water research 43 (2009) 4685–4697

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The contribution of phytoplankton degradation

to chromophoric dissolved organic matter (CDOM)
in eutrophic shallow lakes: Field and experimental evidence

Yunlin Zhanga,b,*, Mark A. van Dijkb, Mingliang Liua, Guangwei Zhua, Boqiang Qina
a
Taihu Lake Laboratory Ecosystem Research Station, State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography
and Limnology, Chinese Academy of Sciences, 73 East Beijing Road, Nanjing 210008, China
b
Netherlands Institute of Ecology (NIOO-KNAW), Centre for Limnology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands

article info abstract

Article history: Eight field campaigns in the eutrophic, shallow, Lake Taihu in the summers from 2005 to 2007,
Received 20 April 2009 and a phytoplankton degradation experiment of 33 days, were carried out to determine the
Received in revised form contribution of phytoplankton degradation to CDOM. Significant and positive correlations
17 July 2009 were found between the CDOM absorption coefficient at 355 nm [aCDOM(355)], normalized
Accepted 21 July 2009 fluorescence emission (QSU) at 450 nm from excitation at 355 nm [Fn(355)], and the chlorophyll
Published online 25 July 2009 a (Chla) concentration for all eight field campaigns, which indicates that the decomposition
and degradation of phytoplankton is an important source of CDOM. In the degradation
Keywords: experiment, the CDOM absorption coefficient increased as phytoplankton broke down during
Chromophoric dissolved the first 12 days, showing the production of CDOM from phytoplankton. After 12 days,
organic matter aCDOM(355) had increased from the initial value 0.41  0.03 m1 to 1.37  0.03 m1 (a 234%
Eutrophic shallow lake increase), and the Chla concentration decreased from the initial value of 349.1  11.2 mg/L to
Phytoplankton 30.4  13.2 mg/L (a 91.3% decrease). The mean daily production rate of CDOM from phyto-
Parallel Factor Analysis plankton was 0.08 m1 for aCDOM(355). Parallel Factor Analysis (PARAFAC) was used to assess
CDOM composition from EEM spectra, and four components were identified: a terrestrial-like
humic component, two marine-like humic components, and a protein-like component. The
rapid increase in marine-like humic fluorophores (C3 and C4) during the degradation experi-
ment suggests that in situ production of CDOM plays an important role in the dynamics of
CDOM. The field campaigns and experimental data in the present study show that phyto-
plankton can be one of the important CDOM producers in eutrophic shallow lakes.
ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction to living organisms. The absorption of ultraviolet radiation, in

The light absorbing component of the dissolved organic matter
(DOM) pool, known as chromophoric dissolved organic matter
(CDOM), can significantly influence the underwater light field.
CDOM strongly absorbs ultraviolet light, restricting the pene-
tration depth of UV-B radiation (280–320 nm), which is harmful
turn, causes CDOM photobleaching (decrease of absorption
coefficient) which subsequently increases the penetration of
ultraviolet radiation, and the release of many carbon and
nitrogen photoproducts which serve as biological substrates
and sources (Moran et al., 2000; Stedmon et al., 2007). Further-
more, CDOM absorption overlaps that of phytoplankton and

