spectroscopic techniques

Optical Characteristics and Chemical Composition of Dissolved Organic Matter (DOM) from Riparian Soil by Using Excitation–Emission Matrix (EEM) Fluorescence Spectroscopy and Mass Spectrometry Yulai Wang,a,b Changming Yang,a,*,  Limin Zou,a,c Hengzhao Cuia a b c

Key Laboratory of Yangtze River Water Environment of the Ministry of Education, Tongji University, Shanghai 200092, China School of Energy and Environment, Anhui University of Technology, Maanshan City 243002, China Water Supply and Drainage Management Station of Shanghai Qingpu Water Authority, Shanghai 201700, China

Understanding the quantity and quality of soil dissolved organic matter (DOM) in riparian buffer zones is critical for explaining the biogeochemical processes of soil DOM in river ecosystems. Here, we investigated the dissolved organic carbon, fluorescent DOM (FDOM), and DOM molecules from riparian soils on Chongming Island in eastern China. Simultaneously, the soil DOM was extensively characterized in terms of the total aromaticity index (TAI) and several optical indices. The excitation (Ex)–emission (Em) matrix parallel factor analysis results showed that two humic-like components were present (Ex/Em ¼ 283(364)/454 nm; 337/410 nm), a fulvic-like component (Ex/Em ¼ 241/426 nm) and a microbial degradation component (Ex/Em ¼ 295/382 nm). The humic-like and fulvic-like substances were the main components in the riparian soil FDOM, accounting for 90% of the FDOM. Mass spectrometry provided more detailed information for the soil DOM molecules. Six chemical fractions, amino acids, carbonyl compounds, fatty acids, lipids, proteins and sugars, were identified using liquid chromatography with quadrupole time-of-flight mass spectrometry. Lipids, proteins, and carbonyl compounds were dominant in the soil DOM, accounting for .85% of the detected molecules (m /z , 1000). Significant differences were observed between the quantities of the six soil DOM chemical fractions at the different sampling locations. Discriminant molecules verified the hypothesis that the chemical soil DOM fractions varied with the land use of the adjacent watersheds. The TAI for the soil DOM could provide more reliable information regarding the biogeochemical processes of DOM. The carbonyl compounds and lipid fractions controlled this index. Overall, the optical indices and TAI values can improve our understanding of soil DOM quality; however, the optical indices did not provide quantitative evidence regarding the sources or properties of the soil DOM. The observations from this study provided detailed information regarding the soil DOM quality and the presence of specific molecules and improved our understanding of the biogeochemical processes of DOM. Index Headings: Chongming Island; Riparian buffer zone; Soil dissolved organic matter; DOM; Excitation–emission matrix; EEM; Received 29 November 2013; accepted 22 October 2014. * Author to whom correspondence should be sent. E-mail: cmyang@ tongji.edu.cn.   Current address: College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China.

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Liquid chromatography with quadrupole time-of-flight mass spectrometry; LC-QTOF-MS.

INTRODUCTION Dissolved organic matter (DOM) is a ubiquitous component of natural ecosystems, including water bodies, soils, the atmosphere, and sediments. In addition, DOM is a heterogeneous mixture of a variety of molecular structures and functional groups. DOM plays important roles in ecosystem functions, such as altering the optical properties and esthetic concerns of natural water bodies1 and influencing the physical and chemical behavior and bioavailability of natural and discharged compounds.2 The chemical compositions of molecules and their origins determine their roles in the ecosystem; hence, interest is growing regarding the chemical compositions and molecular information of DOM at a molecular level to determine its biogeochemical roles. Riparian buffer zones are key elements in watersheds that serve as transitional areas that connect upland and aquatic ecosystems. The importance of transport and biogeochemical hot spots and moments in riparian zones is well recognized.3–5 Riparian zones play important roles in stream shading and bank stabilization, provide habitats for terrestrial and aquatic organisms, and filter nutrients and pollution from agricultural nonpoint pollution.3,4 Previous studies have reported the quantities of soil dissolved organic carbon (DOC) in riparian buffer zones.5,6 However, few reports have presented information regarding the properties or molecular aspects of soil DOM in riparian buffer zones, mainly due to the limitations of the available analytical methods for complex soil DOM. DOI: 10.1366/13-07407R

0003-7028/15/6905-0623/0 Q 2015 Society for Applied Spectroscopy

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FIG. 1. Study area and sampling locations on the Chongming Island. The red circles represent the sampling sites.

