RESEARCH ARTICLE

Absorption and fluorescence characteristics of chromophoricdissolved organic matter in the Yangtze Estuary

Qiyuan Sun & Chao Wang & Peifang Wang & Jun Hou &

Yanhui Ao

Received: 24 June 2013 /Accepted: 24 October 2013 /Published online: 19 November 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The Yangtze Estuary is heavily influenced bycoast-continent geochemical processes and anthropogenic ac-tivity; thus, the source and distribution of chromophoric dis-solved organic matter (CDOM) in the estuary are stronglyimpacted by these processes. Here, a series of samples werecollected from across the Yangtze Estuary to investigate thesource and spatial dynamics of CDOM and its componentsthroughout the system. Three indices (a (355), spectral slope,and fluorescence) were then calculated and interpreted. Theresults indicated that the distribution of CDOM was dominat-ed by allochthonous input, conservative mixing, and phasetransfer. The contribution of biogenic CDOM to total CDOMincreased with salinity, and three individual CDOM compo-nents were identified upon fluorescence excitation emissionmatrix spectroscopy and parallel factor analysis of the watersamples: C1, corresponding to humic substance-like CDOM,C2, corresponding to tryptophan-like CDOM, and C3, corre-sponding to tyrosine-like CDOM. C1 primarily originatedfrom a terrestrial source, C2 had widespread origins, none ofwhich played a dominant role, and C3 mainly originated fromallochthonous input in the medium salinity area. Unexpected-ly, no marine humic-like component was found in the surfacewater of the Yangtze Estuary, possibly because turbidity

decreased the depth of sunlight penetration, limiting produc-tion of this component.

Keywords CDOM .Adsorption spectroscopy . EEM .

Fluorescence indices . PARAFAC . Yangtze Estuary

Introduction

Chromophoric (or colored) dissolved organic matter (CDOM)is defined as an important fraction of total dissolved organicmatter (DOM) that absorbs light in the UVand visible ranges(Coble 2007). Aquatic CDOM has been widely investigatedowing to its impacts on environmental geochemistry andaquatic toxicity (Huguet et al. 2009; Para et al. 2010). Manystudies have shown that CDOM alters the bioavailability andtoxicity of both metals and polycyclic aromatic hydrocarbonsin aquatic environments through ligands controlling the freeion concentration or partition coefficients (KDOC) (Akkanenet al. 2004; Wu et al. 2013). In addition, CDOM plays animportant role in controlling attenuation of UV radiation inaquatic environments (Para et al. 2010).

The Yangtze Estuary is one of the largest subtropical riverestuaries in the world. The estuary consists of a north branch,south branch, north channel, south channel, north passage,south passage, and areas of adjacent coastal water. The estuaryis not only a typical terrestrial–marine transition zone, but alsoan industrialized area. Thus, many factors (e.g., runoff, tides,freshwater–seawater mixing processes, topographic factors,urban sewage, and agricultural nonpoint pollution) stronglyinfluence the source and spatial distribution of CDOM in theestuary, which then affects the environmental geochemistryand leads to aquatic toxicity. Accordingly, it is necessary tostudy the properties of CDOM in the Yangtze Estuary tothoroughly understand these processes.

Responsible editor: Philippe Garrigues

Q. Sun : C. Wang : P. Wang : J. Hou :Y. AoCollege of Environment, Hohai University, Nanjing 210098, China

Q. Sun : C. Wang (*) : P. Wang : J. Hou :Y. AoKey Laboratory of Integrated Regulation and Resource Departmenton Shallow Lakes, Ministry of Education, Hohai University,Nanjing 210098, Chinae-mail: cwang@hhu.edu.cn

Present Address:C. WangXikang Road No.1, Nanjing, Jiangsu 210098, China

Environ Sci Pollut Res (2014) 21:3460–3473DOI 10.1007/s11356-013-2287-4

However, few studies of the characteristics of CDOMin the Yangtze Estuary have been conducted to date. Guoet al. (2007) studied the spectral absorption characteristicsof CDOM in the south branch of this estuary. Yang et al.(2007) focused on developing three cross sections in thesouth branch of this estuary to characterize the CDOMduring both high and low tide by analyzing fluorescencespectra and UV–vis absorption spectrometry. Althoughtheir results characterized the CDOM of this estuary tosome extent, some important regions (e.g., the northbranch and the seawater zone) were not included in theirstudy. Furthermore, some key aspects such as the sourceof the CDOM and spatial variations of the different com-ponents of the CDOM were not fully discussed. Therefore,it is important to investigate the characteristics of theCDOM in the Yangtze Estuary systematically and com-prehensively (Coble 2007).