* Corresponding author at: Taihu Lake Laboratory Ecosystem Research Station, State Key Laboratory of Lake Science and Environment,
Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, 73 East Beijing Road, Nanjing 210008, China. Tel.: þ86 25
86882198; fax: þ86 25 57714759.
E-mail address: ylzhang@niglas.ac.cn (Y. Zhang).
0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2009.07.024
4686 water research 43 (2009) 4685–4697
non-algal particulate matter in the blue part of the visible light
region, which affects the primary productivity of waterbodies,
confounds remote sensing estimation of phytoplankton
biomass and total suspended matter concentration (Carder
et al., 1991; Doxaran et al., 2002). Therefore, CDOM plays a key
role in a broad range of processes and climate-related biogeo-
chemical cycles in aquatic ecosystems, affecting carbon
dynamics, nutrient availability, phytoplankton activity, micro-
bial growth, and ecosystem productivity.
Although the importance of CDOM distribution and cycling
in aquatic ecosystems has been recognized, the sources, trans-
port, and transformation of CDOM are not well understood. In
general, CDOM in aquatic ecosystems derives from two types of
sources: allochthonous and autochthonous. A major source of
allochthonous CDOM is terrestrially derived humic substances
in runoff, and some recent studies have also focused on CDOM
in rainwater (Kieber et al., 2006; Miller et al., 2009). For autoch-
thonous CDOM, important contributors are phytoplankton,
submerged aquatic vegetation, seagrass, and marsh- and
mangrove-derived CDOM (Yamashita and Tanoue, 2004; Wada
et al., 2007; Wang et al., 2007; Henderson et al., 2008; Tzortziou
et al., 2008). A number of physical, chemical and biological
processes can produce CDOM or affect its concentration in the
water column, including release from the sediment (Burdige
et al., 2004), the loss caused by photochemical degradation
(Moran et al., 2000; Tzortziou et al., 2007), products of
zooplankton grazing, bacterial release and uptake, and viral
interactions (Moran et al., 2000; Steinberg et al., 2004).
River input is generally assumed to be the major source of
CDOM in coastal and lake waters. Some studies, however,
have shown that phytoplankton degradation may also be an
important source of CDOM in ocean and coastal waters
(Yamashita and Tanoue, 2004; Vantrepotte et al., 2007; Wada
et al., 2007). Although Rochelle-Newall and Fisher (2002a)
presented little evidence to support phytoplankton as a direct
source of CDOM fluorescence in laboratory incubation
experiments, a significant positive correlation was found
between CDOM absorption and chlorophyll a in field studies in
summer (the phytoplankton growth season), indicating that
phytoplankton accumulation and decomposition were
a contributing source of CDOM (Rochelle-Newall and Fisher,
2002b). However, there are relatively few reports about the
contribution of phytoplankton degradation to CDOM in lake
environments (Hanamachi et al., 2008).
Given that phytoplankton cells are permeable to a variety
of organic compounds, production of CDOM by phytoplankton
blooms is likely an important source of the accumulating
CDOM in eutrophic lakes. In this study, we describe results
from field studies in summer, and from laboratory degrada-
tion experiments using Microcystis, to demonstrate the
contribution of phytoplankton degradation to CDOM in
eutrophic shallow lakes.
2. Materials and methods
2.1. Field study
Lake Taihu (30 550 4000 –31 320 5800 N, 119 520 3200 –120 360 1000 E), is
a large eutrophic shallow lake in China (Zhang et al., 2009),
with CDOM characterized by high temporal and spatial vari-
ability, due to the surrounding complex network of 172 rivers
and channels, frequent algal blooms and sediment
resuspension.
Eight field campaigns were carried out in summer
(June–August) from 2005 to 2007, and 203 samples were
collected from different lake regions and ecological types
(algal-dominated, macrophyte-dominated and transition
regions). In order to discuss the spatial difference of CDOM
absorption, all sampling sites were divided into two cate-
gories. Category 1 included a total of 151 sites distributed over
phytoplankton dominated, macrophytes dominated and
transitional zones. Category 2 included a total of 52 sites near
shore affected by inflowing rivers. Surface water samples
(0.5 m) were collected in 2 L acid-washed bottles, and held on
ice while in the field. The water samples were first filtered over
a pre-combusted Whatman glassfiber GF/C filter (B 47 mm),
followed by filtration over a 0.22 mm Millipore filter (B 25 mm),
precleaned using Milliq water, to obtain the CDOM fraction.
The absorption spectra of the filtered water were measured in
a 4 cm quartz cuvette between 240 and 800 nm at 1 nm
intervals, using a Shimadzu UV-2401PC UV–vis spectropho-
tometer. Milliq water was used as reference.
The absorption coefficients of CDOM were obtained as
follows (Bricaud et al., 1981):
aCDOM ðl0 Þ ¼ 2:303ODðlÞ=r (1)
where aCDOM(l0 ) is the uncorrected CDOM absorption coeffi-
cient at wavelength l, OD(l) is the optical density at wave-
length l, and r is the path length in m. A wavelength depended
correction for backscattering by small particles and colloids
that pass through filters was applied to the absorption coef-
ficients, using the following equation (Bricaud et al., 1981):
aCDOM ðlÞ ¼ aCDOM ðl0 Þ  aCDOM ð7000 Þðl=700Þ (2)
where aCDOM(l) is the absorption coefficient at a given wave-
length (l) corrected for scattering, aCDOM(l0 ) is the uncorrected
absorption coefficient at a given l, and aCDOM(7000 ) is the
uncorrected absorption coefficient at 700 nm. Because of the
chemical complexity of CDOM, the concentration of CDOM is
expressed using the absorption coefficient at 355 nm.
The spectral slope for the interval of 300–500 nm (S300–500)
was determined by nonlinear regression (Matlab software)
using the following equation.
aCDOM ðlÞ ¼ aCDOM ðl0 Þ exp½Sðl0  lÞ (3)
where aCDOM(l) and aCDOM(l0) are the absorption coefficients
at wavelengths l and l0, respectively, and S is the exponen-
tial spectral slope. The use of different methods for calcu-
lating spectral slopes (e.g., nonlinear vs linear fitting,
different spectral ranges) results in differentiating CDOM
sources and composition (Twardowski et al., 2004). In order
to eliminate these possible factors, Helms et al. (2008)
advocated that the ratio of the spectral slopes of two narrow
wavelength ranges (275–295 nm and 350–400 nm) be used as
indicators of molecular weight, source, and photobleaching
of CDOM. Spectral slopes reported here for the intervals of
275–295 nm (S275–295) and 350–400 nm (S350–400) were calcu-
lated using linear regression of the logtransformed aCDOM(l)
 water research 43 (2009) 4685–4697
as in Helms et al. (2008). The spectral slope ratio (SR) was
defined as the ratio of S275–295 to S350–400 (Helms et al., 2008).
In order to compare the present data with other data
reported in the literature, a fluorescence excitation wave-
length at 355 nm was used. Fluorescence was measured in
a 1-cm quartz cell from 380 to 600 nm using a Shimadzu 5301
spectrofluorometer, and the fluorescence spectrum of a Milliq
blank was used for baseline correction. The slit-widths were
set at 5 nm for excitation and emission wavelengths. Fluo-
rescence intensity was calibrated in quinine sulfate units
(QSU), where 1 QSU is the maximum fluorescence intensity of
0.01 mg/L of quinine (qs) in 1 N H2SO4 at the excitation
wavelength (Ex; nm)/emission wavelength (Em; nm) ¼ 350/450
(Hoge et al., 1993; Wada et al., 2007).
Samples for chlorophyll a (Chla) were filtered onto What-
man glassfiber GF/C filters (B 47 mm). Chla was extracted with
ethanol (90%) at 80  C and analyzed spectrophotometrically at
750 nm and 665 nm using a Shimadzu UV-2401PC UV–vis. The
Chla concentration was corrected for phaeopigments (Pa),
which was quantified after acidification using 1 N HCl (Chen
and Gao, 2000; Simis et al., 2005). Total pigment concentration
is the sum of Chla and Pa.
2.2. Degradation experiment
A laboratory experiment was conducted from 20 July to
22 August in 2008 to determine the contribution of phyto-
plankton degradation to CDOM. A 30 L water sample was
collected from the Lake IJsselmeer (52 450 N; 5 200 E), the
Netherlands. Microcystis sp. dominated the plankton
community in Lake IJsselmeer in summer just like in Lake
Taihu. The sample was centrifuged to obtain the particulate
matter and separate it from the CDOM containing lake water.
The phytoplankton concentration in the sample was relatively
low and therefore 10 L pure lab-culture of a colony-forming
Microcystis strain was also centrifuged to obtain enough
phytoplankton. The particulate matter from both the field
sample and the laboratory culture was resuspended in an
isotonic solution (0.5& salinity, no N and P).
The suspension was divided into 3 aliquots of 5 L and
poured into clean bottles. The three suspensions were incu-
bated at room temperature, under gentle shaking, in the dark
for 33 days. Bottles were aerated to prevent anaerobic condi-
tions. Absorption of the total suspension was measured daily
during the experiment, and every 3 days samples were taken
for CDOM and particulate matter absorption, CDOM fluores-
cence, pigment analyses and dissolved total nitrogen (DTN),
soluble reactive phosphorus (SRP), and particulate matter
concentrations. Nutrient concentrations in the GF/F filtrate
were analyzed with a continuous flow analyzer (QuAAtro, Seal
Analytical Limited).
All optical measurements were performed using a Lambda
800 UV–vis spectrophotometer (PerkinElmer, Wellesley, MA,
USA). The absorption coefficients of untreated suspensions
were measured between 350 and 800 nm, with a 2-nm slit
width inside the integrating sphere unit in a 1-cm quartz cell.
The mean optical density value between 750 and 800 nm was
used for a scattering correction. In general, this correction was
minimal because the integrating sphere accounted for most of
the scattered light.
4687
Water filtered over cellulose acetate filters (Schleicher &
Schuell, Germany) was used to measure the CDOM absorption
coefficient. The measurement was obtained in a 5-cm optical-
grade quartz cell, without the integrating sphere accessory,
against a reference of Milliq water. The methods for CDOM
absorption correction and spectral slope calculation were the
same as used for the field samples.
The coefficients of total particulate matter [ap(l)], tripton
[ad(l)], and phytoplankton [aph(l)] were determined with the
quantitative filter technique. Water samples (5–10 ml) were
filtered onto 25 mm diameter Whatman glassfiber GF/F filters
(B 25 mm) under low vacuum pressure. The optical density
maximum of particulate matter on the filters was kept below
0.3. Filters were positioned in the sphere at a 100 angle from
the beam and measured every 1 nm from 350 to 800 nm.
A blank filter, rinsed with Milliq, was used as reference.
Sample and blank filters were wetted in their own filtrate prior
to measurement to ensure equal moisture content. All spectra
were corrected using the mean value of the optical density
between 750 and 800 nm to minimize differences between
sample and reference filter. Measured filter optical density
was corrected for the increase in path length caused by
multiple scattering in the GF/F filter, using the equation of
Simis et al. (2005).
After measuring ap(l), the phytoplankton pigments were
bleached from the filters using sodium hypochlorite. Sodium
hypochlorite was used because it removes the phycobilins in
Microcystis more efficiently than ethanol (Tassan and Ferrari,
1995). The absorption spectra of bleached filters were then
measured again to obtain ad(l). Phytoplankton absorption was
defined as the difference between total particulate matter and
tripton.
aph ðlÞ ¼ ap ðlÞ  ad ðlÞ (4)
Three-dimensional excitation emission matrix (EEM)
spectra of CDOM were measured by a Hitachi F-7000 fluores-
cence spectrometer (Hitachi High-Technologies, Tokyo, Japan)
with a 700-V xenon lamp. The scanning ranges were
200–430 nm for excitation, and 250–550 nm for emission.
Readings were collected at 5 nm intervals for excitation, with
1-nm emission wavelengths, using a scanning speed of
2400 nm/min. The bandpass widths were 5 nm for both exci-
tation and emission. A Milliq water blank of the EEM spectrum
was subtracted to eliminate the water Raman scatter peaks.
The fluorescence intensities were normalized to quinine
sulfate units (QSU), as in the field study.
2.3. The PARAFAC modeling
PARAFAC statistically decomposes the complex mixture of
DOM fluorophores into components, without any assump-
tions about their spectral shape or number. The data signal is
decomposed into a set of three linear terms and a residual
array (Stedmon et al., 2003):
X
F
xijk ¼ aif bjf ckf þ eijk i ¼ 1; 2; .; I j ¼ 1; 2; .; J k ¼ 1; 2; .; K
f ¼1
(5)
where xijk is the fluorescence intensity for the ith sample at
4688 water research 43 (2009) 4685–4697
emission wavelength j and excitation wavelength k; and aif is
directly proportional to the concentration of the fth analyte in
the ith sample. Both bjf and ckf are linearly related to the
emission and excitation spectra at wavelengths j and
k respectively for the fth analyte, and eijk is the residual noise,
representing the variability not accounted for by the model.
Since the pioneering work of Stedmon et al. (2003), the
combination of EEM and PARAFAC has widely been applied to
characterize DOM extracted from soils (Ohno and Bro, 2006),
from terrestrial and marine aquatic environments (Cory and
McKnight, 2005; Murphy et al., 2008; Yamashita et al., 2008;
Kowalczuk et al., 2009), and in laboratory and mesocosm
experiments (Stedmon and Markager, 2005a; Stedmon et al.,
2007). The number of fluorescent components found using
PARAFAC ranges from 4 to 13 for diverse terrestrial and
marine aquatic environments (Cory and McKnight, 2005;
Stedmon and Markager, 2005a,b; Murphy et al., 2008;
Yamashita et al., 2008; Kowalczuk et al., 2009).
Stedmon and Bro (2008) have described how to charac-
terize dissolved organic matter fluorescence with PARAFAC,
including a split-half analysis used to validate the identified
fluorescent components. The PARAFAC analysis in our study
was carried out in MATLAB with the DOMFluor toolbox for
MATLAB according to Stedmon and Bro (2008). For PARAFAC
modeling, excitation wavelengths from 200 to 240 nm and
emission wavelengths from 250 to 300 nm were deleted from
each EEM because of the unreliable data in this region. Two
samples were removed through comparing them to the others
to determine whether they contained measurement error
(Stedmon and Bro, 2008).
2.4. Statistical analyses
Statistical analyses (mean value, linear and nonlinear fitting)
were performed with SPSS 11.0 software (Statistical Program
for Social Sciences). Differences in parameters between two
categories were assessed with a paired t-test, using a p value
of 0.05 to determine significance. Regression and correlation
analyses were used to examine the relationships between
variables using SPSS software.
3. Results
3.1. Field study
A wide range of variability in CDOM absorption and Chla
concentration was found in Lake Taihu between different lake
regions, ranging from 1.23 to 8.33 m1 for aCDOM(355), and 3.8
to 155.1 mg/L for Chla, suggesting temporal–spatial heteroge-
neity in the large shallow lake (Table 1). In fact, different
ecosystem types coexist in Lake Taihu; algal-dominated,
macrophyte-dominated and transition states. Higher CDOM
absorption coefficients were recorded in the northern lake
regions than in the southern lake bays with submerged
aquatic vegetation in previous study (Zhang et al., 2007).
Because of frequent algal blooms in summer in Lake Taihu,
the Chla concentration in summer was very high, with a mean
value of 41.9  33.3 mg/L. S300–500 and SR were in the range of
15.11–25.7 mm1 (19.8  1.9 mm1) and 0.64–1.80 (1.11  0.17)
respectively.
In all eight field campaigns, there were consistent signifi-
cant correlations between the Chla concentration and CDOM
absorption, and fluorescence, with the determination coeffi-
cients (r2) ranging from 0.29 to 0.81 (Table 1). When the data
from all eight cruises were combined, the correlations were
also significant with r2 of 0.42 and 0.41 for aCDOM(355) and
Fn(355), respectively (Table 1, Fig. 1), suggesting that phyto-
plankton degradation was an important source of CDOM in
this eutrophic shallow lake.
The slopes and intercepts between Chla concentration and
aCDOM(355), Fn(355) were very similar (0.026, 1.95 for aCDOM(355)
and 0.027, 1.66 for Fn(355), respectively) indicating that the
changes in aCDOM(355) and Fn(355) were almost synchronous.
Furthermore, the range and mean values of aCDOM(355)
(1.23–8.33, 3.05  1.34 m1) and Fn(355) (1.00–7.13, 2.78  1.39
QSU) were very similar. The correlation between aCDOM(355) and
Fn(355) was as follows:
2
Fn ð355Þ ¼ 0:947aCDOM ð355Þ  0:114 r ¼ 0:84; p < 0:001;