The development of fluorescence spectrometry has allowed researchers to reveal the properties of FDOM.7–10 In addition, the parallel factor (PARAFAC) was introduced to decompose the excitation–emission matrix (EEM) into its individual fluorescent components.11 This method is simple, rapid, and accurate for obtaining the structural properties of soil fluorescent DOM (FDOM) by combining PARAFAC with EEM.12,13 In addition, this method could be widely used to characterize FDOM.7,14 However, little is known about the transformations of DOM in natural systems from EEM results. Fortunately, a large amount of fingerprint information is available for the transformation and transport process. For example, the fluorescence index (FI), the index of the recent autochthonous contributions (BIX), and the humification index (HIX) can reflect the FDOM source and its properties.15,16 In addition, Cory et al.17 used these optical indices (FI, BIX, and HIX) to trace the source of humic-like substance and assess their properties. Regarding the functional groups, chemical structures (or proposed structure), and molecular information of the DOM, liquid chromatography with quadrupole time-offlight mass spectrometry (LC-QTOF-MS) can open a new avenue for analyzing DOM molecules. Due to its unambiguous identification, high sensitivity, and fair reproducibility for quantitatively identifying complex compounds, LC-QTOF-MS has been used to probe the chemical compositions of pesticides,18 pollutants,19 and metabolites at a molecular level.20 In this study, we measured the quantity, fluorescence, and molecular information of DOM extracted from

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riparian soils on Chongming Island in eastern China. The main objectives of this study were to (i) determine the quantity of soil DOM (DOC contents) across a range of riparian soil samples from Chongming Island, (ii) characterize the soil FDOM fluorescence components and analyze their potential sources using the PARACFAC model and the optical indices, and (iii) probe the DOM molecular information and classify the chemical DOM fractions based on the LC-QTOF-MS platform. These findings may be useful for determining the spatial distribution patterns of soil DOM fractions and their molecular properties.

MATERIALS AND METHODS Study Site Descriptions and Soil Collection. As the third largest island in China and the largest alluvial island in the word, Chongming Island (31825 0 –31838 0 N and 121850 0 –122805 0 E; Fig. 1) is located at the Yangtze River estuary in eastern China. This island is representative of a plains watershed with a water surface area of up to 9%. Overall, 8.05% of this watershed involves an intense river network. Chongming Island has a northern subtropical monsoon climate, with an average annual temperature of 15.3 8C, and an average temperature of 13.7 8C in April. The average annual precipitation is 1022 mm, with 60% of the rainfall occurring between May and September. The average humidity is 82%.21 Riparian soil samples were collected from 10 rivers on Chongming Island (Fig. 1) that have experienced minimal disturbance from human activities. The soil samples were divided into four categories based on their

geographical locations. The first category was the north region and included four sampling sites from the North Hengyin River, B4, and B8–B10. The second category (west region) included 14 sites: Cangfang River (C1 and C4), Jie River (J3-1 to J3-6), Miao River (M1–M3), and the Gelong River (G4–G6). The third category (middle region) included nine sites: Dongping River (D1–D3), Zhi River (Z1–Z4) and Xinjian River (XJ1 and XJ3). Finally, the last category was the east region, and included seven sites from the Bayao River (BY1, BY2, and BY4) and the Tuanwang River (T1–T4). Three replicate surface soils (0–15 cm) were collected from 1 3 3 m buffer zones of the different rivers in April 2011. Three replicates of one soil profile with a depth of 60 cm were collected at the J3 site of the Jie River. Samples were collected at 10 cm intervals from the top of the profile to the bottom. After stones and woody debris were removed from the collected soils, the soil samples were mixed onsite and preserved in sterilized bags at 4 8C for less than 24 h. All soil samples were air-dried, gently ground with a roller, and passed through 2 and 0.107 mm sieves. Soil Sample Preparation and Physiochemical Analysis. The DOM was extracted according to Ohno’s method.22 First, the DOM from 5 g of soil (sieved at 0.107 mm) was extracted using 50 mL of deionized distilled water by shaking for 24 h at 20 8C in the dark. The suspensions were centrifuged at 8000 rpm at 4 8C for 20 min and filtered using 0.45 lm glass fiber filters (GF/F; Whatman). The extract was analyzed for DOC by using the high-temperature combustion method on a TOC-VCPH carbon analyzer (Shimadzu). Three to five replicates were performed for each sample, resulting in a typical coefficient of variation of ,2%. The soil bulk density was determined from oven-dried undisturbed cores as the mass per volume of the ovendried soil. The moisture content was determined using a WET-2 sensor (Delta-T Devices Ltd., Cambridge, UK), and the soil pH (soil water extract) was measured using an HQ40 apparatus (Hach Co., Loveland, CO). The tested samples were calcareous alluvial type soils with a sandy texture. The pH of the tested soils ranged from 7.34 to 8.91 (1 : 5 w/w soil/water), the moisture contents ranged from 46.02 to 52.78%, the bulk densities ranged from 1.38 to 1.61 g/cm3, and the organic nitrogen ranged from 0.41 to 1.95 g/kg. Optical Analysis of Soil Dissolved Organic Matter. The optical FDOM properties were determined by EEM fluorescence spectroscopy. The EEM spectra were measured using an F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) with an excitation range of 220–400 nm at 3 nm intervals and emissions of 250–550 nm at 2 nm intervals. The measured EEMs were Raman calibrated to the signal from a Milli-Q water (Millipore) sample that was run the same day by normalizing the signal to the integral of the Raman band from excitation at 350 nm.11 The Raman-normalized Milli-Q EEM was subtracted from the data to remove the Raman scatter effects. The fluorescence intensity spectra were obtained using Raman normalization and correction procedures and are expressed in Raman units (nm1). In our laboratory, no instrumental correction was used for the spectra of each EEM determination; however, the spectra were

