Drained water quality in sludge treatment wetlands: Effects of earthworm densities and plant species

Journal Pre-proof Drained water quality in sludge treatment wetlands: Effects of earthworm densities and plant species Shanshan Hu, Xingtao Zuo, Zuopeng Lv, Jiajie He, Yupeng Wu, Hongbo Liu, Zhongbing Chen PII:

S0959-6526(19)33998-8

DOI:

https://doi.org/10.1016/j.jclepro.2019.119128

Reference:

JCLP 119128

To appear in:

Journal of Cleaner Production

Received Date: 11 June 2019 Revised Date:

4 October 2019

Accepted Date: 31 October 2019

Please cite this article as: Hu S, Zuo X, Lv Z, He J, Wu Y, Liu H, Chen Z, Drained water quality in sludge treatment wetlands: Effects of earthworm densities and plant species, Journal of Cleaner Production (2019), doi: https://doi.org/10.1016/j.jclepro.2019.119128. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Drained water quality in sludge treatment wetlands: effects of earthworm

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densities and plant species

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Shanshan Hua, Xingtao Zuob, Zuopeng Lvc, Jiajie Heb, Yupeng Wub, Hongbo Liud,

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Zhongbing Chena*

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a

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University of Life Sciences Prague, Kamýcká 129, 16521, Prague, Czech Republic

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b

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China

Department of Applied Ecology, Faculty of Environmental Sciences, Czech

College of Resources and Environment, Huazhong Agricultural University, Wuhan,

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c

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Jiangsu Normal University, Shanghai Road 101, 221116, Xuzhou, China

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d

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Technology, 516 Jungong Road, 200093 Shanghai, China

The Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province,

School of Environment and Architecture, University of Shanghai for Science and

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*Corresponding author: Zhongbing Chen, Faculty of Environmental Sciences, Czech

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University of Life Sciences Prague, Email address: [email protected];

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[email protected]

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Abstract

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Earthworms can improve sludge dewatering and stabilization in sludge treatment

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wetlands (STWs). However, drained water quality in STWs with earthworm addition

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is not well known. In this study, the combination of two plant species (Phragmites

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australis and Typha angustifolia) and earthworm (Eisenia foetida) densities of 2.7, 5.4,

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8.1, 10.8, 13.5 and 16.2 kg/m3 in six STWs were investigated to evaluate their effects

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on drained water quality. Meanwhile, clogging characterization of the six STWs was

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evaluated. The results showed that filtration rates in the three earthworm STWs were

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0-0.4 cm/s higher than those in the STWs without earthworm addition. P. australis and

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T. angustifolia had positive effects on chemical oxygen demand (COD), total nitrogen

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(TN), total phosphorus (TP) and ammonium (NH4+) removal, with removal

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efficiencies all over 80%. Moreover, NH4+ removal efficiency was significantly

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different between P. australis and T. angustifolia; the higher NH4+ removal

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efficiencies of 89.5-90.4% were determined in the two P. australis STWs. Meanwhile,

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canonical correlation analysis and principal component analysis indicated that the

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combination of plants and earthworms was beneficial for drained water treatment in

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the STWs. Furthermore, the optimum drained water treatment was determined in the

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P. australis STWs with an earthworm density of 10.8 kg/m3, with the mass removal

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efficiencies for COD, NH4+, TN and TP being 99.1%, 93.1%, 91.5% and 91.0%,

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respectively. Thus, it can be concluded that P. australis and earthworm addition

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improved drained water quality and alleviated clogging in the STWs.

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Keywords: earthworm density; drained water quality; plant species; sludge treatment

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wetland

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Graphical abstract:

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1. Introduction

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Sewage sludge is a by-product in wastewater treatment process; it has high water

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content (more than 95%), high ratio of volatile solids (VS) to total solids (TS) (about

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45-65%), and even contains a large amount of pollutants such as heavy metals, toxic

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organic compounds and pathogens (Brix, 2017; Uggetti et al., 2010). Therefore,

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further treatment is needed before sewage sludge can be discharged into the

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environment.

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concentration, drying, composting, and anaerobic digestion, have the advantages of

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being rapid and effective (Andrade et al., 2017; Uggetti et al., 2010). However, these

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approaches require huge energy consumption and high costs, which limit sludge

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treatment efficiency in many regions (Kengne et al., 2014). Sludge treatment wetlands

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(STWs), as a low technology, low energy consumption, sustainable and

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environmentally friendly sludge treatment approach, have been widely applied in

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Spain, Poland, Italy, Brazil, China and other countries (Chen et al., 2016; Gagnon et

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al., 2012; Kołecka et al., 2019; Magri et al., 2016; Nassar et al., 2006; Uggetti et al.,

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2012).

