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Characterization and grouping of aquatic fulvic acids isolated from clear-water rivers and lakes in Japan
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
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Characterization and grouping of aquatic fulvic acids isolated from clear-water rivers and lakes in Japan Kumiko Tsuda a, Hisayo Mori b, Daichi Asakawa a,1, Yukiko Yanagi b,2, Hiroki Kodama c, Seiya Nagao d,3, Koyo Yonebayashi e, Nobuhide Fujitake b,* a
Graduate School of Science and Technology, Kobe University, Rokkodai 1, Kobe 657-8501, Japan Graduate School of Agricultural Science, Kobe University, Rokkodai 1 Kobe 657-8501, Japan c Analytical Research Center for Experimental Science, Saga University, Honjo 1, Saga 840-8502, Japan d Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan e Department of Environmental Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan b
article info
abstract
Article history:
Characteristics of aquatic fulvic acids (FAs) from 10 clear waters in Japan (around the
Received 28 December 2009
temperate zone) were revealed by several analytical techniquesdhigh performance size
Received in revised form
exclusion chromatography (HPSEC), elemental analysis, liquid-state 13C NMR spectroscopy,
13 April 2010
isotopic analyses (d13C and d15N), and compared with those of International Humic
Accepted 27 April 2010
Substances Society (IHSS) standard samples including FAs from brown waters (Suwannee,
Available online 4 May 2010
Pony, and Nordic FAs). Generally clear-water FAs were different from brown-water FAs in chemical properties. Weight-average molecular weights (Mw) of the clear-water FAs were
Keywords:
similar to each other, whereas their elemental compositions and carbon species distribu-
Aquatic humic substance (AHS)
tion were different. The clear-water FAs all exhibited a high proportion of alkyl carbons,
Temperate zone
which may be attributed to microbial activity. d13C and d15N values of the FAs indicated
Multivariate statistical analysis
that there would be a huge gap between origin and chemical structure of clear-water FA.
Microbial activity
Results of the chemical structural analyses described above were not always linked to
Residence time
those of the isotopic analyses (d13C and d15N). Multivariate statistical analysis, i.e. cluster and principal component analysis was applied to reveal differences or similarities in a more objective manner. The FAs were always classified into two clear-water groups and one brown-water group. Aryl-C and O-Alkyl-C contents were important for the grouping. We speculate that the grouping might depend on the differences of aquatic microbial activity caused by the differences of residence time of water. ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Dissolved organic matter (DOM) is the major form of organic carbon in almost all aquatic ecosystems (Thurman, 1985). It is composed of a complex mixture of substances, including non-
humic solutes such as amino acids, hydrocarbons, carbohydrates, fats, waxes, resins, low molecular weight acids, and aquatic humic substances (AHSs). Since AHSs have a large sorption capacity towards pollutants due to various active functional groups such as carboxyl groups (Leenheer et al.,
* Corresponding author. Tel./fax: þ81 78 803 5847. E-mail address: [email protected] (N. Fujitake). 1 Present address: Vocational Eco College, Hyogo 660-0083, Japan. 2 Present address: Faculty of Horticulture, Minamikyusyu University, Miyazaki 884-0003, Japan. 3 Present address: Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa 923-1224, Japan. 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.04.038
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1995), considerable efforts to evaluate the degree of their potential effects on pollutants have been undertaken. Thus, characterization of AHS should be important for predicting the fate of local pollutants (McCarthy and Zachara, 1989). AHSs have been studied in many rivers and lakes. For example, Malcolm (1990a) characterized more than 50 AHSsdhumic and fulvic aciddsamples collected from 10 streams in the U.S.A. and Norway, showing more similarities of stream AHSs than humic substances from soils or ocean. However, these streams were almost brown waters that exhibited high DOM concentrations. Surface waters in the cold and wet boreal climatic zones in the northern hemisphere are often rich in DOM. Organic matters in such brown-water rivers seem to be of terrestrial origin rather than from aquatic phytoplankton (Hedges and Oades, 1997). Aiken et al. (1996) investigated dissolved organic carbon (including AHS) collected from a lake and from 10 inflowing rivers in Antarctica that were clear waters and exhibited low DOM concentrations. From the structural analysis data they concluded that these organic matters originated from leaching of the algal and bacterial mats and mosses in the river channels and riparian zones. However, climatic condition of their research field was unique. Most recently two studies on AHSs collected from clear waters around temperate zone were published. Kim et al. (2006) characterized natural organic matter (NOM, including AHS) from a Korean river, and McDonald et al. (2007) characterized fulvic acids from an Australian floodplain river, but each of their sampling points was only one. As such, there have been up to now no descriptions about differences or similarities among AHSs isolated from a number of clear-water rivers and lakes around temperate zone. One reason for lack of knowledge about clear-water AHS is the difficulty of collecting sufficient amounts of the sample from these waters for several chemical analyses. Lower concentrations of DOM (AHS) in clear waters, as compared to brown waters, soils, and sediments, require more timeconsuming and laborious processes for their isolation and purification. To solve this problem, Fujitake et al. (2009) developed a large-scale preparative isolation apparatus of AHS. This is an on-flow type AHS adsorption apparatus that sequentially carry out the water pumping, the filtration, the acidification of the filtrates, and application to the nonionic resin (DAX-8). About 500 L of water per hour can be treated by the apparatus. Collecting a number of clear-water AHS samples in sufficient volumes by this apparatus would be expected to gain various analyzed datasets. It is important to analyze each of the samples in various ways to reveal differences or similarities of chemical properties among the AHSs. Furthermore, there is a need to evaluate and harmonize a large number of analyzed datasets to obtain simple and consistent methods for grouping of the samples (Artinger et al., 1999). Statistical techniques like cluster analysis and principal component analysis are often used to classify different types of humic substances (Thomsen et al., 2002; Pena-Mendez et al., 2005; Moreda-Pineiro et al., 2006; Moros et al., 2008). However, these techniques have never been applied for the grouping of a number of AHSs. In this study we characterized AHSs collected from clearwater rivers and lakes in Japan, around temperate zone (from the subtropical to the subarctic zone, Kira, 1971; Fig. 1) by our
apparatus (Fujitake et al., 2009). AHS has been classified traditionally as either fulvic or humic acid based on solubility characteristics, and in this study we focused on fulvic acid (FA) fraction that predominates in aquatic ecosystem and represents the main chemical properties of AHS (Malcolm, 1985; Maurince and Namjesnik-dejanovic, 1999). Ten FA samples were investigated by high performance size exclusion chromatography (HPSEC), elemental analysis, liquidstate 13C NMR spectroscopy, isotopic analyses, and grouped by cluster and principal component analysis. Moreover, we referred to differences or similarities between brown- and clear-water FAs by taking 3 international humic substances society (IHSS) standard FAs, Pony (clear-water), Suwannee and Nordic (brown-water) FAs, into the statistical analysis.
2.
Materials and methods
2.1.
Isolation and purification of AHS
Thirteen FA samples included in the present study are listed in Table 1. Nine clear-water river or lake FAs are our original
Fig. 1 e Location of the sampling sites of 13 aquatic fulvic acids (FAs) including in the present study. The climatic division is illustrated by referring to Kira (1971).
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Table 1 e Types, names, origins, sampling sites and times of 13 aquatic fulvic acid (FAs) including in the present study. Type Clear-water river
Brown-water river Clear-water lake
Brown-water lake
Name Teshio Tomuraushi Ado Fukuchi Chikugo Sunoura Yona Suwannee Biwaa Tankai Kawashiro Pony Nordic
Origin
Lat/Long
Japan, Teshio river, Headwater Japan, Tokachi river, Headwater Japan, Ado river, Headwater Japan, Fukuchi river, Headwater Japan, Chikugo river, Headwater Japan, Sunoura river, Headwater Japan, Yona river, Headwater (IHSS standard sample) Japan, Lake Biwa, North basin Japan, Lake Tankai Japan, Kawashiro-Dam (IHSS reference sample) (IHSS reference sample)
44 06’ 43 27’ 35 29’ 35 07’ 33 11’ 31 58’ 26 45’
52” 48” 47” 58” 40” 34” 28”
N, N, N, N, N, N, N,
142 42’ 55” 142 51’ 40” 135 47’ 07” 134 39’ 25” 130 59’ 36” 130 54’ 13” 128 13’ 04”
Time of collection E E E E E E E
35 13’ 49” N, 135 57’ 42” E 35 27’ 2” N, 135 58’ 50” E 35 19’ 11” N, 135 13’ 44” E
September 2006 June 2005 November 2004 November 2006 November 2005 December 2005 December 2004 October 2001 October 2006 October 1997
a Fujitake et al. (2009).
samples that were isolated and purified according to the conventional XAD isolation protocol (Leenheer, 1981; Thurman and Malcolm, 1981). First, using a large-scale preparative isolation apparatus developed by Fujitake et al. (2009), approximately 500 L of water per hour was continuously filtrated (<0.45 mm), adjusted to pH 2 using HCl, and passed through DAX-8 resin (Supelite DAX-8, Sigma-Aldrich Co., St. Louise, USA); subsequently, the DAX-8 resin absorbing the AHS was carried to the laboratory under cool temperature (5 C). The AHS was eluted from the resin using 0.1 M NaOH aqueous and HA and FA were then separated by adjusting the solution to pH 1.5. The FA (supernatant) was converted to the hydrogen form by passing through an Hþtype cation-exchanger (Amberlite IR120-B, Organo Co., Tokyo, Japan). Finally, powdered FA sample was obtained by freezedrying. One clear-water lake FA, Biwa was offered by Fujitake et al. (2009). Moreover, we used three IHSS samples as references: Suwannee (Cat. No. 1S101F, a standard FA from brown-water river), Pony (1R109F, a reference FA from clearwater lake), Nordic (1R105F, a reference FA from brown-water reservoir).
