Influence of Leaching Solution and Catchment Location on the

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Influence of leaching solution and catchment location on the fluorescence of water-soluble organic matter Rachel S Gabor, Margaret A Burns, Robert H Lee, Jordan B Elg, Cayla J Kemper, Holly R Barnard, and Diane Marie McKnight Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504881t • Publication Date (Web): 11 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Environmental Science & Technology

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Influence of leaching solution and catchment location on the fluorescence of water-soluble

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organic matter

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Rachel S Gabor1,2,6*, Margaret A. Burns3,6, Robert H. Lee4,6, Jordan B. Elg5,6, Cayla J. Kemper2,6,

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Holly R. Barnard3,6, Diane M McKnight1,2,6

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*[email protected] phone: (303)492-4687, Fax: (303)492-6388

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1

Department of Environmental Studies, University of Colorado Boulder

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2

Department of Civil, Environmental, and Architectural Engineering, University of Colorado

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Boulder

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3

Department of Geography, University of Colorado Boulder

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4

Department of Geology, University of Colorado Boulder

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Department of Ecology and Evolutionary Biology, University of Colorado Boulder

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INSTAAR, University of Colorado Boulder

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ABSTRACT

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Organic matter (OM) plays a significant role in biogeochemical processes in soil and water

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systems. Water-soluble organic matter (WSOM), leached from soil samples, is often analyzed as

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representative of potentially mobile OM. However, there are many WSOM extraction methods

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in the literature with no clear guidelines for method selection. In this study, four common

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leaching solutions -- 0.5 M K2SO4, 0.01 M CaCl2, 2 M KCl, and H2O -- were used to extract WSOM

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from various locations within a forested catchment. Fluorescence spectroscopy was used to

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analyze the impact of extraction method on WSOM chemistry. While all four methods

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consistently identified chemical differences between WSOM from a north-facing slope, south-

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facing slope, and riparian zone, there were clear differences in fluorescence signal between the

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leaching methods. All three salt solutions contained WSOM with a higher fluorescence index

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(FI) and humification index (HIX) than WSOM leached with H2O, suggesting the presence of salts

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releases different fractions of the soil organic matter. A PARAFAC model developed from the

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leachates identified a distinctive soil humic fluorophore observed in all samples, as well as

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fluorescent artifacts present in H2O-leached samples.

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INTRODUCTION

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Soil organic matter (OM) influences many chemical and biological processes in terrestrial

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systems and also strongly impacts the quantity and chemistry of dissolved organic matter

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(DOM) in aquatic environments. In soil, organic matter can be found in particulate form, bound

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to mineral surfaces, or as DOM in soil pore water. Water soluble organic matter (WSOM) can

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transition back and forth in a dynamic equilibrium between these fractions1 and may be flushed

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during rainstorms and sustained high infiltration, such as during snowmelt. While DOM and

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WSOM are only a small fraction of the total soil OM pool, they play a significant role in soil

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processes. For example, the chemistry of WSOM has been found to determine its ability to bind

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metals such as iron and aluminum, impacting metal transport and bioavailability2. The

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composition of soil OM also impacts how much OM is lost in response to acid rain3 and can

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indicate the degree of soil desertification4.

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The equilibrium between WSOM and DOM in soil pore water is driven by the chemical

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composition of the WSOM. In turn, WSOM chemistry is influenced by the surrounding

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environment. Conditions such as aridity5, soil salinity4, forest fires6, acidification3,7,8, vegetation

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and elevation9, and land use practices1 have all been found to influence WSOM chemistry.

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Many of these processes have also been found to impact DOM in streams10,11, likely because

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the changes in soil WSOM chemistry propagate through the watershed. Thus understanding

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WSOM and pore-water DOM chemistry can help to reveal drivers for stream DOM chemistry.

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Analysis of soil pore water can be an effective means to study this chemically-important mobile

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fraction of SOM. However, in some regions there is minimal soil pore water and only WSOM is

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readily accessible for analysis.



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There are a variety of techniques for extracting WSOM from soil systems12–14. In order to

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effectively utilize these extraction techniques we need to understand the differences between

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them from a chemical perspective. Common solutions used to leach WSOM include water

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(sometimes heated), 0.5 M K2SO4, 2 M KCl, 0.01 M CaCl2, NaH2PO4, and NaNO39,14–19. These

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extractions have been done with field-moist and dried soils, unsieved and sieved soils, at

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different temperatures, and for different times. The variety of extraction techniques can make

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it difficult to compare results between studies, and to choose an optimal technique for a new

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study, as there are no clear guidelines for method selection18,20.