n ¼ 203
3.2. Degradation experiment
The phytoplankton community in the degradation experi-
ment was dominated by Microcystis. The temporal changes in
nine chemical parameters during the degradation experiment
are shown in Table 2.
Absorption of the untreated suspension decreased during
the first 12 days of the experiment. Thereafter it remained
more or less constant (Fig. 2). During the decrease, there were
three distinct phases: for days 0–2, absorption decreased
gradually, between days 3 and 7 absorption decreased rapidly,
indicating a sudden collapse of Microcystis and from days 7 to
12, absorption decreased gradually again (Fig. 2b). The mean
daily decrease rates of asus(440) and asus(675) during the three
periods were 0.26 and 0.43 m1, 1.08 and 1.73 m1, and 0.10
and 0.22 m1, respectively.
In the first 6 days, distinct phytoplankton absorption peaks
were visible near 440 nm and 675 nm due to Chla, and 625 nm
due to phycobilin (Fig. 2a). With the passage of time, phyto-
plankton decomposed and degraded, and the percentage of
phytoplankton in the total particle pool decreased markedly.
There was a sharp decrease in the phytoplankton absorption
coefficient, and a simultaneous distinct increase in absorption
by CDOM (Fig. 3). From day 0 to day 12, aph(440) and aph(675)
decreased from 10.94  0.22 to 1.11  0.10 m1, and from
6.31  0.09 to 0.66  0.03 m1, respectively. During this period,
aCDOM(355) increased from 0.41  0.03 to 1.37  0.03 m1,
a 234% increase. Therefore, only the 675 nm absorption peak
remained visible in the suspension.
During the 3-day period (days 3–6) that Microcystis
collapsed, the decrease in phytoplankton absorption, and the
increase in CDOM absorption, were more marked than during
any other 3 days. For example, the mean daily decrease of
aph(440) and aph(675) were 2.39 and 1.44 m1, and the mean
daily increase of aCDOM(355) was 0.15 m1 during this period,
respectively. During days 12–18, aCDOM(355) decreased quickly,
and after 21 days it remained at approximately the same level,
 water research 43 (2009) 4685–4697 4689