Conventional

sludge

treatment

approaches,

including

sludge

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Sludge treatment processes by STWs mainly include sludge dewatering and

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sludge mineralization (Uggetti et al., 2010). Previous studies reported that water

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content in accumulated sludge was decreased by 10-60% through evaporation,

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transpiration and draining in STW systems (Brix, 2017; Stefanakis and Tsihrintzis,

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2011; Uggetti et al., 2010); meanwhile, the ratio of VS/TS was decreased by 25-30%

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due to biochemical reactions of rhizosphere microorganisms (Brix, 2017; Nielsen and

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Bruun, 2015). Furthermore, pollutant content (e.g. heavy metals, toxic organic

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compounds and pathogens) in accumulated sludge can also be decreased after the

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STW treatment (Chen et al., 2009; Chen and Hu, 2019; Hu and Chen, 2018;

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Stefanakis and Tsihrintzis, 2012). Although it has be proven that accumulated sludge

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can be treated efficiently in STWs, few studies have focused on drained water

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treatment in STW system. Thus, it is necessary to evaluate the drained water quality 4

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in STW systems.

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Previous research has shown that sludge treatment efficiency in STWs is mainly

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affected by wetland plant species (Hu et al., 2017; Nielsen and Bruun, 2015; Uggetti

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et al., 2012). However, the effects of wetland plant species on drained water treatment

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in STWs are still unclear. Previous study has shown that the highest pollutants

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(chemical oxygen demand (COD), total phosphorus (TP), and ammonium (NH4+))

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removal efficiencies in drained water were determined in STWs planted with

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Phragmites australis, followed by Typha angustifolia (Gagnon et al., 2012).

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Conversely, some studies also found that similar COD and TP removal efficiencies

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were achieved in drained water with T. angustifolia and P. australis STWs (Gagnon et

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al., 2013; Uggetti et al., 2012). Therefore, the plant effects on sludge drained water

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treatment in the STWs need to be investigated under local climate condition.

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In general, outflow concentrations of COD, TP, NH4+ and total nitrogen (TN) in

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drained water after STW treatment were still high, and even do not meet discharge

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requirements (Gagnon et al., 2013; Stefanakis et al., 2011; Stefanakis and Tsihrintzis,

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2011). Moreover, as a sludge and drained water treatment system, STWs still face

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certain difficulties, such as clogging (Chen et al., 2016). Therefore, how to enhance

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drained water treatment efficiency and prevent clogging in STWs has become

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important issues.

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Earthworms are widely used to treat wastewater and leachate in constructed

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wetland (CW) systems, due to their ability to breakdown organic material, as well as

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increase enzyme activity and nitrification potential (Xu et al., 2013a; Xu et al., 2015).

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Previous research showed that the presence of earthworms in CWs can increase COD,

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NH4+, TN and TP removal efficiencies in wastewater (Lavrnić et al., 2019; Singh et al.,

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2018; Xu et al., 2016; Xu et al., 2013b). Meanwhile, they can alleviate clogging and

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restore already clogged CWs by transporting or transforming clog matter (Li et al.,

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2011; Nivala et al., 2012; Ye et al., 2018). Our previous studies have shown that

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earthworms can improve sludge dewatering and stabilization in STWs (Chen and Hu, 5

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2019; Hu and Chen, 2018). However, it is unclear whether it is possible to enhance

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drained water treatment efficiency in STWs by earthworm addition.

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Therefore, the aim of this work were to (i) evaluate plant species and earthworms

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effects on drained water treatment in STWs and (ii) determine optimum plant and

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earthworm densities for drained water treatment in STWs.

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2. Material and methods

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2.1 Experimental setup

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Plexiglass was used to manufacture STWs systems, with a length × width ×

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height of 30 × 20 × 50 cm (Supplementary Material Fig. 1). Three layers of filter

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media were filled in each STW, with size increasing from top to bottom. The layers

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consisted of 10 cm of 4-8 mm gravel, 10 cm of 8-16 mm fine gravel and 10 cm of

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25-40 mm coarse gravel; detail according to Hu and Chen (2018). Uniform and

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healthy rhizomes of P. australis (average height of 70 cm) and T. angustifolia (average

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height of 120 cm) were used in this study; they were selected from a natural pond in

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Huazhong Agricultural University, Wuhan, China. Some STWs were planted with 10

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strains of selected plants. Meanwhile, earthworms (Eisenia foetida) were used in

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some STW systems, they were purchased from a local farm market. Therefore, 6

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STWs were established according to earthworms and plant species, the treatments of

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the 6 STWs were: T. angustifolia + E. foetida (TE), T. angustifolia (T), P. australis + E.

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foetida (PE), P. australis (P), Unplanted + E. foetida (UE) and Unplanted (U).

120

This experiment was carried out in a greenhouse (temperature 25 °C, relative

121

humidity 65%), located at Huazhong Agricultural University, Wuhan, China. Raw

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sewage sludge was taken from a domestic wastewater treatment plant in Wuhan; the

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main characteristics of the sewage sludge are summarized in Supplementary Material

124

Table 1. The experiment was carried out 6 months, every month 80g (about 170) E.