corrected for instrumental components after 10 measurements by using the correction protocol recommended by the manufacturer (F-4500, Hitachi). The inner filtering effects were determined by ultraviolet (UV)absorbance at 240 nm. All of the DOM solutions were diluted with distilled deionized water to set the UVabsorbance at 240 nm to 0.1, and the correction of the inner filtering effects were considered negligible.22 To obtain reliable and valid PARAFAC model results for the EEM analysis, the 34 soil DOM samples in this study were analyzed with the other 72 soil or sediment DOM samples. The EEM data array (106 3151 3 61) consisted of 106 DOM samples with 151 emission wavelengths and 61 excitation wavelengths. PARAFAC modeling was conducted with Matlab using the N-way toolbox for Matlab following the procedures of Stedmon et al.11 The data were split into two halves, and the number of components was validated using spilt-half validation. The FI was calculated as the ratio of emission intensity at 470 nm to that at 520 nm for an excitation wavelength of 370 nm (Eq. 1). The HIX was measured using an excitation wavelength of 254 nm and was calculated as the ratio of the area under the emission spectra (435– 480 nm) divided by the area under the 300–345 nm region (Eq. 2). The BIX was calculated as the ratio of the emission intensity at 380 nm to that at 430 nm for an excitation wavelength of 310 nm (Eq. 3): FI ¼ Icor ð370 : 470Þ=Icor ð340 : 520Þ

ð1Þ

HIX ¼ ðRFcor435:480 Þ=ðRFcor300=345 Þ

ð2Þ

BIX ¼ Icor ð310 : 380Þ=Icor ð310 : 430Þ

ð3Þ

where Icor(i:j) is the corrected fluorescence intensity at i:j (excitation–emission wavelengths in nanometers), and RFcor(i:j) is the area under the emission spectra between the emission wavelengths i and j for an excitation wavelength of 254 nm. Liquid Chromatography with Quadrupole Time-ofFlight Mass Spectrometry Analysis of Soil Dissolved Organic Matter. An off-line extraction was used for a pre-analysis of the soil DOM molecules. First, 10 mL of the crude DOM extract was diluted with 10 mL of nbutanol. After vortexing-mixing for 30 s, the supernatant was evaporated to near dryness and re-dissolved in 2 mL of 50% methanol for LC-QTOF-MS analysis. Liquid chromatography with quadrupole time-of-flight mass spectrometry analysis was performed on an Agilent-1200 LC system that was coupled with an electrospray ionization (ESI) source (Agilent Technologies, Palo Alto, CA) and an Agilent-6520 Q-TOF mass spectrometer. The separation of all samples was performed on an Eclipse Plus C18 column (1.8 mm, 2.1 mm 3 100 mm, Agilent) with a column temperature of 45 8C. The flow rate was 0.3 mL min1 and the mobile phase consisted of (a) distilled deionized water and (b) acetonitrile. The following gradient program was used: 0–3 min, 3–20% B; 3–6 min, 20–60% B; 6–12 min, constant 60% B; 12–13 min, 60–80% B; 13–14 min, 80–98% B; 13– 14 min, and constant 60% B. This program was followed by a re-equilibration step of 4 min. The sample injection

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TABLE I. Quantity and quality of riparian soil DOM from Chongming Island. Index

North region

DOC (gkg1) Comp.1 (nm1) Comp.2 (nm1) Comp.3 (nm1) Comp.4 (nm1) FI BIX HIX a b

0.14 0.28 0.33 0.37 0.11 2.14 0.64 7.75

6 6 6 6 6 6 6 6

0.02a 0.21 (0.24 0.18 (0.30 0.17 (0.36 0.06 (0.10 0.09 0.05 2.06

6 6 6 6

West region 0.04)b 0.02) 0.04) 0.01)

0.15 0.41 0.47 0.51 0.18 2.17 0.61 8.25

6 6 6 6 6 6 6 6

0.05 0.23 0.22 0.19 0.11 0.08 0.05 1.72

(0.25 (0.30 (0.34 (0.11

Middle region

6 6 6 6

0.04) 0.01) 0.05) 0.02)