The present investigation included the entire Yangtze Es-tuary. The specific objectives of this study were (a) to inves-tigate the distribution of CDOM in the subregions of theYangtze Estuary, especially the variations in CDOM duringmixing of fresh and marine waters, and (b) to analyze thesource of CDOM and variations in its components along thesalinity gradient.

Material and methods

Study area

Our study area included the entire Yangtze Estuary (includingthe adjacent coastal waters, which are part of the East ChinaSea). In this area, sand bars are well developed and the water isextremely turbid. A series of alluvial islands (ChongmingIsland, Changxing Island, Hengsha Island, and JiuduanshaIsland) are also present, which has a profound impact on theestuarine terrain. The study area is divided into threesalinity regions: a low-salinity region (salinity<5), amedium-salinity region (5<salinity<15), and a high-salinity region (salinity>15). The seawater zone includesthe area near sample site nos. 17 through 30, while thefreshwater zone refers to the remaining areas.

As one of China's developed regions, many industrialand urban centers are located along the Yangtze Estuary.In recent decades, high loads of anthropogenic nutrientshave been discharged into the estuary. Huangpu River,which is a highly polluted river that flows throughShanghai and empties into the Yangtze Estuary, is animportant source of dissolved organic matter that con-tributes to changes in the optical properties of the estu-arine water (Guo et al. 2007). Agricultural areas nearChongming Island are also an important source of non-point pollution in the estuary.

Sampling and pretreatment

Water samples were collected between 10 and 16 May, 2012.Figure 1 illustrates the sampling locations. Sample point nos.1–16 were located in the south branch, nos. 17–30 were locatedin the adjacent coastal water area, and nos. B1–B5were locatedin the north branch. The geographic coordinates and salinitylevels measured at these sampling points are shown in Table 1,and 35 samples were collected from each site. The salinity andturbidity were measured in situ at the time of collection using aYSI 6600 V2 Multi-Parameter Sonde.

All samples were filtered using GF/F filters (0.7 μm,Whatman) that had been pre-combusted by exposure to450 °C for 5 h. The filtrate was then collected into glass bottlesthat had been pre-combusted at 550 °C for 6 h and frozen at−20 °C until analysis, which was conducted within 2 weeks ofcollection. Immediately prior to analysis, the samples werewarmed to 21 °C in a water bath.

Optical measurements, fluorescence indices, and parallelfactor analysis modeling

CDOM absorption

Absorbance (D) was measured in a 5-cm quartz cell using aTU-1901 dual-beam UV–vis spectrophotometer with an ab-sorbance precision of ±0.003. Milli-Q water was used in thereference quartz cell. The absorption coefficients (a ) werecalculated between 240 and 800 nm at 1-nm intervals, andthe absorption coefficients at 700 nmwere subtracted from theabsorption coefficients at each wavelength (λ ) for baselinecorrection.

The following formula was used to calculate the absorptioncoefficients (Hu et al. 2002):

a λð Þ ¼ 2:303D λð Þ=r ð1Þ

where a (λ ) represents the CDOM absorption coefficient atwavelength λ , and r represents the cuvette path length, whichwas 0.05 m.

In this study, the absorption coefficient (meter) at a wave-length of 355 nm (a (355)) was used to represent the concen-tration of CDOM to facilitate comparison with other studies.

The spectral slope (S ) of the CDOM absorption curve wasobtained through use of a nonlinear least square regressionover a wavelength range of 300 to 500 nm (Stedmon et al.2000):

a λð Þ ¼ a λ0ð Þexp S λ0−λð Þð Þ þ K ð2Þ

where λ0 is a reference wavelength (here, 440 nm) and Krepresents the baseline shifts or attenuation due to factorsother than CDOM.

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CDOM fluorescence

The fluorescence was measured using a Hitachi F-7000fluorescence spectrophotometer to obtain the three-dimensional excitation emission matrix (EEM) spectra.The PMT voltage was 700 V. Emission spectra werecollected at 2-nm intervals between 250 and 500 nm,while excitation spectra were measured between 200 and420 nm at 5-nm intervals. The band pass widths were10 nm for emission and 5 nm for excitation and the scanspeed was 1,200 nm min−1. A Milli-Q water blank wasanalyzed in the EEM spectrum to eliminate peaks due toRaman scattering in water (Coble et al. 1998). Prior tomeasurement, the water samples needed to be dilutedusing Milli-Q water to minimize the fluorescence innerfilter effect (Ohno 2002). The fluorescent intensities wereconverted to quinine sulfate units (QSUs), where 1 QSUis equal to the fluorescence emission at 350/450 nm(exCitation/emission) of 1 μg L−1 quinine sulfate in a0.1 M H2SO4 solution (pH=2).