2.2. High performance size exclusion chromatography (HPSEC) HPSEC was conducted with a Waters 600E pump, a Waters 717 plus autosampler, a Waters 2487 UV-visible detector, and a Waters 2410 refractive index detector (Nihon Waters, Co., Ltd., Japan). The column was a Shodex SB803 HQ, 8.0 300 mm (f L), exclusion limit of 100,000 Da, and the guard column was a Shodex Ohpak SB-G, 6.0 50 mm (f L) (Showa Denko Co., Ltd., Japan). The mobile phase was 0.01 M phosphate buffer, pH 7, with 25% acetonitrile. Sample (30 mL), with a concentration of approximately 0.2 mg L1, was injected into the HPSEC system. The flow rate of the eluent was 0.8 mL min1. The absorbance of the sample was recorded at 260 nm. Weight-average molecular weight (Mw) of each sample was calculated by GPC analysis software (Millenium 32-J, Nihon Waters Co., Ltd., Japan) as standard samples of polystyrene sulfonates (PSSNa). Details of the procedure are presented by Asakawa et al. (2008).
2.3.
Elemental analysis
Elemental analysis of FA was performed on a CHNS/O analyzer (2400II, PerkinElmer Co., Ltd., Japan), using 2 mg of the dry sample per measurement. Ash content of FA was determined after combustion of 10 mg of the dry sample at 550 C in a muffle furnace for 4 h.
2.4.
Liquid-state
13
C NMR
Liquid-state 13C NMR was recorded at 125.757 MHz on a Bruker Avance 500 spectrometer using sample tubes 5 mm in diameter (PS-001, Shigemi Co., Ltd., Japan). FA sample solution was prepared by dissolving 30e50 mg of sample in 400e500 mL of 0.5 M NaOD/D2O (99.9%, Aldrich Chemical Co., Ltd., USA) solution. Sodium 3-trimethylsilylpropionate 2,3,3,3, D4 (TMSP; Euriso-top, Saint Aubin, France) was added as reference (0 ppm) for the chemical shifts. To obtain quantitative conditions for the integration of the spectrum, 13C signals were proton-decoupled by the inverse gated decoupling technique as follows: pulse width 45 , acquisition time 0.2 s. A total repetition time of 2.5 s was applied to permit relaxation of all the spins. A 50 Hz line broadening was used to improve the signal-to-noise ratio, and scan numbering from 8000 to 28,000 was accumulated. Chemical shift assignments were referred from Wilson (1980) and Fujitake and Kawahigashi (1999). To calculate the carbon distributions by considering the spectrum features, every 13C NMR spectra was divided into the following five areas: 5e60 ppm, Alkyl-C; 60e110 ppm, O-AlkylC; 110e165 ppm, Aryl-C; 165e190 ppm, Carboxyl-C; and 190e230 ppm, Carbonyl-C. Aromaticity (AR) was calculated by expressing the amount of Aryl-C (110e165 ppm) as a percentage of Alkyl- þ O-Alkyl- þ Aryl-C (5e165 ppm) (Hatcher et al., 1981; Watanabe and Fujitake, 2008).
2.5.
Isotopic analysis
Carbon (13C) and nitrogen (15N) isotope analyses were performed to evaluate carbon sources and nitrogen uptake and/ or decomposition, respectively. Isotopic analyses on FA were accomplished with an elemental analyzer coupled with an
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w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
IsoPrime EA stable isotope ratio mass spectrometer (GV Instruments, UK). Carbon and nitrogen isotopic values are reported according to the equation: dx Eð&Þ ¼
Rsample =Rstandard 1 103
Cluster analysis
Cluster analysis was performed on the elemental composition (EA), carbon distribution (NMR), d13C and Mw data of the 13 FA samples. Similarities-dissimilarities were quantified through squared Euclidean distance measurements, and the distance between two objects (FAs), i and j, is given as: sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi m X 2 de ¼ Xik Xjk
(2)
k¼1
where de denotes the squared Euclidean distance, Xik and Xjk are the values of variable k for object i and j, respectively, and m is the number of variables. The dendrograms shown in Fig. 5 are based on the Ward method.
2.7.
Principal component analysis
Datasets containing the 13 FA samples were subjected to principal component analysis. The main goal of the principal component analysis was to quantify the significance of a variable that explains the observed groupings and patterns of the inherent properties of individual FAs. Through a liner combination of the original property variables (measured characterization properties) in the data matrix X, the property space is reduced and explained by a set of principal components. Principal component analysis in matrix form is a leastsquare model and is expressed by: X ¼ A$F þ E
(3)
where X is the original data matrix, F is the value of the object in the projection space, A is the loading of the original variables in the hyperspace projected by the principal components, and E contains the residuals. The principal components account for the maximum explainable variance of all original property parameters in descending order, and are non-correlated: Principal components j ¼ aj1 x1 þ aj2 x2 þ . þ ajn xn
Results
3.1.
HPSEC
(1)
where E is the given element, x is the heavy isotope of the element, and R ¼ the abundance ratio of the heavy to light isotopes of the element in the standard or sample. Internal laboratory reference gases for carbon and nitrogen were calibrated against the respective internal standards USGS-40 (L-glutamic acid). The results were reported in delta notation (d) as per mil deviation (&) from corresponding international standards of Pee Dee Belemnite (13C/12C, PDB) and atmospheric N2 (15N/14N, air). Analytical precision was typically within 0.07& for carbon and 0.25& for nitrogen.
2.6.
3.
(4)
The loadings, a, of each original characterization variable (x1exn) in principal components number j, reflects the importance of variable 1 to n for describing the score patterns in the direction of the principal components j.
Fig. 2 shows a typical HPSEC chromatogram of clear-water FA (Teshio). All of the FAs eluted from the HPSEC column as a broad peak with several trace shoulder peaks between the void volume (V0 ¼ 7.0 min) and the total permeation volume (V0 þ Vi ¼ 13.7 min). Mw values of our original FAs (relative to the standard samples of PSSNa at pH 7 and 260 nm) are given in Table 2. Although the Mw of clear-water river FAs were similar to each other, the average Mw of clear-water river FAs was higher than the Mw of clear-water lake FAs. On the whole, these values and the range were smaller than those of soil humic acids (from 3160 Da to 26,400 Da; Asakawa et al., 2008) or that of Suwannee FA (2310 Da; Chin et al., 1994) analyzed by the same standard samples. The absolute comparison of our data with other literature data is difficult because the value of the molecular weight by HPSEC changes on the condition (solvents, pHs, standards, etc.) (Piccolo et al., 2002; Hoque et al., 2003). However, using the data as the relative value of a parameter for grouping of the FAs is possible.
3.2.
Elemental analysis
The elemental compositions of the 13 FAs are given in Table 2. N% showed the feature of decreasing in order of clear-water lake FAs, clear-water river FAs, brown-water FAs. On the other hand, it was difficult to recognize the patterns with the proportion of C, H, or O in the FAs. Therefore, the 13 FAs in Table 2 were plotted over the Diagrams of H/C vs. O/C ratio, called van Krevelen diagrams (van Krevelen, 1961) (Fig. 3). H/C ratio indicates the degree of unsaturation (a small value) or aliphaticity (a large value) of a substance, and O/C ratio indicates the carbohydrate content and degree of oxidation (Steelink, 1985). Surrounded areas are constructed by referring to Abbt-Braun et al. (2004). Most of the clear-water river FAs occurred in the area of soil/sediment FAs, whereas clear-water lake FAs occurred in the area of wastewater FAs. Suwannee and Nordic occurred distant from the clear-water FAs including Pony, namely, in the area of lower H/C and higher O/C ratios, indicating that the brown-water FAs exhibit higher degree of unsaturation and oxidation than clear-water FAs.
3.3.
