Science of the Total Environment 907 (2024) 168092

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Adding Corbicula fluminea altered the effect of plant species diversity on

greenhouse gas emissions and nitrogen removal from constructed wetlands
in the low-temperature season
Luping Yang a, Kai Shen a, Xile Xu a, Derong Xiao a, b, c, Huijuan Cao a, Yishi Lin a,
Xiangyong Zheng a, b, c, Min Zhao a, b, c, Wenjuan Han a, b, c, *
a
College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, Zhejiang, People's Republic of China
b
National & Local Joint Engineering Research Center for Ecological Treatment Technology of Urban Water Pollution, Wenzhou University, Wenzhou 325035, People's
Republic of China
c
Institute for Eco-environmental Research of Sanyang Wetland, Wenzhou University, Wenzhou 325035, Zhejiang, People's Republic of China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Two-species mixture has the lowest CH4

emissions in systems with C. fluminea.
• The presence of H. fulva increased CH4
emissions in systems with C. fluminea.
• Species richness did not affect green­
house gas emissions in systems without
C. fluminea.
• The presence of H. fulva increased CO2
emissions in systems without
C. fluminea.
• Adding C. fluminea increased cumulative
emissions of CH4 and N2O.

A R T I C L E I N F O A B S T R A C T

Editor: Paola Verlicchi Plant species diversity is crucial in greenhouse gas emissions and nitrogen removal from constructed wetlands
(CWs). However, previous studies have overlooked the impact of benthos on cumulative greenhouse gas emis­
Keywords: sions during the low-temperature season in CWs. In this study, we established 66 vertical flow CWs with three
Methane levels of species richness (1, 2, and 4 species) and eleven species compositions. The Corbicula fluminea was added
Nitrous oxide
or not added at each diversity level and monitored greenhouse gas emissions and effluent nitrogen concentration.
Carbon dioxide
Our findings indicated that (1) in microcosms without C. fluminea, high species richness significantly increased
Benthos
Species richness effluent nitrogen concentrations (NO−3 -N, NH+ 4 -N, and TIN), but plant species richness did not affect cumulative
Species identity CH4, N2O, and CO2 emissions. The presence of Hemerocallis fulva significantly increased cumulative CO2 emis­
sions, while the presence of Iris tectorum significantly increased effluent nitrogen (NO−3 -N and TIN) concentra­
tions and cumulative N2O emissions; (2) in microcosms with C. fluminea, the lowest cumulative CH4 emissions
occurred when there were two species, but plant species richness did not affect cumulative CO2 and N2O
emissions. The presence of H. fulva significantly increased cumulative CH4 emissions, while the presence of

* Corresponding author at: College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, Zhejiang, People's Republic of China.
E-mail address: hanwj@wzu.edu.cn (W. Han).

