**2. Materials and methods**

#### **2.1 Study area and sampling**

The case studies were conducted at two sediment cores from Estonia: Lake Peipsi and Lake Rõuge Tõugjärv.

Lake (L.) Peipsi is the largest transboundary lake in Europe shared between Estonia and Russia. It is the fourth biggest lake in Europe. Its surface area is 3,500 km2 with an average depth of 7 metres. The maximum depth is only 15 metres. The catchment area covers more than 47,000 km2.. The catchments area has been used for agricultural purposes for several millennia. On the northern side of the lake, extensive mining areas and several electric power plants operating on oil shale exist. As a consequence, enhanced delivery of nutrients to L. Peipsi has induced an increase in primary productivity within the lake and anthropogenic eutrophication during the last few decades. Today the lake is classified as eutrophic.

A 43-cm long sediment core was collected from L. Peipsi in March 2007 from location 58°47′13″N and 27°19′18″E. The sampling point was located in the middle of the lake. The water depth at the sampling site was 9.8 m. Sediment samples were taken by a Willner corer. The core was cut into 1-cm thick subsamples, packed into plastic bags, and transported to the laboratory. The chronology of the core was established via correlation of its loss-on-ignition (LOI) curve with that of the 2002 year core, which was previously dated by the 210Pb radiometric method using gamma spectrometry (Appleby et al., 1986). For calculations of the 210Pb dates the Constant Rate of Supply model (Appleby & Oldfield, 1978) was applied and the results were compared to two other independent dating approaches the sediment distribution of artificial radionuclide 137Cs and spheroidal fly-ash particles (Alliksaar et al., 1998). The methodology and results of the dating methods used and the reliability of the chronology are explained in detail in Heinsalu et al. (2007).

The second lake investigated is situated in South Estonia, where the anthropogenic pressure is not too high and is expressed mainly through the agricultural activity. L. Rõuge Tõugjärv (57°44'30"N; 26°54'20"E) is a small-size stratified hard-water mesotrophic lake with a surface area of 4.2 ha and a maximum depth of 17 m. The main source of pollutants in L. Tõugjärv sediments is the catchments area. The studied sediment core was visibly laminated, reflecting the annual changes in the lake. Annual laminations, or varves, typically consist of two visible layers (a clastic inorganic layer and a darker organic humic layer), and each varve can be considered as representing one year's deposition.

The topmost 13 cm of the sediment was loose unconsolidated dark gyttja (dated until 1986 AD), while the rest of the sediment sample was laminated gyttja with well-developed varves (dated until the year 1852 AD). The L. Rõuge Tõugjärv sediment core was taken in May 2006 with a Willner-type sampler. The core was transported in a tightly closed Plexiglas tube to the laboratory, immediately sliced into 1-cm thick sub-samples, and packed into plastic bags to maximally avoid oxygen exposure.

The age-scale for the sediment sequence was obtained by correlating marker varve horizons and LOI values with another sediment core sampled in 2001, which had been carefully dated by several parallel dating methods (varve counting, 210Pb, 137Cs, 241Am, and spheroid fly-ash particles) (Alliksaar et al., 2005; Veski et al., 2005; Poska et al., 2008). According to this correlation the obtained sediment core covered about 150 years (1850–2005).

*Pw* samples for analysis were obtained by extraction of unfrozen sediments by centrifugation at 3,500 rpm for 30 minutes and filtration through 0.45 μm filters (Millex, Millipore). Samples were stored at 4 oC in the dark.