0.14 0.45 0.48 0.54 0.16 2.21 0.57 8.97

6 6 6 6 6 6 6 6

0.03 0.21 0.13 0.13 0.06 0.12 0.05 3.65

(0.27 (0.30 (0.34 (0.10

6 6 6 6

East region 0.04) 0.01) 0.02) 0.02)

0.14 0.35 0.39 0.50 0.12 2.29 0.62 8.91

6 6 6 6 6 6 6 6

0.05 0.13 0.11 0.12 0.04 0.08 0.08 2.43

(0.25 (0.29 (0.38 (0.09

6 0.04) þ 0.03) 6 0.07) 6 0.02)

Values represent means 6 SD. Values in parentheses are contributions of each component to total soil DOM.

volume was 5 lL. Mass detection was performed in both positive and negative ion modes with the following parameters: drying gas (N2), 8 L min1; gas temperature, 330 8C; nebulizer pressure, 35 psi; capillary voltage, 4000 V; fragmentor voltage, 160 V; skimmer voltage, 65 V; and the LC-MS accurate mass spectra, which were recorded across the range 50–1000 m/z. The tandem mass spectrometry (MS/MS) analysis was acquired in targeted MS/MS mode by using collision energies of 5– 40 V. The LC-QTOF-MS data from all samples were processed using the MassHunter Qualitative Analysis software (Agilent). The following filter parameters were used: restricted retention time of 0.3–20 min, restricted m/z of 80–1000 Da, relative peak height 1.5%, mass tolerance of 0.05 Da, and a retention time window of 0.1 min. Before multivariate analysis, the data from each sample were normalized to total area. The aromaticity index (AI) can provide reliable information regarding the C=C double bond and rings for the aromatic molecule.23 In this study, we assessed the aromaticity of soil DOM by using the TAI. The TAI was revised according to Koch23 and was calculated according to Eqs. 4 and 5 (CcHhNnOoSsPp is the supposed chemical formula of a soil DOM molecule, which is determined by MS). AI ¼ ð1 þ c  0:5o  s  0:5 hÞ 4ðc  0:5o  s  n  pÞ

TAI ¼

iX max

Fi AIi

ð4Þ

ð5Þ

i¼1

Here, AIi is the aromaticity index for molecule i in the soil DOM, TAI is the total aromaticity of the soil DOM, and Fi is the percentage of molecule i to the total soil DOM. Statistical Data Analysis. Statistical analysis, including the mean value, linear regression, difference, and correlation, were performed using the Statistical Program for Social Sciences 13.0 (SPSS Inc.). The differences of the various parameters among the four site groups were determined using a t-test with p values of 0.05, 0.01, and 0.001. Correlation analysis was used to examine the relationships between the absorption, fluorescence and chemical fractions of the soil DOM using p values of 0.05, 0.01, and 0.001. A discriminant analysis of the different site groups of the soil DOM was

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performed using the SIMCA-P 11 software (Umetrics, Umea˚, Sweden) with variable importance projection (VIP)  1.

RESULTS Amount of Soil Dissolved Organic Matter. The amounts of soil DOC extracted from the riparian soils ranged from 0.10 to 0.20 g/kg (95% confidence interval [CI]) in the buffer zone of Chongming Island (Table I). The average soil DOC contents in the north, west, middle, and east regions were 0.14, 0.15, 0.14, and 0.14 g/kg, respectively. The highest DOC content of 0.23 g/kg occurred in the middle region, and the lowest DOC content of 0.07 g/kg occurred in the east and west regions. Fluorescence Spectra for Soil DOM. For the 34 soil DOM samples that were extracted from the riparian zones, four fluorescent components were identified by PARAFAC (Fig. 2). The four components identified from the fluorescence spectra included two humic-like components (Comp.1 and Comp.2), a fulvic-like component (Comp.3), and a microbial degradation component (Comp.4). Comp.1 displayed two excitation maxima (at 283 and 364 nm) and a single emission maximum at 454 nm that were considered to result from humic-like fluorescence.7,9,16 Comp.2 (Ex/Em = 337/410 nm) was considered to be old autochthonous humic-like fluorescence,24,25 and Comp.3 (Ex/Em = 241/426 nm) was attributed to fulvic-like fluorescence.24,26,27 Comp.4 (Ex/ Em = 295/382 nm) was similar to the N peak reported by Zhang.16 However, Comp.4 was not attributed to marine humic-like substance due to phytoplankton degradation. Instead, we considered Comp.4 as a new microbial degradation component (as described by Chen24) that was composed of protein-like substances that were similar to biological and soil microbial by-product substance. The fluorescence intensities of the four components and their contributions to the total soil DOM fluorescence intensity differed among the different site groups (Table I). The total fluorescence intensities for the soil DOM were 1.09 6 0.61, 1.22 6 0.68, 1.80 6 0.56, and 1.35 6 0.33 for the north, west, middle, and east regions, respectively. Each of the fluorescence intensities from the four components followed the same trends as the total fluorescence intensities for the different site groups, and the contributions of each component to the total FDOM fluorescence intensities were (24 6 4) to (27 6 4)%, (29 6 3) to (30 6 1)%, (33 6 3) to (38 6 7)%, and (9

FIG. 2. Four fluorescent components of soil DOM according to the PARAFAC model. (a–d) Contour plots present the spectral shapes of excitation and emission. (e–h) The line plots present the split-half validation results.