with no significant changes during the last 12 days (days

14
14
32
14
49
31
18
31
203
n
21–33). However, aCDOM(355) was still significantly higher
during these 12 days than at the start of the experiment (t-test,
p < 0.001), showing a 78.1% increase compared to the initial
value.

<0.005

<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.05

<0.05
Fn(355) and Chla

p
The spectral slope S300–500 decreased, accompanying the
production of CDOM during the first 9 days, and reached
a minimum value of 9.5 mm1, then increased during days
12–18 and after 21 days it remained at approximately the same
level without significant changes during days 21–33 (Table 2).
0.57
0.29
0.64
0.39
0.52
0.52
0.68
0.43
0.41
2
r

SR, on the other hand, increased gradually during the first

9 days to 2.38, and then decreased suddenly to 0.82 at
day 12. After that, SR fluctuated slightly and kept a value
significantly lower compared to the initial value (t-test,
<0.001

<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
aCDOM(355) and Chla

p < 0.001) (Table 2).

<0.05

<0.01
p

The nutrient concentrations in the solutions increased

with time during the degradation process, and final values at
day 33 were significantly higher than the initial values (t-test,
p < 0.001) (Table 2). DTN and SRP concentrations increased
from the initial values of 0.20 mmol/L and 0.74 mmol/L to
0.81
0.39
0.37
0.48
0.51
0.53
0.58
0.45
0.42
2

150.21 mmol/L and 9.05 mmol/L respectively, at the rates of

r

6.25 mmol/(L d) and 0.35 mmol/(L d) in the first 24 days respec-

tively, and then remained relatively constant (Table 2). Unlike
Table 1 – Variations in, and correlations between CDOM absorption, fluorescence and Chla concentration.

CDOM absorption, which showed a marked decrease after

61.2  40.6
54.9  28.4
29.1  18.7
44.5  30.6
44.3  37.5
47.1  39.8
38.2  32.1
32.5  25.3
41.9  33.3

12 days, the DTN and SRP concentrations remained high,

Mean

suggesting that they were not microbially consumed.

In agreement with the changes in phytoplankton absorp-
Chla (mg/L)

tion, the Chla concentration decreased rapidly in the first 12

days, followed by a slight decrease over a longer period of
7.2–149.8
12.2–131.5

8.8–109.9
4.8–155.1
4.5–149.2
7.8–109.9
3.8–104.5
3.8–155.1

time. Along with the decrease in Chla concentration, the Pa

4.6–77.8
Range

concentration also decreased, although at a much lower rate.

