Environ. Sci. Technol. 2007, 41, 7864-7869
Spectromicroscopy Mapping of Colloidal/Particulate Organic Matter in Lake Brienz, Switzerland T H O R S T E N S C H A¨ F E R , † V I N C E N T C H A N U D E T , ‡,§ F R A N C I S C L A R E T , |,⊥ A N D M O N T S E R R A T F I L E L L A * ,‡ Institut fu ¨ r Nukleare Entsorgung (INE), Forschungszentrum Karlsruhe, P.O. Box 3640, D-76021 Karlsruhe, Germany, Department of Inorganic, Analytical, and Applied Chemistry, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland, Institut F.-A. Forel, University of Geneva, Route de Suisse, 10, CH-1290 Versoix, Switzerland, and CEA Saclay, CEA/DPC/SECR/LSRM, Gif sur Yvette, France
Transmission electron microscopy (TEM), soft X-ray scanning transmission X-ray microscopy (STXM), and µ-FTIR spectromicroscopy were used to map colloidal/ particulate material in an ultra-oligotrophic lake, Lake Brienz, Switzerland, with a special focus on organic functionality. Within the statistical margin of error and the uncertainties arising from the representativeness of the results, the research reveals that organic material was associated with potassiumrich inorganic colloids present in surface and deep water (depths of 1 and 100 m, respectively), which indicates a vertical transfer of aggregates by sedimentation. Pure organic colloids could only be detected in surface waters. In addition, correlation map analysis of synchrotronbased µ-FTIR and carbon K-edge STXM spectromicroscopic data using spectra from the Lu¨ tschine and Aare Rivers as target spectra revealed spectral similarities with organic components from both tributary rivers in deeper regions (100 m) of the lake. The results prove that STXM and µ-FTIR can characterize colloidal and particulate organic material in low organic carbon systems.
Introduction The application of chemical microscopy to biological systems has benefited greatly from synchrotron light, particularly through scanning transmission X-ray microscopy (STXM) and synchrotron-based infrared microscopy (µ-FTIR) (1). STXM, which uses X-ray absorption spectroscopy (NEXAFS) as the contrast medium, is a very powerful tool for analyzing fully hydrated samples such as colloids (2-4). Dynes and co-workers have recently applied this method to investigating microbial biofilms and the metal/chlorhexadine speciation in them (5, 6). The combination with transmission electron microscopy (TEM) gives additional structural information at the highest resolution (7). TEM has been extensively used to * Corresponding author phone: (+41 22) 379 6046; fax: (+4122) 3796069; e-mail:
[email protected]. † Institut fu ¨ r Nukleare Entsorgung. ‡ Department of Inorganic, Analytical, and Applied Chemistry, University of Geneva. § Institut F.-A. Forel, University of Geneva. | CEA Saclay. ⊥ Present address: BRGM, Environment and Process Division, 3 Avenue Claude Guillemin, F-45060 Orleans Cedex 2, France. 7864
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investigate floc/aggregate structures in freshwater systems (8, 9) in particular, as well as to obtain morphological information on the colloidal fraction (1-1000 nm) in freshwater (10, 11) and groundwater systems (12, 13). Low nutrient or oligotrophic alpine lakes have attracted increased interest over the past few years as they are sensitive indicators of climate change. Recent publications have shown that the properties of the catchment have a clear impact on the macroinvertebrate communities in these lakes, while the influence of geographical patterns is minor (14). Large inputs of suspended sediments and glacial silt/colloids may also limit the spring phytoplankton peak in alpine lakes, which occurs in a number of other temperate lakes (15, 16). As part of a large project focused on understanding the severe productivity limitation conditions existing in a peri-alpine lake (17)sLake Brienz, Switzerlands,submicrometer natural 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. The focus of this study is on (i) the characterization of the NOM found in the lake and in its tributary rivers, both alone and in association with inorganic colloids; (ii) intercomparison of the data obtained by the three different methods used, namely, TEM, STXM, and µ-FTIR microscopy; and (iii) comparison of organic functional group spectral signatures found in the lake with those of the two rivers.
Materials and Methods 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, 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 at depths of 1 and 100 m using a membrane pump, with the end of the tube directly attached to a multi-parameter Zu ¨ llig HPT-D probe. River water samples were collected directly into bottles at a depth of about 10 cm. Samples for dissolved organic carbon (DOC) determination were collected in precombusted (3 h at 550 °C) glass bottles. Samples for ROM and carbohydrate analysis were collected in clean polyethylene 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 GF/C filters) by vacuum filtration. All samples were stored in a cooler in double plastic bags and kept in a refrigerator until measured. All standard and sample solutions were prepared with 18 MΩ cm Milli-Q water. Methods. DOC was determined by a high-temperature combustion method using a TOC 5000-A Shimadzu analyzer. Milli-Q water was used as the blank (0.00 mg C L-1 with a SD less than 0.005 mg C L-1). Total dissolved carbohydrates were analyzed using a modified MBTH (3-methyl-2-benzothiazolinone hydrochloride) method (21, 22). Calibration was performed with glucose. Results are expressed as mg C L-1. Polysaccharide morphology was assessed by TEM with specifically stained (0.1 mmol of Ruthenium Red) TEM grids (18). ROM was measured by following the adsorptive stripping voltammetry response of the complex formed by these compounds in the presence of trace amounts of Mo(VI) (23). This method is particularly well-suited to the quantitative determination of low concentrations of humic-type compounds in fresh water. The same concentrations were obtained when using standard river fulvic (IHSS Suwannee River fulvic acid standard (1S101F)) or humic (IHSS Suwannee 10.1021/es071323z CCC: $37.00
2007 American Chemical Society Published on Web 10/09/2007
FIGURE 1. Mineralogical composition of inorganic colloids in Lake Brienz and its tributaries as determined by TEM-EDS-SAED analysis.