#### **2.2 Chemical analyses**

312 International Perspectives on Global Environmental Change

palaeolimnological research (Leeben et al., 2008a; Lepane et al., 2004, 2010a; Makarõtševa et al., 2010) it is not widely used for evaluating the long-term changes in aquatic ecosystems. At present, no comparative investigations of *pw*DOM from lake sediments are available. Coupling of HPSEC as a separation method with diode-array detection (DAD) allows DOM fingerprints and spectra of DOM molecular fractions to be obtained for qualitative and semi-quantitative analysis. The non-destructive analysis, small sample volume, and minimal sample pretreatment are great advantages of the HPSEC-DAD approach, making the method suitable for environmental studies. HPSEC-DAD has been adapted and optimized for analysis of *pw* samples under various conditions (Lepane et al., 2004; 2010a; O'Loughlin & Chin, 2004). The advantage of the usage of this chromatographic system is a better understanding of the qualitative and quantitative *pw*DOM properties by detecting aromatic fractions (chromophoric compounds). This method has recently been applied for monitoring and detection of organic matter from surface waters after oxidation treatment

 This study aims: (1) to investigate temporal changes in *pw*DOM components' qualitative and quantitative characteristics by exploring different sediment core records; (2) to find the similarities and differences in HPSEC-DAD fingerprints of *pw*DOM after applying the statistical data treatment methods; (3) to explore the potential impact of environmental

The case studies were conducted at two sediment cores from Estonia: Lake Peipsi and Lake

Lake (L.) Peipsi is the largest transboundary lake in Europe shared between Estonia and Russia. It is the fourth biggest lake in Europe. Its surface area is 3,500 km2 with an average depth of 7 metres. The maximum depth is only 15 metres. The catchment area covers more than 47,000 km2.. The catchments area has been used for agricultural purposes for several millennia. On the northern side of the lake, extensive mining areas and several electric power plants operating on oil shale exist. As a consequence, enhanced delivery of nutrients to L. Peipsi has induced an increase in primary productivity within the lake and anthropogenic eutrophication during the last few decades. Today the lake is classified as

A 43-cm long sediment core was collected from L. Peipsi in March 2007 from location 58°47′13″N and 27°19′18″E. The sampling point was located in the middle of the lake. The water depth at the sampling site was 9.8 m. Sediment samples were taken by a Willner corer. The core was cut into 1-cm thick subsamples, packed into plastic bags, and transported to the laboratory. The chronology of the core was established via correlation of its loss-on-ignition (LOI) curve with that of the 2002 year core, which was previously dated by the 210Pb radiometric method using gamma spectrometry (Appleby et al., 1986). For calculations of the 210Pb dates the Constant Rate of Supply model (Appleby & Oldfield, 1978) was applied and the results were compared to two other independent dating approaches the sediment distribution of artificial radionuclide 137Cs and spheroidal fly-ash particles (Alliksaar et al., 1998). The methodology and results of the dating methods used

and the reliability of the chronology are explained in detail in Heinsalu et al. (2007).

change on *pw*DOM records in investigated sediment cores.

(Liu et al., 2010).

Rõuge Tõugjärv.

eutrophic.

**2. Materials and methods 2.1 Study area and sampling** 

> Absorbance spectra of the *pw* samples were collected using a Jasco V-530 UV/VIS Spectrophotometer (Japan), with 1-cm-pathlength fused silica cells and ultrapure water as the blank. Spectra were measured over the range of 200–500 nm with a 2.0-nm bandwidth. The dissolved organic carbon (DOC) concentration in *pw* samples was calculated from absorption spectra using the equation given by Højerslev (1988). The absorbance ratio at 250 and 360 nm (A250/A360), which reflects the aromaticity of dissolved molecules (Peuravuori & Pihlaja, 1997), was calculated from the spectra.

#### **2.3 HPLC analyses**

The molecular characteristics of DOM in sediments were determined using an HPLC system. The HPLC system comprised a Dionex P680 HPLC Pump, Agilent 1200 Series (Agilent Technologies, UK) diode array absorbance detector (DAD), and a Rheodyne injector valve with a 20-μL sample loop. A BioSep-SEC-S 2000 PEEK size exclusion analytical column (length 300 mm, diameter 7.50 mm, Phenomenex, USA) preceded by a suitable guard column (length 75 mm, diameter 7.50 mm, Phenomenex, USA) was used for separation. The applied flow rates were 0.5 mL min-1 (L. Peipsi samples) and 1.0 mL min-1 (L. Rõuge Tõugjärv samples). The column packing material was silica bonded with a hydrophilic diol coating, with a particle size of 5 µm and a pore size of 145 Å. The mobile phase consisted of 0.10 M NH4H2PO4 - (NH4)2HPO4 buffer at pH 6.8. The HPLC system was calibrated using five different molecular mass protein standards (Aqueous SEC 1 Std, Phenomenex, USA) (see Fig. 1). All solutions for HPLC measurements were prepared using ultrapure water passed through a MilliQ water system, filtered with 0.45-µm pore size filters