Fluorescence indices

Three fluorescence indices (fluorescence index, humificationindex, and biological index) were used to further explore theorigins of the CDOM. The fluorescence index (FIX) can beused to distinguish the derivation of the CDOM. For thisanalysis, FIX values of 1.4 or less correspond to terrestrial-derived CDOM, while values of 1.9 or greater refer tomicrobially derived CDOM. If the values of FIX are between1.4 and 1.9, the components of the CDOM are affected byboth terrigenous and biogenic DOM (McKnight et al. 2001).However, this index cannot discern the relative contributionsof allochthonous and autochthonous substances. The follow-ing formula was used to calculate the fluorescence index:

FIX ¼ I370=450=I370=500 ð3Þ

where I370/450 represents the fluorescence intensity at excitation/emission wavelengths of 370/450 and I370/500 is the fluorescenceintensity at excitation/emission wavelengths of 370/500.

Fig. 1 Location of samplingstations in Yangtze Estuary

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The humification index (HIX) can be used to assess thedegree of CDOM humification. A high HIX value indicates ahigh degree of CDOM humification. The HIX was calculatedas follows (Zsolnay et al. 1999):

HIX ¼X

I435→480=X

I300→345 ð4Þ

where ∑I x→y is the sum of the fluorescence intensity atemission wavelengths x→ynm at an excitation wavelengthof 255 nm.

The biological index (BIX) can be used to estimate therelative contribution of biogenic CDOM (Huguet et al. 2009).A BIX value greater than 0.8 indicates that CDOM is mainlyderived from microbial and other biological sources, whereasa value below 0.6 indicates a low amount of biogenic CDOM.The formula for calculating BIX is as follows:

BIX ¼ I310=380=I310=430 ð5Þ

where I 310/380 is the fluorescence intensity at excitation/emission wavelengths of 310/380 and I 310/430 is thefluorescence intensity at excitation/emission wavelengthsof 310/430.

Parallel factor analysis modeling

Parallel factor analysis (PARAFAC) is used here to removethe overlapping effects between various fluorescence peaksand decompose the EEMs into individual components. Thefluorescence of each component is represented by the maxi-mum fluorescence intensity, Fmax (Stedmon and Markager2005). A split-half validation procedure was used to determinethe number of components (Stedmon and Bro 2008).

Parallel factor analysis is a form of bilinear principal anal-ysis generalized to a higher-order array that decomposes N-way arrays into N loading matrices (Wu et al. 2011). There-fore, the fluorescent spectra of CDOM in water samples,which are expressed as a three-dimensional EEM, can bearranged in a three-way array (H ) of dimensions I ×J ×K . Inthis array, I represents the number of samples, while J and Kare the number of emission wavelengths and excitation wave-lengths, respectively. H is then decomposed into three matri-ces, X (the score matrix), Y, and Z (loading matrices), withelements x if, y jf, and zkf (Bro 1997; Stedmon and Bro 2008).

MATLAB R2010a (Mathworks, Natick, MA) with the tool-box DOMFluor (www.models.life.ku.dk) was used to conductthe PARAFAC analysis. Given the actual situation, a non-negativity constraint was applied to the parameters. In addition,some pretreatment was carried out to eliminate the influence ofscattering peaks. Specifically, Raman scattering was removed bysubtracting the EEM spectrum of the Milli-Q water blank, andRayleigh scattering was removed using interpolation (Bahramet al. 2006). The toolbox EEMscat was used to remove Rayleighscattering (www.models.life.ku.dk/algorithms).

Results

Distribution of DOC, CDOM, and the adsorption spectralslope

The values of DOC and CDOMvaried between sampling sites(Fig. 2a, b), but generally decreased from the upstream part of

Table 1 Geographic coordinates and salinity of the 35 samples collectedin the Yangtze Estuary

Sample # Latitude (N) and longitude (E) Salinity (‰)

