The effect of loading frequency and plants on the degradation of sulfamethoxazole and diclofenac in vertical-flow constructed wetlands

Ecological Engineering 122 (2018) 187–196

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The effect of loading frequency and plants on the degradation of sulfamethoxazole and diclofenac in vertical-flow constructed wetlands

T

⁎

Adam Sochackia,b,c, , Monika Nowroteka,b, Ewa Felisa,b, Joanna Kalkaa,b, Aleksandra Ziembińska-Buczyńskaa,b, Sylwia Bajkaczd, Sławomir Ciesielskie, Korneliusz Mikscha,b a

Silesian University of Technology, Environmental Biotechnology Department, ul. Akademicka 2, 44-100 Gliwice, Poland Silesian University of Technology, Centre for Biotechnology, ul. B. Krzywoustego 8, 44-100 Gliwice, Poland c Czech University of Life Sciences Prague, Faculty of Environmental Sciences, Department of Applied Ecology, Kamýcká 129, 165 00 Prague, Czech Republic d Silesian University of Technology, Faculty of Chemistry, Department of Inorganic, Analytical Chemistry and Electrochemistry, ul. M. Strzody 7, PL-44-100 Gliwice, Poland e University of Warmia and Mazury in Olsztyn, Department of Environmental Biotechnology, Faculty of Environmental Sciences, ul. Słoneczna 45G, 10-709 Olsztyn, Poland b

A R T I C LE I N FO

A B S T R A C T

Keywords: Constructed wetlands Ecotoxicity Microbial biodiversity Pharmaceuticals Transformation products

The aim of this study was to investigate the effect of loading frequency (1 pulse per day and 4 pulses per day) and vegetation (monoculture of Phalaris arundinacea ‘Picta’) on the removal and transformation of a mixture of pharmaceuticals diclofenac (DCF) and sulfamethoxazole (SMX) in lab-scale unsaturated vertical-flow constructed wetlands. Additionally, the system performance, the ecotoxicity of the wastewater (Microtox bioassay), and the microbial community structure and diversity in the top layer of the system were assessed. In the experiment, elevated concentrations of the pharmaceuticals (0.5 mg/L) were used to reflect the quality of wastewater from single houses or small settlements. The removal of DCF and SMX was deteriorated by the presence of Phalaris arundinacea ‘Picta’. The lower loading frequency enhanced the removal of DCF and SMX only in the planted columns, suggesting combined effect of the two factors. For the unplanted systems no significant effect of the loading frequency was observed. The observed removal efficiency of SMX and DCF in the experiment was in the range 52.8–91.2% and 47.3–74.2%, respectively. The pharmaceuticals did not affect markedly the removal of DOC, TKN and N-NH4, but they reduced the bacterial diversity within the rhizosphere and in the adjacent substrate of the systems. The main transformations of SMX were (mono-, di-, tri-)hydroxylation, demethylation, deamination and formation of glutathione conjugates, whereas for DCF only trihydroxylation was observed. The toxicity of the raw and treated wastewater containing the pharmaceuticals was at a level comparable with the control samples. This study showed that vertical-flow constructed wetlands can be efficient in the removal of DCF and SMX. Given that the constructed wetlands are inherently planted systems, it is suggested that the loading frequency is the operational variable that could be adjusted to enhance the removal of these pharmaceuticals.

1. Introduction Pharmaceutical compounds (PhCs) are biologically active and persistent substances, which because of their widespread occurrence in the biosphere have been recognized as a threat to the environment (Santos et al., 2010). Many studies reported the presence of pharmaceuticals in the effluents from wastewater treatment plants (Ternes, 1998; Andreozzi et al., 2003; Miao et al., 2004; Castiglioni et al., 2006; Vieno et al., 2007). One of the most often detected PhCs in influents and effluents of wastewater treatment plants, surface water and groundwater

are diclofenac (DCF) and sulfamethoxazole (SMX) (Gao et al., 2014; Vymazal et al., 2017; Sousa et al., 2018). DCF belongs to the group of non-steroidal anti-inflammatory drugs (NSAIDs) administered either orally or topically and is characterised by high consumption (Vieno and Sillanpää, 2014). SMX is a prominent short-acting representative of sulfonamide antibiotics used in high amounts in human and veterinary applications (Baran et al., 2011). The concentration of DCF in raw municipal wastewaters reached up to 95 µg/L (Luo et al., 2014) and for SMX it was found to be up to 2 µg/L (Hirsch et al., 1999). For single-house effluents it was predicted that the