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6 2) to (11 6 1)% for Comp.1, Comp.2, Comp.3, and Comp.4, respectively (Table I). The results indicated that the soil DOM was weakly derived from the microbial degradation materials, which accounted for 10% of the total soil FDOM. The three optical indices (FI, BIX, and HIX) varied between the different site groups (Table I). The FI values ranged from 1.97 to 2.39, with a mean of 2.20 6 0.1 for all soil DOM samples. Significant differences were observed between the east region and the north and west regions (p , 0.05). The BIX values ranged from 0.47 to 0.74 (0.61 6 0.06), and the HIX values ranged from 5.05 to 17.97 (8.52 6 2.46). According to previous reports,15 the BIX and HIX values indicated that the soil DOM exhibited important humic character and a weak and recently derived microbial component. This finding was consisted with the EEM-PARAFAC results. However, the FI values suggested that the soil DOM was mainly derived from a microbial source according to Cory and McKnight.17 However, this assumption was false and contradicted the BIX and HIX results. It is important to use caution when discriminating between soil DOM sources by FI.15 Liquid Chromatography with Quadrupole Time-ofFlight Mass Spectrometry Analysis for Soil Dissolved Organic Matter. For the 34 riparian soil DOM samples, .12 000 peaks were detected in the mass range from 80 to 1000 m/z on the LC-QTOF MASS platform (Fig. S1). Of these peaks, 9021 were analyzed in ESIþ mode and 3306 were analyzed in ESI mode. The complexity of the soil DOM molecules occurred because the soil DOM contained several molecules, including natural products and their metabolites. This finding was similar to the findings of previous studies.28,29 Based on the intensities of the 12 327 peaks from the soil DOM samples, we clustered the sampling sites into four groups using principal component analysis (data not shown). The first group (group A) included seven soil samples from the Bayao and Tuanwang rivers, both of which are located in the eastern region of Chongming Island. This region consisted of an ecological agricultural district rather than a traditional agriculture district, where few commercial fertilizers and pesticides were rapidly transported to the riparian zones. Group B included four soil samples that were collected from the North Hengyin River in the northern region of Chongming Island. Intensive agriculture and livestock breeding plants were located near the sampling sites of the group B samples, resulting in the transport of large amounts of nonpoint pollution and wastewater to the adjacent buffer zones. Group C consisted of eight soil samples, including C2 and the six soil profile slices from the Cangfang and Jie rivers. In the group C district, distributed agriculture was the main land use. However, some riparian zones were cultivated for crop growth. The last group (group D) included 15 samples that were collected from the Miao, Gelong, Dongping, Zhi, and Xinjian rivers, located in the western and middle regions of Chongming Island. This district accounts for 50% of the islands population, with large amounts of raw domestic wastewater entering the riparian buffer zones due to the insufficient number of sewage treatment

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plants on Chongming Island. The four site groups generally agreed with our man-made sampling regions. Furthermore, we identified the signals with peak relative heights 1.5% based on the accurate MS and MS/MS fragments by searching for these compounds in databases (http://metlin.scripps.edu; http://www. chemspider.com) and by comparing them. In this study, 76 compounds were identified. Of these compounds, 37 were identified using ESIþ mode and 39 were identified using ESI mode. We divided these compounds into six fractions (Table S1): amino acids, carbonyl compounds, fatty acids, lipids, proteins, and sugars. Significant differences were observed between the standing quantities of the six soil DOM fractions and their contributions to the total soil DOM (Fig. 3a). The lipid-like fraction was the main fraction in the soil DOM, accounting for 28.87– 43.87% of the total soil DOM. The protein-like and carbonyl compounds fractions had contribution ratios of 17.46–36.54 and 19.13–31.28%, respectively. The smallest fractions included the fatty acids, sugars, and amino acids, accounting for 12% of the soil DOM. We observed significant differences between the six soil DOM fractions at the different site groups (Fig. 3).

