR range (800-4000 cm-1) per point in transmission mode at a resolution of 4 cm-1 using Atlµs software (Thermo Nicolet Instruments). STXM measurements yield information on optical density (OD). OD is defined as the product of sample thickness d, sample density F, and mass absorption coefficient µ(E), which is related to the quotient of the incident flux on the sample I0(E) and the flux detected behind the sample I(E) via )- ) F OD ln[I(E)/I0(E)] µ(E) d (1)

Image stacks were obtained by taking images at different energies across the absorption-edge and aligning them using cross-correlation. After stack alignment, the XANES spectra were extracted (30). Image regions that contained no particles gave the I0(E) information. Given the time and cost constraints involved in STXM and µ-FTIR measurements, it is at present not possible to analyze enough samples to evaluate statistically the signifi- cance of the results obtained. However, since in this study STXM and µ-FTIR analysis were performed in the same sample grids used for TEM-EDS-SAED measurements and FIGURE 2. STXM carbon K-edge and potassium L2,3-edge spectra that TEM-EDS-SAED results fit well with the known com- 1 of cluster analysis from (A) Lutschine River and (B) Aare River position of the inorganic colloids in the lake, it can be assumed sample grids. Regions of extracted cluster spectra are indicated that STXM and µ-FTIR map reasonably well the main types by arrows in the images shown, which were taken at 280 eV. Number of organic matter present in this system. of colloids/particles was 80 for the Lu1tschine River clusters and 40 for the Aare River clusters. Results and Discussion River humic acid standard II (2S101H)) acids for calibration. TEM-EDS-SAED and NOM Analysis. The colloid mineral- Results are expressed as milligrams of C per liter. ogical compositions identified by TEM-EDS-SAED analysis The chemical and mineralogical composition of inorganic are shown in Figure 1. The inorganic minerals present in colloids was assessed by TEM coupled with energy dispersive Lake Brienz and its tributaries are dominated by clay-like, spectroscopy (EDS) and selected area electron diffraction flattened colloidal particles (Figure S1, Supporting Informa- (SAED). Specimen grids were prepared on-site by using a tion): illite, chlorite, biotite, and Ti-rich biotite, but also by non-perturbing procedure based on the centrifugation of albite, orthose, and quartz. The main difference in the the samples directly onto TEM grids (24, 25). The chemical colloidal mineralogical composition of the Aare and Lu¨tschine elemental composition of randomly chosen particles (100 in Rivers is the higher proportion of Ti-rich biotite and albite each system (26)) was measured by EDS and classified into in Aare River waters and the higher concentration of illite in homogeneous chemical classes. SAED analysis was carried the Lu¨tschine River. Measured organic matter concentrations out on some typical particles from each class to either confirm are given in Table 1. At present, Lake Brienz is an ultra- or determine their mineralogy. A detailed description of this oligotrophic lake, as confirmed by the very low concentrations procedure can be found in ref 26. of DOC and MBTH carbohydrates measured in various STXM measurements were performed at the National sampling campaigns (18). Some of the carbohydrates are Synchrotron Light Source (NSLS) at Brookhaven National present in the form of thin fibrils as shown in Figure S1B of

TABLE 1. Organic Matter Concentrations (mg C L-1) in Lake Brienz Samplesa (Error: 1 SD)a system DOCb MBTH carbohydratesb ROMb Lake Brienz, 1 m depth 0.42 ( 0.11 0.16 ( 0.01 0.11 ( 0.05 Lake Brienz, 100 m depth 0.76 ( 0.03 0.09 ( 0.01 0.11 ( 0.08 Aare River 0.22 ( 0.02 0.08 ( 0.04 0.07 ( 0.03 Lu¨ tschine River 0.32 ( 0.02 0.10 ( 0.05 0.05 ( 0.04 a Particulate organic matter concentration (average 0-10 m): 0.24 ( 0.03 (31). b Filtered at 1.2 µm.