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Several studies have utilized fluorescence spectroscopy techniques to identify chemical

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properties of WSOM. While the complex mixture of molecules that make up organic matter

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only contains a fraction of fluorescently active material, the optical properties of this mixture

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correspond well to OM biogeochemistry11, making fluorescence an increasingly common

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technique for identifying OM chemistry. However, few studies have identified the impact of

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leaching method on the chemistry of fluorescently active WSOM. Corvasce et al.16 used 0.01 M

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CaCl2 to leach WSOM from soil pits and, useing fluorescence, found decreasing aromaticity with

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depth. Fest et al.17 compared DOM in soil pore water to WSOM extracted with water and 0.01

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M CaCl2, finding less WSOM in the salt extraction, but no consistent difference in amount of

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humic and fulvic acids within the WSOM extracts. Rennert et al.18 extracted WSOM with water,

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0.01 M CaCl2, and 0.5 M K2SO4 and found that the K2SO4 extracted the most WSOM, the CaCl2

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extracted the least and the H2O extracts had the highest molar absorptivity at 254 nm. They


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suggested that 0.5 M K2SO4 is the best surrogate for DOM in soil pore water due to its molar

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absorptivity. Provenzano et al.20 extracted soil with H2O and 0.01 M CaCl2 and found that CaCl2

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extracted less total organic carbon, but it displayed greater fluorescent intensity.

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For a more complete understanding of the impact of leaching solution on WSOM chemistry,

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this study utilized surface soil samples from a forest ecosystem, collected weekly during and

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after snowmelt from eight catchment locations. Soil samples were leached with water and 0.5

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M K2SO4. An additional set of samples was also leached with 0.01 M CaCl2 and 2 M KCl to

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include these commonly used solutions. Because other studies have compared in detail the

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total leached organic matter due to these methods, our study focused on the differences in the

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fluorescent characteristics, based on fluorescence indices as well as an 8 component PARAFAC

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model. The first part of this study compared the samples to see if the methods of leaching

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WSOM produces different fluorescent signatures. The second part investigated whether the

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methods identified similar trends in WSOM chemistry during and after the snowmelt period21,22

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and between catchment locations.

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EXPERIMENTAL (MATERIALS AND METHODS)

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Site Description and Sample Collection

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Soil was collected from Gordon Gulch (105.47 W, 40.04 N), a headwater catchment in the

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Colorado Front Range with an average elevation of 2627 m and area of 2.75 km2. The north-

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facing slope is characterized by dense lodgepole pines (Pinus contorta) with minimal understory



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vegetation and ground covered by pine needle duff. A persistent snowpack is typical in winter

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and soil is usually moist. The south-facing slope, a meadow with sparse ponderosa pines (Pinus

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ponderosa) and shallower bedrock23, has a transient snowpack and drier soil. The riparian zone

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is larger on the north-facing side and contains grasses and aspen (Populus tremuloides) stands.

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(SI Table 1). Previous studies identified the microbial populations in this region and found

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greater microbial biomass on the north-facing slope and riparian zone than the south-facing

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slope24,25.

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Plots 2 m x 2 m square were chosen along a transect with four plots on the south-facing slope

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and four plots on the north-facing slope, including one in the riparian zone. Beginning the last

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week of snowmelt, samples were taken at 1-2 week intervals from April 14, 2010 until July 12,

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2010. Each week three locations were chosen at random from each plot, the litter layer

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brushed aside, and the top 5 cm of soil was sampled. Samples were placed in a cooler until they

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could be stored in a 4°C fridge. Samples were also collected on May 18, 2011 and June 21, 2011.

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Additionally, one soil sample was collected in September 2013 at 10 cm depth from each of the

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south-facing slope, north-facing slope, and riparian zone at locations adjacent to 10 cm

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groundwater lysimeters.