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foetida were added to the three earthworm STWs. Therefore, earthworm densities for 6

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months were 2.7, 5.4, 8.1, 10.8, 13.5 and 16.2 kg/m3, respectively. 3L of raw sludge

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was added into each STWs every 2 days, with the water emptied before raw sludge 6

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addition. Hence, the sludge loading rate was 45.6 kg TS/m2/yr during the whole

129

experiment. Drained water samples in each STW were taken every 2 days.

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Consequently, 15 drained water samples were collected at each earthworm density.

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2.2 Sample analysis

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Drained water samples were taken from each STW, and were analyzed for pH,

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oxidation-reduction potential (ORP), electrical conductivity (EC), dissolved oxygen

134

(DO), COD, TN, NH4+, nitrate (NO3-), TP. The pH, DO, EC and ORP were monitored

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by Multi 3430 (WTW). COD was quantified with the close-reflux dichromate

136

reduction method at 165 ◦C for 15 min followed by a spectrophotometric

137

quantification with a spectrophotometer model HACH DR 2010. TN, NH4+, NO3-, and

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TP concentrations were determined using spectrophotometric method according to

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Chinese standard methods for water and wastewater monitoring and analysis (EPA,

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2002).

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2.3 Data analysis

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Mass removal efficiency (RE) is commonly used to evaluate pollutant treatment

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performance in CWs due to the high transpiration rate of plants. It was calculated

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according to Chen et al. (2016). Mann-Whitney U test was used to compare the

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effects of the plant species, earthworms and earthworm densities on the treatment

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performance of pollutants. The level of significance was set to P < 0.05. Two-way

147

Analysis of variance (ANOVA) was used to assess the effects of plants and

148

earthworms on pollutants removal in STWs. The correlation relationships between pH,

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DO, EC, ORP, COD, NH4+, TN, NO3−, and TP were analyzed by canonical correlation

150

analysis and principal component analysis (PCA). The correlation coefficient r was

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interpreted as: strong correlation (r ≥ |0.7|) and a moderate correlation (|0.5| ≥ r

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≤ |0.7|). The statistic software Origin 2018 for windows was used to perform the test

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and to plot the graphs. The canonical correlation analysis and PCA were conducted

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using the SPSS 21.0 statistical software.

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3. Results and discussion 7

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3.1 General parameters

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The average values of outflow pH in six STWs were about 6.7-6.9, which had

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insignificant difference with the values of inflow pH (p>0.05). Conversely, the

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average values of ORP, DO and EC in the six STWs were 136.6-286.9 mV, 2.6-3.8

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mg/L and 49.5-860.7 µs/cm higher than that of inflow, respectively (Supplementary

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Material Fig. 2). Meanwhile, two-way ANOVA analysis showed that earthworms had

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significant effects on pH and ORP values (p≤0.001). However, the combination

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effects of plants and earthworms were insignificant for the outflow pH, ORP and DO

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values (p>0.05) (Table 1). This indicated that the influences of earthworms on pH,

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ORP and DO in STWs might be ignored when plants are involved in the STWs,

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although earthworms had the functions of buffering pH and allowing air entering the

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filter media (Ndegwa et al., 2000; Singh et al., 2019). Instead, the value of EC was

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significantly affected by plants and earthworms (p<0.05). Kengne et al. (2014)

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revealed that pH (6.4-7.6) and EC (121-1010 µs/cm) values in the effluent were

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beneficial for biological processes in faecal STWs. In addition, DO values in this

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study were still lower than those in previous studies (Olsson et al., 2014; Uggetti et al.,

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2012). Pedescoll and Sidrach (2013) also reported that outflow ORP value was

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positively correlated with evapotranspiration in wetlands. Therefore, the probably

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reason for low ORP and DO values was caused by the low evapotranspiration in this

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study (water loss below 32.5% in all STWs).

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Table 1: Two-way ANOVA analysis for the effects of plants and earthworms in STWs

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on water parameters and COD, NH4+, NO3-, TN and TP removal efficiencies Parameters pH ORP DO EC Water loss

plants 0.70 0.87 0.41 <0.001 0.001

earthworms <0.001 0.001 0.07 0.002 0.001

Plants x earthworms 0.43 0.43 0.19 <0.001 0.002

COD

0.15

0.86

0.92

NH4+

<0.001

0.82

<0.001

8

NO3-

0.83

0.04

0.67

TN

0.04

0.001

0.001

TP

0.006

0.70

0.02

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the values in bold show significant difference (p < 0.05)

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3.2 Chemical oxygen demand

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Average outflow COD concentrations in the six STWs were below 150.0 mg/L,

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which were significantly lower than inflow. The average COD mass removal

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efficiencies in the six STWs were over 98.0%; an insignificant difference was

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obtained between the six STWs (p>0.05) (Table 2). Two-way ANOVA analysis also

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showed that plants and earthworms had an insignificant effect on COD removal

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efficiency in the STWs (p>0.05) (Table 1). Despite that, COD mass removal

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efficiencies in the four planted STWs were higher than those in the two unplanted

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STWs. In addition, the lowest outflow COD concentration (about 80 mg/L) was

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monitored in the TE STWs under the earthworm density of 10.8 kg/m3 (Fig. 1). COD

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mass removal efficiencies in each earthworm STWs showed insignificant differences

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between the earthworm densities, except earthworm density of 2.7 kg/m3 in the TE

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and UE STWs. Moreover, the highest COD removal efficiency of 99.1% was

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determined in the TE and PE STWs under the earthworm density of 10.8 kg/m3.