DISCUSSION Correlations Between the Soil Dissolved Organic Matter Fractions and the Quality Indices. The correlations between the soil DOM fractions and the quality indices were investigated in this study (Table II). The soil DOM fractions consisted of fluorescence components (Comp.1, Comp.2, Comp.3, and Comp.4), DOM chemical fractions (amino acids, carbonyl compounds, fatty acids, lipids, proteins, and sugars), and quality indices and fluorescence optical indices (FI, BIX, and HIX). Negative correlations were observed between the %humic-like component (%Comp.1) and the FI (p , 0.05) and BIX (p , 0.001). Furthermore, the HIX was positively correlated with %Comp.1 but negatively correlated with the percentage of microbial degradation in the humic component (%Comp.4) (p , 0.01). The above-mentioned results were easily understood. The higher contributions of the humic-like materials corresponded with lower contributions of the microbial degradation components. However, positive correlations were also observed between the BIX and %Comp.3 (p , 0.001), and negative correlations were observed between the BIX and proteins fraction (p , 0.05). Furthermore, positive correlations were observed between the HIX and protein fraction (p , 0.05). We hypothesized that the fulvic-like materials could be degraded by soil microorganisms and that the protein fractions in the soil DOM were less degraded. In addition, the chemical compositions of the soil DOM according to LC-QTOF-MS revealed that the protein fraction was mainly composed of peptides, such as Gly-Ala-Trp, Trp-Gly-Val, and TyrHis, which confirmed our hypothesis. Moreover, we observed that %Comp.3 was negatively correlated with the other components (%Comp.1, Comp.2, and Comp.4) and that positive or negative correlations were observed between the soil DOM chemical fractions (data not shown). These results indicated that the fluorescence components and chem-

FIG. 3. Spatial distributions of the soil DOM chemical fractions. (a) Contributions of the different fractions to the total soil DOM. (b–g) Standing quantities of the different fractions of the different site groups. (h) Total aromaticity of the soil DOM for the different site groups of the samples. Error bars represent the standard deviation for the intergroup, and the lowercase letters (a, b, and c) on the bars represented significant differences (p , 0.05) between the different site groups.

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TABLE II. Pearson correlation coefficients for soil DOM fractions and quality indices.a Quality index Fraction %Comp.1 %Comp.2 %Comp.3 %Comp.4 %Amino acids %Carbonyl compounds %Fatty acids %Lipids %Proteins %Sugars

FI

BIX

HIX

TAI

0.350* 0.150 0.225 0.168 0.306 0.253 0.200 0.205 0.134 0.189

0.726*** 0.142 0.572*** 0.056 0.184 0.158 0.174 0.206 0.364* 0.293

0.556*** 0.193 0.244 0.507** 0.218 0.346 0.301 0.057 0.351* 0.070

0.066 0.114 0.135 0.048 0.330 0.426* 0.201 0.946*** 0.468** 0.074

a *, **, and *** denote significant level at p , 0.05, p , 0.01, and p , 0.001, respectively.

ical fractions of the soil DOM resulted from a common source with the same variation, consistent with the findings of Zhang et al.16 Unfortunately, only weak correlations were observed between the DOM chemical fractions and the fluorescence components. Based on the above-mentioned analysis, the EEM and PARAFAC model can be used to reveal the FDOM profile and to calculate the contributions of the different fractions for FDOM at a semi-quantitative level. The optical indices, including the FI, BIX, and HIX, could be used to characterize the FDOM sources, their age, and their degree of humification. However, these indices were only complementary for assessing the transformations of DOM in the soil system. Thus, care should be used when quantitatively evaluating the sources or properties of soil DOM based on the optical indices.15,25

Chemical Compositions of Soil Dissolved Organic Matter. The DOM that entered the riparian soil primarily originated from autochthonous and allochthonous sources. The autochthonous DOM resulted from riparian vegetation in the riparian ecosystems.30,31 The allochthonous DOM resulted from point sources, sewage overflow, and agricultural activities.32,33 In this study, the 76 identified DOM molecules were divided into three sets using an R-cluster analysis (Fig. 4). The first cluster contained 22 molecules identified in the ESIþ mode and was further subdivided into autochthonous and allochthonous sources (Fig. 4a). In addition, the DOM molecules in the second and third clusters originated from autochthonous and allochthonous sources. Similar to the clusters identified in ESIþ mode, the DOM molecules identified in ESI mode originated from autochthonous and allochthonous sources (Fig. 4b). For example, glabric acid was isolated from licorice root34 and belonged to an autochthonous source. Similarly, medicagenic acid, 35 fumaric acid,36 and cucurbitacin B37 were derived from the vegetation, and these molecules all originated from an autochthonous source. Because of frequent sewage overflows, large amounts of human drugs and cosmetics, including 2,4dichlorobenzoate,38 2-oxo-3 methyl valeric acid,39 3methylglutaric acid,40 a-carboxyethyl-hydroxychroman (a-CEHC),41 and tributyl phosphate,42 were flushed into the riparian soil with domestic sewage. Furthermore, we identified several DOM molecules that resulted from agricultural activities, including the methoprene acid,43 4-pyridyl thioamide,44 and 11-oxo tetradecanoic acid refractory pesticides,45 These sewage and refractory pesticides were considered as allochthonous sources.