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 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
OD ) -ln[I(E)/I0(E)] ) µ(E)Fd
FIGURE 2. STXM carbon K-edge and potassium L2,3-edge spectra of cluster analysis from (A) Lu1 tschine River and (B) Aare River sample grids. Regions of extracted cluster spectra are indicated by arrows in the images shown, which were taken at 280 eV. Number of colloids/particles was 80 for the Lu1 tschine River clusters and 40 for the Aare River clusters. River humic acid standard II (2S101H)) acids for calibration. Results are expressed as milligrams of C per liter. The chemical and mineralogical composition of inorganic colloids was assessed by TEM coupled with energy dispersive spectroscopy (EDS) and selected area electron diffraction (SAED). Specimen grids were prepared on-site by using a non-perturbing procedure based on the centrifugation of the samples directly onto TEM grids (24, 25). The chemical elemental composition of randomly chosen particles (100 in each system (26)) was measured by EDS and classified into homogeneous chemical classes. SAED analysis was carried out on some typical particles from each class to either confirm or determine their mineralogy. A detailed description of this procedure can be found in ref 26. STXM measurements were performed at the National Synchrotron Light Source (NSLS) at Brookhaven National
(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 significance 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 that TEM-EDS-SAED results fit well with the known composition of the inorganic colloids in the lake, it can be assumed that STXM and µ-FTIR map reasonably well the main types of organic matter present in this system.
Results and Discussion TEM-EDS-SAED and NOM Analysis. The colloid mineralogical compositions identified by TEM-EDS-SAED analysis are shown in Figure 1. The inorganic minerals present in Lake Brienz and its tributaries are dominated by clay-like, flattened colloidal particles (Figure S1, Supporting Information): illite, chlorite, biotite, and Ti-rich biotite, but also by albite, orthose, and quartz. The main difference in the colloidal mineralogical composition of the Aare and Lu¨tschine Rivers is the higher proportion of Ti-rich biotite and albite in Aare River waters and the higher concentration of illite in the Lu ¨tschine River. Measured organic matter concentrations are given in Table 1. At present, Lake Brienz is an ultraoligotrophic lake, as confirmed by the very low concentrations of DOC and MBTH carbohydrates measured in various sampling campaigns (18). Some of the carbohydrates are 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
a
system
DOCb
MBTH carbohydratesb
ROMb
Lake Brienz, 1 m depth Lake Brienz, 100 m depth Aare River Lu¨ tschine River
0.42 ( 0.11 0.76 ( 0.03 0.22 ( 0.02 0.32 ( 0.02
0.16 ( 0.01 0.09 ( 0.01 0.08 ( 0.04 0.10 ( 0.05
0.11 ( 0.05 0.11 ( 0.08 0.07 ( 0.03 0.05 ( 0.04
Particulate organic matter concentration (average 0-10 m): 0.24 ( 0.03 (31).
b
Filtered at 1.2 µm.
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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 discussed in ref 22, MBTH measurements probably do not reveal all the carbohydrates in the system. This seems to be particularly the case for the lake sample at 100 m. Concentrations of ROM, usually known as fulvic and humic substances, are also extremely low in the water bodies studied. STXM Analysis. C1s STXM of the particulate/colloidal material found in the Lu ¨ tschine River showed major organic association with potassium-containing phases (probably 7866
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illite-type clay minerals, but orthose cannot be ruled out a priori) as indicated by the strong absorption at the potassium L2,3-edge (Figure 2A, solid line) and the high-edge jump indicated by the OD. In addition, a second fraction of low OD (0-0.07), possibly consisting of pure organics (Figure 2A, dashed line), could be separated by cluster analysis (32). A low contribution of the CdC functionality (285.2 eV) is associated with these pure organic colloids. Other than the absence of potassium absorption, the general features at the
TABLE 2. Deconvolution Results of Lake Brienz Samplesa depth: 1 m functional group areab
red shift CdC, C-H phenol aliphaticc carboxyl carbonyl
cluster 1
cluster 2
7 11 5 23 29 24
0 5 9 2 56 28
depth: 100 m cluster 1