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Fig. 1: COD concentrations and its mass removal efficiencies under different earthworm densities (kg/m3) in the three earthworm STWs (a, b; A, B show the significant difference (p < 0.05), respectively)

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Table 2: Average outflow concentrations and mass removal rates of pollutants in six STWs NH4+

COD

NO3-

TN

TP

STWs C (mg/L)

RE (%)

C (mg/L)

RE (%)

C (mg/L)

RE (%)

C (mg/L)

RE (%)

C (mg/L)

RE (%)

TE

113.6±64.8

98.5±1.2a

39.6±18.1

76.2±6.0a

9.2±2.7

-31.3±11.5a

59.1±45.3

85.4±8.9a

43.1±18.9

80.0±1.6a

T

111.9±70.4

98.6±1.1a

22.7±15.2

78.9±7.6a

10.5±2.3

-24.1±9.8a

80.2±23.1

80.1±1.2a

29.9±12.2

80.0±1.7a

PE

133.8±67.9

98.4±1.1a

17.5±9.7

90.4±7.8b

11.3±6.9

-18.1±6.7a

82.7±29.5

85.6±9.0a

27.9±12.7

85.1±3.3a

P

116.1±53.1

98.8±0.7a

11.9±10.0

89.5±5.4b

12.5±8.5

-35.8±10.8a

91.9±21.4

81.8±1.2a

28.6±11.9

83.4±4.2a

E

143.6±98.7

98.3±2.3a

35.8±14.9

75.7±6.5a

6.4±1.3

-8.3±3.4b

35.0±14.3

76.8±6.8a

42.5±17.6

74.5±4.6b

U

104.5±47.3

98.4±1.2a

20.1±13.1

74.9±7.6a

5.7±2.9

-4.8±1.5b

37.2±14.5

74.1±5.7a

35.4±14.4

72.9±1.5b

198

C: concentrations; RE: mass removal efficiencies

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a, b shows significant difference (p < 0.05)

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In general, plants and filtration (via sludge and gravel layers) have major roles in

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COD removal in STWs. Furthermore, the attached bacteria on the gravel layer can also

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remove a large part of dissolved organic matters by biodegradation (Tunçsiper, 2019).

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Gagnon et al. (2012) reported that filtration and outflow volume reduction were the

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main reason for organic matter decreasing in STWs. Plants can enhance organic

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matter degradation by transporting oxygen to the rhizosphere (Gagnon et al., 2012;

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Uggetti et al., 2009). Meanwhile, the growth of rhizomes can increase the porosity of

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STWs system, which also enhance the mineralization of organic matter in sludge and

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drained water (Nielsen et al., 2014). However, the role of plants in STWs was mainly

209

affected by air temperature (Gagnon et al., 2012; Uggetti et al., 2012). Stefanakis et al.

210

(2009) reported that evapotranspiration and microbial activities in sludge treatment

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reed beds (STRBs) decreased significantly at a low temperature. Gagnon et al. (2012)

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also showed that plants had significant impacts on COD removal in STWs in the middle

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of summer. Therefore, plants had insignificant effects on COD removal in our study

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which can be partly explained by the low evapotranspiration. In addition, T.

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angustifolia and P. australis had similar effects on COD removal under the earthworm

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density of 10.8 kg/m3 in our study, indicating that the two plants were helpful for COD

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removal in STWs under the subtropical monsoon climate (e.g. Wuhan, China). Similar

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COD removal efficiency was achieved between T. angustifolia and P. australis STWs

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under the humid continental climate and Mediterranean climate conditions (Gagnon et

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al., 2013; Uggetti et al., 2012). This also showed that the two wetland plants are well

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adapted to different climate conditions. Meanwhile, previous study also reported that

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both wetland plants had strong adaptability to wastewater with high COD concentration

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(e.g. sludge and tannery wastewater) in terms of survival and propagation in CWs

224

(Calheiros et al., 2007). Furthermore, the presence of earthworm increased organic

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matter mineralization in STWs, which was its capability to enhance aeration through

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burrowing activities (Schütz et al., 2008). Moreover, COD removal efficiency can be

227

influenced by earthworm density (Xu et al., 2013a). The low earthworm densities (< 12

228

10.8 kg/m3) did not improve the treatment efficiency in our study, similar result was

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also obtained by Nuengjamnong et al. (2011). Meanwhile, the death of earthworms can

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cause organic matter release in STWs, which may be the reason for decreasing the COD

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removal efficiency under high earthworm densities. Kanianska et al. (2016) reported

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that gravel used as substrate might have negative effect on the earthworm’s survival

233

due to abrasive action of gravel on their skin. Therefore, COD removal efficiency of

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drained water might be improved in T. angustifolia or P. australis STWs with the

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optimum earthworm density addition.