⁎ Corresponding author at: Czech University of Life Sciences Prague, Faculty of Environmental Sciences, Department of Applied Ecology, Kamýcká 129, 165 21 Prague 6 Suchdol, Czech Republic. E-mail address: [email protected] (A. Sochacki).

https://doi.org/10.1016/j.ecoleng.2018.08.003 Received 15 May 2018; Received in revised form 30 July 2018; Accepted 4 August 2018 0925-8574/ © 2018 Elsevier B.V. All rights reserved.

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et al., 2011). In general, the treatment of wastewater containing DCF and SMX in CWs does not provide complete mineralisation of the compounds but only transformation by various metabolic pathways. The knowledge on the transformation products (TPs) for DCF and SMX and many other PhCs occurring in CWs is still limited. This hinders advancement in understanding of transformation routes but also predicting environmental impact of the CW effluents taking into account that some TPs of DCF and SMX can be more toxic or can retransform to the parent compound (Göbel et al., 2007; Majewsky et al., 2014; Osorio et al., 2016). The aim of this study was to investigate the effect of two loading frequencies and the presence of plants in microcosm unsaturated vertical-flow constructed wetlands on the removal efficiency of DCF and SMX and the quality of their transformation products. Additionally, to broaden the picture of the process it was aimed to assess the effect of DCF and SMX on the performance of the system, the toxicity of the effluents, and finally on the bacterial community structure and diversity in the upper substrate layer of the system.

concentrations of PhCs can be several-fold higher than in municipal wastewater (Abegglen et al., 2009). For SMX, the estimated concentration in the single-house wastewater can be as high as 4.4 mg/L (Abegglen et al., 2009). For DCF, no estimates are available, however for the other NSAIDs (naproxen and ibuprofen) a concentration of several mg/L was predicted (Abegglen et al., 2009). The threat that is caused by DCF and SMX to the aquatic environment was assessed to be low, especially in terms of acute toxicity (Osorio et al., 2016), but the effect of their metabolites and mixtures of multiple pharmaceuticals is largely unknown (Lonappan et al., 2016; Osorio et al., 2016). Additionally, the environmental occurrence of SMX may create a threat by inducing antibiotic resistance in bacteria (Chen et al., 2016). The wastewater treatment systems that were evaluated for the attenuation of the load of PhCs were mostly large conventional activated sludge plants (Margot et al., 2015). However, the elimination and transformation of pharmaceuticals were also studied in extensive systems such as constructed wetlands (CWs). Indeed, CWs apart from their important function in the removal of overall organic matter, nitrogen and phosphorus species have been indicated to be feasible to reduce the load of PhCs in the treated wastewater (Li et al., 2014). SMX and DCF have been often studied in CWs (Gorito et al., 2017) and informally gained a status of model compounds in the studies on the fate of PhCs in CWs. The efficiency of SMX removal in CWs ranged from negative values (Rühmland et al., 2015) up to virtually complete elimination (Hijosa-Valsero et al., 2011). SMX removal was enhanced in the systems providing anoxic conditions (especially in horizontal-flow CWs) (Carranza-Diaz et al., 2014; Rühmland et al., 2015), but its removal in well-oxygenated systems such as vertical-flow CWs (VF-CWs) was low (Dan et al., 2013; Nowrotek et al., 2016). DCF removal efficiencies in CWs were reported to lie in the wide range from 0 to 96% (Gorito et al., 2017) and to be favoured by a combination of aerobic and anoxic microbial metabolic pathways (Zhang et al., 2012; Ávila et al., 2014a, Auvinen et al., 2017; Ghattas et al., 2017). Usually, small CW treatment systems are composed of a single bed with vertical flow (VF-CW), which offers limited control over the treatment process. The VF-CWs are often implemented as unsaturated intermittently loaded systems to nitrify the wastewater from single households or small communities (Langergraber and Weissenbacher, 2017). Therefore, by design these systems support mostly aerobic processes with oxygen as the terminal electron acceptor, while anoxic processes play a minor role in these systems. It is especially the loading frequency in VF-CWs that could be controlled to slightly change the redox and oxygen conditions in the system to enhance the removal of organic matter and TKN. However, the effect of loading frequency on the removal of pharmaceuticals has been scantily discussed in the literature. In practice, loading frequencies vary from single pulse per day (large doses) to very frequent doses every 20 min (Nivala et al., 2013). More frequent doses potentially enhance oxygen transfer and increase redox potential (Nivala et al., 2013, Ávila et al., 2014b), however these conditions may vary in the course of a cycle. The conditions imposed by the feeding frequency depend on hydraulic properties of the bed and the presence and type of vegetation (Molle et al., 2006; Giraldi and Iannelli, 2009). Additionally, plants may also be responsible for direct uptake of some PhCs (Li et al., 2014) and may stimulate microbial activity in the system, thereby improving the elimination of PhCs (Zhu and Sikora, 1995, Gagnon et al., 2007, Faulwetter et al., 2009, Wu