https://doi.org/10.1016/j.scitotenv.2023.168092
Received 31 July 2023; Received in revised form 18 October 2023; Accepted 22 October 2023
Available online 23 October 2023
0048-9697/© 2023 Elsevier B.V. All rights reserved.
L. Yang et al.
Reineckea carnea significantly increased effluent nitrogen (NO−3 -N, NH+4 - N, TIN) concentrations; (3) at the same
diversity level, the addition of C. fluminea significantly increased cumulative CH4 and N2O emissions, as well as
effluent nitrogen concentrations. These results demonstrate that C. fluminea alters the effect of plant species
diversity on cumulative greenhouse gas emissions and nitrogen removal from CWs during the low-temperature
season. We recommend using a two-species mixture to reduce greenhouse gas emissions. However, we caution
against using plant compositions with H. fulva or I. tectorum for effective wastewater treatment and greenhouse
gas reduction in CWs.
1. Introduction
Constructed wetlands (CWs) play an essential role in removing pol­
lutants and providing ecosystem services (Vymazal et al., 2021; Kataki
et al., 2021; Wu et al., 2023). Compared to traditional sewage treatment
plants, CWs only emit 7.7 %, 0.08 %, and 3.4 % of CH4, CO2, and N2O
from removing 1 kg of nitrogen (Liu et al., 2012). However, CWs still
contribute to greenhouse gas emissions due to increased wastewater
production. Additionally, during low-temperature seasons, pollutant
removal efficiency, especially nitrogen removal, often decrease, which
limits the application of CWs in cold climates (Kumwimba et al., 2021;
Varma et al., 2021). Therefore, exploring measures that can enhance
nitrogen removal and reduce greenhouse gas emissions in CWs during
low-temperature seasons is crucial.
Some methods, such as extending hydraulic retention time and
increasing aeration intensity, have improved nitrogen removal effi­
ciency in CWs during low-temperature seasons (Molle et al., 2008;
Tunçsiper et al., 2015; Colares et al., 2019). Nevertheless, these methods
often have high operational demands and costs and do not consider
greenhouse gas emissions. Therefore, exploring measures with low cu­
mulative greenhouse gas emissions and high nitrogen removal efficiency
from CWs during low-temperature seasons is crucial.
Biodiversity plays a significant role in ecosystem function (Gonzalez
et al., 2020). High species richness may increase nitrogen removal ef­
ficiency (Geng et al., 2019) and greenhouse gas emissions in CWs (Sun
et al., 2019; Fan et al., 2021). However, some research found that mixed
systems have lower emissions of CH4 and N2O compared to single sys­
tems (Wang et al., 2022; Jiang et al., 2023). The presence of specific
species can either enhance or reduce greenhouse gas emissions and ni­
trogen removal efficiency in CWs (Han et al., 2019; Jiang et al., 2023).
For instance, the presence of Rumex japonicus reduced effluent nitrogen
concentration and N2O emission by 26 % and 62 % (Han et al., 2019),
while the existence of Arundo donax increased total nitrogen removal
content by 8 mg m− 2 day− 1, but reduced N2O emission by 71 % (Jiang
et al., 2023). Work by Han et al. suggested plant species richness may
not significantly affect nitrogen removal and CH4 emissions (Han et al.,
2017). However, these studies conducted in spring and summer, and the
greenhouse gas emissions monitored only once, which may lead to bias
in the estimated relationship between plant species diversity and
greenhouse gas emissions from CWs. Further research is necessary to
explore the relationship between plant species diversity, cumulative
greenhouse gas emissions, and nitrogen removal during low-
temperature seasons in CWs.
Current studies have focused on the effect of plant species diversity
on CW ecosystem functioning, and the relative influence of organisms
from different trophic levels was often neglected (Barnes et al., 2018).
Benthic organisms, such as Corbicula fluminea, exhibit resistance to
pollution and cold temperatures and can directly eliminate water pol­
lutants through feeding and bioaccumulation (Li et al., 2021; Gomes
et al., 2021; Zhao et al., 2022). Their activities can also indirectly impact
pollutant removal and greenhouse gas emissions by modifying the
growth environment of plants and microorganisms (Gomes et al., 2021;
Zhao et al., 2022). Adding C. fluminea to CWs can improve total nitrogen
removal rates by 13.7 % (Li et al., 2010). However, the effect of intro­
ducing C. fluminea into CWs on nitrogen removal and cumulative
greenhouse gas emissions at different plant species diversity requires
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Science of the Total Environment 907 (2024) 168092
further investigation.
This study established 66 micro vertical flow CWs and selected four
common emergent plants from the local area for plant species diversity
configuration, including monoculture, two-species mixture, and four-
species mixture. C. fluminea was added or not added at each diversity
level, and CH4, CO2, and N2O emissions, effluent nitrogen concentra­
tion, plant biomass, and substrate nitrogen accumulation were deter­
mined. The primary aims were to explore (1) the effects of C. fluminea on
nitrogen removal and accumulated greenhouse gas emissions at
different plant species diversity levels; (2) the effects of C. fluminea on
the relationship between plant species diversity and nitrogen removal
and accumulated greenhouse gas emissions; (3) plant-benthic animal
combinations with low cumulative greenhouse gas emissions and high
nitrogen removal efficiency.
2. Materials and methods
2.1. Experiment design
66 micro vertical flow CWs were constructed for the experiment in
October 2022 at Wenzhou University in Wenzhou City, Zhejiang Prov­
ince, China. All microcosms were inside a greenhouse equipped with a
transparent plastic roof, shielding the experiment from rainwater while
maintaining natural light, temperature, and humidity levels. The simu­
lated microcosms were constructed using PP boards, with dimensions of
40 cm × 40 cm × 80 cm (length × width × height). Each microcosm
consisted of a substrate composed of a 55 cm layer of fine sand (diameter
0.5–1 mm) and a 5 cm layer of coarse sand (diameter 3–6 mm) from top
to bottom. Fine sand was more conducive to the survival of C. fluminea,
and the coarse sand at the bottom of the device was used to prevent