Liquid-state
13
C NMR spectroscopy
The 13C NMR spectra of the 13 FAs is shown in Fig. 4. Spectra of IHSS standard samples are referred to Thorn et al. (1989). All
Teshio
6
8 V0
10
12
14 V0 + Vi
Fig. 2 e HPSEC chromatogram of a clear-water fulvic acid (Teshio). V0, void volume; V0 D Vi, total permeation volume.
Table 2 e Characteristics of 13 aquatic fulvic acids (FAs) revealed by HPSEC, elemental analysis, Name
Mwa
1330 925 2146 1459 1592 1237 2131 (1546)
Suwannee
e
Biwae Tankai Kawashiro Pony (average) Nordic a b c d e
884 920 e e e e e
C NMR, isotopic analysis.
Atomic ratios
C species (%)
ARb
d13C
d15N
C
H
N
O
H/C
O/C
N/C
Alkyl
O-Alkyl
Aryl
Carboxyl
Carbonyl
50.1 59.7 54.1 50.7 53.8 53.0 54.4 (53.7)
5.16 5.70 4.37 5.00 4.92 4.95 4.90 (5.00)
1.34 0.66 0.99 1.70 1.64 1.04 1.22 (1.23)
43.4 33.9 40.5 42.6 39.7 41.0 39.5 (40.1)
1.23 1.15 0.97 1.18 1.10 1.12 1.08 (1.12)
0.65 0.43 0.56 0.63 0.55 0.58 0.54 (0.56)
0.023 0.010 0.016 0.029 0.026 0.017 0.019 (0.020)
39.3 43.7 35.6 31.6 34.4 34.8 34.5 (36.3)
18.5 11.2 16.1 17.9 12.3 14.5 13.6 (14.9)
21.4 23.6 28.5 29.1 29.5 28.3 29.0 (27.1)
17.0 16.4 17.1 16.8 19.3 18.7 19.5 (17.8)
3.9 5.1 2.7 4.7 4.5 3.7 3.5 (4.0)
0.27 0.30 0.36 0.37 0.39 0.37 0.38 (0.35)
28.6 26.4 28.7 29.2 26.2 27.9 29.8 (28.1)
52.4
4.31
0.72
42.2
0.99
0.60
0.012
32.8
15.6
23.6
21.2
6.9
0.33
27.6c
1.85c
56.2 55.0 56.9 52.5 (55.1)
6.06 5.34 4.94 5.39 (5.43)
2.31 1.07 2.04 6.51 (2.98)
35.5 38.6 36.1 31.4 (35.4)
1.29 1.17 1.04 1.23 (1.18)
0.47 0.53 0.48 0.45 (0.48)
0.035 0.017 0.031 0.106 (0.047)
48.2 43.0 38.9 51.4 (45.4)
15.6 11.4 16.5 13.7 (14.3)
16.8 21.5 24.0 15.8 (19.5)
14.0 17.1 16.1 15.0 (15.6)
5.4 7.0 4.4 4.0 (5.2)
0.21 0.28 0.30 0.20 (0.25)
25.8 28.8 27.4d ndc e
4.4 0.0
52.3
3.98
0.68
45.1
0.91
0.65
0.011
21.9
17.7
28.0
21.4
11.0
0.41
27.8d
1.3 0.3 0.7 0.0 1.7 0.3 1.0 (0.7)
e ndc e e
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
Teshio Tomuraushi Ado Fukuchi Chikugo Sunoura Yona (Average)
Weight % on ash-free basis
13
Average molecular weight. Aromaticity. IHSS data (http://ihss.gatech.edu/ihss2/elements.html). “nd” means that an item was not determined. Nagao et al. (2007). Data expect d13C and d15N is presented by Fujitake et al. (2009).
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Teshio
clear-water river brown-water river clear-water lake brown-water lake
1.5
EA+NMR
Ado Kawashiro Chikugo Yona
wastewater
Atomic H/C ratio
cluster 1
Fukuchi
Sunoura
Biwa Pony
Yona Kawashiro
cluster 3
Tankai Biwa
Fukuchi Sunoura Chikugo
Pony
lake/river water
1.0
Suwannee groundwater
Ado
0
0.6
20
40
60
80
Nordic
brown water
0.4
cluster 2
Tomuraushi
Teshio
Tankai
Tomuraushi
Suwannee Nordic
soil/sediment
0.8
Atomic O/C ratio Fig. 3 e Diagrams of H/C versus O/C ratios of the 13 aquatic fulvic acids (FAs). Surrounded areas are constructed by referring to Abbt-Braun et al. (2004).
Teshio Fukuchi Ado Sunoura Yona Chikugo Kawashiro
EA+NMR+
13
C
Suwannee Nordic Tomuraushi Tankai Biwa
0
20
40
Teshio Fukuchi
60
EA+NMR+
80
13
C+Mw
Ado Yona Chikugo Sunoura Tomuraushi Tankai Biwa
0
20
40
60
80
Squared Distance
Fig. 5 e Result of hierarchical cluster analysis, showing the Ward method linkage dendrogram of the rangenormalized data, based on combinations of the variable parameters obtained from elemental analysis (EA: H/C, O/C, N/C), liquid-state 13C NMR spectroscopy (NMR: Alkyl-C, O-Alkyl-C, Aryl-C, Carboxyl-C, Carbonyl-C), isotopic analysis (d13C) and HPSEC (Mw).
Fig. 4 e Liquid-state fulvic acids (FAs).
13
C NMR spectra of the 13 aquatic
spectra exhibited a major broad band of alkyl carbons from 5 to 54 ppm, a broad band of aryl carbons from 110 to 165 ppm, and a narrow band of carboxyl carbons from 165 to 190 ppm. The clear-water FAs collected in this study exhibited two sharp peaks around 60 and 120 ppm typical of lignins. A part of clearwater FAs (Tomuraushi, Biwa, Tankai, Kawashiro) exhibited large M-shaped bands in the alkyl carbons region (5e54 ppm). The others exhibited inverted V-shaped bands in the region. Because M-shaped band was also found in another report of Antarctica clear-water FAs (Aiken et al., 1996), this feature might provide clues to group the FAs. However in this study these features could not be reflected in the results of statistical analyses. The carbon distributions of the 13 FAs are listed in Table 2. Alkyl-C contents showed the feature of increasing in order of
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Variate
Z1 a
Alkyl-C O-Alkyl-C Aryl-C Carboxyl-C Carbonyl-C H/C O/C N/C Eigen-value Total variance (%) Cumulative variance (%)
Z2 b
a
E.V.
F.L.
E.V.
F.L.b
0.341 0.371 0.705 0.380 0.291 0.396 0.018 0.502
0.829 0.021 0.927 0.803 0.144 0.761 0.486 0.753 3.595 44.933 44.933
0.183 0.897 0.069 0.129 0.065 0.149 0.580 0.166
0.459 0.961 0.150 0.138 0.100 0.094 0.776 0.039 1.799 22.483 67.416
a Eigenvector. b Factor loading.
clear-water lake FAs, clear-water river FAs, brown-water FAs. On the contrary, Carboxyl-C contents in clear-water FAs (<20%) were distinctly lower than those in brown-water FAs. These results indicate that clear-water FAs do not only exist in lower concentrations than brown-water FAs, but also have the different chemical properties from them.
3.4.
Isotopic analyses
The results of d13C and d15N values are shown in Table 2. The d13C values exhibited a wide range from 29.8 to 25.8&, indicating both C3 plants and freshwater plankton as the original source (Frimmel et al., 2002). d13C values of Tomuraushi, Chikugo and Biwa were high (>27&), implying a large contribution ratio of phytoplankton as the FA sources (Otero et al., 2003). However, Chikugo had many differences in the elemental composition or the 13C NMR spectral property from Tomuraushi and Biwa. This means that Chikugo would have the same origin as them, but the different generation process from them. The d15N values also exhibited a wide range from 0.3 to 4.4&. All d15N values of clear-water FAs were higher than that of brown-water FA (Suwannee).
other except Tomuraushi. Tomuraushi was linked to clearwater lake FAs (cluster 3) except Kawashiro by a small distance. Kawashiro was linked to clear-water river FAs (cluster 1) by a small distance. Suwannee and Nordic were linked by a small distance to each other (cluster 2) but by a larger distance to the other FAs. In cluster 1, Teshio and Fukuchi, Ado and Kawashiro, Chikugo, Yona, Sunoura were subclustered together. In cluster 3, Tomuraushi and Tankai, Biwa and Pony were subclustered together. As shown by the dendrograms “EAþNMRþd13C” “EAþNMRþd13CþMw” in Fig. 5, even if d13C and Mw values were added to the eight parameters, the FAs were grouped into the same three clusters. This means that information about the chemical structure (EA, NMR) of FAs have greater influence on their characterization than that about the origins (d13C) or the sizes (Mw). Namely, the FAs were always classified into two clear-water groups (cluster 1, cluster 3) and one brown-water group (cluster 2), based on their chemical structural information. The 13C NMR spectral features of the 13 FAs (Fig. 4) were examined again based on the clustered groups. The spectral features of Ado, Kawashiro, Chikugo, Yona, Sunoura were similar to each other in that the Aryl-C broad bands were larger than those of Alkyl-C or O-Alkyl-C. The spectral features of Tomuraushi and Tankai were similar to each other in the large M-shaped band in the alkyl carbon region (5e54 ppm), two sharp peaks typical of lignins (60, 120 ppm) and a peak typical of carbohydrates (75 ppm). The Biwa and Pony spectral features were similar to each other in the quite large band in alkyl carbon regions.