2. Materials and methods 2.1. Experimental system The laboratory system used in the experiment (Fig. S1, Supporting Information (SI)) was designed as a microcosm model of VF-CWs. The experimental system consisted of 24 columns (diameter 0.2 m, height 0.8 m each) filled up to 0.7 m with three layers of filtering media – bottom layer: gravel 3–8 mm (0–5 cm), main layer: quartz sand 0.5–1.0 mm (5–65 cm) and upper layer (65–70 cm): a mixture (1:1 by volume) of sand and organic soil (drawing of the column is given in Fig. SI, SI). The composition of the filtering media layers was the same in all the columns. Eight types of columns were used depending on: the loading frequency (1 pulse per day (hydraulic loading rate (HLR) 38 mm/pulse) or 4 pulses per day (HLR 10 mm/pulse)), the presence or absence of plants, and the presence or absence in the feed of the two pharmaceuticals (PhCs): DCF and SMX. Each type of column was operated in triplicate. The wastewater was fed on top of the columns at the specified loading frequencies. The intervals between the loadings were 24 h for 1 pulse per day and 6 h for 4 pulses per day, respectively. The columns were operated in the same manner on every day of the experiment. The loading duration was very short and lasted maximum 30 s. The daily batch volume was 1.2 L for each column. For the columns fed with 4 pulses per day this volume was divided equally into 4 batches of 0.3 L. The plants used in the experiment were Phalaris arundinacea Picta. The types of columns together with symbols used to denote them are listed in Table 1. The influent was prepared in tap water by dissolving the following components (Felis et al., 2016): urea (208.76 mg/L), NH4Cl (62.4 mg/ L), yeast extract (264 mg/L), skim milk powder (118 mg/L), sodium acetate (510.4 mg/L), peptone (40 mg/L), KH2PO4 (41.37 mg/L), KCr (SO4)2·12H2O (0.96 mg/L), CuSO4·5H2O (0.781 mg/L), MnSO4·H2O (0.108 mg/L), NiSO4·7H2O (0.359 mg/L), PbCl2 (0.1 mg/L), ZnCl2 (0.208 mg/L), MgSO4·7H2O (4.408 mg/L), FeSO4·7H2O (11.6 mg/L). The PhCs columns with and without plants were fed with an influent in which DCF sodium salt (Sigma-Aldrich) and SMX (Sigma-Aldrich) were dissolved to yield concentration of 0.5 mg/L of DCF and 0.5 mg/L of

Table 1 Types of columns used in the experiment and the symbols used to denote them.