blockage of the outlet. A PVC pipe with a diameter of 6.3 cm and a length
of 70 cm was installed in each microcosm, with small holes evenly
spaced along the pipe wall to allow for even water seepage. Mesh was
wrapped around the periphery of the microcosms to prevent sand from
entering.
Plant diversity in the experiment varied across three levels of species
richness (1, 2, and 4 species) and eleven plant species compositions,
including four-species mixture (one), two-species mixtures (six), and
monocultures (four). Four species of emergent plants were selected for
the experiment: Reineckea carnea, Hemerocallis fulva, Acorus calamus,
and Iris tectorum (see Appendix Table 1). These four plant species all
have good cold resistance, strong water purification ability, and devel­
oped root systems. C. fluminea was added or not added to the microcosm
of each level of species diversity (Fig. 1). In systems with C. fluminea,
twelve individuals of C. fluminea were added to each microcosm and
dispersed around the microcosm to ensure their uniform distribution.
The experiment followed a randomized complete block design with
three replicates. Eight plants were planted in each microcosm, with an
equal number and similar size of transplanted seedlings for each plant
species, and they were evenly distributed to eliminate any potential
influence of non-uniformity. The experimental system was observed
every two weeks throughout the experiment, and any plant species
outside of the experimental treatment were manually removed.
The simulated wastewater adjusted the concentration of N and P
elements based on the Hoagland nutrient solution, which can meet the
effluent concentration standards of sewage purifying-tank and ensure
L. Yang et al.
the normal growth of plants (Hoagland and Arnon, 1950; Appendix
Table A.2). The TN concentration in the simulated wastewater was 20
mg L− 1, with nitrate nitrogen (NO−3 -N) at 5 mg L− 1, ammonia nitrogen
− 1 − 1
(NH+ 4 -N) at 15 mg L , and chemical oxygen demand (COD) at 5 mg L
(Appendix Table A.2). According to previous studies that the most
effective hydraulic retention time for pollutant removals was 8 days, for
temperatures under 15 ◦ C, a longer hydraulic retention time was
required (Varma et al., 2021), the hydraulic retention time was 10 days
in this study. 22 L simulated wastewater was supplied to each micro­
cosm at an interval of 10 days, from October 2022 to December 2022, for
a total of six times, simulating the batch feeding mode (Faulwetter et al.,
2009; Wu et al., 2015). The water level in the microcosms was main­
tained 5 cm above the sand surface. Before each new supply of simulated
wastewater, the system was emptied.
2.2. Sample collection and measurements
2.2.1. Greenhouse gas collection and measurement
Greenhouse gas samples were collected in mid-December 2022. CH4,
CO2, and N2O emissions from CWs were measured on the first, fifth, and
tenth days after the last addition of simulated sewage. Using the inter­
nationally recognized static chamber method to collect greenhouse gas
samples. The static chamber used in this study was constructed from
opaque PP board, with dimensions of 40 cm × 40 cm × 50 cm (height ×
width × length) (refer to Fig. 1). The opaque PP board can minimize the
effect of rising temperature on the measurement of greenhouse gas
concentrations in the gas samples. A small hole was left at the top of the
chamber to accommodate the temperature sensor. On one side of the
Fig. 1. Illustration of the constructed microcosm. (a) eleven different plant community compositions and a top view of a four-species mixture; (b) A structural
diagram of a CWs microcosm with the sampling chamber. Blue squares indicate microcosms with C. fluminea; gray squares indicate microcosms without C. fluminea.
Different letters indicate different plant species: a, Reineckea carnea; b, Hemerocallis fulva; c, Acorus calamus; d, Iris tectorum. Different shapes represent different
plant species.
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Science of the Total Environment 907 (2024) 168092
chamber, a small gas sampling hole was tightly sealed with a silicone
plug to serve as a sampling port. A small fan was incorporated to ensure
the even distribution of gas inside the static chamber. In order to prevent
gas leakage, the gap between the static chamber and the CWs system was
sealed with water.
Following the method described by Sun et al. (2013), gas samples
were collected at different times after the chamber was enclosed. It was
observed that the concentrations of greenhouse gases increased linearly
only during the first 20 min (R2 > 0.90). Therefore, this experiment
collected greenhouse gas samples after the static chamber closed for 20
min. CH4, CO2, and N2O concentrations were determined using gas
chromatography (Agilent 7890, Agilent Technologies Inc., USA).
2.2.2. Water sample collection and measurement
After collecting gas samples, the temperature, pH, dissolved oxygen
(DO), and oxidation-reduction potential (ORP) values in the microcosms
were measured using a YSI multi-parameter water analyzer (Pro Qua­
tro). Then, water samples (250 mL) were collected from each micro­
cosm. Before analysis, each water sample was filtered using a
microporous filter membrane (0.45 μm). Then, the concentration of
ammonia nitrogen (NH+ 4 -N) was determined using Nessler's reagent
spectrophotometry, and the concentration of nitrate nitrogen (NO−3 -N)
was measured using ultraviolet spectrophotometry. The sum of NH+ 4 -N
and NO−3 -N concentrations was the total inorganic nitrogen (TIN) con­
centration. The total organic carbon (TOC) concentration was measured
using a Total Organic Carbon analyzer (TOC-L CPH).
L. Yang et al.
2.2.3. Plant sample collection and measurement
At the end of the experiment, all plants in each microcosm were
harvested using the whole-plant harvesting method. The harvested
plants were then washed and divided into aboveground and below­
ground parts. Subjected to quenching at 105 ◦ C for 20 min, the plants
were dried at 65 ◦ C until constant weight to achieve aboveground and
belowground biomass. The sum of the aboveground and belowground
biomass was plant total biomass.
2.2.4. Substrate sample collection and measurement
After the plants were harvested, the substrate of each microcosm was