3.6.
Principal component analysis
A principal component analysis was conducted for the elemental analysis and liquid-state 13C NMR spectroscopy
PC1 -1
0
1 O-Alkyl-C
4
3
Teshio
O/C
Fukuchi
2
Nordic
Cluster analysis
Objective differences or similarities between the 13 FAs were revealed by cluster analysis and principal component analysis, based on combinations of the variable parameters obtained from elemental analysis (EA; atomic ratios), liquidstate 13C NMR spectroscopy (NMR; carbon distributions), isotopic analysis (d13C) and HPSEC (Mw). In Fig. 5, “EAþNMR” illustrates the result of hierarchical cluster analysis, showing the Ward method dendrogram of the range-normalized data, based on the following eight parameters: H/C, O/C, N/C (EA), Alkyl-C, O-Alkyl-C, Aryl-C, Carboxyl-C, Carbonyl-C (NMR). There were roughly three clusters: cluster 1 contained Teshio, Fukuchi, Ado, Kawashiro, Chikugo, Yona, Sunoura; cluster 2 contained Suwannee, Nordic; and cluster 3 contained Tomuraushi, Tankai, Biwa, Pony. Clear-water river FAs were relatively similar to each
1
Z2
3.5.
1
N/CH/C
Biwa
Suwannee Kawashiro
0 Carbonyl-C -1
0
Carboxyl-C
Alkyl-C
Pony
Ado Sunoura Aryl-C
PC2
Table 3 e Explained X-variance of two-component principal component analyses, and total explained variance, based on 13C NMR and elemental analyses derived descriptors.
Yona Chikugo
Tankai -2
clear-water river
Tomuraushi -3
brown-water river clear-water lake
-4
brown-water lake -1 -4
-3
-2
-1
0
1
2
3
4
Z1 Fig. 6 e Bi-plot of loadings and scores, showing PC1 on the abscissa and PC2 on the ordinate. PC1 explains 44.9%, and PC2 22.5% of the variation in X.
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w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
data sets (EAþNMR) of the 13 FAs. Two principal components were extracted using the eight parameters (H/C, O/C, N/C, Alkyl-C, O-Alkyl-C, Aryl-C, Carboxyl-C, Carbonyl-C) as the input variables. The cumulative explained variance with the first two principal components, which was about 67.4% of the total variance (Table 3), indicating the usefulness of principal component analysis for providing a simple summary of the parameter correlation patterns. The first component contained 44.9% of the variance, whereas the second component explained 22.5% of the variance. The first component, PC1 (factor 1) was mainly related to Aryl-C, and the second component, PC2 (factor 2) was mainly related to O-Alkyl-C. The score plot for the first two components (13 observations) is shown in Fig. 6. In the horizontal direction, PC1 reflects mainly an increase in Aryl-C from left to right. In the vertical direction, PC2 mainly reflects an upward increase in O-Alkyl-C. The scores are normalized to have zero mean and unit variance. The plot shown in Fig. 6 could be classified based on the abovementioned clustered groups; the Teshio and Fukuchi scores were plotted in the direction of high O-Alkyl-C and O/C values, the Ado and Kawashiro scores were plotted in the center of all variances, and the Chikugo, Yona, and Sunoura scores were plotted in the direction of high Aryl-C and Carboxyl-C values. The Tomuraushi and Tankai scores were plotted in the direction of high Alkyl-C values, and the Biwa and Pony scores were plotted in the direction of high N/C and H/C values.
4.
Discussion
There is little information on the characterization and grouping of AHS or FAs collected from clear-water rivers or lakes around temperate zone. Aquatic FA can be considered to be composed of allochthonous FA (terrestrial origin) and autochthonous FA (aquatic origin). According to Thurman (1985), river AHS (FA) generally of allochthonous origin, would have spatial differences in surrounding soils, vegetation, and climatic conditions. The soil cover of terrestrial environment in the temperate zones, in particular Japan, is often dominated by Cambisol or Andosol, which has a higher organic matter retention capacity than Histosol or Podozol in the cold and wet boreal climatic zones (Yamane et al., 1978). Thus, clear waters in the temperate zones receive less organic input from the watershed than brown waters in the cold and wet boreal climatic zones, and autochthonous FA production in such clear waters would be more sufficient than allochthonous FA production. It would become presumptuous to say based on the biased datasets in the previous study that AHS (FA) from all types of waters appear to be remarkably similar, regardless of climatic conditions or vegetation (Malcolm, 1990b). The results in the present study revealed the uniqueness of FAs collected from clear-water rivers and lakes around temperate zone. The Mw of clear-water FAs were similar to each other. Differences of the elemental compositions and 13C NMR spectral properties of the FAs were more remarkable than those of the Mw values. The high proportion of alkyl carbons common in all of the clear-water FAs may be related to the microbial activities (McKnight et al., 1994). Differences in elemental composition and carbon distribution among the clear-water FAs were sometimes larger than differences
between the clear- and brown-water FAs, which may be related to other factors different from those of terrestrial environment such as watershed vegetation or soil covers. The fact that some FAs had a lot of differences in their structural properties but similarities in their origin properties (d13C and d15N values), indicates that not only terrestrial materials but also degraded and modified materials in water would be important in clear waters around temperate zone. Cluster and principal component analysis applied to group and reveal differences or similarities of 13 FAs in this study in an objective manner. The cluster analysis showed that even if the other parameters were added to the parameters of atomic ratios (EA) and carbon distributions (NMR), the result were never changed. This means that information about chemical structural properties (EA, NMR) of the FAs have great influence on their grouping. The FAs were always classified into roughly three groups. Considering the environment of each sampling points, it can be said that first group (cluster 1) is composed of FAs mainly from small clear-water rivers, second group (cluster 2) is brown-water FAs, and third group (cluster 3) is composed of FAs mainly from large clear-water rivers and lakes. Differences between the two clear-water FA groups (cluster 1 and 3) may have a close relationship to their transition times in rivers or lakes. The former are from rushing rivers, therefore this group can be called “fleeting clear-water FAs” that have short residence time in the waters. The latter are from gentle rivers or lakes, therefore this group can be called “retaining clear-water FAs” that have long residence time in the waters. If residence time of water were longer, FA inflowing into the water would have more opportunities for contacting with the residues and the metabolites of aquatic microorganisms. That is to say, sufficient residence time of water would enhance influence from not terrestrial but aquatic origin on FA. From chemical structural data, Fujitake et al. (2009) suggested that FA from Lake Biwa that has long residence time of water (average of 5.5 years) is mainly derived from degraded and modified material of microbial residues and microbial metabolites, which support the prediction. Water temperatures or mineral conditions may also be important in the aquatic microbial activity. However, considering almost all clear-water FA samples were collected at the points never affected by anthropogenic inputs in autumn (Table 1), residence time of water would mainly define the microbial impact to the FA. The results of the principal component analysis revealed that Aryl-C and O-Alkyl-C contents were important for the grouping. We cannot refer to the relationship between microbial activity, residence time of water and Aryl-C, O-Alkyl-C contents in this study. If the proper parameters for evaluating transition time in rivers or lakes are found in the future and applied for the grouping, such a relationship may become clear.
5.
Conclusion
Clear-water FAs not only existed in lower concentrations than brown-water FAs, but also had the different chemical properties from them. Clear-water FAs exhibited higher proportion of alkyl carbons than brown-water FAs generally, which may be related to the microbial activities.
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
Results of the elemental and the 13C NMR analyses were not always linked to those of the isotopic analyses, meaning that it may be difficult to decide origins or generation processes of clear-water FAs only by these analyses. Multivariate statistical analysis revealed spatial diversity of clear-water FAs. The FAs were always classified into two clear-water groups and one brown-water group, based on the difference of their chemical structures, especially Aryland O-Alkyl-C contents. The grouping of the FAs might depend on the differences of aquatic microbial activity caused by the differences of residence time of water. It may become possible in the future to evaluate a lot of environmental effects of FA more easily by investigating the functions not individually but as a group, applying the grouping method in this study.
