2. Methods
2.1 Study area and sampling
sites
The Yangtze River (also called Changjiang River), originating from the
Qinghai-Tibet Plateau, is the third longest river in the world, with a
total mainstream length of about 6300 km, a basin area of
1.8×106 km2 and an average annual
discharge of 892 km3 (Yan et al. , 2010)(Fig. 1) . In China, the river is divided based on watershed
boundaries into three reaches, the upper reaches (upstream of Yichang),
middle reaches (between Yichang and Hukou) and lower reaches (downstream
of Hukou) (Wang et al. , 2008). The lower reaches areas of Yangtze
River have dense river networks, numerous lakes, extensive plains and
dense cities, forming a prosperous industrial belt, but also facing
ecological environment problems such as the increasing concentrations of
nutrient in the watershed, frequent occurrence of harmful algal blooms
in lakes and severe red tide in estuaries (Yi et al. , 2011; Qinet al. , 2010; Tang et al. , 2006; Liu et al. , 2018).
In addition, the lower reaches of Yangtze River can be subdivided into
three parts, the Anhui section, Jiangsu section and estuary, according
to the provinces and tidal (Fig. 1) .
In this study, 44 surface water samples (0.5 m below the water surface)
were collected from the river-estuary continuum of the Yangtze River
coastal zone in July, 2018, to investigate the spatial changes of REGs
and Upots (Fig. 1) . There were 19 (CJ1
~ CJ19), 15 (CJ20 ~ CJ34) and 10 (CJ35
~ CJ44) sites belonging to the Anhui section, Jiangsu
section and estuary, respectively.
2.2 Sample collection and
analysis
Surface water for NH4+ recycling
experiments were collected in 1 L carboys. Water samples for nutrient
concentrations (NO3−,
NO2−, and
NH4+) analyses were filtered through
0.7 μm fiberglass filters (Whatman GF/F) immediately following
collection in the field. Water temperature, dissolved oxygen (DO), and
pH were measured in situ using a multi-parameter water quality
analyzer (YSI Professional Plus, 6600V2, USA). All samples were
collected in triplicate and were immediately stored in a dark cooler.
Total dissolved nitrogen (TDN), total dissolved phosphorus (TDP),
ammonium (NH4+), nitrate
(NO3−),
nitrite (NO2−), and phosphate
(PO43−) concentrations were analyzed
in filtered samples (Jin and Tu, 1990). Concentrations of TDN and TDP
were determined using the potassium persulfate digestion and
spectrophotometric method (detection limits of 4 μmol N
L−1 and 1 μmol P L−1 for TDN and
TDP, respectively). NH4+ was
determined using the nesslerization colorimetric method (detection limit
1 μmol N L−1). NO3−and NO2− were determined using the
phenol acid ultraviolet colorimetric method (detection limit 3 μmol N
L−1) and N-(1-naphthyl)-ethylenediamine colorimetric
method (detection limit 0.2 μmol N L−1), respectively.
Total nitrogen (TN) and total phosphorus (TP) were determined on
unfiltered water samples using the potassium persulfate digestion and
spectrophotometric method (Jin and Tu, 1990). Particulate nitrogen (PN)
was calculated as the difference between TN and TDN, and the standard
deviation for PN was obtained using a propagation of error analysis.
NOx− was the sum of
NO3− and
NO2−. Urea was measured using
diacetylmonoxime reagent, and the detection limit was 0.04 μmol Urea-N
L−1 (Mulvenna and Graham, 1992). Chl-aconcentrations, chemical oxygen demand (COD), and suspended solid (SS)
were determined using standard methods (Jin and Tu, 1990). Dissolved
organic carbon (DOC) concentrations were determined using TOC-V CPN
(Shimadzu, Tokyo, Japan) analyzer at high temperature (680 °C) after
being acidified with 10 μL of 85%
H3PO4.
2.3 NH4+regeneration and uptake
experiment
Water column REGs and Upots were determined using
isotope dilution methods. Isotope dilution experiments are usually
conducted with low trace amendment level (about 10% of ambient)
(Glibert et al. , 1982). However, low
NH4+ concentrations and fast
NH4+ recycling rates in summer flood
season of Yangtze River may lead to depleted
NH4+ pool before the end of incubation
(Blackburn, 1979), excess15NH4+(approximately 20 μmol N L−1), as the reaction product
rather than as a potentially limiting substrate, was added at the
beginning of incubation (Mccarthy et al. , 2007a). Excessive15NH4+ addition can
promote NH4+ uptake rates, so the
NH4+ uptakes obtained in this study
were potential rates. On the other hand, because
NH4+ is the end product rather than
the substrate, excess additions will not affect regeneration rates
(Blackburn, 1979).
Water from each site was enriched with 98%15NH4Cl and decanted into duplicate
clear polystyrene culture bottles (70 ml; Coring) after thoroughly
mixed. Initial samples were filtered through a rinsed 0.2 μm syringe
filter immediately after enrichment and mixing for total
NH4+ concentrations and
NH4+-15N analysis.
Bottles were incubated in a transparent bucket containing Yangtze River
water to provide near-ambient light and temperature for 24 h. After
incubation, final samples were collected in the same way as the initial
samples.
NH4+-15N
was measured using NH4+ oxidation
membrane inlet mass spectrometry (OX/MIMS) (Yin et al. , 2014).
Water column REGs and Upots were calculated using a
modified isotope dilution method (Glibert et al. , 1982;
Blackburn, 1979). The relative abundance of
NH4+-15N (R )
is required to calculate the REGs and Upots, which can
be calculated as:
\begin{equation}
R=^{15}N/(^{15}N+^{14}N)\nonumber \\
\end{equation}where 15N and 14N are the
concentrations of
NH4+-15N and
NH4+-14N (μmol N
L−1), respectively.
The REGs can be calculated as
follows
(Bruesewitz et al. , 2015), which was derived from the logarithmic
equations of Blackburn (1979):
\begin{equation}
\text{REG}=(R_{0}-R_{t})/t\ \times(C_{0}/R_{t})\nonumber \\
\end{equation}where R0 and Rt are the
relative abundances of
NH4+-15N at the
initial and finial point, respectively, and t is the incubation
time (h). C0 is the initial concentrations of
NH4+ (μmol N L−1).
REGs (μmol N L−1 h−1) are absolutely
positive values, indicating the actual regeneration rates of
NH4+.
The Upots (μmol N L−1h−1) were calculated with the
NH4+ concentrations change and
regeneration rates (Bruesewitz et al. , 2015):
\begin{equation}
\ U_{\text{pot}}=(C_{t}-C_{0}-REG\ \times t)/t\nonumber \\
\end{equation}where C0 and Ct are the
initial and finial concentrations of
NH4+ (μmol N L−1).
CBAD characterizes internal NH4+recycling by representing the difference between measured potential
NH4+ uptake rates and actual
NH4+ regeneration rates in aquatic
systems (Gardner et al. , 2017). Therefore, CBAD was calculated
as:
\begin{equation}
\text{CBAD}=\ \ U_{\text{pot}}-REG\nonumber \\
\end{equation}2.4 Statistical analysis
Statistical analyses were performed using SPSS 22.0. One-way analysis of
variance (one-way ANOVA) combined with the Independent-Samples T-test
were used to evaluate statistically significant differences between
group average values. Pearson correlation analyses were applied to
analyze the relationship between NH4+recycling rates and environmental factors. Differences and
correlations
were considered statistically significant at p < 0.05.