Feeding frequency Plants Pharmaceuticals (PhCs)

PhCs-1P-Plant

PhCs-1P-noPlant

PhCs-4P-Plant

PhCs-4P-noPlant

CTRL-1P-Plant

1 pulse/d Yes Yes

No

4 pulses/d Yes

No

1 pulse/d Yes No No (control columns; CTRL)

188

CTRL-1P-noPlant

CTRL-4P-Plant

CTRL-4P-noPlant

4 pulses/d Yes

No

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2.4. Bacterial community structure analysis

SMX (in a mixture), respectively. These concentrations were assumed to reflect the DCF and SMX concentrations in wastewater from single house or small communities, which is often treated in VF-CW. The duration of the present experiment was 100 days and it was preceded by the initial 7-month adaptation period (no DCF and SMX in the feed), subsequent 86-day period when DCF and SMX were initially added to the feed (described by Nowrotek et al. (2016)) and next by 100 day transition period (when the dosing of DCF and SMX was suspended). The columns that were previously used as PhCs or CTRL retained their attributed role in this experiment. The vegetation was planted at the beginning of the 7-month adaptation period, this is, more than a year before the present experiment. The plants were harvested 60 days before the onset of this experiment and subsequently regrew. The harvested above-ground biomass in the CTRL columns was significantly (p < 0.05) greater (1548 ± 130 g DW/m2; median ± MAD, n = 6) than in the PhCs columns (1062 ± 38 g DW/m2; median ± MAD, n = 6) suggesting negative effect of the PhCs on the growth of plants. The growth light was provided using high pressure sodium lighting system (photosynthetic photon flux was 750 µmol/s) with light/dark conditions of 14 h and 10 h, respectively.

In order to describe the microbial community in the wetland columns total bacterial genomic DNA was extracted from the samples using DNA Bead-Beat Micro AX Gravity Isolation Kit (A&A Biotechnology, Poland). The samples of the substrate (sand-soil mixture) and below-ground parts of the plants were taken only from the columns fed with 4 pulses/d. All the samples were taken only from the uppermost layer of the substrate (10 cm in depth). This was motivated by the findings of Tietz et al. (2007) indicating that 95% of the bacterial activity was found in the 10-cm upper layer of unsaturated VF-CWs. Three categories of samples should be distinguished: samples of the substrate from the rhizosphere of the planted columns, samples of the substrate from the unplanted columns and samples of the below-ground plant biomass. In the case of the samples of the below-ground parts of the plants the bacteriological material was flushed with sterile 1 × phosphate-buffered saline (PBS, pH 7.4 in order to obtain bacteriological material of the surface bacterial community. All samples were washed thrice before DNA isolation procedure in order to remove potential PCR inhibitors. Samples were vortexed intensively after last washing and left for sedimentation. The sampling was performed at the end of the experiment on day 100. The samples were taken from one replicate of a given type of a column. Isolated DNA was kept at 4 °C for further analyses. Isolated DNA was fragmented by sonication and the DNA libraries were prepared with NEBNext DNA Library Prep Master Mix Set for Illumina (New England Biolabs), according to the manufacturer’s instruction. The sequencing procedure was performed with Illumina MiSeq Sequencer with the MiSeq Reagent Kit v2 (500 cycles, Illumina) in the paired-end mode. The sequencing data were automatically demultiplexed with MiSeq Reporter v 2.3 Software, resulting in two FASTQ files with paired reads per sample. The analysis consisted of: the linking of overlapping paired sequences; the removal of unwanted sequencing artefacts (Gomez-Alvarez et al., 2009); the analysis of the reads quality and the removal of the reads with a low value (Phred Quality Score ≤ 15) (Cox et al., 2011) and the classification based on the available reference databases.