thoroughly mixed. Substrate samples were then collected and stored at
− 20 ◦ C for further analysis. Prior to analysis, the substrate samples were
thawed at 4 ◦ C. Each microcosm contained 150 g of fresh sand extracted
with 100 mL of a 2 mol L− 1 KCl solution. The ratio of solution volume to
matrix fresh weight was 1:1.5. The extraction solution was filtered using
a microporous filter membrane (0.45 μm). The filtrate was then used to
determine the concentrations of NH+ 4 -N, NO3 -N, and TOC. The deter­
−
mination method for these parameters in the filtrate was the same as in
the water samples.
2.3. Parameter calculations
(1) Flux of greenhouse gas emission
The greenhouse gas emission flux calculated following Cheng et al.
(2007):
V dc T0
E = ρ× × ×
A dt T0 + T
where E (mg m− 2 day− 1) is the flux of greenhouse gas emission (CH4,
CO2, and N2O); A (m2) is the chamber bottom area; ρ (kg m− 3) is the
greenhouse gas density under standard conditions (The gas densities of
CH4, CO2, and N2O are 0.71 kg m− 3, 1.98 kg m− 3, and 1.96 kg m− 3,
respectively; V (m3) is the sampling chamber volume; dc is the change in
CH4, CO2, and N2O concentration of the chamber. dt is the duration of
sampling (20 min); T0 and T are the absolute temperature (273 K) and
the actual temperature (K), respectively.
(2) Cumulative greenhouse gas emissions were calculated as follows:
n ( ) species (Fig. 2a). Plant species richness did not significantly affect the
∑ Fi + Fi+1
CE = × (ti+1 − ti )
i=1
2 C. fluminea (Fig. 2c, e).
where CE (mg m− 2) is the cumulative greenhouse gas emissions; i is the
i-th greenhouse gas sampling; n represents the total measurement
number of greenhouse gas emission flux; F (mg m− 2 day− 1) is the flux of
greenhouse gas emission (CH4, CO2, and N2O); and ti+1-ti represents the
number of days between adjacent greenhouse gas flux measurements.
(3) According to Engelhardt and Ritchie's research (2002), relative
yield (RY) was an important indicator for determining the
competitiveness of specific species in the mixture. RY was
calculated using the following formula:
Oi
RY i =
Ei.
where Ei is the monoculture average aboveground biomass of species i
divided by the total number of species in the mixture contained species i,
and Oi is the measured aboveground biomass of plant species i in the
mixture. RYi > 1 represents species i is the superior competitor in the
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Science of the Total Environment 907 (2024) 168092
mixture; RYi = 1 represents no difference between species intraspecies
competition and interspecific competition, and RYi < 1 represents spe­
cies i is the inferior competitor in the mixture.
2.4. Statistical analysis
The effects of plant species richness and the presence of C. fluminea,
species composition and the presence of C. fluminea on cumulative
greenhouse gas emissions, effluent nitrogen concentrations, substrate
nitrogen accumulations, plant biomass and environmental factors in
CWs were analyzed using a two-way ANOVA. The effects of plant species
richness on the variables were examined using Linear regression anal­
ysis, while the effects of plant species richness and species compositions
on the variables were analyzed using one-way ANOVA in microcosms
with and without C. fluminea, respectively. If the effect proved to be
significant, multiple comparisons (TukeyHSD) were used to determine
the difference among treatments. The effects of C. fluminea or each
species on the variables were analyzed using independent sample t-tests.
The significant difference between the RY value of each species in each
mixture with one was analyzed using one sample t-test. In order to meet
the assumptions of normality (tested using the Kolmogorov-Smirnov
test), the data were log-transformed before analysis. If the data still
did not meet the assumptions of normality even after log transformation,
nonparametric Kruskal-Wallis tests were used. Furthermore, the corrplot
function from the corrplot package in R was utilized to create a corre­
lation heatmap, which visually represents the strength of correlations
between various parameters. Colors were used to indicate the correla­
tion strength. All analyses were completed using the R program (R
4.2.3), and the significance level was 0.05.
3. Results
3.1. Responses of cumulative greenhouse gas emissions to C. fluminea and
plant diversity
In the microcosms without C. fluminea, there was no significant
difference in the cumulative CH4 emissions among different levels of
plant species richness (Fig. 2a). However, in the microcosms with
C. fluminea, the cumulative CH4 emissions showed a trend of first
decreasing and then increasing with increasing species richness. The
lowest cumulative CH4 emissions were observed when there were two
cumulative emissions of CO2 and N2O in microcosms with or without
The composition of plant species significantly impacted the cumu­
lative emissions of greenhouse gases (CH4, CO2, and N2O), and there was
an interactive effect between plant species compositions and C. fluminea
(Appendix Table A.3). In the microcosms without C. fluminea, the
monoculture of H. fulva had the highest cumulative CH4 emissions. In
comparison, the monoculture of R. carnea had the lowest emissions
(Fig. 2b). The presence of H. fulva significantly increased the cumulative
CH4 emissions (Table 1 and Appendix Table A.4). The cumulative CO2
emissions from the mixture of A. calamus and I. tectorum were signifi­
cantly lower than those from the mixture of R. carnea and A. calamus
(Fig. 2d). The monoculture of I. tectorum had the highest cumulative N2O
emissions (Fig. 2f). However, species identity did not significantly affect
the cumulative N2O emissions and cumulative CO2 emissions
(Table 1and Appendix Table A.4).
In the microcosms with C. fluminea, the mixture of R. carnea and
A. calamus had the lowest cumulative CH4 emissions (Fig. 2b). The cu­
mulative CO2 emissions from the mixture of R. carnea and I. tectorum
were significantly lower than those from the mixture of R. carnea and
H. fulva (Fig. 2d). The presence of H. fulva significantly increased the
cumulative CO2 emissions (Table 2 and Appendix Table A.5). I. tectorum
monoculture had the highest cumulative N2O emissions compared to the
other three monocultures (Fig. 2f). Additionally, the presence of
L. Yang et al. Science of the Total Environment 907 (2024) 168092