The ratio of Pa to Chla is usually used as an indicator of the
health of the cyanobacterial community, with a high ratio
representing the death and degradation of phytoplankton
3.44  1.56
3.63  1.54
2.39  1.14
3.71  1.49
2.19  0.90
2.77  1.38
2.80  1.73
3.02  1.44
2.78  1.39

(Simis et al., 2005). A sharp increase in the ratio of Pa to Chla

Mean

after 6 days indicated a collapse of Microcystis and strong

Fn(355) (QSU)

degradation of Chla during the phytoplankton lysis phase

(Table 2 and Fig. 3).
Four fluorescent components were identified by PARAFAC
based on the split-half validation procedure, using 34 EEM
1.72–6.33
1.56–6.09
1.00–6.29
1.63–6.14
1.06–3.98
1.20–6.21
1.48–6.76
1.23–7.13
1.00–7.13
Range

spectra collected during the degradation experiment (Fig. 4).

Split-half analysis involved dividing the data set into two
random, typically equal sized groups and, consequently,
making a PARAFAC model on both halves independently.
3.40  1.03
4.22  1.43
2.89  1.00
3.77  1.52
2.75  0.96
2.79  1.34
3.35  2.31
2.79  1.09
3.05  1.34

If the correct number of components was chosen, the loadings

Mean
aCDOM(355) (m1)

from both models would be the same, due to the uniqueness

of the PARAFAC model (Stedmon et al., 2003). Fig. 4 shows the
highly overlapping excitation and emission loadings for the
four components modeled on the two halves of the data set
1.97–5.60
2.24–6.83
1.64–5.18
1.40–6.79
1.42–5.03
1.23–6.39
1.29–8.33
1.40–5.65
1.23–8.33

and the whole data set. All of these fluorescent components

Range

had single emission maxima, with single or multiple excita-

tion maxima. The excitation and emission characteristics of
the CDOM components we identified, and examples of
matching components identified by other researchers who
Jul.29–Agu.1, 2006

have modeled CDOM EEMs in various marine, oceanic and

Agu.15–18, 2005

Agu.10–13, 2006

Aug.12–13, 2007

estuarine environments around the world, are given in

Jun.15, 2005
Jul.14, 2005

Jul.18, 2006

Jun.3, 2007

Table 3.
Two distinct types of fluorescent substances, with humic-
like, and protein-like fluorescence, are found in natural waters
All

(Coble et al., 1998). From the fluorescence characteristics, the

4690 water research 43 (2009) 4685–4697
Fig. 1 – Correlations between the Chla concentration and (a) CDOM absorption coefficient [aCDOM(355)],(b) normalized
fluorescence [Fn(355)]. The results of the linear regression are displayed as solid line.
four components we identified could be distinguished as
terrestrial-like humic components (C1 and C3), biological
degradation or marine-like humic components (C3 and C4), and
protein-like component (C2) (Table 3). Component 1 displayed
two excitation maxima (at 270 and 365 nm) corresponding to the
same emission maximum (at 453 nm), which is similar to the
humic-like fluorophores in the ultraviolet region (peak A) and
the visible region (peak C) as defined by Coble (1996) and Coble
et al. (1998). Component 2 had excitation and emission charac-
teristics similar to a fluorescent protein-like compound that has
been observed previously (Coble, 1996; Coble et al., 1998;
Yamashita et al., 2008; Kowalczuk et al., 2009), which was
confirmed as an autochthonous tyrosine-like fluorophore.
Many previous EEM–PARAFAC studies have identified similar
protein-like components as tyrosine- and tryptophan-like flu-
orophores (Cory and McKnight, 2005; Stedmon and Markager,
2005a,b). For component 3, two excitation maxima (at 255 nm
and 330 nm) were observed in the EEM (Fig. 4, Table 3). Although
the excitation and emission maxima basically fell in the range of
the terrestrial-like humic fluorophores defined by Coble (1996),
Stedmon et al. (2003) thought it was similar to marine-like
humic fluorophores defined as peak M by Coble (1996). In this
study it originates from microbially processed phytoplankton
degradation products. Component 4 was characterized by peaks
at 315 nm for excitation and 372 nm for emission, similar to
peak M (Coble, 1996; Coble et al., 1998). Peak fluorescence in this
region has been thought to be coupled to plankton productivity
as it is primarily observed in the open ocean environment, and
has also been produced and altered by microbial reprocessing
during a mesocosm experiment with CDOM produced by the
plankton community (Stedmon and Markager, 2005b).
The mean fluorescence intensity of the four components
and changes in their respective contribution to total CDOM
fluorescence intensity derived by the PARAFAC model during
the degradation experiment are shown in Fig. 5. At the
beginning of the experiment, the fluorescence intensity,
especially of components 3 and 4 was low (12.2% and 1.1%
respectively). With the degradation of phytoplankton and
production of CDOM, components 3 and 4 increased rapidly to
values significantly higher than the initial values (t-test,
p < 0.001). Component 3 increased by a factor 2.2 and reached
its maximum at day 6 and component 4 by a factor 26.1 with
the maximum at day 12. Thus, the contributions of compo-
nents 3 and 4 to the total fluorescence increased. From day 15,
the fluorescence intensities of components 3 and 4 remained
at the same level, or slightly decreased. The contribution of
components 3 and 4 accounted for a half of the total fluores-
cence since 12 days. The composition of CDOM fluorescence,
and the relative contribution of the four components show
that phytoplankton degradation mainly produces component
3 and 4 fluorophores.
4. Discussion
Phytoplankton degradation has previously been shown to be an
important source of CDOM, contributing to the carbon budgets
of both coastal and lake waters, in field studies and laboratory
algal culture experiments (Yamashita and Tanoue, 2004;
Stedmon and Markager, 2005a; Wada et al., 2007; Wetz and
Wheeler, 2007; Hanamachi et al., 2008). A significantly positive
correlation was found between CDOM absorption and Chla in
many field studies in summer, indicating that phytoplankton
accumulation and decomposition were a contributing source of
CDOM (Kahru and Mitchell, 2001; Rochelle-Newall and Fisher,
2002b). Etheridge and Roesler (2004) observed that CDOM and
the colloidal organic matter (0.2–0.7 mm) increased markedly at
the height of the bloom during a Long Island (USA) brown tide.
CDOM absorption did not increase simultaneously with Chla,
but Sasaki et al. (2005) observed that the CDOM increase lagged
relative to Chla concentration in Funka Bay (Japan) during the
spring bloom, suggesting that phytoplankton degradation
started after the spring bloom; and then CDOM absorption
increased.
In agreement with these previous studies, we have deter-
mined, through field and laboratory studies, that phyto-
plankton blooms are indeed an important source of CDOM in
eutrophic lakes with high phytoplankton biomass. For all
eight field campaigns in summer, significant correlations were
found between aCDOM(355), fluorescence and the Chla
concentration. Monthly mean aCDOM(355) decreased from 3.37
in June to 3.20 in July and further to 2.83 m1 in August,
accompanying the decrease of Chla concentration from 48.3 to
46.2 and further to 36.2 mg/L, respectively, reflecting the
 water research 43 (2009) 4685–4697 4691