1 121°01′54.7″, 31°46′26.8″ 0.13

2 121°07′50.2″, 31°44′42.7″ 0.13

3 121°16′10.8″, 31°38′46.9″ 0.13

4 121°24′43.4″, 31°32′25.5″ 0.12

5 121°37′48.7″, 31°29′53.8″ 0.12

6 121°46′40.0″, 31°25′22.5″ 0.12

7 121°57′18.2″, 31°22′53.2″ 0.12

8 122°06′45.8″, 31°22′13.8″ 0.12

9 121°32′27.6″, 31°24′15.3″ 0.12

10 121°41′30.4″, 31°19′47.9″ 0.18

11 121°48′39.7″, 31°16′59.8″ 0.13

12 121°56′49.7″, 31°15′38.6″ 0.18

13 122°05′35.7″, 31°13′33.5″ 0.22

14 121°51′19.7″, 31°11′04.0″ 0.22

15 121°57′28.9″, 31°04′47.4″ 0.19

16 122°01′12.6″, 30°58′07.6″ 0.20

17 122°01′43.0″, 31°34′18.9″ 12.44

18 122°10′41.1″, 31°33′29.5″ 17.35

19 122°16′45.7″, 31°34′04.4″ 5.71

20 122°27′06.8″, 31°34′03.2″ 17.97

21 122°20′12.0″, 31°22′02.2″ 16.73

22 122°29′11.0″, 31°21′33.2″ 25.67

23 122°22′03.9″, 31°09′01.5″ 19.47

24 122°32′22.7″, 31°07′40.7″ 29.41

25 122°23′57.4″, 30°57′59.3″ 21.44

26 122°36′25.9″, 30°58′25.2″ 28.72

27 122°01′55.4″, 30°49′59.8″ 15.43

28 122°12′28.4″, 30°50′00.1″ 23.69

29 122°22′09.0″, 30°48′58.0″ 25.63

30 122°32′47.0″, 30°48′52.0″ 22.7

B1 121°17′13.9″, 31°52′22.1″ 0.17

B4 121°25′12.1″, 31°49′44.5″ 0.22

B2 121°33′16.5″, 31°45′49.0″ 1.83

B5 121°40′27.5″, 31°42′35.6″ 3.76

B3 121°52′36.0″, 31°38′11.2″ 7.80

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the estuary to the coastal water. In addition, the DOC valueswere significantly higher in the freshwater zone than in theseawater zone (t text, p <0.001). The maximum value wasobserved in the north branch (sampling site 3), while theminimum value was observed in the seawater zone of theestuary (sampling site 24). The maximum DOC value was3.03 times higher than the minimum value.

The variation in a (355) differed from that of DOC(Fig. 2b). Specifically, the highest value of a (355) was ob-tained in the south channel (sampling site 10) near the Huang-pu River outlet, while the lowest value was observed in theseawater zone (sampling site 26). The maximum value was28.2 times higher than the minimum value, indicating a pro-nounced decreasing trend (t text, p <0.001).

There was a significantly negative correlation betweena(355) and the absorption spectral slope (S)(r2=0.526, p <0.001) (Fig. 3a). The variable trend of S is shown in Fig. 2c.Clearly, there were significantly higher values of S in thefreshwater zone than in the seawater zone (t text, p <0.001).The maximum and minimum values were obtained at sampling

sites 24 and 10, respectively, and the maximum value wasapproximately 1.3 times higher than the minimum value.

Table 2 presents the regional distribution and mean valuesof the three indices and the subregional values (freshwaterzone and seawater zone) in the Yangtze Estuary.

Fluorescence indices

Figure 4 shows the spatial variations of FIX, BIX, and HIX.The FIX values in the freshwater zone were clearly muchlower than in the seawater zone; however, the areas nearsampling sites 2 and 10 did not exhibit the expected lowvalues, but instead showed high values of 2.22 and 2.08,respectively. The trend in BIX is similar to that of FIX;however, the BIX value near sampling site 2 was not differentfrom that of the surrounding area, and its value was 1.26 atsampling site 10. The HIX values in the freshwater zone werehigher than those in the seawater zone, except for areas nearsampling site 10 and the south passage (sampling sites 14, 15,and 16), where the values are much lower than those in any

Fig. 2 Spatial distribution of a DOC (μM), b a(355) (m-1), and c S (μm-1)

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other area in the freshwater zone. The HIX values at samplingsites 10, 14, 15, and 16 were 0.396, 0.625, 0.696, and 0.726,respectively. The mean values and the regional values of thethree indices in the freshwater zone, seawater zone, and theentire estuary are shown in Table 3.

Fluorescence indices are expressed in arbitrary units. Sub-panels a, b, and c of Fig. 4 show the spatial distributions ofFIX, BIX, and HIX, respectively.