HPLC Fingerprints of Porewater Organic Compounds as Markers for Environmental Conditions 315

Cluster analysis using the Ward method was applied to reveal age-related periods in the analysed samples (Brereton, 2003). The analysis was performed on the chromatographic data. As descriptors of the DOM, all of the separated peak areas and total chromatogram areas, molecular masses, and their ratios, DOC and A250/A360, for all samples were included in the analysis. The Euclidean distance was used as a measure of the similarity– dissimilarity of the samples. The statistical analyses were carried out using WinSTAT for

The descriptions and dating values for analysed sediment cores are reported in Table 1. Both sediment cores covered roughly 150 years of sediment deposition. The characteristics of L. Peipsi sediment core solid-phase have been reported previously (Lepane, 2010b). Briefly, the organic matter content was 23 to 25% for the period 1860–1950 and increased up to 27% after

In the second case investigated, L. Rõuge Tõugjärv, the organic component of the particulate sediment matrix had low values (11-13%) between 1850 and the 1880s, followed by a distinct peak (18%) around the 1900s and a progressive rise to 27% thereafter (Alliksaar et al., 2005). The rest of the sediment consisted mainly of terrigenous mineral matter eroded into the lake from the catchment. In L. Rõuge Tõugjärv, the low organic matter values in sediment probably indicated land derived input from human-induced topsoil erosion and dilution of organic matter by addition of clastic mineral particles, while the increase in organic matter presumably reflected a lower contribution of terrestrial mineral material with a reduction in the overall rural activities. The DOC values corresponded to an increase in organic matter from 1850 to the 1890s; thereafter the decrease has been significant up to the present. The low DOC values may be due to a high amount of aliphatic organic compounds resulting from the microbial activity. Since the determination of DOC was based on the spectroscopic

the 1950s. This increase was followed by an increase in DOC values from the 1960s.

method, the aliphatic organic matter fraction was not determined in present study.

The sediment DOM was characterized by *pw* sample analysis using HPLC (HPSEC) with DAD. Absorbance spectroscopy with a single detection wavelength has been verified as a suitable detection method after HPLC separation (Lepane, 2010a). The UV absorbance at 250–280 nm has been widely used to provide an estimation of aromatic compounds (Filella, 2010). In the present study, multi-wavelength HPLC analysis has been carried out to detect changes in *pw*DOM composition and molecular mass profiles down the cores. The absorbance spectra were examined from 200 to 400 nm. Figures 2 and 3 display the HPLC chromatograms of *pw* from studied sediment cores and from different layers, respectively. The UV detector response range from 205 to 400 nm has been plotted against the retention times to obtain the multi-wavelength contour plots. Plot colours provide the visual representation of the relative absorbance intensity. The multi-wavelength plots allowed the most suitable detection wavelength for separated DOM components to be selected. Simultaneously, the chromatograms were registered at 280 nm. The chromatogram patterns suggested that the *pw*DOM molecules included two fractions in both lakes. The first peak, with shorter retention times of 6–7 min and of 8–9 min, corresponded to the

**2.4 Statistical analyses** 

Excel software (R. Fitch Software, Germany).

**3.1 Main characteristics of the sediment profiles** 

**3. Results and discussion** 

**3.2 Multi-wavelength HPLC analyses** 

(Millipore), and degassed. Samples were analysed in triplicate. In general, the relative standard deviations for the replicated measurements did not exceed 5% (obtained by comparison of total peak areas). For quality control the aqueous protein standard was analysed each day. The chromatograms were recorded and processed by Agilent ChemStation software. Full details of the method used are described previously (Lepane et al., 2004).

