Impact of seasonal climate change on optical and molecular properties of river water dissolved organic matter by HPLC-SEC and UV-vis spectroscopy
© Lepane et al. 2015
Received: 28 November 2014
Accepted: 4 June 2015
Published: 23 June 2015
The change in the optical parameters of dissolved organic matter (DOM) from natural water samples of Pirita River in Estonia were studied during 2 years of winter–spring season. At present, the relative quantitative and qualitative composition of DOM components leached from soils to water, in relation to air and water temperatures and water level fluctuations, has not been estimated in Estonia. The aim was to characterise and fractionate DOM using high-performance size-exclusion chromatography with multiple wavelength detection (DAD) and ultraviolet-visible (UV-vis) spectroscopy. Changes in several chromatographic/molecular and spectroscopic/optical parameters were investigated.
The experimental data indicated that the optical and molecular properties of DOM depend on the climate conditions, but the effect can be diverse. In cold climates, the concentration of DOM in river water can be lower (2013) or higher (2014) than in warmer climate conditions. Optical properties indicate that the quantity of humic substances (HS) is minimal under an ice cover and DOM contains more microbial-derived peptide-like constituents.
The results point out the usefulness of HPLC-SEC and UV-vis spectroscopy for climate change-related DOM studies in real environmental conditions. Used methods enable detailed monitoring of humic molecules dissolved from the catchment soil, as well as microbial constituents (peptide-like components of DOM). The used approach can be additionally applied for monitoring the changes of raw water quality and thus for the design and optimisation of drinking water process technology.
KeywordsDOM HPLC-SEC UV-vis spectroscopy Absorbance ratios Water Climate
Dissolved organic matter (DOM) forms most of the naturally occurring organic matter in water, and its influence on aquatic environments is enormous. Despite the important ecological role, there have been relatively few investigations on the impact of climate change on DOM constituents [1–6] and much is still uncertain about the structure, quality and properties. Organic carbon in soil and water has a great impact on the global carbon cycle . Previously, the importance of organic matter was recognised as a possible indicator of climate change . A considerable part of soil organic matter can be dissolved during the seasonal climate changes because of the extensive floods and runoff, as well as because of the significant changes in the air and the water temperatures. A river with a large catchment area usually receives a considerable amount of soil-derived (usually named allochthonous) organic matter. The DOM, expressed as dissolved organic carbon (DOC) concentration, depends on many hydrological parameters that are highly variable, as noted in recent studies [9, 10]. Increases in DOC concentrations in water are evident and observed in many countries, but the reasons are still unclear. There are seasonal factors like air and water temperatures, runoff and the amount of precipitation, such as rain or snow, as well as the yearly different times of ice-break, and connected to this, the inflow of snowmelt. Generally, factors like climate, land use and anthropogenic input have been outlined. In addition, Klavins et al. stated the importance of wetlands in the river catchment, land usage type and runoff regime .
High-performance size-exclusion chromatography is extensively used for DOM characterisation and fractionation, because it is a non-destructive technique with well-known advantages [9, 11], that has been successfully applied in several studies by the present author [12, 13]. Usually, the most common wavelength used to detect separated DOM fractions is 254 nm, but more studies nowadays apply multi-wavelength detection (diode array detection, (DAD)) . Another means of gaining more information about DOM composition is to calculate the absorbance ratios at several wavelengths. In the present study, these ratios are named as optical/spectroscopic parameters. Several ratios have been found to serve as proxies for DOM compositional changes. The theoretical background is described in Yan et al.’s recent work .
The aim of the current work was to study the change of DOM parameters from natural water samples of Pirita River in Estonia during 2 years of winter–spring seasons, where the most drastic climate change occurs (temperature rises from −10 to +15 °C), using HPLC-SEC-DAD and ultraviolet-visible (UV-vis) spectroscopy.
Hydrological data for Pirita River water samples
March 17, 2013
March 24, 2013
March 31, 2013
April 7, 2013
April 14, 2013
April 21, 2013
April 28, 2013
May 5, 2013
May 12, 2013
January 10, 2014
January 22, 2014
February 12, 2014
March 13, 2014
April 10, 2014
April 28, 2014
May 5, 2014
May 12, 2014
May 19, 2014
May 26, 2014
June 2, 2014
Samples were filtered through 0.45-μm Millipore filters before conducting the HPLC analysis. For this procedure, a 20-mM phosphate buffer with 10 % methanol (pH 6.8) was used as a mobile phase for the HPLC-SEC system. HPLC measurements were carried out on BioSep-SEC-S 2000 size-exclusion column (300 × 7.8 mm, Phenomenex). The HPLC-SEC system was equipped with a multi-wavelength absorbance detector, DAD (Agilent Technologies), and the chromatograms were registered on wavelengths between 200 and 600 nm using isocratic elution at a flow rate of 0.5 mL min−1 . Then, 20 μL of sample was injected for analysis. At least three replicates were carried out for each sample. All solutions for HPLC measurements were prepared using distilled water passed through a Milli-Q water purification system, filtered through 0.45-μm pore size filters (Millipore) and degassed. The chromatograms were recorded and processed by Agilent ChemStation software.