VOL. 41, NO. 22, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7865 FIGURE 3. Lake Brienz 1 m depth sample. Top left: absorption image taken at 280 eV below the C1s-edge showing inorganic colloids and particles. The high-resolution ratio images shown in Figure 4 were taken in the two regions marked by rectangles. Bottom left: PCA and cluster analysis of the TEM grid shows three distinctive clusters (red, yellow, and green) and the background region (blue). Right: corresponding C1s spectra of clusters red and green are shown. Number of particles taken for the respective clusters was 39.

FIGURE 4. Lake Brienz 1 m depth sample. Ratio images of region a (upper row) and region b (lower row) marked in Figure 3. From left to right: absorption image at 280 eV, distribution of aromatics, and distribution of organics. Shades of bright gray indicate high concentrations of organic functionality. the Supporting Information. It should be mentioned that, as illite-type clay minerals, but orthose cannot be ruled out a discussed in ref 22, MBTH measurements probably do not priori) as indicated by the strong absorption at the potassium reveal all the carbohydrates in the system. This seems to be L2,3-edge (Figure 2A, solid line) and the high-edge jump particularly the case for the lake sample at 100 m. Concen- indicated by the OD. In addition, a second fraction of low trations of ROM, usually known as fulvic and humic OD (0-0.07), possibly consisting of pure organics (Figure substances, are also extremely low in the water bodies studied. 2A, dashed line), could be separated by cluster analysis (32). STXM Analysis. C1s STXM of the particulate/colloidal A low contribution of the CdC functionality (285.2 eV) is material found in the Lu¨tschine River showed major organic associated with these pure organic colloids. Other than the association with potassium-containing phases (probably absence of potassium absorption, the general features at the

7866 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 22, 2007 a energies 297.3 and 300 eV, indicative of the potassium L2,3- TABLE 2. Deconvolution Results of Lake Brienz Samples edge, but relatively weak absorption on the carbon-edge. depth: 1 m depth: 100 m This suggests the presence of the previously mentioned potassium-rich phases such as orthose, biotite, or illite functional group cluster 1 cluster 2 cluster 1 cluster 2 associated with low amounts of organic material (OD290eV - b red shift area 70<11OD280eV ) ∼0.05). Finally, higher amounts of organic struc- CdC, C-H 11 5 17 18 tures (OD ∼0.12) can be found, either surrounding these phenol 5 9 2 2 inorganic phases as submicrometer colloidal structures or c aliphatic 23 2 18 17 as separate submicrometer particles: cluster 2 (Figure 3, carboxyl 29 56 42 41 green region). Neither with PCA nor with cluster analysis carbonyl 24 28 21 21 could a C1s f π* transition of inorganic carbonate around a Areas are given as percentage of total area. b Red shift due to 290.2 eV be observed. High-resolution C1s ratio images heteroatom substitution/aromatic ring destabilization or benzoquinone- (Figure 4) of two distinct regions (areas marked in Figure 3) type functional groups. c Energy fixed to 287.2 eV. show that inorganic phases are either coated by organics, as shown by the shades of light gray on the mineral-edges, or carbon K-edge are very similar. In contrast, cluster analysis form mixed aggregates with inorganic structures. of the Aare River particulate/colloidal material revealed the The spectra found applying cluster analysis were decon- highest edge jump (organic concentration) in the areas of voluted (Table 2) by using the procedure described in refs almost no potassium absorption (Figure 2B, dotted line). 4 and 34. Deconvolution of the C1s spectra remains a The spectrum of these purely organic colloids/particulates semiquantitative treatment since, for example, the expected reveals a very different functionality as compared to the energy of C1s f π* d transitions in molecules containing mineral associated organics. Besides showing the C1s f π* C O carbonyl groups can vary as much as 4 eV (33). The transition for CdC groups at 285.2 eV, the strongest absorp- deconvolution results show a relatively high aliphatic/ tion in the spectra is around 288.2-288.4 eV, typical for C1s f π* transitions of CdO bonds in carboxyl- or amide-type aromatic content with lower amounts of oxygen-containing structures (33). Inorganic mineral associated organics could functional groups for organics associated with potassium- also be detected in Aare River material (Figure 2B, solid line). rich inorganic minerals (cluster 1), whereas the areas of pure C1s-edge features were comparable with the Lu¨tschine River organic C are more enriched in carboxyl-type groups (cluster colloids/particles, but the organic concentration was found 2). to be significantly lower. Analysis of the Lake Brienz sample taken at 100 m depth Principal component analysis (PCA) and cluster analysis also showed organic functionalities (Figure 5). Moreover, all (32) of the lake water sample taken at 1 m depth revealed organics are associated with potassium in this sample. The spectroscopically different regions with a cluster (Figure 3, main difference among clusters 1 (Figure 5, yellow region) yellow region) showing a general high OD (spectra not shown) and 2 (Figure 5, red region) was in the potassium and, and no features on the carbon K-edge or potassium L-edge. therefore, in the inorganic mineral content but not in the This is indicative of purely inorganic minerals (i.e., quartz, organic functionality. Detailed C1s deconvolution analysis albite, or chlorite as identified by TEM-SAED (Figure 1)). of the clusters gave very similar results (Table 2). The organic Cluster 1 (Figure 3, red region) has a high absorption at the functionality in deeper parts of the lake (100 m depth) is very