Weight-average and number-average molecular masses of DOM (Mw and Mn, respectively) were determined using the formulae Mw = Σ(hiMi)/Σhi and Mn = Σhi/ Σ(hi/Mi), where hi is the detector output and Mi is the molecular mass, both at the i-th retention time (Mori & Barth, 1999).

As a semi-quantitative DOM characteristic, the total chromatogram peak areas, representing the total UV-absorbing fraction of the specific molecular size fraction of DOM in each sample, were used in the data analysis. The total chromatogram peak areas obtained with DAD actually represent the variations in optical intensities of DOM fractions at the chosen wavelength of 280 nm. The detector response (the height of the chromatogram at the i-th elution volume) refers to the amount of DOM in a specific molecular size fraction. The sum of all peak heights represents the total amount of DOM capable of UV adsorption in the sample (Matilainen et al., 2006; Peuravuori & Pihlaja, 1997; Vartiainen et al., 1997). Peak areas were used as a semi-quantitative characteristic to present age-related variations in the DOM fractions. To obtain qualitative DOM characteristics the chromatograms were divided into two molecular size fractions: 1) high molecular mass (HMW), and 2) humic substances (HS) (Lepane et al., 2010a, 2010b). The polydispersity Mw/Mn, describing the homogeneity or heterogeneity of organic matter, was calculated from the data obtained.

Fig. 1. Separation of calibration standards by HPLC. Protein molecular masses (1) 670 kDa, (2) 150 kDa, (3) 44 kDa, (4) 17 kDa, (5) 244 Da; detection wavelength 280 nm.

#### **2.4 Statistical analyses**

314 International Perspectives on Global Environmental Change

(Millipore), and degassed. Samples were analysed in triplicate. In general, the relative standard deviations for the replicated measurements did not exceed 5% (obtained by comparison of total peak areas). For quality control the aqueous protein standard was analysed each day. The chromatograms were recorded and processed by Agilent ChemStation software. Full details of the method used are described previously (Lepane et

Weight-average and number-average molecular masses of DOM (Mw and Mn, respectively) were determined using the formulae Mw = Σ(hiMi)/Σhi and Mn = Σhi/ Σ(hi/Mi), where hi is the detector output and Mi is the molecular mass, both at the i-th retention time (Mori &

As a semi-quantitative DOM characteristic, the total chromatogram peak areas, representing the total UV-absorbing fraction of the specific molecular size fraction of DOM in each sample, were used in the data analysis. The total chromatogram peak areas obtained with DAD actually represent the variations in optical intensities of DOM fractions at the chosen wavelength of 280 nm. The detector response (the height of the chromatogram at the i-th elution volume) refers to the amount of DOM in a specific molecular size fraction. The sum of all peak heights represents the total amount of DOM capable of UV adsorption in the sample (Matilainen et al., 2006; Peuravuori & Pihlaja, 1997; Vartiainen et al., 1997). Peak areas were used as a semi-quantitative characteristic to present age-related variations in the DOM fractions. To obtain qualitative DOM characteristics the chromatograms were divided into two molecular size fractions: 1) high molecular mass (HMW), and 2) humic substances (HS) (Lepane et al., 2010a, 2010b). The polydispersity Mw/Mn, describing the homogeneity

Fig. 1. Separation of calibration standards by HPLC. Protein molecular masses (1) 670 kDa,

(2) 150 kDa, (3) 44 kDa, (4) 17 kDa, (5) 244 Da; detection wavelength 280 nm.

or heterogeneity of organic matter, was calculated from the data obtained.

al., 2004).

Barth, 1999).

Cluster analysis using the Ward method was applied to reveal age-related periods in the analysed samples (Brereton, 2003). The analysis was performed on the chromatographic data. As descriptors of the DOM, all of the separated peak areas and total chromatogram areas, molecular masses, and their ratios, DOC and A250/A360, for all samples were included in the analysis. The Euclidean distance was used as a measure of the similarity– dissimilarity of the samples. The statistical analyses were carried out using WinSTAT for Excel software (R. Fitch Software, Germany).