Acknowledgements This work was partily supported by a Grant-in-Aid for Scientific Research (No. 16380049, 2004e2007) from the Ministry of Education, Science, Sports and Culture of Japan.
references
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Characterization and grouping of aquatic fulvic acids isolated from clear-water rivers and lakes in Japan Kumiko Tsuda a, Hisayo Mori b, Daichi Asakawa a,1, Yukiko Yanagi b,2, Hiroki Kodama c, Seiya Nagao d,3, Koyo Yonebayashi e, Nobuhide Fujitake b,* a
Graduate School of Science and Technology, Kobe University, Rokkodai 1, Kobe 657-8501, Japan Graduate School of Agricultural Science, Kobe University, Rokkodai 1 Kobe 657-8501, Japan c Analytical Research Center for Experimental Science, Saga University, Honjo 1, Saga 840-8502, Japan d Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan e Department of Environmental Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan b
article info
abstract
Article history:
Characteristics of aquatic fulvic acids (FAs) from 10 clear waters in Japan (around the
Received 28 December 2009
temperate zone) were revealed by several analytical techniquesdhigh performance size
Received in revised form
exclusion chromatography (HPSEC), elemental analysis, liquid-state 13C NMR spectroscopy,
13 April 2010
isotopic analyses (d13C and d15N), and compared with those of International Humic
Accepted 27 April 2010
Substances Society (IHSS) standard samples including FAs from brown waters (Suwannee,
Available online 4 May 2010
Pony, and Nordic FAs). Generally clear-water FAs were different from brown-water FAs in chemical properties. Weight-average molecular weights (Mw) of the clear-water FAs were
Keywords:
similar to each other, whereas their elemental compositions and carbon species distribu-
Aquatic humic substance (AHS)
tion were different. The clear-water FAs all exhibited a high proportion of alkyl carbons,
Temperate zone
which may be attributed to microbial activity. d13C and d15N values of the FAs indicated
Multivariate statistical analysis
that there would be a huge gap between origin and chemical structure of clear-water FA.
Microbial activity
Results of the chemical structural analyses described above were not always linked to
Residence time
those of the isotopic analyses (d13C and d15N). Multivariate statistical analysis, i.e. cluster and principal component analysis was applied to reveal differences or similarities in a more objective manner. The FAs were always classified into two clear-water groups and one brown-water group. Aryl-C and O-Alkyl-C contents were important for the grouping. We speculate that the grouping might depend on the differences of aquatic microbial activity caused by the differences of residence time of water. ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Dissolved organic matter (DOM) is the major form of organic carbon in almost all aquatic ecosystems (Thurman, 1985). It is composed of a complex mixture of substances, including non-
humic solutes such as amino acids, hydrocarbons, carbohydrates, fats, waxes, resins, low molecular weight acids, and aquatic humic substances (AHSs). Since AHSs have a large sorption capacity towards pollutants due to various active functional groups such as carboxyl groups (Leenheer et al.,
* Corresponding author. Tel./fax: þ81 78 803 5847. E-mail address: [email protected] (N. Fujitake). 1 Present address: Vocational Eco College, Hyogo 660-0083, Japan. 2 Present address: Faculty of Horticulture, Minamikyusyu University, Miyazaki 884-0003, Japan. 3 Present address: Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa 923-1224, Japan. 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.04.038
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1995), considerable efforts to evaluate the degree of their potential effects on pollutants have been undertaken. Thus, characterization of AHS should be important for predicting the fate of local pollutants (McCarthy and Zachara, 1989). AHSs have been studied in many rivers and lakes. For example, Malcolm (1990a) characterized more than 50 AHSsdhumic and fulvic aciddsamples collected from 10 streams in the U.S.A. and Norway, showing more similarities of stream AHSs than humic substances from soils or ocean. However, these streams were almost brown waters that exhibited high DOM concentrations. Surface waters in the cold and wet boreal climatic zones in the northern hemisphere are often rich in DOM. Organic matters in such brown-water rivers seem to be of terrestrial origin rather than from aquatic phytoplankton (Hedges and Oades, 1997). Aiken et al. (1996) investigated dissolved organic carbon (including AHS) collected from a lake and from 10 inflowing rivers in Antarctica that were clear waters and exhibited low DOM concentrations. From the structural analysis data they concluded that these organic matters originated from leaching of the algal and bacterial mats and mosses in the river channels and riparian zones. However, climatic condition of their research field was unique. Most recently two studies on AHSs collected from clear waters around temperate zone were published. Kim et al. (2006) characterized natural organic matter (NOM, including AHS) from a Korean river, and McDonald et al. (2007) characterized fulvic acids from an Australian floodplain river, but each of their sampling points was only one. As such, there have been up to now no descriptions about differences or similarities among AHSs isolated from a number of clear-water rivers and lakes around temperate zone. One reason for lack of knowledge about clear-water AHS is the difficulty of collecting sufficient amounts of the sample from these waters for several chemical analyses. Lower concentrations of DOM (AHS) in clear waters, as compared to brown waters, soils, and sediments, require more timeconsuming and laborious processes for their isolation and purification. To solve this problem, Fujitake et al. (2009) developed a large-scale preparative isolation apparatus of AHS. This is an on-flow type AHS adsorption apparatus that sequentially carry out the water pumping, the filtration, the acidification of the filtrates, and application to the nonionic resin (DAX-8). About 500 L of water per hour can be treated by the apparatus. Collecting a number of clear-water AHS samples in sufficient volumes by this apparatus would be expected to gain various analyzed datasets. It is important to analyze each of the samples in various ways to reveal differences or similarities of chemical properties among the AHSs. Furthermore, there is a need to evaluate and harmonize a large number of analyzed datasets to obtain simple and consistent methods for grouping of the samples (Artinger et al., 1999). Statistical techniques like cluster analysis and principal component analysis are often used to classify different types of humic substances (Thomsen et al., 2002; Pena-Mendez et al., 2005; Moreda-Pineiro et al., 2006; Moros et al., 2008). However, these techniques have never been applied for the grouping of a number of AHSs. In this study we characterized AHSs collected from clearwater rivers and lakes in Japan, around temperate zone (from the subtropical to the subarctic zone, Kira, 1971; Fig. 1) by our
apparatus (Fujitake et al., 2009). AHS has been classified traditionally as either fulvic or humic acid based on solubility characteristics, and in this study we focused on fulvic acid (FA) fraction that predominates in aquatic ecosystem and represents the main chemical properties of AHS (Malcolm, 1985; Maurince and Namjesnik-dejanovic, 1999). Ten FA samples were investigated by high performance size exclusion chromatography (HPSEC), elemental analysis, liquidstate 13C NMR spectroscopy, isotopic analyses, and grouped by cluster and principal component analysis. Moreover, we referred to differences or similarities between brown- and clear-water FAs by taking 3 international humic substances society (IHSS) standard FAs, Pony (clear-water), Suwannee and Nordic (brown-water) FAs, into the statistical analysis.
2.
Materials and methods
2.1.
Isolation and purification of AHS
Thirteen FA samples included in the present study are listed in Table 1. Nine clear-water river or lake FAs are our original
Fig. 1 e Location of the sampling sites of 13 aquatic fulvic acids (FAs) including in the present study. The climatic division is illustrated by referring to Kira (1971).
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Table 1 e Types, names, origins, sampling sites and times of 13 aquatic fulvic acid (FAs) including in the present study. Type Clear-water river
Brown-water river Clear-water lake
Brown-water lake
Name Teshio Tomuraushi Ado Fukuchi Chikugo Sunoura Yona Suwannee Biwaa Tankai Kawashiro Pony Nordic
Origin
Lat/Long
Japan, Teshio river, Headwater Japan, Tokachi river, Headwater Japan, Ado river, Headwater Japan, Fukuchi river, Headwater Japan, Chikugo river, Headwater Japan, Sunoura river, Headwater Japan, Yona river, Headwater (IHSS standard sample) Japan, Lake Biwa, North basin Japan, Lake Tankai Japan, Kawashiro-Dam (IHSS reference sample) (IHSS reference sample)
44 06’ 43 27’ 35 29’ 35 07’ 33 11’ 31 58’ 26 45’
52” 48” 47” 58” 40” 34” 28”
N, N, N, N, N, N, N,
142 42’ 55” 142 51’ 40” 135 47’ 07” 134 39’ 25” 130 59’ 36” 130 54’ 13” 128 13’ 04”
Time of collection E E E E E E E
35 13’ 49” N, 135 57’ 42” E 35 27’ 2” N, 135 58’ 50” E 35 19’ 11” N, 135 13’ 44” E
September 2006 June 2005 November 2004 November 2006 November 2005 December 2005 December 2004 October 2001 October 2006 October 1997
a Fujitake et al. (2009).
samples that were isolated and purified according to the conventional XAD isolation protocol (Leenheer, 1981; Thurman and Malcolm, 1981). First, using a large-scale preparative isolation apparatus developed by Fujitake et al. (2009), approximately 500 L of water per hour was continuously filtrated (<0.45 mm), adjusted to pH 2 using HCl, and passed through DAX-8 resin (Supelite DAX-8, Sigma-Aldrich Co., St. Louise, USA); subsequently, the DAX-8 resin absorbing the AHS was carried to the laboratory under cool temperature (5 C). The AHS was eluted from the resin using 0.1 M NaOH aqueous and HA and FA were then separated by adjusting the solution to pH 1.5. The FA (supernatant) was converted to the hydrogen form by passing through an Hþtype cation-exchanger (Amberlite IR120-B, Organo Co., Tokyo, Japan). Finally, powdered FA sample was obtained by freezedrying. One clear-water lake FA, Biwa was offered by Fujitake et al. (2009). Moreover, we used three IHSS samples as references: Suwannee (Cat. No. 1S101F, a standard FA from brown-water river), Pony (1R109F, a reference FA from clearwater lake), Nordic (1R105F, a reference FA from brown-water reservoir).