synchronous trend for variation in the Chla concentration and

0.82  0.03

0.77  0.03

TSM: total suspended matter; OSM: organic suspended matter; Chla: chlorophyll a; Pa: phaeopigment; Pa/Chla: the ratio of phaeopigment to chlorophyll a; DTN: dissolved total nitrogen; SRP: soluble
1.80  0.03

0.62  0.01

0.64  0.04
2.01  0.02

0.79  0.03

0.70  0.01
2.42  0.13

0.74  0.07
2.38  0.09

0.73  0.01
CDOM absorption. However, no significant difference was

SR
found between months from June to August for the Chla
concentration and CDOM absorption, suggesting that there
was no significant difference in allochthonous CDOM input
during these months.
S300–500(mm1)

Spatially, aCDOM(355) was significantly lower in category

16.3  0.6
13.4  0.6

12.5  0.1

15.2  0.6
11.9  0.2

15.0  0.7

17.2  0.8
10.5  0.1

16.3  0.3
9.5  0.2

16.1  0.1
12.0  0.2
1 (the open waters) than in category 2 (the near-shore waters)
(t-test, p < 0.001), suggesting an important contribution of
allochthonous CDOM by river input. Significant linear rela-
tionships were found between aCDOM(355) and the Chla
concentration for categories 1 (r2 ¼ 0.47, p < 0.001) and
aCDOM(355) (m1)

0.74  0.02 2 (r2 ¼ 0.33, p < 0.001), but the higher determination coefficient
0.41  0.03

1.29  0.02

0.76  0.03
0.53  0.01

0.80  0.02

0.72  0.04
0.97  0.03

0.75  0.01
1.15  0.03

0.74  0.06
1.37  0.03

of category 1 suggests a more important contribution of

phytoplankton degradation to the CDOM pool in open water
locations than in near-shore waters.
Furthermore, aCDOM(355) values as high as 20.1 m1 were
observed during the drinking water crisis in Gonghu Bay in the
northeast of Lake Taihu in 2007, which was caused by accu-
mulating algal blooms.1 This value was almost 5 times that of
SRP (mmol/L)
Table 2 – Means and standard deviations of nine chemical parameters during the degradation experiment (n [ 3).

0.74  0.57

8.61  0.25

9.03  0.05
0.39  0.09

8.81  0.47

9.12  0.30
13.28  3.63

8.39  0.92
10.99  0.97

9.05  0.34
8.77  0.28

9.20  0.17

the mean in the northern region of the lake, further indicating

the important contribution of phytoplankton degradation to
CDOM absorption in the field.
In the degradation experiment, the CDOM absorption
reactive phosphorus; aCDOM(355): CDOM absorption coefficient at 355 nm; S300–500: spectral slope; SR: spectral slope ratio.

coefficient first increased rapidly, then decreased and finally

DTN (mmol/L)

stabilized at a level significantly higher than the initial value

133.70  13.67
132.70  28.52
0.20  0.08

130.89  8.45

148.07  7.20
0.09  0.10

148.43  9.00
1.54  0.13
10.22  1.22

150.21  0.74
84.74  7.58

152.56  1.54

(t-test, p < 0.001). The DTN, SRP and Chla concentrations

showed a different pattern. Nutrient concentrations increased
substantially during the first period and remained relatively
stable (Table 2). The Chla concentration decreased rapidly
during the first period until it stabilized at a very low level
compared to the initial concentration (Table 2, Fig. 3). The
0.22  0.02

2.60  1.66

2.92  0.57
0.38  0.04

1.97  0.54

3.01  0.59
2.20  0.39

2.51  0.23
3.83  1.54

4.07  0.61
2.45  2.06

2.95  0.45
Pa/Chla

variation in CDOM, DTN, SRP and Chla concentrations

indicates that two major processes of CDOM production
occur during the degradation experiment. In the first period,
a large amount of CDOM is produced directly from phyto-
49.5  11.5

plankton lysis. During the second period (12–21 days), this

56.5  29.8
Pa (mg/L)

77.7  7.8

38.4  2.9
117.0  6.1

43.2  3.5

42.5  6.4
115.5  4.7

45.1  1.6
89.6  5.1

50.1  1.8
42.0  1.2

CDOM is consumed and modified by microbes resulting in

decreasing CDOM absorption and fluorescence. However,
the DTN and SRP concentrations do not decrease because
they are not easily microbially consumed. On the contrary,
DTN will be produced by microbes accompanying the modi-
Chla (mg/L)