FIG. 4. Sources of the DOM that was derived from the riparian soils on Chongming Island. (a) R-cluster profile for the soil DOM molecules identified in ESIþ mode. (b) R-cluster profile for the soil DOM molecules identified in ESI mode.

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FIG. 5. Molar ratios of the elemental compositions of the soil DOM chemical fractions. (a) H : C-O : C plots for the fatty acids, proteins, and sugars. (b) N : C–H : C plots for the amino acids and lipids.

Special soil DOM chemical fractions have special H:C, O:C, or N:O molar ratios.46 For the 76 identified soil DOM molecules, we calculated the H:C, O:C, and N:O molar ratios and clustered their regions in the H:C–O:C and N:C–H:C plots (Fig. 5). For the fatty acids, proteins, and sugars found in the soil DOM, the molar ratios of H:C/O:C were 1.93–2.03/ 0.13–0.24, 1.18–1.67/0.20–0.26 and 0.82–1.65/0.26–0.46, respectively, with a 95% CI (Fig. 5a). The N:C-H:C plot could be used to distinguish amino acids from lipids (Fig. 5b). The amino acids had N:C/H:C ratios of 0–0.27/0.73– 1.53, whereas the lipids had N:C/H:C ratios of 0.16–0.27/ 1.18–1.67. These results were consistent with those of previous studies.23,47 Unfortunately, we could not distinguish the carbonyl compounds from the other soil DOM fractions based on the H:C–O:C or N:C–H:C plots. It was possible that the carbonyl compounds covered a wide range of ratios that were common for other soil DOM fractions. The aromaticity of the soil DOM that was extracted from the riparian soil was evaluated using the FI and TAI values (Table III). McKnight et al.48 reported that higher FI values corresponded with more microbial sources, suggesting that higher FI values indicated DOM with lower aromaticity because of microbial degradation. Table III showed that the FI values of the soil DOM from group A were higher than those from groups B and C (p , 0.01) and that the FI values of the soil DOM from group D were higher than those from group C (p , 0.05). These results indicated that the aromaticity of the soil DOM from group A was lower TABLE III. Difference of soil DOM quantity and quality between each set of two groups.a

Group A A A B B C

3 3 3 3 3 3

B C D C D D

Amino Carbonyl Fatty acids compounds acids Lipids Proteins Sugars FI TAI *** – – *** *** –

– ** – – – –

– *** *** *** – –

– – *** – ** ***

– – * – *** ***

*** – – *** ***

** ** – – – *

– * *** – ** *

a *, **, and *** denote significant level at p , 0.05, p , 0.01, and p , 0.001, respectively.

than that from groups B and C. In addition, the soil DOM from group D exhibited lower aromaticity than that from group C. We calculated the TAI values to evaluate the total aromaticity of the soil DOM (Fig. 3h). Regardless of whether the aromaticity was due to photochemical or microbial processes, the TAI values could provide more reliable information regarding the affects of C=C double bonds on the chemical compositions of the soil DOM molecules.23,29,47 Our results showed that the total aromaticity of the soil DOM from group D was higher than that from groups A and B (p , 0.001) and that the total aromaticity of the soil DOM from group C was higher than that from group A (p , 0.05). This result suggested that the aromaticity of the soil DOM varied with the land use of the adjacent watersheds. In addition, we identified positive significant correlations between the TAI and the percentages of carbonyl compounds and lipids (p , 0.001). This result suggested that the carbonyl compound and the lipid fractions are mainly attributed to the total aromaticity of the soil DOM. As a complementary tool for EEMs, LC-QTOF-MS revealed molecular information for DOM, including chemical molecules, functional groups, precise measure mass, and the molar ratios (H:C, O:C, or N:O) and TAI values of DOM. The above-mentioned information can improve our understanding of biogeochemical DOM processes in natural ecosystem. However, it is unfortunate that we restricted the m/z to ,1000 because we only revealed chemical information for a small portion of the DOM. In additional, it is possible that we could not observe the correlations between the TAI and BIX or HIX because the m/z was ,1000. Spatial Variations of Soil Dissolved Organic Matter Quantity and Quality. The structure and composition of soil DOM can be studied to understand its biogeochemical processes in the natural environment. In this study, we analyzed the spatial distribution patterns of soil DOM at Chongming Island, including the soil DOM quantity (DOC), fluorescence components (Comp.1, Comp.2, Comp.2, and Comp.4), and their chemical fractions (amino acids, carbonyl compounds, fatty acids, lipids, proteins, and sugars).