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WSOM Extraction

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WSOM was extracted from field-moist, unsieved soil to avoid the effects of drying and sieving

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on WSOM release14. Soil samples were mixed in their sample bag and rocks and large plant

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matter were removed. 30 g of soil was mixed with 150 ml of solvent and agitated on a shaker


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table for 1 hour at 200 rev/min. They were then centrifuged for 15 min, decanted, and vacuum

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filtered through a 0.7 μm GFF filter. Samples from 2010 were leached with nanopure H2O and

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0.5 M K2SO4. Samples from 2011 and 2013 were leached with nanopure H2O and 0.5 M K2SO4 as

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well as 0.01 M CaCl2 and 2 M KCl. Leachates were stored in the dark at 4°C. To ascertain the

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impact of salt on fluorescence, the water-leached samples from 2013 were divided into fourths

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and salt was added to reach the concentrations used in the other leaching solutions. At the

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same time as extraction, 10 g of soil was dried for 48 hours at 105°C to determine soil moisture.

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Dissolved organic carbon (DOC) concentration of the WSOM was measured with a Shimadzu

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TOC analyzer

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Spectroscopic Analysis

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UV-vis analysis was performed using an Agilent 8453 spectrophotometer with measurements

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taken every 1 nm from 190-1100 nm. To enable correction for inner-filter effects on the

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fluorescence scans, any sample with abs254 greater than 0.2 cm-1 was diluted to an abs254

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between 0.1 cm-1 and 0.2 cm-1 26,27. Fluorescence measurements were taken at 20 +/- 2°C in a 1

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cm cell using a Fluoromax-3. EEMs were collected at excitation wavelengths 240-450 nm every

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10 nm and emission wavelengths 300-550 nm every 2 nm with a 5 nm slit width and 0.25 s

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integration time 28. Fluorescence signal was collected in ratio mode (sample/reference) and

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EEMs were corrected for instrument response and the inner-filter effect before being blank-

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subtracted and raman-normalized29. All fluorescence data are presented in Raman Units.

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Several spectroscopic indices were used to quantify the data30. Specific Ultraviolet Absorbance

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(SUVA254), an indicator of OM aromaticity with higher numbers corresponding to more aromatic

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character, was calculated by normalizing the absorbance at 254 nm by the DOC concentration31.

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The humification index (HIX) is used to measure the degree of humification, with a higher value

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corresponding to lower H:C ratios and more ring structure. HIX was calculated using

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interpolated values for excitation at 254 nm, as the area under the emission peak from 435-480

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nm divided by the area under the peak from 330-345 nm27,32. The fluorescence index (FI),

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calculated as em 470 nm / em 520 nm at ex 370 nm, is an indicator of precursor material to the

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humic portion of OM, with higher values indicating microbe-dominated origins and lower

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values indicating plant-dominated33,34.

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Statistical Analysis

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A parallel factor analysis (PARAFAC) model was built35 using 56 WSOM leachates from the soil

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collected from the north- and south-facing slopes in 2011, corresponding to two samples from

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the seven non-riparian plots, each leached four ways. Samples were normalized to their

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maximum emission intensity before modeling and a non-negativity constraint was imposed. An

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eight component model was verified with split-half analysis. This model was then applied to all

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WSOM samples from 2010 and 2011. For analysis of the results, the loading of each component

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fluorophore was normalized by the DOC concentration to account for differences in total

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amount of OM leached.

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RESULTS AND DISCUSSION


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WSOM fluorescence varied by both catchment location and leaching method. Figure 1 shows

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representative soil samples from the south-facing slope, north-facing slope, and riparian zone,

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leached with all four methods. The four soil plots on the south-facing slope all had EEMs that

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were similar to each other in shape and magnitude of the main humic peak, as did the three soil

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plots on the north-facing slope. The riparian zone EEMs were noticeably different in shape and

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intensity. The main humic peak was located at the same wavelength range for the north-facing

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and south-facing WSOM but the north-facing humic peaks had higher fluorescence intensity.

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Also, the WSOM on the north-facing slope displayed more protein-like signal than the WSOM

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on the south-facing slope. The humic peak in the riparian zone WSOM was less defined and had

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lower fluorescence intensity but had high intensity in the protein-like region of the EEM. The

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riparian humic material was also more blue-shifted, indicating more oxidized humic material36.

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There were also visible differences in EEMs leached with different solutions (Figure 1). The

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humic peaks on WSOM leached with K2SO4 appeared slightly red-shifted, or more reduced, and

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the CaCl2 samples had a slightly broader humic peak. WSOM leached with KCl had a more

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distinct peak in the protein-like region than other leachates. Samples leached with water had a

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strongly fluorescing band at 240-250 nm excitation which was not present in those leached with

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a salt solution. These visual differences in the EEMs across catchment locations and leaching

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solution were quantified with results from the fluorescent indices and PARAFAC model.