3D-EEM fluorescence spectral characteristics of CDOM

The bands of the various fluorescence peaks are listed inTable 4 (Coble 1996). To simplify the listing, each type ofpeak is abbreviated as a unique letter. The four main fluores-cence peaks (UV fulvic acid, visible-band fulvic acid, high-excitation tryptophan, high-excitation tyrosine) are designatedas peak A, peak C, peak T, and peak B, respectively (Coble

Fig. 3 Adsorption and fluorescence index analysis chart. Graphs a–bshow the correlation between two parameters: a S versus a(355); b DOCversus a(355). R2 refers to the coefficient of determination of the fitcurve. Graphs c–d show the variation of parameters versus salinity: ca(355) and S versus salinity (superimposed is the modeled behavior of

a(355) (straight line) and S (curved line) under conservation mixing ofthe freshwater and seawater zones identified); d three fluorescence indi-ces (FIX, BIX, and HIX) versus salinity (superimposed are the fittingcurves of the three fluorescence index scatter points)

Table 2 The descriptive statisticsfor values of DOC, a(355), and Sfor two parts of sites (freshwaterzone and seawater zone) in theYangtze Estuary

All Freshwater end member Seawater end member

Min–max Mean ± SD Min–max Mean ± SD Min–max Mean ± SD

DOC 41.71–126.19 87.34±21.36 80.09–123.47 96.81±13.62 41.71–126.19 79.36±23.67

a(355) 0.10–2.82 1.20±0.73 0.80–2.82 1.60±0.50 0.10–2.25 0.86±0.74

S 16.82–20.00 18.38±0.68 16.82–18.64 17.95±0.44 17.78–20.00 18.75±0.64

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1996; Coble et al. 1998). Among these, the UV fulvic acid andvisible-band fulvic acid are humic substance-like CDOM,while high-excitation tryptophan and high-excitation tyrosineare protein-like CDOM.

Based on the literature, the CDOM that produced the peaksdescribed above originated from different sources. High-excitation tryptophan could originate from both autochtho-nous and allochthonous sources. Unlike the high-excitationtryptophan, high-excitation tyrosine always originates fromsediment and is seldom found in river runoff. The UV fulvicacid and visible-band fulvic acid CDOM could be derivedfrom terrestrial autochthonous sources, as well as biogeo-chemical processing of terrestrial particulate organic matter.These two humic substance-like types of CDOM always have

higher concentrations in freshwater than in seawater (Stedmonand Markager 2005; Coble et al. 1998; Coble 1996).

Given the large number of sampling points and overlapsbetween fluorescence peaks, it is difficult to distinguish thereal peaks and their fluorescence intensities in the raw EEMcontour plots. Therefore, PARAFACwas employed to decom-pose the raw EEM contour plots into a series of fluorescentcomponents and obtain the Fmax of each sample. These resultswere then used to analyze the characteristics of the surfacewater CDOM in the various subregions of the estuary.

The EEMs could be decomposed into three componentsusing the PARAFAC models and split-half validation. Figure 5presents the spectral characteristics of the three fluorescent com-ponents decomposed using PARAFAC. To avoid the

Fig. 4 Spatial distribution of a FIX, b BIX and c HIX

Table 3 The descriptive statisticsof FIX, BIX, and HIX for twoparts of sites (freshwater zone andseawater zone) in the Yangtzeestuary

All Freshwater end member Seawater end member

Min–max Mean ± SD Min–max Mean ± SD Min–max Mean ± SD

FIX 1.67–2.28 1.85±0.15 1.67–2.22 1.80±0.14 1.71–2.28 1.90±0.15

BIX 0.88–1.68 1.16±0.23 0.92–1.26 1.02±0.08 0.88–1.68 1.28±0.24

HIX 0.40–1.86 1.10±0.39 0.40–1.63 1.14±0.38 0.52–1.86 1.07±0.40

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interference of noise signals, the fluorescence below 250 nm(exCitation wavelengths) and 300 nm (emission wavelengths)was not considered for further analysis (Stedmon and Markager2005).

The three CDOM components are designated component 1(C1), component 2 (C2), and component 3 (C3). The contourplots of the EEMs of the three components are shown inFig. 5a–c, while Fig. 5d–f presents the excitation (left lines)and emission (right lines) loadings of the components. The ex/em peak wavelengths of C1 through C3 are 250 (330)/430,275/340, and 270/300 nm, respectively. The wavelength in theparentheses represents the longer peak excitation wavelengthas the component contour plot showed two fluorescencepeaks.

Comparison of the ex/em values in Table 4 reveals that C1corresponds to UV fulvic acid and visible-band fulvic acid(humic substance like) CDOM (peaks A and C), C2 corre-sponds to high-excitation tryptophan (protein like) CDOM(peak T) and C3 corresponds to high-excitation tyrosine (pro-tein like) CDOM (peak B) (Coble 1996).

Discussion

The relationship between CDOM and DOC

In the Yangtze Estuary, a (355) and DOC showed a linearrelationship (Fig. 3b), indicating that CDOM is an importantcomponent of DOC (Gao et al. 2010). When a (355) is equalto zero, the value of DOC is positive, indicating that there aresome components of DOC that are not related to the CDOMdynamics, but are instead related to uncolored dissolved or-ganic matter (UDOM).