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3.3 Nitrogen

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3.3.1 Total nitrogen

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TN mass removal efficiency increased in the order of U, UE, T, P, TE and PE

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STWs throughout the experiment, while TN removal efficiencies in the three

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earthworm STWs were 2.7-5.3% higher than those in STWs without earthworm

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addition (Table 2). TN concentrations in the three earthworm STWs were

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2.2-21.1mg/L lower than those in the STWs without earthworm addition. In addition,

243

TN mass removal efficiencies in the planted STWs were 3.3-11.5% higher than those

244

in the unplanted STWs. Two-way ANOVA analysis also showed that plants and

245

earthworms had a significant effect on TN removal efficiency in STWs (p<0.05)

246

(Table 1). The lowest outflow TN concentrations of 40.0 mg/L were determined in the

247

TE and PE earthworm STWs under the earthworm density of 10.8 kg/m3 (Fig. 2). The

248

highest TN mass removal efficiency of 91.5% was obtained in the PE STWs under

249

earthworm densities of 8.1 and 10.8 kg/m3, which was significantly different to those

250

under earthworm densities of 2.7, 5.4 and 16.2 kg/m3 (p<0.05).

13

251 252

Fig. 2: TN and NH4+ concentrations and their mass removal efficiencies under

253

different earthworm densities (kg/m3) in the three earthworm STWs (a, b; A, B; 1, 2

254

show the significant difference (p < 0.05), respectively)

255

Sewage sludge contains large amounts of solid particles, which include a lot of

256

organic nitrogen. Magri et al. (2016) studied the mass balance of pollutants (COD and

257

nitrogen) in STWs, which indicated that pollutants were mainly retained in the

258

accumulated sludge layer, and the remaining pollutants were discharged as water, or

259

retained in the filter, or incorporated in the plants. Therefore, the main reason for TN

260

removal in STWs was that a large number of solid particles were trapped in the sludge

261

layer. TN contents of the accumulated sludge in the six STWs in our study were more

262

than 15g/kg (Hu and Chen, 2018). Wang et al. (2009) reported that TN removal in

263

STWs was mainly dependent on the interception function of the gravel layer and

264

sludge layer, which greatly reduced the outflow TN concentration. Meanwhile, TN

265

removal was also influenced by plants and earthworms. In general, wetland plants in

266

CWs reduce nitrogen in wastewater via nitrogen fixation and absorption (Ding et al., 14

267

2018). On the one hand, plants can uptake nitrogen to meet their own growth needs

268

(Saeed et al., 2018); on the other hand, microorganism activities around the

269

rhizosphere provide a favorable environment for nitrogen removal (Brix, 2017). The

270

main processes of nitrogen removal by microorganisms are ammonification,

271

nitrification, and denitrification (Lutterbeck et al., 2017). Some literature has shown

272

that activities of microorganism near the rhizosphere and oxygen content in the STW

273

are the main reasons for TN removal (Chen et al., 2015; Stottmeister et al., 2003). In

274

addition, TN removal efficiency in the two P. australis STWs were higher than those in

275

the T. angustifolia STWs, probably reason was that some P. australis leaves fell into

276

the STWs, which provided organic matter for denitrifying bacteria utilization during

277

the denitrification process (Wu et al., 2018). Earthworms leave a large number of

278

holes in the sludge layer through their activities, which can increase oxygen content in

279

STWs. Singh et al. (2018) reported that microorganism activities can be increased by

280

earthworm addition to CWs. Moreover, earthworm activity in CWs positively

281

correlated with the total number of bacteria, ammonifying agents, ammonia-oxidizing

282

bacteria and nitrite oxidizing bacteria (Li et al., 2011; Wu et al., 2013). In addition,

283

earthworm can promote the nitrogen uptake by wetland plants (Lavrnić et al., 2019;

284

Li et al., 2019). Xu et al. (2013a) reported that adding earthworms increased the

285

aboveground nitrogen uptake of T. angustifolia and P. australis by 216% and 108%,

286

respectively. Similar results were also determined in our study, which reported that

287

TN content in the T. angustifolia and P. australis with earthworm addition were 1.0

288

and 4.0 mg/kg higher than those in the T. angustifolia and P. australis without

289

earthworm addition, respectively. Therefore, TN removal efficiency of drained water

290

in STWs can be increased by plants and earthworm.

291

3.3.2 Ammonium

292

Outflow NH4+ concentrations in the six STWs were significantly lower than

293

inflow (p<0.05), and the NH4+ mass removal efficiencies were more than 70% (Table

294

2). NH4+ concentrations in the two P. australis STWs were 2.6-27.7 mg/L lower than 15

295

those in T. angustifolia and unplanted STWs. The highest NH4+ mass removal

296

efficiencies of 89.5-90.4% were also determined in the two P. australis STWs, which

297

was significantly different to those in T. angustifolia and unplanted STWs (p<0.05).