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PARAFAC Model



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By using different leaching methods and sampling diverse soil locations, we obtained

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substantial variation in the EEMs, which supported development of an eight component

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PARAFAC model (SI Figure 1 and 2). Many of these components are similar to components

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found in other published PARAFAC models (Table 1). Five of the components are located in the

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region of the EEM associated with humic material. Components GG-SQ1 and GG-SQ2 have peak

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wavelength ranges similar to components identified in the Cory-McKnight model as

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semiquinone-like components, and thus correspond to reduced humic fluorophores34,37. GG-

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SQ2 is also similar to a component abundant in the anoxic bottom of an Antarctic lake38. GG-SH

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is blue-shifted compared to other humic-like components, which may indicate a more oxidized

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state of the OM36, and has been found in several published models built using WSOM39, but has

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not been reported in aquatic PARAFAC models. GG-OH is also blue-shifted, though less strongly,

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and has peaks that match an unidentified component in the Cory-McKnight model and a peak

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from a marine study identified as humic material from marine, terrestrial, or anthropogenic

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sources34,37. Component GG-OQ has peaks which closely align with a component identified by

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the Cory-McKnight model as oxidized quinone-like34.

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GG-PS, a broad band across ex 240 nm, is predominately present in the water-leached samples,

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and has several possible sources. Due to observations of silicon colloids and clays in the

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catchment, it is likely a band of scatter from particles not present in the salt-leached solutions

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where salt ions can lead to flocculation. This component has also been observed in the photic

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zone of an ice-covered Antarctic lake38, in the ballast water of a boat during a marine study

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where it was considered a possible fluorometer artifact37, and identified as a possible amino


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acid in a study of leached WSOM39. GG-P1 and GG-P2 are tyrosine-like and tryptophan-like

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amino acid fluorophores, commonly found in many studies34,37.

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Comparison of Methods for Leaching WSOM

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Soil leached with K2SO4 consistently released more organic matter than soils leached with water

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(Figure 2), leading to a higher DOC concentration in those samples. KCl leached samples had

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comparable DOC to their water-leached counterparts and CaCl2 leached samples had slightly

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lower DOC, similar to results from Provenzano (2010) (figure 3). This difference in leaching

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amount could indicate that higher ionic strength and the presence of divalent ions allows for

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more release of OM sorbed to mineral surfaces.

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SUVA254 values showed a wide range, with water-leached samples often higher (>7) than values

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reported in the literature and considered possible for the index40,41. It is known that

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interference from iron, nitrate, and other commonly occurring environmental compounds can

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create unreasonably high SUVA254 values. In this case, we found a strong correspondence

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between the SUVA254 values and component GG-PS in the PARAFAC model, with samples

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having a DOC-normalized GG-PS loading also tending toward a high SUVA254 value (SI Figure 3).

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If GG-PS is due to particle scatter, this scattering would also create higher absorbance in this

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region and explain the values. This suggests that if WSOM is leached with water, SUVA254 may

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be unreliable as an analytical method.

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Results from fluorescent indices indicate that in addition to different methods releasing varying

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amounts of fluorescently-active DOC, these fluorescent fractions also have different chemical

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compositions (Figure 2 and 3). The HIX values for the KCl leachate were close to double those

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for the water-leached WSOM, and the K2SO4 and CaCl2 leachates yielded increasingly greater

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values of HIX. This greater HIX value indicates organic matter with a lower H:C ratio, likely larger

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molecules with more rings and double bonds. If this more humified material is bound tightly to

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mineral surfaces, it is possible the Ca2+ cation releases them more effectively than the K+.

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Additionally, OM with a higher HIX value usually also has a higher SUVA254 value, since both

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indices utilize the 254 nm excitation wavelength and increase with greater aromaticity. While

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this correlation holds true among the salt leachates, it is not evident in the water leachates.

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This difference furthers the evidence that SUVA254 cannot be reliably used to analyze water-

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leached WSOM. The FI values for K2SO4 were closest to that of water, with KCl higher, and CaCl2

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usually even higher, indicative of more microbially-produced organic matter in salt leaches.