Fig. 2. Difference in cumulative greenhouse gas emissions among species richness and species compositions in CWs with or without C. fluminea. (a, b) cumulative
CH4 emissions; (c,d) cumulative CO2 emissions; (e,f) cumulative N2O emissions. Blue bars or circles indicate microcosms with C. fluminea; gray bars or circles indicate
microcosms without C. fluminea. Blue or gray diamonds represent mean ± standard error. Species abbreviations: Rc, Reineckea carnea; Hf, Hemerocallis fulva; Ac,
Acorus calamus; It, Iris tectorum. Significant differences (P < 0.05) among species richness or compositions are represented by different letters above the circles or
bars. The asterisks between the blue and gray bars or circles indicate significant differences in microcosms with and without C. fluminea, where *:P < 0.05; **:P <
0.01; ***:P < 0.001.

I. tectorum significantly increased the cumulative N2O emissions (Table 2
and Appendix Table A.5).
The addition of C. fluminea significantly affected the cumulative
greenhouse gas emissions (Appendix Table A.3). In the monocultures of
R. carnea and A. calamus, the addition of C. fluminea significantly
3.2. Responses of effluent N concentration and substrate N accumulation
to C. fluminea and plant diversity
In the microcosms without C. fluminea, effluent nitrogen (NO−3 -N,
NH+ 4 -N, and TIN) concentrations and substrate NO3 -N accumulations
−

increased the cumulative CH4 emissions (Fig. 2b). Similarly, in the
mixture of A. calamus and I. tectorum, the addition of C. fluminea also
increased the cumulative emissions of CO2 and N2O (Fig. 2d, f). How­
ever, in the mixtures of R. carnea and A. calamus, as well as R. carnea and
I. tectorum, the addition of C. fluminea significantly reduced the cumu­
lative CO2 emissions (Fig. 2d).
significantly increased with the increase of plant species richness (Fig. 3,
Appendix Table A.6, Appendix Table A.7). However, in the microcosms
with C. fluminea, only effluent TIN concentration significantly increased
with the increased of plant species richness (Fig. 3c), substrate nitrogen
accumulations did not response to plant species richness (Fig. 3d, e, f).
Plant species composition significantly influenced the effluent nitrogen
concentrations and substrate nitrogen accumulations, and there was an
interactive effect between the plant species compositions and C. fluminea
(Appendix Table A.6 and Appendix Table A.7). In the microcosms
without C. fluminea, the effluent nitrogen (NH+ 4 -N and TIN)

5
L. Yang et al. Science of the Total Environment 907 (2024) 168092

Table 1 Table 2
The effects of the presence or absence of specific plant species on cumulative The effects of the presence or absence of specific plant species on cumulative
greenhouse gas emissions, N concentration in effluent, N accumulation in sub­ greenhouse gas emissions, N concentration in effluent, N accumulation in sub­
strate, plant biomass, and environmental factors in the microcosms without strate, plant biomass, and environmental factors in the microcosms with
C. fluminea. C. fluminea.
Response variable Source of variation Response variable Source of variation

d. R. carnea H. fulva A. calamus I. tectorum d. R. carnea H. fulva A. calamus I. tectorum

f. f.