2.2. Chemical analysis The influent and effluent samples were collected fortnightly (for standard parameters) and weekly for PhCs. The samples were taken at the end of a cycle governed by the feeding frequency, this is directly before the next loading. All the samples were filtered using qualitative filter paper (MN 615 filter for medium fast filtration from MachereyNagel GmbH & Co. KG, Germany) prior to analysis. Standard wastewater parameters: orthophosphate phosphorus (PPO4), ammonium nitrogen (N-NH4), nitrate nitrogen (N-NO3) were analysed photometrically using Spectroquant tests from Merck (tests no. 1.00798.0001, 1.00683.0001, 1.09713.0001, respectively); total Kjeldahl nitrogen (TKN) was determined in digested samples using the Kjeltec System purchased from Foss Tecator and dissolved organic carbon (DOC) was determined by TOC analyser Shimadzu TOC–L using the non-purgeable organic carbon method. High-performance liquid chromatography (HPLC) method was used to determine the SMX and DCF concentrations in the influent and effluents during the experiment. The analytical protocol for this method is provided in Section S1.2 (SI). LOQ for of SMX and DCF was equal to 0.2 mg/L and the limit of detection (LOD) was 0.02 mg/L. The values between LOD and LOQ were estimated based on the signal and the calibration curve (equation in the form y = ax). The analyses of the SMX and DCF and their transformation products (TPs) were performed using UHPLC-MS/MS system (UltiMate 3000 RS; Dionex) coupled with a triple quadrupole mass spectrometer (AB SCIEX 4000 Q TRAP). The identification of the by-products was performed using LightSight Software. Further details of the HPLC-MS/MS analysis are given in Section 1.3 in SI.

2.5. Statistical analysis The Shapiro-Wilk W test was employed to test for normality of the data. Median and the median absolute deviation (MAD) were used as descriptors of the central tendency and a measure of dispersion for all the data, respectively. Independent groups of the data were compared using the Mann-Whitney U because the analysed data sets had predominantly non-normal distribution. Differences were considered statistically significant when p < 0.05. The outliers were detected and removed based on median absolute deviation (MAD) according to the protocol of (Leys et al., 2013). Statistical testing was performed using the Dell Statistica 13 software (Dell Inc., 2016). 3. Results and discussion

2.3. Ecotoxicological analysis – microtox bioassay 3.1. The effect of PhCs on the standard wastewater parameters The samples of influents and effluents for the ecotoxicity assessment were sampled in seven sampling campaigns on days 12, 26, 33, 54, 75, 89, 100 day of the experiment. The samples of the effluent were taken from all three replicates of a given type of a column, but prior to the toxicity testing samples were averaged for each type of column. The ecotoxicity of the samples was measured by means of Microtox system – based on Aliivibrio fischeri luminescence inhibition. The tests were performed in the Microtox M500 toxicity analyzer, according to the standard procedure – EPA whole effluent toxicity (EPA). The lyophilized bacteria Aliivibrio fischeri were purchased from Modern Water (Cambridge, UK). A 2% solution of NaCl was used as a negative controls as well as diluent. The Aliivibrio fischeri luminescence inhibition test was performed in 5 replicates for a given sample (replicate).

The concentrations and removal efficiency of standard wastewater quality parameters and the PhCs determined in the course of the experiment are shown in Table 2 with indication of statistical significance of the differences between counterpart columns. The removal efficiencies of DOC in all the types of columns were above 94%. The presence of PhCs was noted to negatively affect the removal of DOC in PhCs-1P-Plant and in the PhCs-4P-noPlant columns in comparison to respective counterpart CTRL columns. Although, the differences were significant (p < 0.05) the values of the removal efficiency were only less than 2% lower. The effect of PhCs on the nitrogen transformations in the experimental system was found significant (p < 0.05) only for N-NO3 effluent concentrations in the case of the 189

190

N-NO3

23.1±1.7

32± 3

165±3

no

no

no

no

23.2±0.7

34± 10

174±13

2±1

302.6±36.4

n.a.

n.a.