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PARAFAC results also varied between the different leachates (Figure 4, SI Figure 4). Loadings for

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GG-SQ1 and GG-SQ2 were lowest for KCl leachates and highest for CaCl2 leachates, with K2SO4

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values falling between the others. This pattern suggests that CaCl2 and K2SO4 leach a greater

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portion of humic material containing quinone-like moieties in a reduced state. GG-SH and GG-

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OH, humic-like components, both have slightly higher values for CaCl2 leachates. However for

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GG-SH the K2SO4 leached samples have the smallest loadings while for GG-OH the KCl samples

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are smallest. The higher loading in humic fluorophores in the CaCl2 leached WSOM accounts for

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the observed broader humic peak in these samples. Loadings for GG-OQ all occur within the


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same range for the salt leachates, demonstrating no clear difference due to method in release

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of this oxidized quinone-like fluorphore. Loadings for GG-P1 are slightly higher for KCl leachates

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and loadings for GG-P2 are comparable for salt and water leachates. The higher values for GG-

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P1 loadings cause the KCl leachates to have a higher overall total protein signal visible in the

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EEMs.

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Component GG-PS appeared almost exclusively in the water-leached WSOM, with loadings

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close to zero for all the salt leachates. The addition of salts to water-leached WSOM lessened

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the intensity of the fluorescence scatter at the location of GG-PS (SI Figure 5,6 and 7). This

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result suggests that GG-PS is a scatter peak resulting from particles that flocculate in the

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presence of salt. Additional evidence that GG-PS results from scatter can be found in the

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Rayleigh scatter peaks from these samples (SI Figure 8). The salt-leached samples all had a

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single Rayleigh peak, while the water-leached samples had a more intense double peak,

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suggesting increased scatter due to particulates that split the peak. The addition of salt

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decreased the doubling of the Rayleigh peak, but did not completely remove it. Centrifuging the

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samples with split Rayleigh peaks significantly reduced the degree of splitting in the water-

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leached samples and removed the split in the salt-leached samples, supporting the evidence of

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particles causing this spectroscopic feature (SI Figure 9).

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While the addition of salt to water-leached samples altered the GG-PS scatter peak, the humic

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peak did not change significantly with addition of salt (SI Figure 5,6 and 7). This result indicated

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that the differences in the DOM character with leaching process were due to the solutions



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leaching different portions of the SOM pool and not changing the fluorescence itself. The one

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exception is that the addition of K2SO4 in all three cases increased the intensity in the protein-

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like region, suggesting this salt could denature proteins in a way that may make them more

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fluorescently active42. None of the extraction methods match the lysimeter samples (SI Figure

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5,6,7), all containing a soil humic component that doesn’t appear to be present in the

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groundwater samples. However, it is important to note that lysimeters are also imperfect

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representations of soil pore water DOM12 and, in this case, we did not have enough lysimeter

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data to do a thorough analysis.

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WSOM Chemistry Across the Landscape

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Regardless of the leaching method, there was no significant change in the chemistry of surface

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WSOM during the time period from snowmelt to midsummer. This consistency was observed in

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all catchment locations. However, differences in WSOM chemistry between the north-facing

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slope, south-facing slope, and riparian zone were maintained during the snowmelt transition

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(Figure 2 and 3). For the water-leached WSOM, the south-facing slope released the least WSOM

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and the more organic soils of the north-facing slope and riparian zone released more WSOM.

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There is a less pronounced difference between the slopes in the salt-leached samples, though

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the south-facing slope DOC is still slightly lower overall.

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The fluorescence indices (Figure 2 and 3) also demonstrated a clear difference in the WSOM

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chemistry among the locations. For all leaching methods, the HIX was highest and varied the

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most in the south-facing slope and was lowest in the riparian zone. This indicated a greater


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proportion of large, complex, potentially older, molecules on the south-facing slope. The north-

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facing slope, with less sun exposure and greater soil moisture, tended toward a higher FI value

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than the dry south-facing slope, indicating a greater degree of microbial contribution to the

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humic material. The north-facing slope also had more intensity in the protein-like components

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(Figure 1). The dark, grass-dominated, soil of the riparian zone had some of the lowest FI values,

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comparable to those found in wetlands, indicating OM dominated by plant decomposition. All

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these values suggest the WSOM on the north-facing slope had a greater proportion of input to

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the organic matter pool from microbial processes. Thus, overall the characteristics of leached

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OM indicated more microbial contribution to humic organic matter on the slope with more

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moisture and a deeper weathering profile. The riparian zone EEMs had a distinct protein-like

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peak (Figure1) with greater intensity than either slope. This finding suggests that while plant

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decomposition is the dominant source of humic material, there was still significant microbial

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contribution in the riparian zone.