349.1  11.2
307.2  13.1

30.4  13.2
22.1  7.2

13.4  1.9
22.8  4.9

14.2  0.8
53.8  8.1

18.0  1.0
25.5  8.0

12.5  1.6
14.4  2.0

fication of CDOM, resulting in an increase in the DTN

concentration.
The results of the experiment also show that both ‘‘labile’’
and ‘‘refractory’’ fractions of CDOM are produced. The ‘‘labile’’
fraction of CDOM derived from phytoplankton is easily
OSM (mg/L)

consumed and decomposed by bacteria. However, the

40.4  1.1

2.9  0.2

4.7  0.5
33.2  0.7

1.4  0.2

5.5  0.6
17.5  0.7

2.4  0.1
10.8  1.0

4.6  1.7
10.3  1.7

4.2  0.5

‘‘refractory’’ fraction, which appears resistant to microbial

processing, is decomposed slowly and thus remains almost
constant or only slightly reduces. Decomposition experiments
using phytoplankton cells collected from a eutrophic lake,
Lake Kasumigaura, Japan (Hanamachi et al., 2008), also
TSM (mg/L)

45.5  1.0

16.5  0.2

15.2  0.3
36.8  2.7

15.6  0.5

16.0  0.3
19.9  1.2

14.5  0.2
18.2  0.8

15.3  0.2
16.0  0.6

14.8  0.3

showed that the ‘‘labile’’ fraction of organic matter, such as

glucose, was rapidly decomposed within a few days, whereas
the ‘‘refractory’’ fraction was decomposed more slowly.
The variation in S300–500 and SR reflects the changes in
composition and molecular weight of the CDOM in the
Day

15

30
18

33
21
24
12

27
0
3
6
9

1
Unpublished data.
4692 water research 43 (2009) 4685–4697

Fig. 2 – Decrease in the absorption coefficient of the suspension [asus(l)] during the degradation experiment. (a) Mean full
spectra of asus(l) (n [ 3), sampling days indicated in the plot; (b) mean absorption coefficients with standard deviation of the
suspension at the Chla absorption maxima at 440 and 675 nm (n [ 3).

degradation experiment. As mentioned in Belzile and Guo
(2006), a low spectral slope is typical of high molecular weight
and less-altered CDOM, while a steep slope is indicative of low
molecular weight, highly degraded CDOM (Twardowski et al.,
2004). S300–500 decreased in the first 9 days, indicating the
production of a large amount of high molecular weight CDOM.
Next, it increased as microbial activity altered the CDOM
composition, thereby decreasing the molecular weight.
Helms et al. (2008) reported that aerobic microbial incu-
bations in the dark resulted in a statistically significant
decrease in SR over timescales of days to weeks, either due to
microbial production or selective preservation of long-wave-
length-absorbing substances. Therefore, the decrease in SR
after 12 days also confirms that high molecular weight CDOM
originating from phytoplankton was degraded into low
molecular weight CDOM, which is consistent with the former
discussion on the ‘‘labile’’ fraction of CDOM. Compared with
the field study, S300–500 and SR were lower in the last stage of
laboratory experiment. This difference can be explained by
a combination of processes occurring in the field, including

Fig. 3 – Dynamics of (a, b) the mean absorption spectra (n [ 3) of (a) phytoplankton pigment absorption [aph(l)]; and (b) CDOM
absorption [aCDOM(l)], sampling days are indicated in the plot; (c, d) means and standard deviations (n [ 3) of
(c) concentrations of total pigment, Chla and Pa; and (d) phytoplankton pigment absorption at the Chla absorption maxima
at 440 and 675 nm, and CDOM absorption at 355 nm during the degradation experiment.
 water research 43 (2009) 4685–4697 4693

Fig. 4 – The PARAFAC model output showing fluorescence signatures of four components identified. Contour plots present
spectral shapes of excitation and emission. Line plots at right side of each contour plot present split-half validation results
for the corresponding component. Excitation (left) and emission (right) spectra were estimated from two independent halves
of the data set (black lines) and the complete data set (red lines).(For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

mixing of CDOM from different sources, photochemical
degradation, and microbial decomposition.
Several studies have suggested that marsh plants and
phytoplankton might be important sources of nutrients
(Moran et al., 2000; Wang et al., 2007; Wang and Chen, 2008).
Our results indicate that the release of nutrients from
degrading phytoplankton is a very rapid process and can
cause a significant accumulation of nutrients. Our study has
shown the major contribution of phytoplankton to the
production of CDOM and dissolved nutrients in eutrophic Lake
Taihu. The organic nutrients released from the phytoplankton
bloom can be remineralized and regenerated as nutrient
input, to sustain new biomass in the next phytoplankton
bloom.
The number of components identified by the PARAFAC
modeling, and the spectral characteristics of CDOM EEM data
from the degradation experiment are similar in terms of the
number of fluorescence peaks and their position to previously
identified components in aquatic environments (Table 3)
(Cory and McKnight, 2005; Stedmon and Markager, 2005a;
Murphy et al., 2006, 2008; Yamashita et al., 2008; Kowalczuk
et al., 2009). For example, Stedmon et al. (2003) observed five
peaks from terrestrial and autochthonous organic matter
sources in their study on the Baltic Sea and the spectral
characteristics are comparable to components 1 and 3 found
in this study (Table 3). The characteristics of the excitation
and emission spectra of component 3 fall in the transition
zone between terrestrial- and marine-like humic compo-
nents. Compared to component 1, component 3 has excitation
and emission maxima at shorter wavelengths and is similar to
peak M, which is believed to belong to marine-like fluo-
rophores. Based on the observed trends in the abundance of
component 3 during the phytoplankton degradation, this
study further supports the fact that component 3 (and
therefore also peak M), is of biological origin and is not
exclusively a marine component.
Actually, the appearance of component 3 is the result of
blue-shifting (toward shorter wavelengths) of the Exmax and
Emmax peak of terrestrial-like humic fluorophore (com-
ponent 1) dominated CDOM, resulting from the increase of
autochthonous humic substances during the degradation
experiment. Comparable blue-shifting was observed in other
studies. Blue-shifting of a humic-like fluorescence peak,
with Exmax/Emmax of 340/448 nm in the river, 342/442 nm in
the near-shore area, and 310/423 nm in the transition zone
of a shallow sea, has previously been observed by Coble
(1996). Similar results have been observed in open water
areas compared to near-shore areas (Her et al., 2003; Boehme
and Wells, 2006).
In order to further analyze which fluorophores are
produced by the degrading phytoplankton, and the mutual
relationships between the four components, regression anal-
yses between aCDOM(355) and fluorescence intensity of the
four components were carried out. Significant linear correla-
tions were found between the CDOM fluorescence intensities
of components 3, 4 and the natural logarithm of the CDOM
absorption coefficient [ln aCDOM(355)] (Fig. 6). However, no
significant correlations were found between CDOM absorption
and fluorescence for components 1 and 2. Because the varia-
tion in CDOM absorption was attributed to phytoplankton
degradation in the experiment, we conclude that fluorophores
3 and 4 were produced during the degradation experiment,
further confirming that these components are marine-like
humic fluorophores and originated from phytoplankton.
Table 4 shows that components 3 and 4 were strongly
linearly correlated, suggesting that they have a common
source. Compared with component 4, component 3 was
strongly linearly correlated with component 1, indicating that
 4694
Table 3 – Spectral characteristics of excitation and emission maxima of the four fluorescent components identified by PARAFAC modeling for the whole EEM data set,
compared with previously identified sources.
Component no. Exmax (nm) Emmax (nm) Coble (1996) Comparison with Description and probable source
other studies
using PARAFAC

