Storage effects on quantity and composition of dissolved organic carbon and nitrogen of lake water, leaf leachate and peat soil water
Graphical abstract
Introduction
Dissolved organic matter (DOM) is a mixture of various soluble compounds differing in their molecular weight, structure and complexity (Leenheer and Croué, 2003). The composition of DOM can determine its bioavailability (Parr et al., 2015, Petrone et al., 2009) and consequently strongly influences the fate and persistance of DOM in aquatic ecosystems. Changes of environmental conditions such as alterations of pH or ion density, as well as freezing and thawing can affect the structure of these compounds (Dryer et al., 2008, Giesy and Briese, 1978, Pace et al., 2012) and as a consequence thereof, also DOM concentration and the optical properties of chromophore DOM (Fellman et al., 2008, Gao et al., 2015, Peacock et al., 2015, Spencer et al., 2007, Thieme et al., 2016). Acidification can be applied to arrest biological activity during cold storage (Schneider-Zapp et al., 2013), and is a common sample preservation method for later analysis of bulk dissolved organic carbon (DOC). However, for subsequent fluorescence and absorbance analysis (Schneider-Zapp et al., 2013, Spencer et al., 2007) or analysis with size exclusion chromatography (SEC; Sandron et al., 2015) it is not recommended, since acidification may result in drastic alterations of the molecular structure and confirmation of DOM molecules (Dryer et al., 2008, Pace et al., 2012). When optical properties of DOM are to be addressed and immediate sample analysis is not possible, freezing samples may constitute an appropriate preservation method. For freezing various effects on chromophoric DOM composition, as well as on bulk DOC and dissolved organic nitrogen (DON) concentration have been observed so far (Fellman et al., 2008, Otero et al., 2007, Peacock et al., 2015, Spencer et al., 2007, Thieme et al., 2016). For instance, Fellman et al. (2008) reported that DOC and DON concentration decreased due to freezing, whereas Peacock et al. (2015) reported that DOC concentration in peatland samples was mostly unaffected by freezing. Similarly, Otero et al. (2007) did not observe effects of freezing for sediment pore water samples in an estuary. Previous findings on fluorescence and absorbance properties of DOM were likewise inconsistent, reporting either no effects (Otero et al., 2007), variable responses (Spencer et al., 2007) or sometimes strong effects (Fellman et al., 2008, Peacock et al., 2015, Thieme et al., 2016) of freezing. Thereby factors like freezing and/or thawing temperature (Chen et al., 2016, Xue et al., 2015), ionic strength (Müller et al., 2011), DOC concentration (Fellman et al., 2008, Thieme et al., 2016) and DOM composition influence the physico-chemical processes during freezing and thawing and thus may explain the various results observed in literature. Size exclusion chromatography (SEC) can be used to determine bulk DOC and DON concentration and DOM composition, in particular the C:N ratio and the distribution of DOC and DON in different molecule size classes of DOM (Huber et al., 2011). SEC applied in parallel with analysis of optical properties enables to detect changes of DOM composition and concentration (Graeber et al., 2012a, Graeber et al., 2015, Heinz et al., 2015). While acidification affects size fractionation with SEC (Sandron et al., 2015) and is not a suitable preservation method for this analysis, the effects of freezing and cold storage at 4 °C on DOM size fractions determined with SEC have not been investigated yet at least in freshwater samples. For salt water from the Baltic sea no freezing effects were detected (Müller et al., 2011). However, the sometimes strong effects of freezing on spectral DOM properties (Fellman et al., 2008, Peacock et al., 2015, Thieme et al., 2016) indicate structural DOM alterations and suggest that SEC fractioning may be likewise vulnerable to freezing. Thieme et al. (2016) demonstrated that even if bulk concentration is not affected by fast freezing with liquid nitrogen, alterations of DOM structure and hence optical properties cannot be precluded.
In order to present a recommendation for storage and preservation of DOM samples for later SEC analysis as well as fluorescence and absorbance analysis, this study aims to evaluate the effects of freezing and cold storage on bulk DOC and DON concentration and SEC fractions. To account for differences in DOM composition we analyzed storage effects for three different sample types (freshwater DOM, leaf litter leachate, peatland pore water). To assess also the effects of freezing on DOC and DON concentration and SEC fractions for a set of different samples of the same sample type, but covering a range of DOM concentrations, we analyzed a set of peatland pore water samples from two oligotrophic nutrient poor bogs in different geographic regions.
We hypothesized that leaf leachates are more vulnerable to storage and freezing than lake samples due to the more ‘labile’ nature of leachate samples. The other way around, we expect that the peatland pore water samples constituting a less reactive humic sample type behave more or less conservative, independent from the geographical region where they derive from and independent of DOM concentration. In our study we aim to give a recommendation for storage and sample preservation for three different types of natural samples.
Section snippets
Sampling and preparation of the leaf leachate
To test the effects of cold storage and freezing on different types of DOM samples we used water from Lake Müggelsee (52.446°N, 13.640°E). For lake details see (Recknagel et al., 2016) representing a freshwater DOM source (hereafter referred to as lake sample). The leaf leachate from Phragmites australis grown in an inundated peatland ‘Polder Stangenhagen’ south of Berlin (52.199°N, 13.086°E) represents a purely terrestrial, but microbially unaltered, ‘fresh’ DOM source (hereafter referred to
DOC and DON concentration
Permutational ANOVA for lake and leachate samples revealed neither effects of DOM source nor of storage treatment on changes of DOC and DON concentration (perm. ANOVA, p < 0.05). Overall, changes of DOC and DON concentration in the samples due to freezing were lower than changes due to cold storage. This was in particular true for leachate samples where comparatively high changes of DOC and DON concentration occurred after cold storage (Fig. 1a and b). Cold storage resulted in stronger changes
Discussion
We have selected three different types of DOM samples to test if freezing and storage at 4 °C for one week alter DOC and DON concentration and DOM composition. We expected larger effects on leaf leachates compared to lake and peatland samples, since leachate DOM is not microbially processed so far, and thus supposed to be of more labile nature, i.e. more vulnerable to cold storage and freezing.
In accordance with our expectations effects on DOC and DON concentration were stronger for leachate
Conclusions
Overall, freezing seemed to constitute an appropriate preservation method for the analysis of bulk DOC concentration with SEC for lake, leachate and peatland samples analyzed here, but maybe not apply for samples from other geographical regions or preserved under other freezing conditions. If initial DOC concentrations in samples are high (>7 mg C L−1) e.g. in peat samples, freezing can affect the individual SEC fractions as well as DON concentration and should therefore be avoided. Likewise
Acknowledgement
We thank Sarah Schell, Mirjam Schneider and Thomas Rossoll for support in field work and laboratory analyses. This research was conducted within the project DONCOPRA (PU 136/7-1), we want to thank German Science Foundation (DFG) for funding the project.
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