CTRL

no

no

15.9±3.9 (31%) no

no

13±10 (59%)

16±5 (90%)

132±30

yes

0.2040±0.1621 (69.0%) 0.1252±0.0598 (74.2%) 15.2±1.8 (94.2%)

PhCs-1PPlant

17.6±1.3 (24%)

12± 6 (64%)

31±26 (82%)

115±34

12.5±0.5 (95.9%)

n.a.

n.a.

CTRL1P-Plant

no

yes

14.2±4.9 (39%) no

no

20±9 (36%)

21±7 (87%)

119±16

no

0.0576±0.0376 (91.2%) 0.1532±0.0759 (68.5%) 13.9±1.6 (94.7%)

PhCs-1PnoPlant

16.3±2.1 (29%)

10±5 (70%)

17± 3 (90%)

144±20

11.0±2.4 (96.4%)

n.a.

n.a.

17.3±2.2 (25%)

1±1 (96%)

9±3 (95%)

136±19

no

no

no

no

no

0.3100±0.2350 (52.8%) 0.2562±0.1264 (47.3%) 10.7±1.2 (95.9%)

PhCs-4PPlant

type of the columns

CTRL-1PnoPlant

Effluent

16.7±1.9 (28%)

3± 2 (91%)

9±3 (95%)

142±21

13.4±3.9 (95.6%)

n.a.

n.a.

CTRL4P-Plant

14.2±3.5 (39%)

3±2 (92%)

11± 3 (94%)

158±11

no

no

no

yes

yes

0.1178±0.0978 (82.1%) 0.1541±0.1292 (68.3%) 9.8±2.9 (96.3%)

PhCs-4PnoPlant

15.5±3.7 (33%)

2±1 (93%)

9±4 (95%)

134±17

7.7±1.0 (97.4%)

n.a.

n.a.

CTRL-4PnoPlant

n.a. – not added to the influent; nitrite nitrogen (N-NO2) was monitored but the results are not shown because the observed concentrations were deemed negligible.

n.a. not added to the influent; nitrite nitrogen (N-NO2) was monitored but the results are not shown because the observed concentrations were deemed negligible

P-PO4: CTRL/ PhCs difference

P-PO4

N-NH4: CTRL/ PhCs difference

N-NH4

TKN: CTRL/ PhCs difference

TKN

N-NO3: CTRL/ PhCs difference

2±1

262.7±7.5

DOC no

0.4863±0.2376

DCF

DOC: CTRL/PhCs difference

0.6573±0.0727

PhCs

Influents

SMX

Parameter

Table 2 Concentrations (mg/L; median ± MAD) and removal efficiencies in brackets, n = 4–36 (depending on data set), the differences for the counterpart CTRL and PhCs columns assessed by Mann-Whitney U test, “yes” indicates significant difference at p < 0.05).