2.2. High performance size exclusion chromatography (HPSEC) HPSEC was conducted with a Waters 600E pump, a Waters 717 plus autosampler, a Waters 2487 UV-visible detector, and a Waters 2410 refractive index detector (Nihon Waters, Co., Ltd., Japan). The column was a Shodex SB803 HQ, 8.0 300 mm (f L), exclusion limit of 100,000 Da, and the guard column was a Shodex Ohpak SB-G, 6.0 50 mm (f L) (Showa Denko Co., Ltd., Japan). The mobile phase was 0.01 M phosphate buffer, pH 7, with 25% acetonitrile. Sample (30 mL), with a concentration of approximately 0.2 mg L1, was injected into the HPSEC system. The flow rate of the eluent was 0.8 mL min1. The absorbance of the sample was recorded at 260 nm. Weight-average molecular weight (Mw) of each sample was calculated by GPC analysis software (Millenium 32-J, Nihon Waters Co., Ltd., Japan) as standard samples of polystyrene sulfonates (PSSNa). Details of the procedure are presented by Asakawa et al. (2008).
2.3.
Elemental analysis
Elemental analysis of FA was performed on a CHNS/O analyzer (2400II, PerkinElmer Co., Ltd., Japan), using 2 mg of the dry sample per measurement. Ash content of FA was determined after combustion of 10 mg of the dry sample at 550 C in a muffle furnace for 4 h.
2.4.
Liquid-state
13
C NMR
Liquid-state 13C NMR was recorded at 125.757 MHz on a Bruker Avance 500 spectrometer using sample tubes 5 mm in diameter (PS-001, Shigemi Co., Ltd., Japan). FA sample solution was prepared by dissolving 30e50 mg of sample in 400e500 mL of 0.5 M NaOD/D2O (99.9%, Aldrich Chemical Co., Ltd., USA) solution. Sodium 3-trimethylsilylpropionate 2,3,3,3, D4 (TMSP; Euriso-top, Saint Aubin, France) was added as reference (0 ppm) for the chemical shifts. To obtain quantitative conditions for the integration of the spectrum, 13C signals were proton-decoupled by the inverse gated decoupling technique as follows: pulse width 45 , acquisition time 0.2 s. A total repetition time of 2.5 s was applied to permit relaxation of all the spins. A 50 Hz line broadening was used to improve the signal-to-noise ratio, and scan numbering from 8000 to 28,000 was accumulated. Chemical shift assignments were referred from Wilson (1980) and Fujitake and Kawahigashi (1999). To calculate the carbon distributions by considering the spectrum features, every 13C NMR spectra was divided into the following five areas: 5e60 ppm, Alkyl-C; 60e110 ppm, O-AlkylC; 110e165 ppm, Aryl-C; 165e190 ppm, Carboxyl-C; and 190e230 ppm, Carbonyl-C. Aromaticity (AR) was calculated by expressing the amount of Aryl-C (110e165 ppm) as a percentage of Alkyl- þ O-Alkyl- þ Aryl-C (5e165 ppm) (Hatcher et al., 1981; Watanabe and Fujitake, 2008).
2.5.
Isotopic analysis
Carbon (13C) and nitrogen (15N) isotope analyses were performed to evaluate carbon sources and nitrogen uptake and/ or decomposition, respectively. Isotopic analyses on FA were accomplished with an elemental analyzer coupled with an
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w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
IsoPrime EA stable isotope ratio mass spectrometer (GV Instruments, UK). Carbon and nitrogen isotopic values are reported according to the equation: dx Eð&Þ ¼
Rsample =Rstandard 1 103
Cluster analysis
Cluster analysis was performed on the elemental composition (EA), carbon distribution (NMR), d13C and Mw data of the 13 FA samples. Similarities-dissimilarities were quantified through squared Euclidean distance measurements, and the distance between two objects (FAs), i and j, is given as: sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi m X 2 de ¼ Xik Xjk
(2)
k¼1
where de denotes the squared Euclidean distance, Xik and Xjk are the values of variable k for object i and j, respectively, and m is the number of variables. The dendrograms shown in Fig. 5 are based on the Ward method.
2.7.
Principal component analysis
Datasets containing the 13 FA samples were subjected to principal component analysis. The main goal of the principal component analysis was to quantify the significance of a variable that explains the observed groupings and patterns of the inherent properties of individual FAs. Through a liner combination of the original property variables (measured characterization properties) in the data matrix X, the property space is reduced and explained by a set of principal components. Principal component analysis in matrix form is a leastsquare model and is expressed by: X ¼ A$F þ E
(3)
where X is the original data matrix, F is the value of the object in the projection space, A is the loading of the original variables in the hyperspace projected by the principal components, and E contains the residuals. The principal components account for the maximum explainable variance of all original property parameters in descending order, and are non-correlated: Principal components j ¼ aj1 x1 þ aj2 x2 þ . þ ajn xn
Results
3.1.
HPSEC
(1)
where E is the given element, x is the heavy isotope of the element, and R ¼ the abundance ratio of the heavy to light isotopes of the element in the standard or sample. Internal laboratory reference gases for carbon and nitrogen were calibrated against the respective internal standards USGS-40 (L-glutamic acid). The results were reported in delta notation (d) as per mil deviation (&) from corresponding international standards of Pee Dee Belemnite (13C/12C, PDB) and atmospheric N2 (15N/14N, air). Analytical precision was typically within 0.07& for carbon and 0.25& for nitrogen.
2.6.
3.
(4)
The loadings, a, of each original characterization variable (x1exn) in principal components number j, reflects the importance of variable 1 to n for describing the score patterns in the direction of the principal components j.
Fig. 2 shows a typical HPSEC chromatogram of clear-water FA (Teshio). All of the FAs eluted from the HPSEC column as a broad peak with several trace shoulder peaks between the void volume (V0 ¼ 7.0 min) and the total permeation volume (V0 þ Vi ¼ 13.7 min). Mw values of our original FAs (relative to the standard samples of PSSNa at pH 7 and 260 nm) are given in Table 2. Although the Mw of clear-water river FAs were similar to each other, the average Mw of clear-water river FAs was higher than the Mw of clear-water lake FAs. On the whole, these values and the range were smaller than those of soil humic acids (from 3160 Da to 26,400 Da; Asakawa et al., 2008) or that of Suwannee FA (2310 Da; Chin et al., 1994) analyzed by the same standard samples. The absolute comparison of our data with other literature data is difficult because the value of the molecular weight by HPSEC changes on the condition (solvents, pHs, standards, etc.) (Piccolo et al., 2002; Hoque et al., 2003). However, using the data as the relative value of a parameter for grouping of the FAs is possible.
3.2.
Elemental analysis
The elemental compositions of the 13 FAs are given in Table 2. N% showed the feature of decreasing in order of clear-water lake FAs, clear-water river FAs, brown-water FAs. On the other hand, it was difficult to recognize the patterns with the proportion of C, H, or O in the FAs. Therefore, the 13 FAs in Table 2 were plotted over the Diagrams of H/C vs. O/C ratio, called van Krevelen diagrams (van Krevelen, 1961) (Fig. 3). H/C ratio indicates the degree of unsaturation (a small value) or aliphaticity (a large value) of a substance, and O/C ratio indicates the carbohydrate content and degree of oxidation (Steelink, 1985). Surrounded areas are constructed by referring to Abbt-Braun et al. (2004). Most of the clear-water river FAs occurred in the area of soil/sediment FAs, whereas clear-water lake FAs occurred in the area of wastewater FAs. Suwannee and Nordic occurred distant from the clear-water FAs including Pony, namely, in the area of lower H/C and higher O/C ratios, indicating that the brown-water FAs exhibit higher degree of unsaturation and oxidation than clear-water FAs.
3.3.