The aliphatic fraction of DOM is not measured in our experiments because of the absence of conjugated double bonds, and thus, the obtained data represent only the UV-absorbing fraction. The registered chromatogram peak area at the selected wavelength (210, 254 and 280 nm) corresponds to the relative quantity of DOM in a specific molecular size fraction that is not determined in the present study. The changes in the molecular structure were assessed following the changes in the retention times. Respectively, the peak with the lowest retention time refers to the highest molecular size fraction of DOM, and the peak with the highest retention time refers to the lowest molecular size fraction. Quantitative molecular properties of DOM were evaluated using a comparison of chromatographic parameters: A TOT210 (total peak area at 210 nm), A TOT254 (total peak area at 254 nm), A TOT280 (total peak area at 280 nm), A PL210 (peptide-like components, peak area at 210 nm) and ratio A 254/A PL210. Peak identification is carried out by extracting the UV-vis spectra of the chromatographic peak from the DAD data and comparing it with the existing database records of protein (bovine thyroglobulin, human gamma globulin, ovalbumin, myoglobin, uridine and tryptophan, Phenomenex), peptide, nitrate and humic substance (HS) (NOM, IHSS Nordic Reservoir 1R108N; HA, IHSS Nordic Aquatic 1R105H; FA, IHSS Nordic Aquatic 1R105F) standards. The total peak area recorded at 210 nm represents DOM (both humic and peptide-like constituents), and the total peak area recorded at 254 and 280 nm corresponds to the HS fraction of DOM. The peak at higher retention times is named peptide-like because the absorption spectra coincide with a 94–98 % probability with respective spectra of protein standards, but the molecular size is even smaller than that of HS. The UV spectrum of a peptide-like peak is characteristic for polypeptides (e.g. tripeptides). The peptide bonds found in the amino acids absorb at 205 nm. The use of HPLC-SEC in studies of polypeptides of a molecular mass less than 5000 Da is possible with a stationary phase used in column BioSep-SEC-S2000  that enables excellent resolution in the molecular mass range from a few hundred to a few thousand Da. Peptides emerge from a column near the lower limit of the separation range.
UV-vis absorption spectra were recorded using a UV-vis spectrophotometer (SPECORD 250 PLUS, Analytik Jena) with a wavelength area of 210–900 nm in a 1-cm quartz cuvette. Milli-Q water served as a blank. Absorbances at several wavelengths (A 205, A 254, A 280, A 365, A 470, A 665) and their ratios (A 254/A 205, A 365/A 470, A 470/A 665)  were applied for the characterisation of the optical properties.
Results and discussion
Chromatograms of water
Trends in absorbance ratios are different: The A 254/A 205 ratio decreased in January thus reflecting the minimum number of DOM aromatic constituents in relation to the peptide-like substances of microbial origin that exhibited the maximum at the same time. The UV-vis absorbing functional groups ratio of DOM (A 365/A 470) decreased slightly from 6 to 5. The degree of the aromatic groups condensation of DOM (A 470/A 665), or else degree of humification , decreased, with the exception of January and April. This absorbance ratio varied between 4 and 8. This variation may be the result of dilution. The high values (up to 12) are explained by the presence of low molecular mass fulvic acids in the water . The low values indicate input of DOM originating from the soil.
Effect of water and air temperatures
Temperature effects on DOM characteristics were evaluated by studying how retention times and areas of different peaks change as a function of air and water temperatures. Air temperatures in 2013 (March to May) varied between −3.9 and 14.5 °C and in 2014 between −10.8 and 19.8 °C (January to June) (Table 1). The long time averages for the months of January to June are as follows: −4.0, −4.7, −1.3, 3.9, 10.1 and 13.2 °C, respectively . Water temperatures varied between 0 and 14.5 °C (2013) and between 0.1 and 19.0 °C (2014). In the 2014 samples, both water and air temperatures were highly correlated (0.89 and 0.75, respectively) to UV ratio A 254/A 205. The increase of temperature caused increases of this ratio and constant decreases of peptide-like constituents in the water. Pirita River water is intensively coloured because of the high concentrations of HS originating from the catchment area. DOM sources are known to be marshlands and forests. During spring, usually, the snow melts, causing an increase of the water level, and DOM components from the soil are washed into the river water. This explains the increase of HS (A 254). The increasing water temperature possibly causes a rise in the microbial activity, and thus, the relative content of peptide-like DOM components in water should decrease.
The year 2013 samples show a very interesting temperature dependence; at low water temperatures (0–6 °C), peptide-like substance (A PL210) concentrations increase, but at warmer climates (7–15 °C), the opposite trend—a decrease—is obvious. A similar effect is noticed for A TOT210 and A TOT280. We explain the results by assuming domination of DOM degradation processes in cold climates and enhanced microbial activity in warmer climate conditions. The increase of peptide-like components in water can also indicate an anthropogenic impact, like wastewater input .
Effect of water level
The results point out the usefulness of HPLC and UV-vis spectroscopy for studying DOM in relation to climate change. The methods used enabled detailed monitoring of humic molecules dissolved from the catchment soil, as well as microbial derivatives (peptide-like components of DOM). The new data obtained in the present study may help to clarify the role of humic molecules and peptide-like components of DOM on bioactivity changes due to the effects of temperature and water level. The approach used can be applied for monitoring changes in raw water quality, and thus, for the design and optimisation of drinking water process technology.
diode array detection
dissolved organic carbon
dissolved organic matter
high-performance liquid chromatography
high-performance size-exclusion chromatography
natural organic matter
The authors are grateful to Dr. Tiiu Alliksaar (Institute of Geology, Tallinn University of Technology) for the kind permission to use the UV-vis spectrometer. Language was corrected by A. Shardell.
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