309 310

The PARAFAC components were also distributed differently between the slopes. The reduced

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humic components, GG-SQ1 and GG-SQ2, both had lower values on the north-facing slope than

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the south-facing slope for the water leachates, with the riparian zone soil also in the lower

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range. There is less differentiation in the salt leachates, but the lower loading values are still

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from the south-facing slope. In addition, GG-SQ1 and GG-SQ2 strongly relate to the FI values

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(Figure 5) indicating that GG-SQ1 is a plant-derived semiquinone-like component and GG-SQ2 is

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a microbially-derived semiquinone-like component. This indicates that most of the humic



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material in the riparian zone is of plant origin while on the north-facing slope there is a greater

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proportional input from microbes.

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GG-SH and GG-OH, the more oxidized humic components, have more location-based variation

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in the salt leachates than the water leachates. On the north-facing slope, GG-SH has a greater

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contribution to the fluorescence than on the south-facing slope, with riparian zone values

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falling between the two. The opposite is true for GG-OH, with the WSOM from the south-facing

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slope having higher DOC-normalized loadings than the north-facing slope. In the water

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leachates, GG-OQ is most prevalent in the south-facing slope and least in the riparian zone,

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showing less oxidized quinone-like character in this likely reducing environment. GG-PS,

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dominated by the scatter peak, is found in all catchment locations. The protein-like

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components, GG-P1 and GG-P2, both have generally higher values in the riparian zone,

329

accounting for the greater protein signal in this region. The greater protein signal in the north-

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facing slope appears to be due to a greater contribution from GG-P2 compared to the south-

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facing slope. The difference in WSOM character across the landscape suggests stream DOM

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chemistry would be partially influenced by the proportional input from each slope.

333 334

There was a difference in the fluorescence characteristics of the WSOM depending on the

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leaching method, indicating that salt solutions leach different fractions of fluorescently active

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OM. However, all four leaching methods identified consistent differences in the chemical

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character of WSOM from various catchment locations, as measured by fluorescence (SI Table 2).

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Regardless of the method, WSOM from the north-facing slope, with more moisture and a


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deeper weathering profile, had a fluorescence signature indicating more microbial contribution

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and fresher humic material than the WSOM from the dry, shallow soil of the south-facing slope.

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The riparian zone consistently demonstrated a higher protein-like character and humic material

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dominated by recent plant decay. As noted by other studies, given that there is no standardized

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method for extracting WSOM from soil18,20, with the increasing use of fluorescence to analyze

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WSOM chemistry, it is important to understand the differences in the fluorescently active

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WSOM leached by various solutions. In general, WSOM leached with 0.5 M K2SO4, 0.01 M CaCl2,

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and 2 M KCl are more similar to each other than either is to WSOM leached with water. While

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none of the leachates is a perfect match for the DOM in soil pore water as extracted by

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lysimeters, the possible artifacts/interference contributing to the GG-PS fluorescence and high

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SUVA254 values indicate salt leachates are preferable methods to pure water if fluorescence

350

analysis is to be used. Additionally, the CaCl2 method may be preferable due to the other salts

351

producing an increased signature in the protein-like region of the EEM.

352 353

ACKNOWLEDGEMENTS

354

We would like to thank Caitlin Crouch, Mike SanClements and Phil Taylor for help with sample

355

collection and study design. We would also like to thank Wendy Roth for assistance with soil

356

processing and Eric Parrish for assistance with the figures. This study was funded by the Boulder

357

Creek Critical Zone Observatory (NSF-0724960, NSF-1239281).

358 359

SUPPORTING INFORMATION AVAILABLE

360 361

Supplementary Tables 1–2, Supplemental Figures 1–9. This material is available free of charge via the Internet at http://pubs.acs.org/



362 363


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Component Excitation maxima (nm) GG-SQ1 260

Emission Identification maxima (nm) 498 Reduced semiquinonelike humic peak 436 Reduced semiquinonelike humic peak

GG-SQ2

260 (340)

GG-SH

330 (250)

394

Soil humic-like

GG-OH

260 (300)

420

Unidentified humiclike

GG-OQ

Influence of Leaching Solution and Catchment Location on the

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