Cumulative GHG emissions Cumulative GHG emissions

CH4 (mg m− 2) 31 0.786 0.044↑ 0.575 0.270 CH4 (mg m− 2) 31 0.575 0.503 0.731 0.270
CO2 (g m− 2) 31 0.073 0.152 0.760 0.175 CO2 (g m− 2) 31 0.924 0.001↑ 0.379 0.679
N2O (mg m− 2) 31 0.600 0.053 0.786 0.286 N2O (mg m− 2) 31 0.111 0.176 0.301 < 0.001↑

N concentration in effluent N concentration in effluent

NO−3 -N (mg L− 1) 31 0.009↑ 0.943 0.078 0.936 NO−3 -N (mg L− 1) 31 0.906 0.182 0.643 <0.001↑
− 1 − 1
NH+4 -N (mg L ) 31 0.004↑ 0.377 0.127 0.428 NH+4 -N (mg L ) 31 0.083 0.973 0.040↑ 0.304
TIN (mg L− 1) 31 <0.001↑ 0.657 0.044↑ 0.771 TIN (mg L− 1) 31 0.383 0.269 0.119 0.028↑

N accumulation in substrate N accumulation in substrate

NO−3 -N (mg g− 1) 31 0.885 0.563 0.031↑ 0.121 NO−3 -N (mg g− 1) 31 0.142 0.090 0.102 0.316
− 1 − 1
NH+4 -N (mg g ) 31 0.018↓ 0.529 0.806 0.851 NH+4 -N (mg g ) 31 0.034↓ 0.025↓ 0.016↑ 0.038↑
TIN (mg g− 1) 31 0.084↓ 0.649 0.006↑ 0.257 TIN (mg g− 1) 31 0.033↓ 0.011↓ 0.016↑ 0.974

Plant biomass Plant biomass

Aboveground biomass 31 < 0.228 0.002↓ 0.347 Aboveground biomass 31 < 0.212 0.025↓ 0.140
(g m− 2) 0.001↑ (g m− 2) 0.001↑
Belowground biomass 31 0.010↓ 0.001↑ 0.500 0.226 Belowground biomass 31 0.008↓ < 0.384 0.451
(g m− 2) (g m− 2) 0.001↑
Total biomass (g m− 2) 31 0.622 0.001↑ 0.015↓ 0.632 Total biomass (g m− 2) 31 0.922 < 0.024↓ 0.722
0.001↑

Environmental factor
Temperature (◦ C) 31 0.954 0.877 0.877 0.487 Environmental factor
DO (mg L− 1) 31 0.212 0.338 0.786 0.704 Temperature (◦ C) 31 0.252 0.347 0.157 0.018↑
pH 31 0.448 0.333 0.471 0.391 DO (mg L− 1) 31 0.024↑ 0.192 0.034↑ 0.024↓
ORP (mv) 31 0.772 0.717 0.942 0.124 pH 31 0.971 0.294 1.000 0.526
ORP (mv) 31 0.365 0.356 0.828 0.128
Note: Arrows (↑) or (↓) represent the significant increase or decrease in variables
when a certain specific species is present. Significant P values (P < 0.05) are Note: Arrows (↑) or (↓) represent the significant increase or decrease in variables
represented in bold black font. when a certain specific species is present. Significant P values (P < 0.05) are
represented in bold black font.
concentrations from R. carnea monoculture were significantly higher
than those from I. tectorum monoculture (Appendix Fig. A.1b, c). The H. fulva + I. tectorum, the addition of C. fluminea significantly reduced
presence of R. carnea significantly increased the effluent nitrogen (NO−3 - effluent NH+4 -N concentrations (Appendix Fig. A.1b). Additionally, in
N, NH+ 4 -N, and TIN) concentrations (Table 1 and Appendix Table A.4).
the mixture of R. carnea + I. tectorum, the addition of C. fluminea reduced
Additionally, the presence of A. calamus significantly increased the substrate NO−3 -N accumulation (Appendix Fig. A.2a).
substrate nitrogen (NO−3 -N and TIN) accumulations (Table 1and Ap­
pendix Table A.4), and the substrate TIN accumulation from A. calamus 3.3. Responses of plant biomass to C. fluminea and plant diversity
monoculture was significantly higher than that from I. tectorum mono­
culture (Appendix Fig. A.2c). Plant species richness did not significantly affect either aboveground
In the microcosms with C. fluminea, the effluent NH+ 4 -N concentra­ biomass, belowground biomass, or overall total biomass in microcosms
tion and the substrate NH+ 4 -N accumulation from H. fulva monoculture with or without C. fluminea (Fig. 4 and Appendix Table A.8). However,
were the lowest among the four monocultures (Appendix Fig. A.1b, plant species composition significantly impacted plant biomass. There
Appendix Fig. A.2b). The presence of H. fulva significantly reduced the was an interactive effect between plant species composition and
substrate nitrogen (NH+ 4 -N and TIN) accumulations, while the presence C. fluminea (Appendix Table A.8). In the microcosms without
of A. calamus significantly increased the substrate nitrogen (NH+ 4 -N and C. fluminea, the aboveground, and total biomass were highest in the
TIN) accumulations (Table 2, Appendix Table A.5). The substrate ni­ mixture of R. carnea and H. fulva (Fig. 4b, f). In microcosms with
trogen (NH+ 4 -N, NO3 -N, and TIN) accumulations in the mixture of
−
C. fluminea, the aboveground biomass was highest in the R. carnea
A. calamus and I. tectorum were the highest among all the plant species monoculture (Fig. 4b), while the belowground and total biomass was
compositions (Appendix Fig. A.2). highest in the H. fulva monoculture (Fig. 4d, f). Regardless of the pres­
The addition of C. fluminea had a significant influence on effluent ence or absence of C. fluminea, the presence of R. carnea significantly
NO−3 -N and TIN concentrations (Appendix Table A.6). In the mono­ increased aboveground biomass and reduced belowground biomass. The
culture of A. calamus, the addition of C. fluminea significantly increased presence of H. fulva significantly increased belowground and total
effluent nitrogen (NH+ 4 -N, NO3 -N, and TIN) concentrations (Appendix
−
biomass. The presence of A. calamus significantly reduced aboveground
Fig. A.1). Similarly, in the mixtures of R. carnea + I. tectorum and and total biomass. The presence of I. tectorum did not have a significant
H. fulva + I. tectorum, the addition of C. fluminea also increased effluent effect on plant biomass (Table 1 and Table 2, Appendix Table A.4 and
nitrogen (NO−3 -N and TIN) concentrations (Appendix Fig. A.1a, c). In the Appendix Table A.5). In the R. carnea monoculture and the mixture of
monoculture of I. tectorum, the addition of C. fluminea significantly A. calamus and I. tectorum, the addition of C. fluminea significantly
increased substrate nitrogen (NO−3 -N and TIN) accumulations (Appendix increased aboveground biomass. However, in the I. tectorum mono­
Fig. A.2a, c). However, in the mixtures of H. fulva + A. calamus and culture, the addition of C. fluminea significantly decreased aboveground