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TABLE IV. Discriminant DOM molecules among different groups and their VIP values. Comparison among groups Molecule

A with B

Gly-Ala-Trp Trp-Gly-Val Tyr-His Antanapeptin A 2,4-Dichlorobenzoate Sphinganine p-Aminobenzoic acid PC(P-18:0/20:5(5Z,8Z,11Z,14Z,17Z)) Neoabietic acid Dillapiole Atracurium 3,4-Dihydroxybenzoic acid Nonic acid 4-Pyridylthioamide Dihydro isorescinnamine Oleamide 2-Methyl-16-heptadecenoic acid Tributyl phosphate a,a 0 -Diethyl-3,4,3 0 ,4 0 -stilbenetetraol Cucurbitacin B a b

VIPaAB

A with C

VIPAC

A with D

VIPAD

C with D

VIPCD

C,D

2(þ)

C.D

3()

C,D

2()

C,D C,D C,D

6() 4() 5()

C.D C.D

1(þ) 1()

b

A.B A.B A.B A.B A,B A,B

1(þ) 2(þ) 3(þ) 4(þ) 5(þ) 6(þ)

A.B A.B A.B A.B A,B A,B A,B

7(þ) 1() 2() 3() 4() 5() 6()

A,C

A.C A.C A,C A,C A,C A,C A,C A,C A,C

1()

3() 2() 6() 4() 5() 1(þ) 2(þ) 3(þ) 4(þ)

A.D A,D

4(þ) 4()

A,D A.D

6(þ) 5(þ)

A.D A,D A,D A,D A,D A,D A,D

1() 5() 2() 3() 1(þ) 2(þ) 3(þ)

The higher values of VIP obtained in loading plots, the more the molecules derived from the origin. (þ) and () represent ESIþ mode and ESI mode, respectively.

We did not find any significant differences between the site groups (A, B, C, and D) in terms of their DOC contents or the contributions of the fluorescence components to the total fluorescence intensity (%Comp.1, %Comp.2, %Comp.3, and %Comp.4). However, significant differences in the DOM chemical fractions were observed among the four site groups (Table III). This result indicates that LC-QTOF-MS could provide more detailed information for soil DOM. Overall, we observed that 20 soil DOM molecules contributed to the classification among the four site groups based on the VIP values (Table IV). For example, soybean was cultivated in the eastern and middle regions of Chongming Island across large areas. In addition, Marcus et al.49 reported that multiple peptides, such as Gly-Ala-Trp, Trp-Gly-Val, and Tyr-His, were derived from soybean. Therefore, we hypothesized that the soybean crop could explain why the standing quantities of the peptides (Gly-Ala-Trp, Trp-Gly-Val, and Tyr-His) in group A were more intensive than those in group B and why those in group C were less than those in group D. Cucurbitacin B is a triterpene hydrocarbon that is derived from the Cucurbitaceae family of plants.50 It is likely that the land use change of the riparian zone caused the greater signal intensity of group C relative to group D. More than 50% of the population lived in the middle of Chongming (group D). Thus, cosmetics with paminobenzoic acid where transported to the riparian soil in this region.51 Therefore, the p-aminobenzoic acid in the central region was greater in the central region relative to the eastern region (A , D for p-aminobenzoic acid). 4-Pyridylthioamide, a pesticide that originates from agricultural nonpoint pollution, corresponded with agricultural land use. Based on the above-mentioned analysis, the discriminant molecules verified our hypothesis that landscape variations altered the soil chemical fractions between the four different site groups, which included land use changes and human activities.

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CONCLUSIONS This study simultaneously evaluated the quantities and qualities of soil DOM in riparian buffer zones on Chongming Island by using optical and mass spectrometry. In the riparian buffer zones, the soil DOM was derived mainly from humic-like or fulvic-like substances and a weak microbial degradation source. The optical indices, such as FI, BIX, and HIX, showed complicated correlations that could be used to understand the biogeochemical processes of soil DOM. However, data from these optical indices did not provide direct evidence for the sources of the soil DOM. No significant differences (p . 0.05) were determined for the soil DOM concentrations and fluorescence components among the different site groups. However, distinct differences in the soil DOM chemical fractions were observed among the four site groups, demonstrating that LC-QTOF-MS could provide more detailed molecular information regarding soil DOM and that the landscape of the riparian buffer zone could affect the chemical compositions of the soil DOM. We first introduced the TAI to reflect the aromatic structure of soil DOM. This index was successfully applied to evaluate the comprehensive aromaticity of soil DOM. The TAI can provide more reliable information regarding aromaticity due to photochemical and microbial degradation and should be used as a conservative criterion for assessing the aromatic structure of DOM. ACKNOWLEDGMENTS The authors acknowledge financial support from the Research Fund of State Key Laboratory of Soil and Sustainable Agriculture at the Nanjing Institute of Soil Science, Chinese Academy of Science (Y412201426). In addition, we acknowledge partial financial support from the National Special Item on Water Resource and Environment (2011ZX07303-001). Furthermore, we thank Dr. Zhang from Shanghai Crick Biomedical Technology Ltd. Co. for assistance with analyzing the data from LC-QTOF-MS.

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