298

However, an insignificant difference emerged between the earthworm STWs with the

299

corresponding STWs without earthworm addition (p>0.05), which was in agreement

300

with the results of two-way ANOVA analysis. In addition, two-way ANOVA analysis

301

also showed that the combination effect of plants and earthworms had a significant

302

impact on the NH4+ removal in STWs (Table 1). In addition, the lowest outflow NH4+

303

concentration of 6.2 mg/L was monitored in the PE STWs under the earthworm

304

density of 10.8 kg/m3 (Fig. 2). Meanwhile, the highest NH4+ mass removal

305

efficiencies of 93.1% were determined in the PE STWs under the earthworm density

306

of 10.8 kg/m3, which had significant differences with those under the earthworm

307

densities of 2.7, 5.4, 8.1 and 16.2 kg/m3. Therefore, the best NH4+ removal in drained

308

water was determined in the P. australis STWs with earthworm density of 10.8 kg/m3.

309

The major pathway for NH4+ removal in STWs is nitrification of aerobic

310

microorganisms (nitrify bacteria), which is mainly caused by plant transfer oxygen to

311

the rhizosphere (Nielsen, 2015; Nielsen and Bruun, 2015). Lu et al. (2016) reported

312

that NH4+ removal efficiency in CWs increased with rise in microbial biomass. NH4+

313

removal efficiencies in the two P. australis STWs were significantly higher than those

314

in the two T. angustifolia and unplanted STWs during the experiment (p<0.05), which

315

reported that a favorable aerobic environment was provided around the rhizosphere of P.

316

australis, thus resulting in stronger nitrification in the P. australis STWs. In addition,

317

outflow NO3- concentrations in the two P. australis STWs were 0.8-6.8 mg/L higher

318

than those in the other STWs in our study (Table 2). Collison and Grismer (2015) also

319

revealed that NO3- concentration in the planted CWs was 9.5 mg/L higher than that in

320

the unplanted CWs. Moreover, the nutrients (TN and TP) in P. australis and T.

321

angustifolia were increased significantly during the whole experiment; among them,

322

the highest TN contents of 12.5 mg/kg was determined in P. australis (Supplementary 16

323

Material Fig. 3). This indicated that nitrogen removal in the STWs was partly

324

explained by plant uptake, and P. australis had a higher potential to uptake nitrogen to

325

meet its own nutrient requirements. Gagnon et al. (2012) also reported that the lower

326

outflow NH4+ concentration in P. australis system was due to the higher uptake of

327

ammonia by P. australis, whose biomass was determined to account for 26% of TKN

328

input by the sludge, while T. angustifolia accounted for 12%. In addition, the combined

329

effect of earthworms and plants also had a significant influence on NH4+ removal in

330

our study, especially in the earthworm density of 10.8 kg/m3. Earthworms can secrete

331

mucus, as well as proteins, polysaccharides, and other nitrogenous end-products, which

332

help to mineralize available nitrogen and provide biological resources to plants in the

333

form of nutrients (Bajsa et al., 2003). Wu et al. (2013) also reported that urease and

334

protease activities in the earthworm assisted wetlands were enhanced, thus improving

335

the nitrogen removal efficiency. Meanwhile, the activity of earthworms can increase the

336

content of DO in the system, which provides favorable conditions for nitrification

337

process (Huang et al., 2016). Moreover, nitrification process was also influenced by

338

earthworm density. Singh et al. (2019) and Lavrnić et al. (2019) revealed that the

339

increase of earthworm density had a positive impact on nitrification process. However,

340

earthworms are hardly survival under a high earthworm density due to some special

341

environmental conditions (e.g. high water content, special substrate) in CWs

342

(Kanianska et al., 2016; Xu et al., 2013a). Hence, NH4+ removal efficiency in drained

343

water was improved under the P. australis STWs with the optimum earthworm density

344

addition.

345

3.4 Phosphorus

346

Outflow TP concentrations in the six STWs were significantly lower than inflow

347

(p<0.05); the lowest TP concentration of 27.9 mg/L was determined in the PE STWs,

348

with the highest TP mass removal efficiency of 85.1% (Table 2). TP removal

349

efficiencies in the planted STWs were significantly higher than that in the unplanted

350

STWs (p<0.05); however, an insignificant difference was measured between T. 17

351

angustifolia STWs and P. australis STWs (p>0.05). Moreover, TP removal

352

efficiencies in the three earthworm STWs were 0-1.6 % higher than those in the

353

STWs without earthworm addition. Meanwhile, two-way ANOVA analysis showed

354

that plants, as well as the combined effect of earthworms and plants had significant

355

effects on TP removal in STWs during the whole experiment (p<0.05) (Table 1). In

356

addition, the lowest outflow TP concentrations of 22.2 mg/L in the PE and TE STWs

357

were achieved under the earthworm density of 10.8 kg/m3; meanwhile, the highest TP

358

removal efficiencies of 91.0% were also determined in this condition (Fig. 3).

359

Moreover, TP removal efficiencies in two planted STWs under the earthworm density

360

of 10.8 kg/m3 had a significant difference to those under the earthworm densities of

361

5.4 and 16.2 kg/m3 (p<0.05).