water research 43 (2009) 4685–4697

C1 270 (365) 453 A peak 230–260/380–460 C3: 270 (360)/478 (Stedmon et al., 2003) Terrestrial-like humic substances
C peak 320–360/420–480 C4: 250 (360)/440 (Stedmon and Markager, 2005b)

C2 275 <300 B peak 275/305–310 C4: 275/306 (Stedmon and Markager, 2005a) Autochthonous tyrosine-like fluorescence.
C8: 275/304 (Stedmon and Markager, 2005b)
C1: 275/<300 (Murphy et al., 2008)
C7: 270/299 (Yamashita et al., 2008)

C3 255 (330) 412 A peak 230–260/380–460 C4: 325 (250)/416 (Stedmon et al., 2003) Marine-like and terrestrial-like
C peak 320–360/420–480 C8: 250 (380)/416 (Murphy et al., 2008) humic materials,
M peak 290–310/370–420 C3: 250 (310)/400 (Kowalczuk et al., 2009) possible
microbial activity

C4 315 372 M peak 290–310/370–420 C3: 295/398 (Stedmon and Markager, 2005a) Marine-like humic
C2: 315/418 (Murphy et al., 2008) materials (phytoplankton degradation)
C6: 325 (<260)/385 (Yamashita et al., 2008)

Secondary excitation band is given in brackets.

 water research 43 (2009) 4685–4697 4695

Fig. 5 – (a) Composition of CDOM fluorescence of four components and (b) changes in their respectively percent contribution
(%) to total CDOM fluorescence intensity derived by the PARAFAC model during the degradation experiment.

component 3 was partly derived from production and alter-
ation by microbial reprocessing, which also could be inferred
from the lower determination coefficient between the fluo-
rescence intensity of component 3 and CDOM absorption
(Fig. 6). Tyrosine-like fluorescence was represented by
component 2, which has been observed in other similar
studies (Murphy et al., 2008). Tyrosine-like fluorescence is
frequently attributed to autochthonous production associated
with biological degradation of DOM (Coble et al., 1998; Sted-
mon and Markager, 2005b). However, component 2 was
significantly correlated with terrestrial-like humic fluores-
cence (component 1), but only weakly correlated or not
correlated with marine-like humic fluorescence derived from
phytoplankton degradation, suggesting it is produced from
both terrestrial and autochthonous substrates.
McKnight et al. (2001) used the ratio of the emission
intensity of 450:500 nm, obtained with an excitation of
370 nm, as a simple index to distinguish sources of isolated
aquatic fulvic acids. This index has a value of approximately
1.9 for microbially derived fulvic acids, and a value of
approximately 1.4 for terrestrially derived fulvic acids. In our
study, the value of this index ranged from 1.97 to 2.40, with
a mean value of 2.13  0.09, suggesting that CDOM was
microbially derived fulvic acids from phytoplankton. This is
comparable to the value observed for an autochthonous
CDOM source in Antarctic Lakes (McKnight et al., 2001).

Fig. 6 – Correlations between the fluorescence intensities of components 3, 4 and the natural logarithm of CDOM absorption
coefficients [ln aCDOM(355)].
4696 water research 43 (2009) 4685–4697
Table 4 – Determination coefficients and statistical
significance of the linear regression analysis between
fluorescence intensities of the four components derived
from the PARAFAC model.
C1 C2 C3 C4
C1 1.000
C2 0.582** 1.000
C3 0.206** 0.133* 1.000
C4 0.012 NS 0.000 NS 0.831** 1.000
C1, C2, C3 and C4 represent components 1, 2, 3 and 4, respectively.
Statistical significance is reported as either NS ( p > 0.05),
*(0.05 > p > 0.01), or **( p < 0.01).
5. Conclusion
Our field and experimental evidence shows that phyto-
plankton degradation plays an important role in contributing
CDOM in eutrophic lakes, and thus affects nutrient cycling
and the optical properties of the lake water. Although labo-
ratory experiments do not duplicate the actual environmental
conditions in nature, results from our degradation experiment
suggest that the production of CDOM and nutrients from
phytoplankton degradation can be a very rapid process
(3–6 days) that could also occur under field conditions.
Within the degradation experiment, the PARAFAC model
enabled us to identify four distinct components in the CDOM
fluorescence excitation emission spectra: a terrestrial-like
humic component (C1), a protein-like component (C2), and
two marine-like humic components (C3 and C4). The changes
in the CDOM fluorescence composition, and the correlations
between fluorescence intensity and CDOM absorption, show
that production of components 3 and 4 accompanies phyto-
plankton degradation. Therefore it can be determined that
local CDOM is produced from phytoplankton, based on anal-
ysis of the intensities and contribution of components 3 and
4 in field samples.
Acknowledgements
This study was jointly supported by the National Natural
Science Foundation of China (Grant No. 40730529, 40601099)
and the Knowledge Innovation Project of the Chinese
Academy of Sciences (KZCX1–YW–14, KZCX2–YW–419). We
would like to thank Ji Jiang, Qian Rongshu, Li Junsheng for
their help for sample collection in the field.
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