The distribution of CDOM in the Yangtze Estuary

As shown in Fig. 2b, the values of a (355) in the freshwaterzone are much higher than those in the seawater zone ingeneral, indicating that the freshwater zone has a higher levelof CDOM than the seawater zone. However, CDOM is widelydistributed in the freshwater zone, while in the seawater zone,it exhibits a banded distribution. This pattern occurs because

Table 4 Major bands of CDOMfluorescence peaks for surfacewater (Coble 1996; Coble et al.1998; Stedmon and Markager2003; Stedmon and Markager2005)

Peak Compound type Excitation max. (nm) Emission max. (nm)

A UV fulvic acid 230–260 380–480

C Visual fulvic acid 320–360 420–460

T High-excitation tryptophan 270–280 320–350

B High-excitation tyrosine 270–280 300–310

M Marine humic like 310–315 380–420

Fig. 5 Spectral characteristics ofthe three fluorescent componentsidentified by PARAFAC. Graphsa–c show the EEM contours ofindividual components; graphsd–f present split-half validationresults of three components; theexcitation (left lines) andemission (right lines) loadings ofthe components are estimatedfrom two independent halves ofthe dataset (red and olivaceouslines). The standard of a perfectvalidation is that the loadingsfrom the two halves are identical.The fluorescence intensities of thethree components are expressedas quinine sulfate units (QSU)

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the distribution of CDOM in the freshwater zone is stronglyinfluenced by discharge of wastewater input along the banksof the Yangtze River (e.g., Huangpu River). The HuangpuRiver is a heavily contaminated river that contains high loadsof urban sewage from Shanghai, where a great deal of protein-like CDOM is produced. The water at sampling site 10, whichis near the outlet of the Huangpu River, is highly polluted andhas the highest content of CDOM in the entire estuary. Theseresults are similar to those obtained by Zhu et al. (2010).

The distribution map, mixing curve of a (355), and adsorp-tion spectral slope are used as the basis for a discussion of thedistribution of CDOM in the seawater zone. The mixing curveof a (355) is shown in Fig. 3c. The straight line between thefreshwater zone and the seawater zone is the theoretical dilu-tion line. It is evident that most of the points fall on the linewhen the salinity is above 15. This trend indicates that CDOMis well conserved among most sampling sites and is wellcorrelated with salinity in the seawater area. The main sourceof CDOM in the seawater area comes from water in thefreshwater area of the estuary, whereas CDOM generated inthe seawater zone contributes little to the total CDOM. Thus,decreases in the CDOMvalues in the seawater area are mainlydue to the freshwater–seawater mixing process.

The data from the two points at the freshwater–seawaterinterface (sampling sites B3 and 17, situated in the northbranch of the estuary) markedly deviate from the theoreticaldilution line (with values lying above the line), indicating thatthere are other CDOM sources in these areas in addition to thefreshwater area. One possible source is the exogenous inputfrom Qidong, as the two points are close to the outlet of thenorth branch near Qidong, which is an industrialized city. Thepresence of the estuarine turbidity maximum (ETM) may alsobe a factor leading to this phenomenon, as the suspendedsediments promote phase transformation of organic matterfrom particulate matter to the aqueous phase (Blough et al.1993). The salt marshes in Dongtan on Chongming Island areanother possible source of the CDOM (Coble 2007; Gao et al.2011).

It was found that there is a negative correlation between thevalue of S and the molecular weight of CDOM (Carder et al.1989). Figure 3c shows the trend of S with salinity variations.The S was lower in the freshwater area and higher in theseawater area and seawater–freshwater mixing area. In addi-tion, the values of S increased with salinity; thus, a highersalinity was associated with a smaller molecular weight ofCDOM. This trend coincides with the freshwater–seawatermixing process of the CDOM. Overall, these findings indicatethat decreases in the molecular weight of the CDOMprimarilyresult from the freshwater–seawater mixing process. In addi-tion, the values of S at sampling sites B3 and 17 were higherthan the regression curve of S , indicating that the molecularweights of CDOM at these two sites are smaller than those inareas only affected by the freshwater–seawater mixing

process. Hence, there might be a certain amount of exogenouslow molecular weight matter being input into the area nearsites B3 and 17. This conclusion is in agreement with theearlier discussion. Furthermore, these results indicate that theCDOM released from the ETM and Dongtan marshes as wellas city sewage might consist of lowmolecular weight CDOM.

In summary, the mixing process between freshwater andseawater is the determinant factor in the distribution ofCDOM in the seawater zone of the estuary. The CDOM fromthe freshwater zone is the main source of CDOM in theseawater zone of the estuary. Moreover, the allochthonousCDOM (from the ETM, salt marshes, and city sewage) mayincrease the low molecular weight components of the CDOMin the freshwater–seawater transition zone.