Liquid-state
13
C NMR spectroscopy
The 13C NMR spectra of the 13 FAs is shown in Fig. 4. Spectra of IHSS standard samples are referred to Thorn et al. (1989). All
Teshio
6
8 V0
10
12
14 V0 + Vi
Fig. 2 e HPSEC chromatogram of a clear-water fulvic acid (Teshio). V0, void volume; V0 D Vi, total permeation volume.
Table 2 e Characteristics of 13 aquatic fulvic acids (FAs) revealed by HPSEC, elemental analysis, Name
Mwa
1330 925 2146 1459 1592 1237 2131 (1546)
Suwannee
e
Biwae Tankai Kawashiro Pony (average) Nordic a b c d e
884 920 e e e e e
C NMR, isotopic analysis.
Atomic ratios
C species (%)
ARb
d13C
d15N
C
H
N
O
H/C
O/C
N/C
Alkyl
O-Alkyl
Aryl
Carboxyl
Carbonyl
50.1 59.7 54.1 50.7 53.8 53.0 54.4 (53.7)
5.16 5.70 4.37 5.00 4.92 4.95 4.90 (5.00)
1.34 0.66 0.99 1.70 1.64 1.04 1.22 (1.23)
43.4 33.9 40.5 42.6 39.7 41.0 39.5 (40.1)
1.23 1.15 0.97 1.18 1.10 1.12 1.08 (1.12)
0.65 0.43 0.56 0.63 0.55 0.58 0.54 (0.56)
0.023 0.010 0.016 0.029 0.026 0.017 0.019 (0.020)
39.3 43.7 35.6 31.6 34.4 34.8 34.5 (36.3)
18.5 11.2 16.1 17.9 12.3 14.5 13.6 (14.9)
21.4 23.6 28.5 29.1 29.5 28.3 29.0 (27.1)
17.0 16.4 17.1 16.8 19.3 18.7 19.5 (17.8)
3.9 5.1 2.7 4.7 4.5 3.7 3.5 (4.0)
0.27 0.30 0.36 0.37 0.39 0.37 0.38 (0.35)
28.6 26.4 28.7 29.2 26.2 27.9 29.8 (28.1)
52.4
4.31
0.72
42.2
0.99
0.60
0.012
32.8
15.6
23.6
21.2
6.9
0.33
27.6c
1.85c
56.2 55.0 56.9 52.5 (55.1)
6.06 5.34 4.94 5.39 (5.43)
2.31 1.07 2.04 6.51 (2.98)
35.5 38.6 36.1 31.4 (35.4)
1.29 1.17 1.04 1.23 (1.18)
0.47 0.53 0.48 0.45 (0.48)
0.035 0.017 0.031 0.106 (0.047)
48.2 43.0 38.9 51.4 (45.4)
15.6 11.4 16.5 13.7 (14.3)
16.8 21.5 24.0 15.8 (19.5)
14.0 17.1 16.1 15.0 (15.6)
5.4 7.0 4.4 4.0 (5.2)
0.21 0.28 0.30 0.20 (0.25)
25.8 28.8 27.4d ndc e
4.4 0.0
52.3
3.98
0.68
45.1
0.91
0.65
0.011
21.9
17.7
28.0
21.4
11.0
0.41
27.8d
1.3 0.3 0.7 0.0 1.7 0.3 1.0 (0.7)
e ndc e e
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
Teshio Tomuraushi Ado Fukuchi Chikugo Sunoura Yona (Average)
Weight % on ash-free basis
13
Average molecular weight. Aromaticity. IHSS data (http://ihss.gatech.edu/ihss2/elements.html). “nd” means that an item was not determined. Nagao et al. (2007). Data expect d13C and d15N is presented by Fujitake et al. (2009).
3841
3842
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
Teshio
clear-water river brown-water river clear-water lake brown-water lake
1.5
EA+NMR
Ado Kawashiro Chikugo Yona
wastewater
Atomic H/C ratio
cluster 1
Fukuchi
Sunoura
Biwa Pony
Yona Kawashiro
cluster 3
Tankai Biwa
Fukuchi Sunoura Chikugo
Pony
lake/river water
1.0
Suwannee groundwater
Ado
0
0.6
20
40
60
80
Nordic
brown water
0.4
cluster 2
Tomuraushi
Teshio
Tankai
Tomuraushi
Suwannee Nordic
soil/sediment
0.8
Atomic O/C ratio Fig. 3 e Diagrams of H/C versus O/C ratios of the 13 aquatic fulvic acids (FAs). Surrounded areas are constructed by referring to Abbt-Braun et al. (2004).
Teshio Fukuchi Ado Sunoura Yona Chikugo Kawashiro
EA+NMR+
13
C
Suwannee Nordic Tomuraushi Tankai Biwa
0
20
40
Teshio Fukuchi
60
EA+NMR+
80
13
C+Mw
Ado Yona Chikugo Sunoura Tomuraushi Tankai Biwa
0
20
40
60
80
Squared Distance
Fig. 5 e Result of hierarchical cluster analysis, showing the Ward method linkage dendrogram of the rangenormalized data, based on combinations of the variable parameters obtained from elemental analysis (EA: H/C, O/C, N/C), liquid-state 13C NMR spectroscopy (NMR: Alkyl-C, O-Alkyl-C, Aryl-C, Carboxyl-C, Carbonyl-C), isotopic analysis (d13C) and HPSEC (Mw).
Fig. 4 e Liquid-state fulvic acids (FAs).
13
C NMR spectra of the 13 aquatic
spectra exhibited a major broad band of alkyl carbons from 5 to 54 ppm, a broad band of aryl carbons from 110 to 165 ppm, and a narrow band of carboxyl carbons from 165 to 190 ppm. The clear-water FAs collected in this study exhibited two sharp peaks around 60 and 120 ppm typical of lignins. A part of clearwater FAs (Tomuraushi, Biwa, Tankai, Kawashiro) exhibited large M-shaped bands in the alkyl carbons region (5e54 ppm). The others exhibited inverted V-shaped bands in the region. Because M-shaped band was also found in another report of Antarctica clear-water FAs (Aiken et al., 1996), this feature might provide clues to group the FAs. However in this study these features could not be reflected in the results of statistical analyses. The carbon distributions of the 13 FAs are listed in Table 2. Alkyl-C contents showed the feature of increasing in order of
3843
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
Variate
Z1 a
Alkyl-C O-Alkyl-C Aryl-C Carboxyl-C Carbonyl-C H/C O/C N/C Eigen-value Total variance (%) Cumulative variance (%)
Z2 b
a
E.V.
F.L.
E.V.
F.L.b
0.341 0.371 0.705 0.380 0.291 0.396 0.018 0.502
0.829 0.021 0.927 0.803 0.144 0.761 0.486 0.753 3.595 44.933 44.933
0.183 0.897 0.069 0.129 0.065 0.149 0.580 0.166
0.459 0.961 0.150 0.138 0.100 0.094 0.776 0.039 1.799 22.483 67.416
a Eigenvector. b Factor loading.
clear-water lake FAs, clear-water river FAs, brown-water FAs. On the contrary, Carboxyl-C contents in clear-water FAs (<20%) were distinctly lower than those in brown-water FAs. These results indicate that clear-water FAs do not only exist in lower concentrations than brown-water FAs, but also have the different chemical properties from them.
3.4.
Isotopic analyses
The results of d13C and d15N values are shown in Table 2. The d13C values exhibited a wide range from 29.8 to 25.8&, indicating both C3 plants and freshwater plankton as the original source (Frimmel et al., 2002). d13C values of Tomuraushi, Chikugo and Biwa were high (>27&), implying a large contribution ratio of phytoplankton as the FA sources (Otero et al., 2003). However, Chikugo had many differences in the elemental composition or the 13C NMR spectral property from Tomuraushi and Biwa. This means that Chikugo would have the same origin as them, but the different generation process from them. The d15N values also exhibited a wide range from 0.3 to 4.4&. All d15N values of clear-water FAs were higher than that of brown-water FA (Suwannee).
other except Tomuraushi. Tomuraushi was linked to clearwater lake FAs (cluster 3) except Kawashiro by a small distance. Kawashiro was linked to clear-water river FAs (cluster 1) by a small distance. Suwannee and Nordic were linked by a small distance to each other (cluster 2) but by a larger distance to the other FAs. In cluster 1, Teshio and Fukuchi, Ado and Kawashiro, Chikugo, Yona, Sunoura were subclustered together. In cluster 3, Tomuraushi and Tankai, Biwa and Pony were subclustered together. As shown by the dendrograms “EAþNMRþd13C” “EAþNMRþd13CþMw” in Fig. 5, even if d13C and Mw values were added to the eight parameters, the FAs were grouped into the same three clusters. This means that information about the chemical structure (EA, NMR) of FAs have greater influence on their characterization than that about the origins (d13C) or the sizes (Mw). Namely, the FAs were always classified into two clear-water groups (cluster 1, cluster 3) and one brown-water group (cluster 2), based on their chemical structural information. The 13C NMR spectral features of the 13 FAs (Fig. 4) were examined again based on the clustered groups. The spectral features of Ado, Kawashiro, Chikugo, Yona, Sunoura were similar to each other in that the Aryl-C broad bands were larger than those of Alkyl-C or O-Alkyl-C. The spectral features of Tomuraushi and Tankai were similar to each other in the large M-shaped band in the alkyl carbon region (5e54 ppm), two sharp peaks typical of lignins (60, 120 ppm) and a peak typical of carbohydrates (75 ppm). The Biwa and Pony spectral features were similar to each other in the quite large band in alkyl carbon regions.