362 363 364 365

Fig. 3:TP concentrations and its mass removal efficiencies under different earthworm densities (kg/m3) in the three earthworm STWs (a, b; A, B show the significant difference (p < 0.05), respectively)

366

TP in sewage sludge mainly exists in the form of particulate (>90%), which is

367

easily retained by sludge and gravel layers (Chen et al., 2016; Hu et al., 2017; Wang 18

368

et al., 2009). Meanwhile, previous studies reported that TP removal in CWs was

369

mainly through adsorption or precipitation by filter materials containing calcium, iron,

370

and aluminum contents (Vohla et al., 2011; Vymazal, 2007). However, Ma et al. (2019)

371

revealed that plant uptake was the major pathway for TP removal in CWs filled with

372

gravel. A significant difference for TP removal was obtained between plants and

373

unplanted STWs in our study (p<0.05). However, plant species had an insignificant

374

impact on TP removal. Similar research also reported that there was no significant

375

difference in TP removal between different plant species (I. pseudacorus, P. australis

376

and T. angustifolia) in STWs (Wang et al., 2009). Gagnon et al. (2012) revealed that the

377

lowest TP removal efficiency was measured in P. australis STWs compared to other

378

species and unplanted control, which was due to a new STW design, with the presence

379

of a saturated layer enabling a longer contact time for TP removal through physical,

380

chemical and biological processes. The positive effect of plants on TP removal was

381

directly due to their uptake process (Kengne et al., 2014; Kołecka et al., 2019). TP

382

contents in the plants were 3-4 times higher than those in the blank control after the

383

experiment (Supplementary Material Fig. 3). Meanwhile, Hu et al. (2019) also reported

384

that the interaction between plants and microorganisms in CWs also promoted TP

385

removal. Furthermore, earthworm addition also provided a potential capability to

386

increased TP removal efficiency in STWs. The possible reason was that porosity in the

387

earthworm STWs was increased by earthworm peristalsis, which enhanced the effects

388

of plants and microorganisms on TP degradation. TP contents in the two plants with

389

earthworm addition was 0.2-0.4 higher than those in the control without earthworm

390

addition (Supplementary Material Fig. 3). This also indicated that plant nutrient

391

uptake was increased by earthworm addition in STWs. Xu et al. (2013a) also showed

392

that TP removal efficiency in CWs was increased by earthworm addition, which could

393

be connected with the higher photosynthetic activity and TP uptake. Therefore, plants

394

and earthworm provide a possibility to improve TP removal efficiency in STWs.

395

3.5 Clogging 19

396

Filtration rate is usually used to evaluate clogging in wetland systems. Generally,

397

clogging in wetland systems can be alleviated in the condition of high filtration rate.

398

Filtration rates in the TE, T, UE and U STWs were decreased to 1.2, 1.1, 0.9 and 0.5

399

cm/s in the feeding period, respectively (Table 3). However, the filtration rates in the

400

two P. australis (PE and P) STWs during the feeding period were the same as when the

401

experiment began. The main reason for the filtration rate decreased in STWs was that

402

sewage sludge with high water content (about 98%) was continuous injected into each

403

STWs during the feeding period, resulting in a gradual decrease in the porosity of

404

each STW. Meanwhile, the filtration rates of the two P. australis (PE and P) STWs

405

remained constant due to the continuous growth of P. australis roots, resulting in

406

increased porosity in these STWs. Gagnon et al. (2013) also reported that STWs

407

planted with P. australis exhibited the best sludge dewatering due to their special root

408

systems. In addition, filtration rates in the three earthworm STWs were 0-0.4 cm/s

409

higher than those in the STWs without earthworm addition. This indicated that

410

permeability in the three STWs were enhanced by earthworm addition, which provided

411

a process to alleviate clogging in STWs. Previous studies reported that earthworms can

412

transfer the subsurface clogging matter to the surface of the CWs and reduce about 56%

413

dry weight of the clog matter (Davison et al., 2005; Jaime et al., 2012). Chen et al.

414

(2016) studied earthworm and vegetation effects on sludge treatment in STRBs, their

415

results revealed that water residence time on sludge surface in STRBs without

416

earthworm addition was increased, with 6 times longer than that at the beginning of

417

experiment. Li et al. (2011) also reported that porosity of STWs was increased by

418

earthworm peristalsis, thereby increasing the water filtration rate. Therefore,

419

earthworms had a positive effect on alleviating clogging in STWs.

420

Table 3: Filtration rates in the six STWs during the whole experiment Filtration rate (cm/s) Experiment began Feeding period

TE 1.3 1.2

20

T 1.2 1.1

PE 1.4 1.4

P 1.4 1.4

E 1.0 0.9

U 0.8 0.5

421 422 423 424 425 426 427

Fig. 4: Correlation relationships between pH, ORP, DO, EC, water loss and mass removal efficiencies of COD, NH4+, TN, and TP in the four STWs. Red color represents positive correlation coefficients, green color represents negative correlation coefficients, and smaller ovals represent lower correlations, while bigger ovals represent stronger correlations; * and ** show significant difference of p<0.05 and p<0.001, respectively.