Sources and component transformation of CDOM

To assess the origin and degree of transformation of CDOM inthe Yangtze Estuary, three fluorescence indices were used toextract information from the 3D-EEM fluorescence spectrum.

As shown in Fig. 4a, the values of FIX decrease from thefreshwater zone to the seawater zone. However, no conclusionregarding FIX can be derived from Fig. 3d. Huguet et al.(2009) reported that flocculation of humic substances withincreasing salinity may lead to a bathochromic shift, whichthen affects the FIX value. Thus, it is difficult to use FIX todetermine the source of CDOM in the seawater zone of anestuary.

However, it is feasible to use FIX in the freshwater zone ofan estuary. The FIX values near the outlet of the HuangpuRiver (sampling site 10) and the west side of ChongmingIsland (sampling site 2) were higher than those in the adjacentareas in the freshwater zone. This phenomenon may havebeen due to the input of CDOM from adjacent land. The westend of Chongming Island and its adjacent area, which belongto Jiangsu Province, are mainly agricultural areas. The organicfertilizer used in this area is mainly microbial-derived andalways flows into the adjacent water with each rainfall runoffevent. Sampling site 10, which is close to the outlet of theHuangpu River, receives discharges of wastewater and sew-age that contain abundant microbial-derived CDOM; there-fore, the FIX values are high in these areas.

The BIX values (Fig. 4b) increase from the freshwater zoneto the seawater zone in general, which coincides with the trendshown in Fig. 3d (in which the values of BIX increase linearlywith salinity), indicating that the relative contribution of bio-genic CDOM grows with increasing salinity. This may havebeen due to the allochthonous CDOM from the upstream partsof the Yangtze River, which decreases with the freshwater-seawater mixing process, while the relative amount of biogen-ic CDOM increases. The BIX value at sampling site 10 ishigher than the values in adjacent areas, indicating that a large

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portion of components in this area are composed of newbiogenic CDOM from the Huangpu River.

The HIX values (Figs. 4c and 3d) in the freshwater zone aremuch higher than those in the seawater zone and decrease assalinity increases, demonstrating that CDOM in the freshwaterzone has a higher degree of humification than that in theseawater zone in general. The explanation for this findingmay be the dilution of freshwater in the freshwater–seawatermixing area (freshwater with high-humification CDOM isdiluted by seawater with low-humification CDOM) andphotobleaching in the high-salinity area (Vodacek et al.1997). The high HIX value near sampling site 10 may bedue to a contribution from allochthonous CDOM that is of lowhumification.

The three fluorescence indices reveal a great deal of infor-mation regarding the origin and transformation of the CDOM.However, their indices represent raw data and cannot explainthe sources of the various CDOM components and theirdistributions. The 3D-EEM fluorescence spectral characteris-tics of the CDOM coupled with PARAFAC and split-halfvalidation are employed here to further study the sourcesand component transformation of CDOM.

Figure 6 displays the Fmax distribution of the three FDOMcomponents in the Yangtze Estuary. As shown in Fig. 6a, C1has higher values in the freshwater area of the estuary than inthe seawater area. When combined with the ex/em peakwavelengths in the PARAFAC decomposed contour plot(Fig. 5a), these results indicate that C1 primarily representstwo types of terrestrial humic-like substances. River flow isthe main source of C1. In addition, there are four areas withhigher Fmax in the freshwater area than in the surroundingarea, and these areas are always near the outlets ofanabranches or tidelands. Thus, anabranch input and tidelandsare also important sources of C1.

Because it is a protein-like CDOM, the distribution of C2differs from that of the other components of the CDOM.Specifically, C2 is widely distributed across the entire estuary,indicating that it has widespread sources (e.g., seawater,suspended sediment, and terrestrial water; see Fig. 6b)(Holbrook et al. 2006; Coble et al. 1998). The outlet of theHuangpu River near the upstream end of the estuary shows asignificantly higher value of C2 than other upstream areas,indicating that urban sewage is also an important source of C2.

In general, the Fmax value of C3 is lower in the upstreamparts of the estuary and the seawater zone, while it is higher inthe medium-salinity areas (see Fig. 6c). It is worth noting thathigher values of C3 are always distributed in the ETM of theestuary and the outlet of Huangpu River, indicating that C3primarily originates from resuspended sediment and urbansewage.