3.6.
Principal component analysis
A principal component analysis was conducted for the elemental analysis and liquid-state 13C NMR spectroscopy
PC1 -1
0
1 O-Alkyl-C
4
3
Teshio
O/C
Fukuchi
2
Nordic
Cluster analysis
Objective differences or similarities between the 13 FAs were revealed by cluster analysis and principal component analysis, based on combinations of the variable parameters obtained from elemental analysis (EA; atomic ratios), liquidstate 13C NMR spectroscopy (NMR; carbon distributions), isotopic analysis (d13C) and HPSEC (Mw). In Fig. 5, “EAþNMR” illustrates the result of hierarchical cluster analysis, showing the Ward method dendrogram of the range-normalized data, based on the following eight parameters: H/C, O/C, N/C (EA), Alkyl-C, O-Alkyl-C, Aryl-C, Carboxyl-C, Carbonyl-C (NMR). There were roughly three clusters: cluster 1 contained Teshio, Fukuchi, Ado, Kawashiro, Chikugo, Yona, Sunoura; cluster 2 contained Suwannee, Nordic; and cluster 3 contained Tomuraushi, Tankai, Biwa, Pony. Clear-water river FAs were relatively similar to each
1
Z2
3.5.
1
N/CH/C
Biwa
Suwannee Kawashiro
0 Carbonyl-C -1
0
Carboxyl-C
Alkyl-C
Pony
Ado Sunoura Aryl-C
PC2
Table 3 e Explained X-variance of two-component principal component analyses, and total explained variance, based on 13C NMR and elemental analyses derived descriptors.
Yona Chikugo
Tankai -2
clear-water river
Tomuraushi -3
brown-water river clear-water lake
-4
brown-water lake -1 -4
-3
-2
-1
0
1
2
3
4
Z1 Fig. 6 e Bi-plot of loadings and scores, showing PC1 on the abscissa and PC2 on the ordinate. PC1 explains 44.9%, and PC2 22.5% of the variation in X.
3844
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
data sets (EAþNMR) of the 13 FAs. Two principal components were extracted using the eight parameters (H/C, O/C, N/C, Alkyl-C, O-Alkyl-C, Aryl-C, Carboxyl-C, Carbonyl-C) as the input variables. The cumulative explained variance with the first two principal components, which was about 67.4% of the total variance (Table 3), indicating the usefulness of principal component analysis for providing a simple summary of the parameter correlation patterns. The first component contained 44.9% of the variance, whereas the second component explained 22.5% of the variance. The first component, PC1 (factor 1) was mainly related to Aryl-C, and the second component, PC2 (factor 2) was mainly related to O-Alkyl-C. The score plot for the first two components (13 observations) is shown in Fig. 6. In the horizontal direction, PC1 reflects mainly an increase in Aryl-C from left to right. In the vertical direction, PC2 mainly reflects an upward increase in O-Alkyl-C. The scores are normalized to have zero mean and unit variance. The plot shown in Fig. 6 could be classified based on the abovementioned clustered groups; the Teshio and Fukuchi scores were plotted in the direction of high O-Alkyl-C and O/C values, the Ado and Kawashiro scores were plotted in the center of all variances, and the Chikugo, Yona, and Sunoura scores were plotted in the direction of high Aryl-C and Carboxyl-C values. The Tomuraushi and Tankai scores were plotted in the direction of high Alkyl-C values, and the Biwa and Pony scores were plotted in the direction of high N/C and H/C values.
4.
Discussion
There is little information on the characterization and grouping of AHS or FAs collected from clear-water rivers or lakes around temperate zone. Aquatic FA can be considered to be composed of allochthonous FA (terrestrial origin) and autochthonous FA (aquatic origin). According to Thurman (1985), river AHS (FA) generally of allochthonous origin, would have spatial differences in surrounding soils, vegetation, and climatic conditions. The soil cover of terrestrial environment in the temperate zones, in particular Japan, is often dominated by Cambisol or Andosol, which has a higher organic matter retention capacity than Histosol or Podozol in the cold and wet boreal climatic zones (Yamane et al., 1978). Thus, clear waters in the temperate zones receive less organic input from the watershed than brown waters in the cold and wet boreal climatic zones, and autochthonous FA production in such clear waters would be more sufficient than allochthonous FA production. It would become presumptuous to say based on the biased datasets in the previous study that AHS (FA) from all types of waters appear to be remarkably similar, regardless of climatic conditions or vegetation (Malcolm, 1990b). The results in the present study revealed the uniqueness of FAs collected from clear-water rivers and lakes around temperate zone. The Mw of clear-water FAs were similar to each other. Differences of the elemental compositions and 13C NMR spectral properties of the FAs were more remarkable than those of the Mw values. The high proportion of alkyl carbons common in all of the clear-water FAs may be related to the microbial activities (McKnight et al., 1994). Differences in elemental composition and carbon distribution among the clear-water FAs were sometimes larger than differences
between the clear- and brown-water FAs, which may be related to other factors different from those of terrestrial environment such as watershed vegetation or soil covers. The fact that some FAs had a lot of differences in their structural properties but similarities in their origin properties (d13C and d15N values), indicates that not only terrestrial materials but also degraded and modified materials in water would be important in clear waters around temperate zone. Cluster and principal component analysis applied to group and reveal differences or similarities of 13 FAs in this study in an objective manner. The cluster analysis showed that even if the other parameters were added to the parameters of atomic ratios (EA) and carbon distributions (NMR), the result were never changed. This means that information about chemical structural properties (EA, NMR) of the FAs have great influence on their grouping. The FAs were always classified into roughly three groups. Considering the environment of each sampling points, it can be said that first group (cluster 1) is composed of FAs mainly from small clear-water rivers, second group (cluster 2) is brown-water FAs, and third group (cluster 3) is composed of FAs mainly from large clear-water rivers and lakes. Differences between the two clear-water FA groups (cluster 1 and 3) may have a close relationship to their transition times in rivers or lakes. The former are from rushing rivers, therefore this group can be called “fleeting clear-water FAs” that have short residence time in the waters. The latter are from gentle rivers or lakes, therefore this group can be called “retaining clear-water FAs” that have long residence time in the waters. If residence time of water were longer, FA inflowing into the water would have more opportunities for contacting with the residues and the metabolites of aquatic microorganisms. That is to say, sufficient residence time of water would enhance influence from not terrestrial but aquatic origin on FA. From chemical structural data, Fujitake et al. (2009) suggested that FA from Lake Biwa that has long residence time of water (average of 5.5 years) is mainly derived from degraded and modified material of microbial residues and microbial metabolites, which support the prediction. Water temperatures or mineral conditions may also be important in the aquatic microbial activity. However, considering almost all clear-water FA samples were collected at the points never affected by anthropogenic inputs in autumn (Table 1), residence time of water would mainly define the microbial impact to the FA. The results of the principal component analysis revealed that Aryl-C and O-Alkyl-C contents were important for the grouping. We cannot refer to the relationship between microbial activity, residence time of water and Aryl-C, O-Alkyl-C contents in this study. If the proper parameters for evaluating transition time in rivers or lakes are found in the future and applied for the grouping, such a relationship may become clear.
5.
Conclusion
Clear-water FAs not only existed in lower concentrations than brown-water FAs, but also had the different chemical properties from them. Clear-water FAs exhibited higher proportion of alkyl carbons than brown-water FAs generally, which may be related to the microbial activities.
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 3 7 e3 8 4 6
Results of the elemental and the 13C NMR analyses were not always linked to those of the isotopic analyses, meaning that it may be difficult to decide origins or generation processes of clear-water FAs only by these analyses. Multivariate statistical analysis revealed spatial diversity of clear-water FAs. The FAs were always classified into two clear-water groups and one brown-water group, based on the difference of their chemical structures, especially Aryland O-Alkyl-C contents. The grouping of the FAs might depend on the differences of aquatic microbial activity caused by the differences of residence time of water. It may become possible in the future to evaluate a lot of environmental effects of FA more easily by investigating the functions not individually but as a group, applying the grouping method in this study.
Acknowledgements This work was partily supported by a Grant-in-Aid for Scientific Research (No. 16380049, 2004e2007) from the Ministry of Education, Science, Sports and Culture of Japan.
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