6
L. Yang et al. Science of the Total Environment 907 (2024) 168092

Fig. 3. Difference in effluent N concentrations and substrate N accumulations among species richness in CWs with or without C. fluminea. (a) NO−3 -N concentration in
effluent; (b) NH+
4 -N concentration in effluent; (c) TIN concentration in effluent; (d) NO3 -N accumulation in substrate; (e) NH4 -N accumulation in substrate; and (f)
− +

TIN accumulation in substrate. Blue circles indicate microcosms with C. fluminea; gray circles indicate microcosms without C. fluminea. Blue or gray diamonds
represent mean ± standard error. Significant differences (P < 0.05) in richness are represented by different letters above the circles. The asterisks between the blue
and gray bars or circles indicate significant differences in microcosms with and without C. fluminea, where *:P < 0.05; **:P < 0.01; ***:P < 0.001.

biomass (Fig. 4b). In the A. calamus and I. tectorum monocultures, the
addition of C. fluminea significantly decreased belowground and total
biomass (Fig. 4d, f).
4. Discussion
4.1. Responses of cumulative N2O emissions to C. fluminea and plant
species diversity in CWs
The nitrification and denitrification processes of microorganisms
were the main pathways for producing N2O (Maucieri et al., 2017; Sun
et al., 2022; Hu et al., 2023). Previous studies reported that high plant
species richness can promote or reduce N2O emissions in CWs. Some
studies suggest that high species richness increases N2O emissions by
enhancing gas transport capacity or reducing nitrogen absorption (Han
et al., 2016), while others indicate that it reduces N2O emissions by
increasing nitrogen absorption or substrate/soil nitrogen accumulation
(Du et al., 2020; Jiang et al., 2023). However, in this study, plant species
richness did not significantly affect cumulative N2O emissions in mi­
crocosms with or without C. fluminea (Fig. 2e). One possible reason for
this finding is that species richness did not influence substrate nitrogen
accumulation and plant biomass (Appendix Table A.7 and Appendix
Table A.8). Furthermore, these previous studies have primarily
concentrated on the impact of species diversity on a single time point

7
L. Yang et al. Science of the Total Environment 907 (2024) 168092

Fig. 4. Difference in plant biomass between species richness and species compositions in CWs with or without C. fluminea. (a,b) aboveground biomass; (c,d)
belowground biomass; (e,f) total biomass. Blue bars or circles indicate microcosms with C. fluminea; gray bars or circles indicate microcosms without C. fluminea. Blue
or gray diamonds represent mean ± standard error. Species abbreviations: Rc, R carnea; Hf, H fulva; Ac, A calamus; It, I tectorum. Significant differences (P < 0.05) in
richness or composition are represented by different letters above the circles or bars. The asterisks between the blue and gray bars or circles indicate significant
differences in microcosms with and without C. fluminea, where*:P < 0.05; **:P < 0.01; ***:P < 0.001.

N2O emission (Han et al., 2019; Wang et al., 2022; Jiang et al., 2023),
rather than considering the impact of species diversity on longer-term
cumulative emissions. Abalos et al. (2014) found that cumulative N2O
emissions were not related to plant species richness in grassland systems,
suggesting that the effect of plant species richness on cumulative N2O
emissions may be limited.
Plant species composition is critical to ecosystem functioning (Zhao
et al., 2023). In this study, plant species compositions significantly
affected cumulative N2O emissions (Appendix Table A.3). The cumula­
tive N2O emissions of I. tectorum monoculture was higher than other
species compositions in the microcosms with C. fluminea (Fig. 2f). N2O
emissions are influenced by multiple factors, with dissolved oxygen
(DO) being one of the critical factors (Wu et al., 2009; Meng et al., 2014;
Chen et al., 2022). This study observed a significant negative correlation
between cumulative N2O emissions and DO (Fig. 6). Therefore, the
presence of I. tectorum significantly increased N2O emissions by reducing
DO levels (Table 2 and Appendix Table A.5). Highly competitive species
can strongly affect ecosystem function in mixed species systems
(Engelhardt and Ritchie, 2002), which may promote N2O emissions in
mixtures. However, in the microcosms without C. fluminea, the presence
of I. tectorum did not affect cumulative N2O emissions. The addition of
C. fluminea significantly increased cumulative N2O emissions in the
mixtures of R. carnea + H. fulva and A. calamus + I. tectorum (Fig. 2f).
This may be due to the oxygen consumed by C. fluminea being utilized by
microorganisms in the intestinal microenvironment, resulting in
anaerobic conditions and incomplete denitrification, leading to
increased N2O emissions (Kang et al., 2017). The above results indicate
that C.fluminea may be able to alter the relationship between cumulative
N2O emissions and plant species diversity, and I. tectorum may not be a
suitable choice for reducing cumulative N2O emissions in CWs.