428

3.6 Factors influencing pollutants removal

429

Canonical correlation analysis and PCA were carried out to assess the

430

relationships between pH, ORP, EC, DO, water loss, and mass removal efficiencies of

431

COD, NH4+, TN, NO3- and TP throughout the experiment (Fig. 4, Fig. 5). Compared

432

to the PE STWs, removal efficiency of TN had a stronger negative correlation with

433

EC in the P STWs (r = -0.85), and a significant difference was found between them

434

(p<0.01). Moreover, there was a moderate positive correlation between the removal

435

efficiencies of NH4+ and TN in the PE STWs (r = 0.55), and the pH was also

436

moderately correlated with DO (r = 0.5, p<0.05). Nevertheless, compared to the

437

planted STWs, significant correlations between these parameters were increased in

438

the unplanted STWs, especially for water loss in UE STWs, which reached a

439

significant correlation with DO, EC, removal efficiencies of TN, NH4+ and TP 21

440

(p<0.05). This may be related to a few factors affecting these parameters in the

441

unplanted STWs. In addition, PCA carried out on these parameters indicated that the

442

variation in pH, ORP, DO, EC, water loss, and mass removal efficiencies of COD,

443

NH4+, NO3−, TN, and TP under the planted and unplanted STWs were captured by

444

two main axes, together explaining 37.9% and 43.3% of the variation, respectively.

445

No matter in which STW, water loss, mass removal efficiencies of TN, TP, COD and

446

NH4+ were largely determined in the PCA axis 1 (first component), positive

447

correlation was also shown between these parameters. Moreover, a similar variation

448

trend of component was determined in the P and PE STWs, with an insignificant

449

difference obtained. However, the distribution of components in the E STWs was

450

independent of U STWs, which was distributed in the PCA axis 2 (second component).

451

This suggested that earthworms had an effect on the removal of pollutants in STWs,

452

while the earthworm effect was limited in STWs compared with the removal of

453

pollutants by plants. Nevertheless, the combined effects of plants and earthworms had

454

a significant impact on pollutant removal in STWs.

455 456

Fig. 5: Principle component analyses of pH, ORP, DO, EC, water loss and mass 22

457

removal efficiencies of COD, NH4+, TN, NO3−, and TP in the four STWs

458 459

4. Conclusion

460

Earthworms and plants play an important role in drained water treatment in

461

STWs; meanwhile, earthworms had a positive effect on alleviating clogging in the

462

STWs. Plants had significant effects on NH4+, TN and TP removal in STWs, while an

463

insignificant difference was obtained between P. australis and T. angustifolia except

464

for NH4+removal; the highest NH4+ removal efficiency of 90% was determined in P.

465

australis STWs. Furthermore, COD, NH4+, TN and TP removal efficiencies in STWs

466

were influenced by earthworm densities; the optimal pollutants removal efficiencies

467

were determined in the P. australis STWs with the earthworm density of 10.8 kg/m3,

468

with the highest removal efficiencies of COD, NH4+, TN and TP were 99.1%, 93.1%,

469

91.5% and 91.0%, respectively. Meanwhile, the combined effect of plants and

470

earthworms was significant for drained water quality in STWs. However, further

471

studies are needed on earthworm behavior especially under extreme conditions such

472

as hot summers and cold winters.

473

Acknowledgements

474

Shanshan Hu would like to thank the China Scholarship Council for the PhD

475

scholarship (CSC, No. 201706760061). This work was supported by the Natural

476

Science Foundation of Hubei Province (Grant No. 2014CFB928). The authors are

477

grateful to Mark Francis Sixsmith for revising the English language.

478

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Ye, J., Xu, Z., Chen, H., Wang, L., Benoit, G., 2018. Reduction of clog matter in constructed wetlands by metabolism of Eisenia foetida: Process and modeling. Environ. Pollut. 238, 803-811.

28

657

Appendix:

658

Fig. SM1: Experimental setup of earthworm assistant STW system.

659

Fig. SM2: pH, ORP, DO and EC in the six STWs during the whole experiment (n =

660

15)

661

Fig. SM3: TN and TP contents in the P. australis and T. angustifolia at the end of

662

experiment (a, b, c and d show the significant difference (p < 0.05))

663

Table SM1: Characteristics of raw surplus sludge, n=48

29

Appendix Fig. SM1

30

Fig. SM2

31

Fig. SM3

32

Table SM1: Characteristics of raw surplus sludge, n=48 Parameters pH ORP DO EC

Unit mV mg/L µs/cm

Mean 6.5- 7.3 -336 - -127 0.01-0.95 568-1721

Standard

COD

mg/L

6315.7

2345.4

NH4+

mg/L

87.8

23.1

NO3-

mg/L

6.8

3.0

TN

mg/L

251.5

116.5

TP

mg/L

127.7

45.7

33

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: