Water Research 201 (2021) 117349

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Water Research
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Removal of micropollutants and biological effects by conventional and

intensified constructed wetlands treating municipal wastewater
Nadine A. Sossalla a, b, *, Jaime Nivala c, Thorsten Reemtsma d, e, Rita Schlichting f, Maria König f,
Nicolas Forquet c, Manfred van Afferden a, Roland A. Müller a, Beate I. Escher f, g
a
Centre for Environmental Biotechnology, Helmholtz Centre for Environmental Research (UFZ), Permoserstrasse 15, Leipzig 04318, Germany
b
Institute of Urban Water Management, Dresden University of Technology, Bergstrasse 66, Dresden 01069, Germany
c
Research Unit REVERSAAL, French National Research Institute for Agriculture, Food and Environment (INRAE), 5 rue de la Doua, CS 20244, Villeurbanne Cedex
69625, France
d
Department of Analytical Chemistry, Helmholtz Centre for Environmental Research (UFZ), Permoserstrasse 15, Leipzig 04318, Germany
e
Institute of Analytical Chemistry, University of Leipzig, Linnéstrasse 3, Leipzig 04103, Germany
f
Department of Cell Toxicology, Helmholtz Centre for Environmental Research (UFZ), Permoserstrasse 15, Leipzig 04318, Germany
g
Center for Applied Geoscience, Eberhard Karls University Tübingen, Schnarrenbergstraße 94-96, Tübingen 72076, Germany

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

Keywords: Seven treatment wetlands and a municipal wastewater treatment plant (WWTP) were weekly monitored over the
Effect-based method course of one year for removal of conventional wastewater parameters, selected micropollutants (caffeine,
Effect-based trigger values ibuprofen, naproxen, benzotriazole, diclofenac, acesulfame, and carbamazepine) and biological effects. The
Emerging organic contaminant
treatment wetland designs investigated include a horizontal subsurface flow (HF) wetland and a variety of
In vitro bioassay
Nature-based solution
wetlands with intensification (aeration, two-stages, or reciprocating flow). Complementary to the common
Treatment wetland approach of analyzing individual chemicals, in vitro bioassays can detect the toxicity of a mixture of known and
unknown components given in a water sample. A panel of five in vitro cell-based reporter gene bioassays was
selected to cover environmentally relevant endpoints (AhR: indicative of activation of the aryl hydrocarbon
receptor; PPARγ: binding to the peroxisome proliferator-activated receptor gamma; ERα: activation of the es­
trogen receptor alpha; GR: activation of the glucocorticoid receptor; oxidative stress response). While carba­
mazepine was persistent in the intensified treatment wetlands, mean monthly mass removal of up to 51% was
achieved in the HF wetland. The two-stage wetland system showed highest removal efficacy for all biological
effects (91% to >99%). The removal efficacy for biological effects ranged from 56% to 77% for the HF wetland
and 60% to 99% for the WWTP. Bioanalytical equivalent concentrations (BEQs) for AhR, PPARγ, and oxidative
stress response were often below the recommended effect-based trigger (EBT) values for surface water, indicating
the great benefit for using nature-based solutions for water treatment. Intensified treatment wetlands remove
both individual micropollutants and mixture effects more efficiently than conventional (non-aerated) HF wet­
lands, and in some cases, the WWTP.

1. Introduction as for operation and maintenance of public infrastructure (van Afferden

Treatment wetlands are nature-based solutions for water treatment
that can be used to treat different types of wastewater in areas where
decentralized treatment technologies are the most cost-effective option
(Kadlec and Wallace, 2009). This is the case, for example, in developing
countries where infrastructure is lacking or high costs are incurred, such
et al., 2015). Conventional subsurface horizontal flow (HF) wetlands
have been used for decades to treat domestic wastewater. Due to stricter
regulations regarding discharge standards and the knowledge that ox­
ygen is a limiting factor for removal of certain pollutants, various
treatment wetland designs have been developed that utilize pumping of
air or water to increase the oxygen transfer efficiency and thus pollutant

* Corresponding author.
E-mail addresses: nadine.sossalla@magenta.de (N.A. Sossalla), jaime.nivala@inrae.fr (J. Nivala), thorsten.reemtsma@ufz.de (T. Reemtsma), rita.schlichting@ufz.
de (R. Schlichting), maria.koenig@ufz.de (M. König), nicolas.forquet@inrae.fr (N. Forquet), manfred.afferden@ufz.de (M. van Afferden), roland.mueller@ufz.de
(R.A. Müller), beate.escher@ufz.de (B.I. Escher).

https://doi.org/10.1016/j.watres.2021.117349
Received 31 March 2021; Received in revised form 4 June 2021; Accepted 7 June 2021
Available online 12 June 2021
0043-1354/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
N.A. Sossalla et al.
removal (Wallace et al., 2008). Treatment wetland technology varies
from completely passive systems to highly engineered systems that
employ aeration, recirculation, or reciprocation. In general, intensified
treatment wetlands have operating costs but also have a smaller area
footprint and higher pollutant removal capacity per person equivalent
per square meter (PE/m2) compared to passive (non-aerated) wetlands
(Nivala et al., 2019).
Wastewater treatment plants (WWTPs) are major sources of micro­
pollutants entering aquatic ecosystems (Verlicchi and Zambello, 2014)
and the removal of micropollutants in wastewater treatment has become
an increasing focus of research in recent decades. Micropollutants are
anthropogenic chemicals from the substance classes of e.g. personal care
products, hormones, and pharmaceuticals. Micropollutants occur in low
(ng/L) to very low (pg/L) concentrations in the environment and pose
risks to aquatic and terrestrial ecosystems (Ilyas et al., 2020).
Recent studies on micropollutant treatment wetlands have examined
a range of wetland designs, operational conditions, and influent com­
positions to efficiently remove micropollutants (Hijosa-Valsero et al.,
2016; Kahl et al., 2017; Matamoros et al., 2007). Biodegradation is one
of the most important micropollutant degradation processes in treat­
ment wetlands (Jekel et al., 2015). It has become accepted practice to
analyze representative chemicals of a substance class with so-called in­
dicator substances in order to draw conclusions about other micro­
pollutants with the similar property (Dickenson et al., 2011). In previous
studies several micropollutants were identified as suitable indicator
compounds for identifying water quality along small rivers (Müller
et al., 2018), drinking water treatment (Jin and Peldszus, 2012) and
surveilling hospital effluents (Helwig et al., 2013). Jekel et al. (2015)
also identified process and source indicator substances and recom­
mended micropollutants with varying degrees of biodegradability
(recalcitrant, moderately biodegradable and readily biodegradable) in
order to be able to make a conclusive statement on treatment efficacy of
engineered treatment systems. Seven of such indicator substances have
already be successfully applied to monitor treatment efficacy of inten­
sified treatment wetlands (Kahl et al., 2017).
The measurement of indicator substances reflects the concentrations
of the micropollutants analyzed but does not give a holistic view of all
the chemicals present in a given water sample. However, even if the
parent compounds are degraded, the resulting transformation products
and metabolites can lead to environmental risks. The use of cell-based in
vitro bioassays (also known as effect-based methods) can assess the sum
effect of all biologically active pollutants (both quantitatively and
qualitatively) in a water sample (Escher et al., 2020). Bioassays provide
complementary information to chemical analysis in that they can be
used to detect unknown compounds, transformation products, and me­
tabolites with similar modes of action. Active pollutants, even if present
below their chemical analytical detection limits, can produce mixture
effects (Neale et al., 2020a; Välitalo et al., 2017). Cell-based in vitro
bioassays are designed to cover relevant steps of cellular toxicity path­
ways (such as xenobiotic metabolism, hormonal activity, and adaptive
stress response) to address different groups of chemicals (Neale et al.,
2020a).
Bioassays have been used in several individual studies to examine the
removal efficacy of biological effects from WWTPs as well as constructed
wetlands for wastewater treatment (Lundqvist et al., 2019; Neale et al.,
2020b; Nivala et al., 2018). Lundqvist et al. (2019) assessed the removal
of biological effects in five WWTPs with a bioassay test battery (estro­
genicity, androgenicity, aryl hydrocarbon receptor activity, oxidative
stress response and the ability to activate nuclear factor
kappa-light-chain-enhancer of activated B cells) and found a removal
efficacy between 16%− 99%, depending on the bioassay. Ávila et al.
(2014) reported a reduction in biological effects of 70 – 100% (diox­
in-like activity, estrogenicity and apical effects in whole organisms) by a
hybrid constructed wetland. In the proof-of-concept study by Nivala
et al. (2018) at the same research site as the current study, seven
different treatment wetlands and a municipal WWTP were evaluated
2
Water Research 201 (2021) 117349
once in summer and once in winter for their removal efficacy of selected
micropollutants and biological effects. The municipal WWTP consis­
tently showed lower (52 – 98%) removal of biological effects than the
intensified treatment wetlands (77 – 99%).
The present study advances on previous research by comparing
seven different treatment wetland designs with an adjacent municipal
wastewater treatment plant for their removal efficacy of conventional
pollutants, indicator micropollutants and biological effects over the
course of one year. The overarching aims of this study were i) to identify
whether removal of mixture effects is influenced by certain treatment
wetland design aspects (aerated vs. non-aerated; planted vs. non-
planted; single-stage vs. two-stage), and ii) to determine the relation­
ship between removal of indicator micropollutants and biological
effects.
2. Materials and methods
2.1. Site description
The study was conducted at a treatment wetland research platform in
Langenreichenbach, Germany. The platform is located adjacent to a
municipal WWTP (activated sludge technology followed by biological
and chemical phosphorous precipitation) that serves approximately
16,000 inhabitants. At the research platform, a two-chamber septic tank
serves as primary treatment of raw wastewater with a nominal hydraulic
residence time (nHRT) of two days, before it is sent to the pilot-scale
wetland systems. The horizontal flow (HF) wetland (50 cm saturated
depth; H50p) served as a baseline technology (non-aerated, single-
stage). The intensified systems included saturated horizontal flow wet­
lands with aeration (HA, aerated and unplanted; HAp, aerated and
planted), saturated vertical flow wetlands with aeration (VA, aerated
and unplanted; VAp, aerated and planted), a two-stage wetland system
consisting of VAp followed by an unsaturated sand filter (VAp+VSp), as
well as a two-cell reciprocating (R) system (Table 1). All systems started
operation in June 2010. Further details on the research facility are
provided in Nivala et al. (2013).
2.2. Experimental methods
2.2.1. Sampling and water quality analysis
Grab samples were collected from the influent of the treatment
wetlands (e.g., outlet of the septic tank) and of the effluents of the seven
treatment wetlands, according to the procedure published by Nivala
et al. (2013). Grab samples were also collected from the WWTP influent
(WWTPin) and effluent (WWTPout). For practical reasons, grab sam­
pling was conducted (as opposed to time- or time and flow-weighted
composite samples). A comprehensive discussion about uncertainties
related to grab sampling at the research facility, the reader is referred to
Kahl et al. (2017).
Samples were analysed for field water temperature (T), pH, Oxida­
tion Reduction Potential (ORP) (SenTix® ORP sensor), electrical con­
ductivity (EC) and dissolved oxygen (DO) (ConOx®, WTW Weilheim)
using a WTW Multi 350i Multimeter and a WTW pH 96 meter; Total
Organic Carbon (TOC, DIN EN 1484, TOC-VCSN device, Shimadzu,
Duisburg, Germany), Total Nitrogen (TN, DIN EN 12,260, TNM-1
module), Nitrate Nitrogen (NO3–N, DIN EN ISO 13,395, Eppendorf
EPOS ANALYZER 5060), Nitrite Nitrogen (NO2–N, DIN EN 26,777,
Eppendorf EPOS ANALYZER 5060), Ammonia Nitrogen (NH4–N, DIN
38,406 E5, Eppendorf EPOS ANALYZER 5060), Total Suspended Solids
(TSS, DIN 38,409 H2–3), and five-day Carbonaceous Biochemical Oxy­
gen Demand (CBOD5, DIN 38,409 H52, OxiTop, WTW Weilheim, Ger­
many). Escherichia coli (E. coli) was determined with the IDEXX Colilert-
18 test (IDEXX, USA; ISO 11,133:2014). Samples were analysed for
conventional wastewater parameters within 24 h of collection.
N.A. Sossalla et al. Water Research 201 (2021) 117349

Table 1
Design, operational details, and abbreviations of the treatment wetlands at the research platform in Langenreichenbach (May 2018 – April 2019).
System System Effective depthc Saturation Status Filter Media Total Surface Average Inflow Nominal Hydraulic retention
Abbreviationa Typeb (cm) Area (m3/d) time (d)
(m2)
Conventional
H50p HF 50 Saturated 8 – 16 mm gravel 5.6 0.2 5.5
Intensified
VA, VAp VF + Aeration 85 Saturated 8 – 16 mm gravel 6.2 0.6 3.5
HA, HAp HF + Aeration 100 Saturated 8 – 16 mm gravel 5.6 0.6 3.7
R Reciprocating 95 Alternating 8 – 16 mm gravel 13.2 1.4 3.0
Two-Staged
VAp+VSp VF + Aeration VF sand 85 (VAp) Saturated (VAp) 8 – 16 mm gravel 5.6 (VAp) 0.6 3.5
filter 85 (VSp) Unsaturated (VAp) 5.6 (VSp)
(VSp) 1 – 3 mm sand
(VSp)
a
Systems planted with Phragmites australis are denoted with “p” in the system abbreviation.
b
HF: horizontal flow; VF: vertical flow.
c
Effective depth refers to the depth of the media involved in treatment.
d
VAp+VSp operational as a two-stage system since 2016; the retention time did not increase substantially as a two-stage filter since the second stage was unsaturated.

2.2.2. Chemical analysis
After filtration (syringe filters, ProFill, 0.45 µm, LABSOLUTE®)
samples were stored by − 18 ◦ C until analysis. Samples were spiked with
isotope-labelled internal standards which were used for quantification.
Samples were directly injected into a high-performance liquid chroma­
tography instrument coupled to tandem mass spectrometry (HPLC-MS/
MS, Agilent 1260 Infinity series HPLC, Agilent Technologies, Wald­
bronn, Germany) according to Kahl et al. (2017).The seven indicator
compounds were micropollutants commonly found in wastewater:
ibuprofen (IBU), naproxen (NPX), diclofenac (DCL) and carbamazepine
(CBZ) are common pharmaceuticals; acesulfame (ACE) an artificial
sweetener, benzotriazole (BTZ) a corrosion inhibitor and caffeine (CAF)
a stimulant. The compounds, ranked from most readily biodegradable to
most recalcitrant under aerobic conditions are: CAF, IBU, NPX, BTZ,
DCL, ACE, CBZ.
protein by microscopic imaging (Escher et al., 2019). The reference
compounds and their effect concentrations (EC) are listed in Table 2,
LODs of each bioassay are derived in the Supplementary Information,
Section A3 (Table A1). A battery of five bioassays was applied to
quantify the mixture effects present in the water samples. The bioassays
cover three different stages of the cellular toxicity pathway: xenobiotic
metabolism analysed by the bioassays AhR CALUX (activation of the aryl
hydrocarbon receptor, AhR) and PPARɣ-GeneBLAzer (binding to the
peroxisome proliferator-activated receptor gamma; PPARɣ); hormonal
activity analyzed by the bioassays ERα-GeneBLAzer (indicative of acti­
vation of the estrogen receptor alpha; ERα) and GR-GeneBLAzer (acti­
vation of the glucocorticoid receptor; GR); oxidative stress response was
evaluated with AREc32 (mediated by the transcription factor Nrf2).
2.3. Data analysis

2.2.3. Bioanalysis
Bioassay samples were collected in clear glass bottles (cleaned with
methanol, ethyl acetate and LC-MS-grade water) and transported to the
laboratory at 4 ◦ C. The 250 mL samples were acidified with 6 M hy­
drochloric acid to pH 2 and filtrated with a glass fiber microfilter (GF/F,
0.7 µm) followed by solid-phase extraction (Enrichment Factor, EF: 200)
as described by Sossalla et al. (2020) within 24 h of collection. Extracts
were stored at − 18 ◦ C until bioanalysis. A detailed description of the
quantification of the mixture effects are described in König et al. (2017)
and Neale et al. (2017). In short, the appropriate amount of extract was
transferred to a dosing vial, the solvent (methanol) was blown down
under a gentle nitrogen stream and the residue was resuspended in test
medium to a maximum Relative Enrichment Factor (REF) of 50 in the
bioassay. A serial dilution (1000-fold; 211) of each water extract and, for
quality control, blank extracts prepared from LC-grade water, methanol,
ethyl acetate were also run in duplicate. Each plate included a positive
control (reference compound of the bioassay) for reproducibility con­
trol. Cytotoxicity was assessed prior to quantification of the reporter
Limits of Detection (LOD) and Limits of Quantification (LOQ) are
given in the Supplementary Information, Sektion A3 (Table A1). For
bioassays, cell viability was also determined. Effect Concentrations (EC)
causing more than 10% cytotoxicity were excluded from the
concentration-effect modeling. Further information on the data evalu­
ation method for each bioassay is published in Escher et al. (2018b). For
bioassays indicative of xenobiotic metabolism and receptor-mediated
effects, effect concentrations triggering 10% (EC10) of the investigated
maximum effect were used to calculate EC10. For oxidative stress
response, effect concentrations causing an induction ratio of 1.5
(ECIR1.5) were used.
With the concept of bioanalytical equivalent concentrations (herein
referred to as BEQ), an observed effect can be translated into the
equivalent concentration of the reference compound for a given
bioassay. For this, the EC of the reference compound (ECreference) is
divided by the EC of the sample (ECsample) (Equation 1).

Table 2
Summary of the bioassay, mode of actions, reference compounds, effect concentrations (EC) and bioanalytical equivalents (BEQ) used in this study.
Bioassay Mode of action Reference EC Reference compound EC Bioanalytical equivalent Reference
compound benchmark (M) (BEQ)

10
AhR CALUX Aryl hydrocarbon-receptor Benzo[a]pyrene EC10 (8.38 ± 0.55) × 10− Benzo[a]pyrene-EQ (B[a]P- Brennan et al.
induction EQ) (2015)
− 6
AREc32 Oxidative stress response Dichlorvos ECIR1.5 (7.70 ± 0.30) × 10 Dichlorvos-EQ Escher et al.
(2012)
11
ERα-GeneBLAzer Estrogenicity 17β-Estradiol EC10 (1.57 ± 0.03) × 10− 17β-Estradiol-EQ (EEQ) König et al. (2017)
10
GR-GeneBLAzer Glucocorticogenic activity Dexamethasone EC10 (4.69 ± 0.06) × 10− Dexamethasone-EQ König et al. (2017)
10
PPARγ- Peroxisome proliferation Rosiglitazone EC10 (4.97 ± 0.08) × 10− Rosiglitazone-EQ Neale et al. (2017)
GeneBLAzer activation

3
N.A. Sossalla et al. Water Research 201 (2021) 117349

EC10 (reference) ECIR1.5 (reference) rate Qin (L/d), inflow concentration Cin (µg/L or mg/L), inflow BEQ
BEQ (ng / L) = or (1)
EC10 (sample) ECIR1.5 (sample) (BEQin in ng/L or µg/L), outflow rate Qout (L/d), outflow concentration
Cout (µg/L or mg/L), and outflow BEQ (BEQout in ng/L or µg/L) (Equa­
Outliers were identified first by manual inspection and removal of
tion 2 and Equation 3) (Müller et al., 2020).
data corresponding to dates of sitewide maintenance followed by iden­ ( )
tification of recommended upper and lower bounds for outliers ac­ Cout × Qout
Mass Load Removal (%) = 1 − × 100 (2)
cording to von Sperling et al. (2020). Further information is given in the Cmin × Qin
Supplementary Information, Section A1. ( )
The use of chemical concentrations and BEQs to determine the BEL Removal (%) = 1−
BEQout × Qout
× 100 (3)
pollutant removal efficacy of small-scale treatment wetlands is not BEQin × Qin
particularly accurate because the water loss due to evapotranspiration
can amount to a significant fraction of the daily inflow during the
summer months (Kadlec and Wallace, 2009). Therefore, the Mass Load 2.4. Statistical analysis
(ML) for chemical concentrations and the Bioanalytical Equivalent Load
(BEL) for bioanalysis were calculated on a monthly basis by using inflow Calculations were conducted in Microsoft Excel (Office 2013) and
KNIME Analytical Platform (Version 4.1.3). Analysis was conducted in

Fig. 1. Monthly mass removal of selected pollutants and log10 E. coli. H50p: horizontal flow wetland (non-aerated); HA, HAp: horizontal flow wetland with aeration;
VA, VAp: vertical flow wetland with aeration; VAp+VSp: two-stage wetland (vertical flow with aeration + unsaturated vertical flow sand filter); R: reciprocating
wetland; WWTP: wastewater treatment plant.

4
N.A. Sossalla et al. Water Research 201 (2021) 117349

GraphPad Prism (Version 9). The Kruskal-Wallis test was used to identify

Annual mean concentrations and mass load removals (given in parentheses) of selected conventional wastewater parameters from May 2018 to April 2019 (n = 29 – 39). E. coli data is given as geometric means (log10

(MPN/100 mL)

3535,845
177,189 (1.3)
402 (3.9)
610 (3.8)
83,676 (1.6)
22,448 (2.2)
69 (4.7)
46,631 (1.9)

14,760 (2.9)
12,150,633
whether there was a significant difference between the mass removal
and BEL removal of the treatment wetlands, followed by the post-hoc

E. coli
Dunn’s multiple comparisons test to compare the mass removal effi­
cacy of a particular wetland with another.

NO2–N

<LOD
<LOD
<LOD
<LOD

<LOD
0.18
0.05

0.13
<LOD
<LOD
2.5. Multivariate data analysis

(mg/L)
The aim of the multivariate data analysis was to evaluate whether or
not bioassays are representative of concentration changes observed

74 ± 12

<LOQ (100%)
0.1 ± 0.1 (100%)

0.02 ± 0.01 (100%)

59 ± 10
65 ± 10 (34%)

14 ± 4 (81%)
1 ± 1 (98%)

5 ± 3 (93%)

0.1 ± 0.1 (94%)

among pools of micropollutants using Principal Component Analysis

NH4–N
(mg/L)
(PCA). Therefore, PCA was only performed on the micropollutant and
bioassay data (other variables were used as quantitative data providing
orientation). Analysis was conducted in the R software (R Core Team,
2020). Considering the number of missing data, pre-processing steps
were necessary. Variables with more than 30% of missing values among

78 ± 9 (37%)
47 ± 6 (51%)
55 ± 9 (48%)
33 ± 8 (66%)
41 ± 7 (62%)
40 ± 8 (66%)
37 ± 10 (61%)

7 ± 0.8 (91%)
94 ± 10

89 ± 11
observations were removed. The remaining missing data were inferred

(mg/L)
using a regularized iterative multiple correspondence analysis imple­

TN
mented in the package missMDA (Josse and Husson, 2016). The PCA
method implemented in the FactoMineR package (Husson et al., 2011)
was chosen. Two separate PCAs were carried out, one for all observa­

88 ± 85
4 ± 2 (95%)
35 ± 30 (32%)
32 ± 25 (33%)
10 ± 7 (84%)
14 ± 7 (79%)
1 ± 1 (99%)
4 ± 1 (93%)
405 ± 119
6 ± 2 (99%)
tions and one for intensified wetland effluent concentrations and BEQs

(mg/L)
TSS
only. Results were graphically displayed and analyzed using the Fac­
toExtra package (Kassambara and Mundt, 2020).

3. Results and discussion

40 ± 6 (82%)
18 ± 5 (89%)
26 ± 15 (85%)
18 ± 2 (90%)
16 ± 2 (92%)
13 ± 4 (95%)
16 ± 2 (91%)

14 ± 1 (94%)
165 ± 29

249 ± 77
(mg/L)
TOC
3.1. Conventional wastewater parameters

Temperature and rainfall during the study are provided in Figure A1;
air temperatures ranged between +40 ◦ C and − 10 ◦ C and monthly

1 ± 1 (100%)
41 ± 17 (85%)
2 ± 2 (99%)
2 ± 2 (99%)
6 ± 2 (98%)
3 ± 2 (99%)

5 ± 2 (98%)
409 ± 114
4 ± 3 (99%)
300 ± 64
rainfall ranged between 0.9 mm/month (April 2019) and 60.5 mm/

(mg/L)
month (February 2019). Monthly mass pollutant removals are provided CBOD5
in Fig. 1. Annual mean concentrations and mass removals for selected
conventional wastewater parameters are given in Table 3. The high
annual mean mass removal of CBOD5 (>98%) and TOC (>85%), the
3±1
10 ± 2
10 ± 1
5±1
8±1
10 ± 2
5±1
1 ± 0.3

1±2
5±1
(mg/L)
DO

positive redox potential, as well as the higher effluent dissolved oxygen

(DO) concentration (>5 mg/L), is due to intensifications that lead to
increased oxygen transfer in the wetlands. Intensified wetlands per­
1872 ± 171
2088 ± 408
1446 ± 149
1659 ± 269
1469 ± 144
1592 ± 256
1814 ± 451
1425 ± 142
1702 ± 163
1321 ± 102
(µS/cm)

formed similarly to the WWTP in terms of CBOD5 and TOC mass removal
EC

(Fig. 1A and Fig. 1B, respectively). Ammonium nitrogen (NH4–N) mass

removal was high (>93%) in the intensified wetlands HA, HAp, VAp,
VAp+VSp, and R (Fig. 1D). NH4–N mass removal was lower in VA
7.1 ± 0.2
7.3 ± 0.2
7.1 ± 0.5
7.0 ± 0.6
7.2 ± 0.1
7.1 ± 0.2
7.2 ± 0.2
6.9 ± 0.1
7.8 ± 0.2
7.1 ± 0.1
(81%), which could be a result of experiments conducted on VA from
pH

2013 – 2017 that could have caused poor functioning of the aeration
system (Boog et al., 2018, 2014). H50p removed very little NH4–N due
− 65 ± 102
− 169 ± 87

243 ± 37
242 ± 33
154 ± 84
224 ± 52
240 ± 35
221 ± 51
50 ± 145
238 ± 49

to lack of aerobic conditions (34%). Mass removal of total nitrogen (TN)

Redox
(mV)

varied depending on the systems (Fig. 1C). The lowest TN mass removal
was observed in H50p (37%), which can be explained by inefficient
nitrification due to the lack of dissolved oxygen. TN mass removals
435 ± 128
149 ± 42

450 ± 73

487 ± 84

1437 ± 10
–

571 ± 2

565 ± 8

ranged from 48% (HAp) to 66% (VA, VAp+VSp) in the intensified

1147 ± 207 m3/d1147 ± 207 m3/d

wetlands, and was 91% for the WWTP. E. coli was reduced, depending on
the design of the treatment wetlands, by 1.3 log10 units in H50p and up
Outflow

(L/d)
Qout

to 4.7 log10 units in the two-stage system VAp+VSp (Fig. 1E). In

contrast, the single-stage HF aerated wetlands (HAp: 3.8 log10 units; HA:
3.9 log10 units) had higher log reductions than the VF aerated wetlands
(VAp: 2.2 log10 units; VA 1.6 log10 units) and the municipal WWTP (2.9
log10 units).
–
197 ± 0
578 ± 0
578 ± 0
578 ± 0
578 ± 0
578 ± 0
1442 ± 9
Inflow

(L/d)

Image deleted. Please check.Image deleted. Please check.Image

reduction in parentheses).

Qin

deleted. Please check.Image deleted. Please check.Image deleted. Please

check.Image deleted. Please check.Image deleted. Please check.Image
Wetland Influent

deleted. Please check.

WWTP out
VAp+VSp

WWTP in

3.2. Micropollutants
Table 3

H50p

HAp

VAp
HA

VA

All seven micropollutants were detected in the influent to the

5
N.A. Sossalla et al. Water Research 201 (2021) 117349

Table 4
Annual mean concentrations, standard deviations and mass load removal (given in parenthesis; calculated from monthly mass load removal provided in SI Table A2)
for indicator micropollutants from May 2018 to April 2019 (n = 30 – 39). H50p: horizontal flow wetland (non-aerated); HA + HAp: horizontal flow wetland with
aeration; VA + VAp: vertical flow wetland with aeration; VAp+VSp: Two-stage wetland (vertical flow with aeration + unsaturated vertical flow sand filter); R:
Reciprocating wetland; WWTP: wastewater treatment plant.
Caffeine Ibuprofen Naproxen Benzotriazole Diclofenac Acesulfame Carbamazepine
(µg/L) (µg/L) (µg/L) (µg/L) (µg/L) (µg/L) (µg/L)
Wetland Influent 89 ± 53 33 ± 6 5±1 22 ± 5 7±2 33 ± 10 3±1
H50p 17 ± 17 (86%) 21 ± 4 (51%) 2 ± 1 (61%) 17 ± 4 (44%) 6 ± 2 (39%) 29 ± 11 (36%) 3 ± 1 (24%)
HA 2 ± 2 (99%) 0.4 ± 0 (99%) 0.1 ± 0.1 (99%) 3 ± 2 (83%) 3 ± 1 (54%) 2 ± 3 (94%) 3 ± 1 (− 10%)
HAp 0.3 ± 0.1 (99%) 0.6 ± 0.8 (98%) 0.2 ± 0.4 (96%) 3 ± 2 (86%) 1 ± 0.3 (93%) 4 ± 5 (85%) 4 ± 1 (− 1%)
VA 4 ± 5 (96%) 4 ± 1 (89%) 0.4 ± 0.2 (91%) 10 ± 2 (57%) 4 ± 1 (41%) 6 ± 3 (82%) 4 ± 1 (− 10%)
VAp 2 ± 2 (98%) 0.8 ± 0.3 (98%) 0.2 ± 0.1 (95%) 5 ± 1 (82%) 2 ± 0.5 (81%) 4 ± 5 (87%) 4 ± 1 (− 4%)
VAp+VSp 0.3 ± 0.3 (99%) 0.4 ± 0 (99%) 0.1 ± 0.1 (99%) 0.9 ± 0.6 (97%) 0.4 ± 0.4 (95%) 2 ± 4 (94%) 5 ± 1 (3%)
R 2 ± 2 (98%) 1 ± 0.5 (97%) 0.5 ± 0.1 (90%) 7 ± 3 (68%) 4 ± 1 (53%) 4 ± 4 (87%) 4 ± 1 (− 11%)
WWTP in 104 ± 23 32 ± 8 3±1 19 ± 7 6±2 31 ± 11 4±1
WWTP out 0.3 ± 0.1 (100%) 0.4 ± 0 (99%) 0.1 ± 0.1 (96%) 4 ± 1 (79%) 4 ± 0.5 (40%) 2 ± 3 (91%) 4 ± 1 (− 9%)

wetlands (e.g., septic tank effluent) as well as the influent to the WWTP.
Except for H50p, consistently low effluent concentrations were achieved
in all wetlands and the WWTP for the readily biodegradable compounds
CAF (< 4 μg/L), IBU (< 4 μg/L), and NPX (< 0.5 μg/L) (Table 4). Aerobic
conditions in the intensified treatment wetlands HA, HAp, VA, VAp,
VAp+VSp, and R resulted in >89% mass removal of CAF, IBU, and NPX
(Fig. 2A-C), which is consistent with previous studies (Hijosa-Valsero
et al., 2016; Verlicchi and Zambello, 2014). The non-aerated wetland
H50p showed low mass removal of micropollutants due to the prevailing
anaerobic conditions: 86% for CAF, 51% for IBU and 61% for NPX. For
the moderately biodegradable compounds BTZ, DCL, ACE, greater
variability was observed in effluent concentrations and mass removals
depending on the treatment wetland (Fig. 2C-E). BTZ was well removed
in HA, HAp, VAp, VAp+VSp, and the WWTP (<5 µg/L; >82% mass
removal), while moderate mass removal was achieved for BTZ in VA
(57%) and R (68%). H50p had a BTZ annual mass percent removal of
44%, which is significantly less than the BTZ removal observed in the
WWTP (p<0.05) or in VAp+VSp (p<0.0001), p-values are provided in
Table A3. Previous studies showed moderate degradation of BTZ under
anaerobic conditions and increased degradation under aerobic condi­
tions (Liu et al., 2011; Matamoros et al., 2010). VAp, HAp, and the
two-stage system VAp+VSp were most effective in removing DCL (81%,
93%, and 95% mass removal, respectively), which was even higher than
in a previous study conducted on this research side (Kahl et al., 2017).
These wetlands are characterized by high redox potentials, favoring the
biological degradation of DCL (Hijosa-Valsero et al., 2010). The
remaining wetlands achieved DCL mass removal between 39% (H50p)
and 53% (R), and the WWTP achieved 40%.
On an annual basis, mean effluent ACE concentrations for all systems
ranged between 2.0 μg/L (VAp+VSp, HA) and 29 μg/L (H50p). ACE
removal in the treatment wetlands was highly seasonal, which is re­
flected in the higher standard deviations in effluent ACE concentrations
compared to the other monitored micropollutants (Table 4). Although
previously thought to be resistant to biodegradation, ACE has recently
been reported to be moderately biodegradable (Castronovo et al., 2017;
Kahl et al., 2018). Monthly mean mass removal efficacies ranged be­
tween 85% – >99% for the intensified wetlands, 37% – 60% for H50p
and 96% – 99% for the municipal WWTP from May 2018 – November
2018, when effluent water temperatures were 16.7 ± 3.9 ◦ C. From
December 2019 – April 2018, when effluent water temperatures were
lower (7.9 ± 1.8 ◦ C), ACE mean monthly mass removal dropped as low
as 38% for the intensified wetlands, 7% for H50p and 61% for the
municipal WWTP. Monthly mean mass percent removals for each system
are shown in the Supplementary Information (Table A2). Other studies
confirm poorer ACE biodegradation at low temperatures, which may be
due to reduced metabolic activity of the ACE-degrading microorganisms
(Burke et al., 2014; Kahl et al., 2018).
CBZ was not effectively removed in any of the treatment systems
(Fig. 2G), which is consistent with other studies on constructed wetlands
and municipal WWTPs (Ilyas et al., 2020; Kahl et al., 2017). Several
studies suggest that CBZ enters the wetlands as a conjugate and is
metabolized back into the parent compound during biological treatment
(Jekel et al., 2015; Neale et al., 2020b). Thus, the negative removal ef­
ficacies might be explained by the presence of conjugated CBZ (glucu­
ronide conjugate). König et al. (2016) reported from column and batch
experiments that under reducing conditions and by adapted bacteria a
degradation of CBZ into the main degradation product 10,11-dihy­
dro-CBZ could be observed (20% degradation in column experiment;
anoxic bank filtrate used as column influent and spiked with CBZ). Since
reducing conditions prevail in H50p, reductive transformation of CBZ
cannot be excluded. A differentiation between the original and newly
formed parent compound (and the resulting removal efficacy) can
therefore not be made. The observed mass load removal efficacies of
micropollutants are consistently with a previous study on the research
site Langenreichenbach (Kahl et al., 2017). The second stage of the
two-stage system VAp+VSp provided incremental polishing, resulting in
higher micropollutant mass removal than single-stage systems (except
for CBZ mass removal in H50p).
3.3. Bioanalysis
Annual BEL removal efficacies for four of the five investigated bio­
assays are provided in Fig. 3. Either no effect was observed for the
activation of GR, or cytotoxicity masked the effect, therefore, this data is
not presented. Hashmi et al. (2020) reported masking effects of
GR-mediated activity by other cytotoxic compounds in raw water ex­
tracts and recommended fractionation as a sample preparation step to
avoid masking effects. Normalization of BEQs to BELs for treatment
plants was recommended by Neale et al. (2020b). BEQ percent removal
is analogous to concentration percent removal, and BEL removal is
analogous to mass load removal. Comparing BEL removal (as opposed to
BEQ removal) gives a more accurate picture that takes into account flow
dynamics such as those due to evapotranspiration. The EC values in units
of REF for each treatment wetland (influent, effluent), monthly BEL
removals and p-values are provided in the Supplementary Information
in Table A4, Table A5 and Table A6, respectively.
B[a]P-EL (indicative for activation of AhR; Fig. 3A) was well
removed in the intensified systems (78% – 91%), with the two-stage
system VAp+VSp being the most effective system. H50p and the
WWTP showed lower removal efficacy of B[a]P-EL (66% and 60%,
respectively). High variability of BEQ removal of activation of AhR was
reported in the literature (Lundqvist et al., 2019; Neale et al., 2020b).
Between 79% and 94% of the oxidative stress response was removed by
the intensified wetlands and the WWTP, while H50p removed signifi­
cantly less (56%, p<0.05; Fig. 3B). In previous studies, BEQ removal
efficacies varied from 60% to 85% (Neale et al., 2020b; Nivala et al.,
2018). Estrogenic activity (Fig. 3C) was efficiently removed in all
intensified wetlands (>99%), which was significantly higher than

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N.A. Sossalla et al. Water Research 201 (2021) 117349

Fig. 2. Annual mass removal of indicator micropollutants (A – G). Mean (dot), median (line), first and third quartiles (boxes), and minimum and maximum values
(whiskers) are shown. H50p: horizontal flow wetland (non-aerated); HA, HAp: horizontal flow wetland with aeration; VA, VAp: vertical flow wetland with aeration;
VAp+VSp: two-stage wetland (vertical flow with aeration + unsaturated vertical flow sand filter); R: reciprocating wetland; WWTP: wastewater treatment plant.

7
N.A. Sossalla et al. Water Research 201 (2021) 117349

Fig. 3. Annual BEL removal efficacy of investigated bioassays (A) Activation of the aryl hydrocarbon receptor, B) Oxidative stress response, C) Activation of the
estrogen receptor, D) Activation of the peroxisome proliferator activated receptor.). Mean (dot), median (line), first and third quartiles (boxes), and minimum and
maximum values (whiskers) are shown. H50p: horizontal flow wetland (non-aerated); HA, HAp: horizontal flow wetland with aeration; VA, VAp: vertical flow
wetland with aeration; VAp+VSp: two-stage wetland (vertical flow with aeration + unsaturated vertical flow sand filter); R: reciprocating wetland; WWTP:
wastewater treatment plant. Because of masking effects no EC10 could be derived for VAp+VSp (*).

removal observed in VA (88%; p<0.05) and H50p (77%; p<0.05). These
high removal efficacies are consistent with previous studies (Bain et al.,
2014; Lundqvist et al., 2019; Neale et al., 2020b). Because of masking
effects (cytotoxicity), no EEQs could be derived for VAp+VSp. High
removal efficacy for Rosiglitazone-EL (indicative for Activation of
PPARγ) was observed for the intensified treatment wetlands and the
WWTP (>91%), and to a lesser extent for H50p (76%) (Fig. 3D). Similar
removals were also reported in previous studies, where BEQ removal
efficacies for rosiglitazone-EQ ranged from 67% to >99% (Bain et al.,
2014; Neale et al., 2020b).
With the exception of VA, the monthly BEL removal efficacies for the
intensified wetlands did not fluctuate much over the course of the year
(Fig. 4). Previous experiments on VA may have altered the microbial
community or reduced the effectiveness of the aeration system, resulting
in a poorer and more variable system performance (Boog et al., 2018).
BEL removal for the WWTP was comparable to that of the intensified
wetlands, except for activation of AhR. H50p showed lower and more
variable BEL removal efficacies for all bioassays, especially in the colder
months.
Not every observed biological effect in a bioassay results in an
environmental or human health risk. To distinguish between poor and
good water quality, recent studies have derived Effect-Based Trigger
(EBT) values from the Environmental Quality Standards (EQS) of the
European Union Water Framework Directive. EBTs for surface water
have been proposed (Escher et al., 2018a) and updated in a more recent
study (Neale et al., 2020a). But no EBT values currently exist for the
treated wastewater, except for estrogenicity (Jarošová et al., 2014). The
samples for Wetland Influent, WWTPin, and H50p mostly showed high
BEQs that were above the propsed EBTs for surface water. Even though
the current EBTs apply only to surface waters, comparison with the BEQs
of treated effluents in this study shows the BEQs achieved by the
treatment systems (intensified wetlands and the municipal WWTP) were
close to or even below the surface water EBTs (Fig. 5), indicating that no
(or very little) dilution would be required for the treated effluent to be
released to the environment without causing negative environmental
effects. For Activation of AhR, Oxidative Stress Response, and Activation
of PPARy, the effluents from HA, HAp, VA, VAp, VAp+VSp, R, and the
WWTP could be discharged directly to the environment without
adversely affecting the receiving water. The two-stage wetland system
VAp+VSp had the highest removal of biological effects, with effluent
BEQs well below the proposed surface water EBTs. The exception for all
investigated treatment systems (wetlands and WWTP) was EEQ (acti­
vation of ER; estrogenic activity), where effluent BEQs did not fall below
the proposed surface water EEQ-EBT of 0.34 ngE2/L.
A PCA was conducted on all data, with the intention of exploring the
relationship between removal efficacies of conventional pollutants,
micropollutants, and biological effects. Operational conditions were also
used to discriminate the results (categorical complementary data). The
multidimensional dataset of micropollutants and bioassays can be
reduced to two dimensions that explain more than 82% of the total
variance. The first dimension corresponds to micropollutants that were
more easily biodegradable under aerobic conditions (BTZ, ACE, IBU,

8
N.A. Sossalla et al.
Fig. 4. Monthly BEL removal efficacy for the four investigated bioassays: A) Activation of the aryl hydrocarbon receptor, B) Oxidative stress response, C) Activation
of the estrogen receptor, D) Activation of the peroxisome proliferator activated receptor. H50p: horizontal flow wetland (non-aerated); HA, HAp: horizontal flow
wetland with aeration; VA, VAp: vertical flow wetland with aeration; VAp+VSp: two-stage wetland (vertical flow with aeration + unsaturated vertical flow sand
filter); R: reciprocating wetland; WWTP: wastewater treatment plant.
DCL, NPX) while the second axis was mainly associated with CBZ.
Intensified wetlands significantly reduced a pool of micropollutants that
are regarded as biodegradable under aerobic conditions. These are
grouped together on the left part of the score plot (Fig. 6A). The data
points for the intensified wetlands were similarly distributed along the
vertical axis, regardless of the individual system. The ellipse centers of
the effluent concentrations of all intensified wetlands and the WWTP
were similarly close to each other and the major axes of the ellipses had
similar orientations and magnitudes. The poor removal of micro­
pollutants and biological effects in H50p were grouped with the influent
wastewater (SEP and WWTPin) on the highest values of dimension 1.
A second PCA was conducted to reduce the dimensionality of the
variation in data in order to identify which BEQs were associated with
the dominant dimensions. The first three dimensions explained 73% of
the total variance. Micropollutants that are more easily biodegradable
were associated with Dimension 1 and fell into two clusters: BTZ, DCL,
IBU and NPX were associated with the bioassay for Dichlorvos-EQ. CAF
and ACE associated with the bioassay for Rosiglitazone-EQ (Fig. 7B).
These associations do not imply a causal relationship; it is possible that
variables in a cluster were associated simply because they all are sen­
sitive to another common variable. For example, the difference between
these two clusters might be explained by changes in temperature since
this parameter was associated with the second dimension. The contri­
bution of Rosiglitazone-EQ to Dimension 1 was larger (13.5%) than the
one of Dichlorvos-EQ (11.3%). Both CBZ and B[a]P-EQ significantly
contributed to Dimension 2 (28.5% and 43.6%, respectively (Fig. 7B)).
9
Water Research 201 (2021) 117349
Fig. 7A shows that B[a]P-EQ has a large contribution to Dimension 3
(34.7%) as well as CBZ (23.3%). This does not mean that CBZ is acti­
vating the AhR but rather that chemicals that activate the AhR are
similarly recalcitrant as CBZ.
Conclusions
Seven treatment wetlands and a municipal WWTP were monitored
for removal efficacy with respect to conventional wastewater parame­
ters, seven indicator micropollutants and five bioassays. Aerobic con­
ditions, as evidenced by high DO, elevated redox potential, and full
nitrification, in the intensified wetlands promoted biodegradation of
most micropollutants and removal of biological effects. Carbamazepine
was not well-removed in the intensified wetlands or the WWTP but was
somewhat removed in the non-aerated wetland H50p, which exhibited
predominantly anaerobic conditions (low DO, low redox potential, very
little nitrification). The two-stage wetland VAp+VSp had the best
overall removal of biological effects, often with higher BEL removal than
the conventional WWTP. All intensified treatment wetlands had higher
BEL removal than H50p, thus further demonstrating the advantages of
intensified wetland systems. No significant differences were observed
between the aerated HF and VF systems or between the planted and
unplanted pairs, indicating that vegetation plays only a small role in the
removal capacity of intensified treatment wetlands for indicator
micropollutants and selected mixture effects. The two-stage treatment
wetland system VAp+VSp exhibited the highest and most stable mass
N.A. Sossalla et al. Water Research 201 (2021) 117349