FIGURE 5. Lake Brienz 100 m depth sample. Top left: absorption image taken at 280 eV below the C1s-edge showing inorganic colloids and particles. High-resolution ratio images are in the Supporting Information. Bottom left: PCA and cluster analysis of the TEM grid shows two distinctive clusters (yellow and red) and the background region (blue). Right: corresponding C1s spectra of yellow and red clusters are shown. Number of particles taken for the respective clusters was 50.

VOL. 41, NO. 22, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7867 -1 1375 cm and mixed linear -CH3 and cyclic -CH2 vibrations at 1455 cm-1 (38). The rocking absorption of longer chains -1 of -CH2 centered around 720 cm could not be detected due to the frequency range limitations of the detection system used. The CdO vibration around 1670-1630 cm-1 is typical for tertiary amides with the general formula (RCO)3N. Differences in the average spectra of the Aare River particles as compared to those of the Lu¨tschine River are found in the region of ester- or aldehyde-type structures, which are depleted, and in the aliphatic region (νCH). The -1 -1 CH2/CH3 (2915-2940 cm /2950-2975 cm ) intensity ratio measurement technique described in ref 39 can be applied to estimate the length and degree of side-chain branching of the aliphatics. The difference in the ratios between the Aare (1.3) and the Lu¨tschine (3.9) samples indicates that the NOM in the Aare River has shorter and more branched aliphatic chains than in the Lu¨tschine River. Using the correlation between the number of carbon atoms in n-alkanes FIGURE 6. Left: average µ-FTIR spectra of particles isolated from and the determined CH2/CH3 intensity ratio published by Lu1tschine (n ) 15) and Aare (n ) 20) Rivers as shown in the visual ref 40, the average number of carbon atoms varies from six light microscopy (VLM) images of the TEM grid region. The spectra atoms (Aare River) to 12 (Lu¨tschine River). The shorter of a polyamide 6 structure (Hummel polymer sample library, index aliphatic chains found in the Aare River might be interpreted 28) is given for comparison (dotted line). Right: average µ-FTIR as a stronger decomposition of biota-derived materials. spectra of particles isolated from Lake Brienz at1m(n ) 25) and The structure of the particles in the Lake Brienz samples 100m(n ) 60) depths, respectively, as shown in the VLM images. from different depths (1 and 100 m) are highly organic, as × Image size is 100 µm 100 µm. shown in the average spectra of Figure 6. No OH vibrations in the range of 3600-3750 cm-1, indicative of clay minerals similar to the cluster of potassium-rich inorganic particle or micas, could be observed on these large particles. The associated organic matter in lake surface waters and suggests particles show CH vibrations of both aliphatic and aromatic that sedimentation of organic material, particularly via the structures and a high content of carboxyl/ketone-type CdO association with potassium-rich mineral phases, is one of groups. These observations are in good agreement with the the major organic removal processes in the lake, thus C1s results, although the size of the particles/colloids supporting conclusions from a previous mass balance study investigated does not overlap due to the different resolution (18). Almost pure organic material with high amounts of of the techniques used. Investigations at the same location oxygen-containing functional groups and poor aromatic in the grid as performed with STXM could not be carried out content, as