The relationship between Fmax values in the PARAFACcomponents and salinity can be used to determine whethervarious DOM components exhibit conservative behavior

during the freshwater–seawater mixing process. Figure 7 dis-plays the relationships between the Fmax of the three compo-nents and salinity. As shown in Fig. 7a, the C1 (UV fulvic acidand visible-band fulvic acid) vs. salinity relationship is linear,and nearly all points follow the conservation mixing line.Therefore, C1 displays conservative behavior in the seawaterarea, indicating that it primarily originates from the freshwaterarea, whereas the medium-salinity area and the seawater zonecontribute little to this component. In contrast, the relation-ships between the Fmax of C2 and C3 and salinity (Fig. 7b, c)appear to show non-conservative mixing behavior (Stedmonand Markager 2003). The Fmax of C2 shows a fluctuation inthe medium- and high-salinity areas, indicating that the distri-bution of C2 is not dominated by the freshwater–seawatermixing process. Thus, C2 (tryptophan-like CDOM) likelyhas widespread sources from terrestrial input of protein-likeCDOM and protein-like CDOM produced in the medium-salinity areas and seawater zone.

The Fmax of C3 (tyrosine-like CDOM) increases signifi-cantly in the medium-salinity area and decreases to a constantvalue in the seawater area, indicating that the medium-salinityarea is a primary source of C3, while any protein-like CDOMproduced in marine water makes little contribution.

The medium-salinity area is an important source of the twoprotein-like CDOM components, but is not a main source ofthe humic-substance-like CDOM component. This may re-flect allochthonous sources of protein-like CDOM in themedium-salinity areas (salinity 5–15) and chemical/biological conversion, phase transfer in the north branchETM of the estuary (Shen and Pan 2001). At the ETM, largenumbers of bacteria attach to the particles. A high suspendedparticulate matter content promotes the adsorption and desorp-tion process of CDOM between the aqueous phase and parti-cle phase, which then raises the levels of protein-like CDOM(Huguet et al. 2009). Conversely, the transformation betweenUDOM and CDOM always occurs in a complex environment.In addition, the wastewater discharges from Qidong are animportant exogenous source of protein-like CDOM (Huguetet al. 2009).

The relationship between the two protein-like types of theCDOM and salinity differs from the relationship betweena (355) and salinity because a (355) only reflects the totalamount of the CDOM, while it cannot reflect the componentsof the CDOM. The protein-like CDOM also comprises alower proportion of the total CDOM. Thus, although thelevels of protein-like component CDOM increase in themedium-salinity area, the total amounts of CDOM decrease.

No marine humic-like component (peak M identified byCoble (1996)) was identified in the present study, which wassurprising because many previous studies of coastal waterCDOM revealed the presence of this component (Coble1996; Gueguen et al. 2011). According to the results ofprevious studies, this component is always produced by

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Fig. 6 Spatial distribution of theFmax (in QSU) for three CDOMcomponents (C1–C3, labeled a–c)

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Fig. 7 Relationships betweenFmax of individual components(C1, C2, and C3) and salinity(graphs (a–c))

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microbial oxidation and phytoplankton degradation (Cory andMcKnight 2005). Therefore, the absence of peak M from theYangtze Estuary might be because the highly turbid waterdecreased the depth of sunlight penetration, which then de-creased the biomass of the phytoplankton and microbial deg-radation activity.

Conclusions

The Yangtze Estuary is a representative area in which manyfactors such as geochemical and physical mixing processesand wastewater deeply influence the source and distribution ofCDOM. This study revealed that most of the DOC consistedof CDOM, and the distribution of CDOM in the freshwaterzone was dominated by the release of both urban sewage andagricultural nonpoint pollution. In contrast, in the seawaterzone, the distribution was primarily affected by the dilutionprocess. Moreover, the contribution of biogenic CDOM to thetotal CDOM increased with salinity and three PARAFACcomponents (humic substances-like, C1, tryptophan-like,C2, and tyrosine-like, C3) were the main fractions of CDOMin the surface water of the estuary. The distributions of thethree components and the relationships between componentsof CDOM and salinity indicate that the humic substances-likeCDOM mainly originate from a terrestrial source. Thetryptophan-like CDOM has widespread sources in the seawa-ter area (salinity >5), including ETM release and marinesource CDOM; however, neither of these sources were dom-inant. The main source of tyrosine-like CDOM was found tobe phase transfer in the ETM and urban sewage discharges,while terrestrial and marine source CDOMs contribute little.The absence of a marine humic-like component from theYangtze Estuary may be due to limited sunlight penetrationcaused by high turbidity.

Acknowledgments This work was supported by the National KeyBasic Research Development Program (“973” project) of China (no.2010CB429006), China National Funds for Distinguished YoungScientists (51225901), Jiangsu Province Funds for DistinguishedYoung Scientists (BK2012037), and QingLan Project of JiangsuProvince.

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