8
L. Yang et al.
4.2. Responses of cumulative CH4 emissions to C. fluminea and plant
species diversity in CWs
In the microcosms with C. fluminea, the lowest cumulative CH4
emissions observed when there were two species (Fig. 2a). This finding
aligns with previous studies suggesting that higher species richness may
increase CH4 emissions by increasing organic carbon content (Zhang
et al., 2012). In this study, the lowest TOC accumulation in the substrate
was observed when there were two species, and the highest occurred
when there were four species (Appendix Fig. A.3), which follows the
same trend as the cumulative CH4 emissions. In the microcosms without
C. fluminea, cumulative CH4 emissions were not affected by the species
richness of the plant (Fig. 2a). This result agreed with previous research
findings (Mo et al., 2015; Han et al., 2017). Higher concentrations of
NO−3 -N may inhibit the oxidation of methanotrophic bacteria due to
osmotic stress (Bodelier and Laanbroek, 2004; Søvik and Kløve, 2007).
Species richness increased the effluent NO−3 -N concentration (Fig. 3a)
and substrate NO−3 -N accumulation in this study (Fig. 3d). However,
species richness also increased the effluent NH+ 4 -N concentration. NH4 -
+
N can promote the growth and activity of methanotrophic bacteria,
thereby enhancing the oxidation of CH4 (Bodelier et al., 2000).
In the microcosms with C. fluminea, the cumulative CH4 emissions
from the mixture of R. carnea and A. calamus were negative (Fig. 2b),
indicating that CH4 had been oxidized. We observed that the presence of
R. carnea and A. calamus improved the water's dissolved oxygen (DO)
levels (Table 2 and Appendix Table A.5). Therefore, R. carnea and
A. calamus may promote the oxidation of CH4, resulting in lower cu­
mulative CH4 emissions (Fig. 2b). Additionally, the increase of plant
biomass also can promote CH4 emissions (Zhang et al., 2012). Thus, the
presence of R. carnea and A. calamus may also reduce cumulative CH4
Fig. 5. The relative yield (RY) of a specific species in different plant species compositions. Species abbreviations: Rc, R carnea; Hf, H fulva; Ac, A calamus; It, I
tectorum. Blue circles indicate microcosms with C. fluminea; gray circles indicate microcosms without C. fluminea. Values are the mean ± standard error. The sig­
nificant difference between the RY value of each species in each mixture and the RY value equal with one was represented by an asterisk.
9
Science of the Total Environment 907 (2024) 168092
emissions by reducing plant biomass (Table 2 and Appendix Table A.5).
However, in the case of the four-species mixture of R. carnea and
A. calamus, the relative yield of these species did not reach 1 (Fig. 5 and
Appendix Table A.9), indicating that they were inferior competitors
compared to other species in the mixture. Consequently, the cumulative
CH4 emissions from the four-species mixture did not decrease (Fig. 2b).
It is worth noting that in the microcosms without C. fluminea, the
presence of H. fulva significantly increased CH4 emissions by increasing
belowground biomass (Table 1 and Appendix Table A.4), while R. carnea
reduced belowground biomass but did not affect CH4 emissions. This
may be because the presence of R. carnea significantly increased effluent
nitrogen concentration, which promoted CH4 emissions, thereby off­
setting the reduction in belowground biomass. These results indicate
that adding C. fluminea also alters CH4 emissions on different species
richness or species compositions. As a benthic fauna, C. fluminea can
alter the microbial community structure of aquatic ecosystems through
its activities and interactions with microorganisms (Gomes et al., 2021).
Therefore, changes in microbial community structure may increase
methanogenic bacteria, resulting in higher cumulative CH4 emissions.
However, this study did not specifically focus on changes in microbial
community structure. Future research can explore the potential effects
of C. fluminea on microbial community structure in more depth.
4.3. Responses of cumulative CO2 emissions to C. fluminea and plant
species diversity in CWs
A previous study reported no effect of species richness on CO2
emissions from natural wetland microcosms (Mo et al., 2015). However,
Sun et al. (2019) reported that high plant species richness could increase
CO2 emissions from CWs due to the positive response of carbon fixation
L. Yang et al.
by plants to plant species richness (Sun et al., 2019). In this study, cu­
mulative CO2 emissions were unaffected by plant species richness in
microcosms with or without C. fluminea (Fig. 2c). We also found a
positive correlation between cumulative CO2 emissions and the above­
ground biomass of plants (Fig. 6). However, the aboveground biomass of
plant was not affected by plant species richness (Fig. 4a). Accordingly,
the cumulative CO2 emissions from CWs also were not affected.
There were significant differences among plant species in their
regulation of CO2 production, consumption, and transportation (Sun
et al., 2019). In this study, species compositions significantly affected
cumulative CO2 emissions (Appendix Table A.3), and the presence of
H. fulva significantly increased the cumulative CO2 emissions in mi­
crocosms with C. fluminea (Table 2 and Appendix Table A.5). As one of
the main factors affecting CO2 emissions in wetland systems, soil
respiration includes aerobic microbial respiration, root respiration, and
anaerobic respiration (Liu et al., 2019). The microbial communities
associated with different plant species can vary (Leff et al., 2018), and
there are differences in the physiological and morphological traits of
different plant species, such as root surface area, root diameter, and root
tip number (Fox et al., 2020). H. fulva, in particular, has an extensive
root surface area (Appendix Table A.10), which provides more attach­
ment surfaces and secretions for microbial activity, promoting the
conversion of organic pollutants into CO2. Additionally, we observed a
significant negative correlation between cumulative CO2 emissions and
substrate TIN accumulation (Fig. 6). The presence of H. fulva signifi­
cantly increased cumulative CO2 emissions by reducing TIN
Fig. 6. Correlation analysis between various parameters. The number represents the correlation coefficient. The asterisks indicate a significant level, where *:P <
0.05; **:P < 0.01; ***:P < 0.001.
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Science of the Total Environment 907 (2024) 168092
accumulation in the substrate (Table 2 and Appendix Table A.5).
In the microcosms without C. fluminea, the presence of H. fulva did
not significantly affect cumulative CO2 emissions. In this study, there
was an interaction between species combinations and C. fluminea (Ap­
pendix Table A.3), which may have influenced cumulative CO2 emis­
sions. The valve movement behavior of C. fluminea's shell can lead to
CO2 production through respiration, thereby promoting CO2 emissions
in the system. However, C. fluminea also ingests food from the substrate,
converting some pollutants into soluble forms, promoting nutrient up­
take by plant roots, and enhancing plant growth and microbial activity.
Therefore, the presence of C. fluminea may contribute to the different
effects of H. fulva on cumulative CO2 emissions in the two systems.
However, the current research results do not allow for clear conclusions
or provide strong data support. Further research is needed to explore
these interactions in more detail.
4.4. Responses of nitrogen removal to C. fluminea and plant species
diversity in CWs
Nitrogen removal is a crucial ecosystem function in CWs. However,
nitrogen removal efficiency in autumn and winter may not be as high as
in spring and summer due to factors such as temperature, and the ben­
efits of using CWs to treat sewage may be offset by increased CH4 and
N2O emissions (Mander et al., 2014). The effluent TIN concentration in
the monoculture was significantly lower than in the mixtures in mi­
crocosms with or without C. fluminea (Fig. 3c). This finding contradicts
L. Yang et al.
some previous research results. Most studies have shown that promoting
plant nitrogen uptake and substrate nitrogen accumulation is an
important reason why high plant species richness can improve nitrogen
removal efficiency (Chang et al., 2014; Du et al., 2020). However, our
research showed that species richness did not affect plant biomass and
substrate nitrogen accumulation (Appendix Table A.7 and Appendix
Table A.8). The ambient temperature may have led to this observation.
This study was conducted during autumn and winter, when climatic
conditions directly or indirectly influence plant nutrient uptake. The
low-temperature environment is not conducive to the proliferation and
metabolism of bacteria. Microbial nitrification is also inhibited at low
temperatures (Wang et al., 2017). Therefore, the inhibitory effect of
temperature on plants and microorganisms may contribute to an in­
crease in the effluent N concentration of the four-species mixture
system.
In this study, adding C. fluminea significantly increased the effluent
concentrations of NO−3 -N and TIN (Appendix Fig. A.1). This may be
attributed to differences in experimental designs. For example, Li et al.
(2010) used floating CWs with C. fluminea to improve the N removal
rates by 13.7 %. Our experiment used vertical flow CWs, which provide
more attachment points for microbial growth. Additionally, the rela­
tively high N loading in our experiment and the low temperatures during
the cold season may inhibit the growth of C. fluminea, thereby affecting
its N removal efficiency. As a benthic animal, C. fluminea can produce
feces, which may release more nitrogen through biological sediment
processes. Furthermore, it is worth noting that experiment duration
could alter the influence of biodiversity on ecosystem functioning.
Previous studies reported that the effects of plant diversity on biomass
productivity increased and became less saturating over time (Reich
et al., 2012). Future work should aim to conduct long-term period ex­
periments to explore further the effects of plant diversity and benthic
animals on the ecosystem functions of CWs.
5. Conclusions
This study provides novel insight into the effects of plant species
diversity and the addition of C. fluminea on cumulative greenhouse gas
emissions and nitrogen removal. The results indicate that adding
C. fluminea significantly increases the cumulative greenhouse gas (CH4
and N2O) emissions under the same species compositions. Plant species
richness did not significantly influence cumulative greenhouse gas (CO2
and N2O) emissions, but plant species richness significantly increased
the effluent nitrogen concentration. The specific plant species compo­
sition significantly impacted cumulative greenhouse gas (CH4, CO2, and
N2O) emissions. In the presence of C. fluminea, the presence of H. fulva
significantly increased cumulative CO2 emissions, while the presence of
I. tectorum significantly increased cumulative N2O emissions. These
findings suggest that plant compositions with H. fulva or I. tectorum may
not be optimal for wastewater treatment and reducing greenhouse gas
emissions in CWs. Selecting a diverse range of plant species in future
research is recommended to ensure effective wastewater treatment and
minimize greenhouse gas emissions. Additionally, further research can
explore the effects of C. fluminea on microbial communities and the
elemental changes within C. fluminea. This will provide a more
comprehensive understanding of the impacts of plant species diversity
and C. fluminea on cumulative greenhouse gas emissions in CWs.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
11
Science of the Total Environment 907 (2024) 168092
Acknowledgements
We would like to thank Dr. Daniel Petticord at the University of
Cornell for his assistance with English language and grammatical editing
of the manuscript.
Author contributions
Conceptualization, WJH; formal analysis, LPY; investigation, LPY,
KS, XLX, HJC, YSL; writing—original draft preparation, LPY, KS XLX and
WJH; writing—review and editing, WJH, DRX, XYZ and MZ; funding
acquisition, WJH and XYZ. All authors have read and agreed to the
published version of the manuscript.
Funding
This research was funded by the Natural Science Foundation of
Zhejiang Province [grant number LY22C030003] and the National Key
Research and Development Program of China [grant number
2022YFE0106200].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2023.168092.
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