found in surface waters, could not be detected due to the thickness of the large particles. Correlation map in deeper zones. This can be interpreted as (i) colloidal analysis shows that some aggregates show spectral similarities stability of these phases in the epilimnion, (ii) degradation to one river particle input, whereas other aggregates found of these organics in surface waters, or (iii) a problem of the in the correlation maps seem to be the result of aggregating microfocusing method applied being far from statistically the colloidal/particulate material from both river inputs (see representative (37). Supporting Information for details). Taking the cluster spectra found for the Aare and Lu¨tschine Rivers (Figure 2) as target spectra (32), correlation maps of Acknowledgments organic spectral similarities for the Lake Brienz samples were calculated (see Supporting Information). Interestingly, the Funding was provided by the regional government of the correlation maps show that the aggregate structures are built Canton Bern, Kraftwerke Oberhasli (KWO), Bundesamt fu¨r of colloidal/particulate material with spectral signatures from Umwelt (BAFU), and Lake Brienz shoreline communities. both tributaries at both sampling depths. We are grateful for beamtime allotment by BNL/NSLS. STXM The results obtained show that a significant surface of data were collected at endstation X1-A developed by the group inorganic mineral phases is covered by ROM-type material, of Janos Kirz and Chris Jacobsen at SUNY Stony Brook, with thus leading to a change in surface charge and therefore support from the Office of Biological and Environmental potentially influencing colloid/particulate stability. This result Research, U.S. DOE under Contract DE-FG02-89ER60858 and is particularly relevant because it supports laboratory studies from the NSF under Grant DBI-9605045. The zone plates that show that different types of NOM exert contrasting were developed by Steve Spector and Chris Jacobsen of Stony influences on mineral colloid coagulation (ref 35 and Brook and Don Tennant of Lucent Technologies Bell Labs references in the corresponding section of ref 36; ref 19 for with support from the NSF under Grant ECS-9510499. Lake Brienz colloids). µ-FTIR Analysis. Average µ-FTIR spectra of riverine and Supporting Information Available Lake Brienz particulate material are shown in Figure 6. The TEM micrographs of some typical Lake Brienz colloids; STXM larger particles found on TEM grids of both river samples correlation maps of Lake Brienz samples with cluster spectra show an average spectrum very similar to polyamide-type found in Lu¨tschine and Aare Rivers; and visual light mi- material. These larger particles seem to be condensed purely croscopy image µ-FTIR analysis of Lake Brienz samples. This organic materials with almost no absorption in the region material is available free of charge via the Internet at http:// - of Si O vibration bands. The spectra show strong asymmetric pubs.acs.org. and symmetric -CH3 and -CH2 stretching vibrations in the -1 2800-3000 cm region and -CH3 deformation vibration at 1470-1440 cm-1, overlapping with a scissor vibration at Literature Cited - -1 - - 1490 1440 cm and a symmetric CH3 vibration at 1390 (1) Navratil, M.; Mabbott, G. A.; Arriaga, E. A. Chemical microscopy -1 - - 1370 cm . The aliphatic CH2 and CH3 bands can applied to biological systems. Anal. Chem. 2006, 78, 4005- be further separated into exclusive -CH3 absorption at 4019.

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