Fig. 5. Comparison of observed BEQs with the revised surface water EBTs (provided in Supplementary Information Table A7). H50p: horizontal flow wetland (non-
aerated); HA, HAp: horizontal flow wetland with aeration; VA, VAp: vertical flow wetland with aeratio; VAp+VSp: two-stage wetland (vertical flow with aeration +
unsaturated vertical flow sand filter); R: reciprocating wetland; WWTP: wastewater treatment plant. gray crosses represent samples where EC10 was masked by
cytotoxicity, so no valid EC10 could be derived. Therefore, BEQmax was derived from IC10 (calculation is provided in Supplementary Information, section A2).

Fig. 6. A) Score plot of the first two principal components with all treatment systems and the influents. Percent of variance accounted for is given in brackets. H50p:
horizontal flow wetland (non-aerated); HA, HAp: horizontal flow wetland with aeration; VA, VAp: vertical flow wetland with aeration; VAp+VSp: two-stage wetland
(vertical flow with aeration + unsaturated vertical flow sand filter); R: reciprocating wetland; WWTP: wastewater treatment plant. B) Orientation of the variables
(micropollutants and bioassays) on the factor map.

removal for conventional wastewater parameters (CBOD5, TOC, TN,
NH4–N), E. coli (annual average 4.7 log10 unit removal), micro­
pollutants (94 – >99%, except CBZ) and biological equivalent loads (91
– >99%). If cost and required footprint play an important role in a
wastewater treatment project, the aerated wetlands (HAp and VAp) are
recommended, as they also provide high quality effluent. BEL removal
for the intensified treatment wetlands were similar to or higher than
those of the municipal WWTP, further demonstrating the high treatment
capacity of nature-based solutions.
No direct correlation between micropollutants removal and BEL
removal was found, which underlines the importance of the comple­
mentary approach, where indicator chemicals are a measure of the ca­
pacity for biodegradation, but the bioassays give an impression on the
overall water quality. Bioassays provide a sum parameter of risk-scaled
concentrations and are relevant for the overall water quality.
Effect-based trigger (EBT) values have recently been proposed for

10
N.A. Sossalla et al.
Fig. 7. A) Percentage contribution of the micropollutants (acesulfame, ACE; benzotriazole, BTZ; carbamazepine, CBZ; caffeine, CAF; diclofenac, DCL; ibuprofen, IBU;
naproxen, NPX) and BEQs of the bioassays (Activation of AhR, B[a]P-EQ; Oxidative stress response, Dichlorvos-EQ; Activation of PPARγ, Rosiglitazone-EQ) to the
first three PCA dimensions. B) Loading plots of Dimension 2 versus Dimension 1. C) Loading plot of Dimension 3 versus Dimension 2. The quantitative supplementary
variables are indicated in gray.
differentiate good from poor water quality by indicating acceptable risk
for complex mixtures in surface waters (Escher et al., 2018a). While
EBTs have so far only been developed for surface water, the intensified
wetland systems in this study were able to achieve effluent bio­
equivalent concentrations (BEQs) near to or below three proposed sur­
face water EBTs (B[a]P-EQ, Dichlorvos-EQ, and Rosiglitazone-EQ),
indicating an immense potential for the use of enhanced nature-based
solutions for water treatment and reuse. Discharge of treated effluent
to a receiving surface water would result in a dilution of effects, effec­
tively reducing further the BEQs. Future studies should include internal
sampling in saturated treatment wetland systems to better understand
the spatial dynamics of pollutant and mixture effect removal. Since
aerobic conditions are an important factor in removal of biological ef­
fects, future studies could try to gain a better understanding of the role of
redox potential and removal of biological effects. The effect of inter­
mittent or spatially zoned aeration on removal of micropollutants and
biological effects could also be investigated.
Availability of data
Data is available in Supplementary Material.
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.
Acknowledgements
N.S. acknowledges the Helmholtz Interdisciplinary Graduated
School for Environmental Research (HIGRADE) and the UFZ for funding
and support. We gratefully acknowledge access to the platform CITEPro
(Chemicals in the Environment Profiler) funded by the Helmholtz
11
Water Research 201 (2021) 117349
Association. The authors are grateful to Katy Bernhard for technical
support at Langenreichenbach; Grit Weichert, Monika Möder, Petra Keil
and Jürgen Steffen for analytical support; Lisa Glauch for the help with
bioassays; Mayara Oliveira dos Santos and Luc Dejonghe for laboratory
support. Ted Linke (AZV Heidelbach) and his team are acknowledged for
support at the Langenreichenbach wastewater treatment plant.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.watres.2021.117349.
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