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loading frequency. The underlying mechanistic explanation of the effect of the plants (specifically Phalaris arundinacea ‘Picta’) requires further study. Notably, the presence of plants in the PhCs columns did not significantly affect the removal of standard wastewater contaminants (Section 3.1), but nevertheless the removal of DOC was slightly lower in the planted columns. It can be hypothesized that the plant exudates or DOC of plant origin could have competed with SMX and DCF as a source of microbially available organic carbon. The other indirect effects exerted by plants such as the influence on the hydraulic properties (e.g. infiltration rate) of the filtering bed cannot also be ruled out (Molle et al., 2006). The observation regarding the negative effect of Phalaris arundinacea ‘Picta’ cannot serve to generalise about all the emergent hydrophytes used in VF-CW as those plants exhibit wide variety of properties such as e.g. roots penetration depth, the magnitude of the release of oxygen and exudates in the rhizosphere (Vymazal, 2011). Also the growth conditions specific to indoor column studies such as limited space for rhizome propagation, artificial light conditions and others factors could have magnified the effect of plants on the removal of DCF and SMX. As stated by Li et al. (2014) plants played positive role in the removal of SMX and DCF in CWs but this conclusion was based on the performance of system providing anoxic conditions such as horizontalflow systems (Hijosa-Valsero et al., 2010). The effect of plants on the removal of SMX and DCF in unsaturated VF-CWs was not studied to the best of authors’ knowledge. In comparison to our previous study in the same experimental system (Nowrotek et al., 2016), the removal efficiencies of SMX increased by 45% and 61% for planted and unplanted columns, respectively, and for DCF by 23% and 16% for planted and unplanted columns, respectively. This comparison refers to the columns fed by 1 pulse/d because only this feeding frequency was applied in the previous experiment. The observed increase may suggest that the microbial community gradually adapted to the removal of the compounds in the course of the experiment. The discrepancy between the increase in removal efficiency for SMX and DCF may suggest different removal mechanisms (biodegradation or adsorption) or microbial adaptation mechanisms, but also the occurrence of spatial zones supporting the removal of SMX and DCF. As suggested by Banzhaf et al. (2012) the mobility of SMX implies that this compound is rather degraded in the bottom part of the bed, whereas DCF is not as mobile as SMX and may adsorb to organic matter in the upper part of the bed. Taking into account that the inoculation of the columns was performed by applying activated sludge mixed liquor on the surface of the columns (Nowrotek et al., 2016), the increase in SMX removal may also indicate the ongoing colonisation of the bed by adapted bacterial biomass in the downward direction. The results presented in the previous study indicated contrary to the present experiment that the plants had no effect on the removal of PhCs. This could have resulted from increased plant biomass in the course of the present experiment but also from indirect factors such as increased amount of detrital plant biomass.

PhCs-1P-noPlant and the PhCs-4P-noPlant columns. The character of this effect was, however, ambiguous because the effluent concentration was either higher (in PhCs-4P-noPlant) or lower (in PhCs-1P-noPlant) than in the counterpart CTRL columns. The main processes responsible for the production of N-NO3 in CWs, which are ammonification and nitrification (assessed based on TKN and N-NH4 concentrations), were not significantly (p ≥ 0.05) affected by the PhCs. This could suggest that the PhCs affected denitrification but this process plays negligible role in unsaturated VF-CW. This aspect requires further study. Finally, the P-PO4 removal was observed not to be affected by the PhCs. The removal of P-PO4 in CWs is governed mostly by adsorption or plant uptake (Vohla et al., 2011). The latter process in the experimental system was negligible and in fact, all the planted columns provided lower removal of P-PO4 than the counterpart unplanted columns (Table 2). Hence, adsorption is believed to be the main mechanism for P-PO4 removal in the experimental system. Competition between phosphorus compounds and organic (micro)pollutants for the sorption sites was indicated in several studies, especially concerning specific mineral phases (Remucal and Ginder-Vogel, 2014), but this phenomenon would require further evaluation in the case of CWs. 3.2. Removal of SMX and DCF The influent and effluent concentrations and the removal of DCF and SMX in the experimental period are shown in Table 2 (and additionally in Fig. S3, SI). In the unplanted columns no significant differences (p ≥ 0.05) in SMX removal were found between the 1 pulse/d (91.2%) was the 4 pulses/d (82.1%) columns (Table S3, SI). By comparison, the SMX removal in the planted fed at 1 pulsed/d (69.0%) was significantly higher (p < 0.05) than in the columns fed at 4 pulses/d (52.8%). Additionally, the removal of SMX was significantly lower (p < 0.05) than in the counterpart unplanted columns. The removal efficiency of DCF in the unplanted columns was equal to 68.5% and 68.3%, for the columns fed by 1 pulse/d and 4 pulses/d, respectively, and was not significantly different (p < 0.05) (Table S4, SI). In contrast, in the planted columns the removal of DCF was significantly (p < 0.05) higher for the columns fed by 1 pulse/d (74.2%) than in the columns fed at 4 pulses/d (47.3%). The effect of plants was statistically significant (p < 0.05) only for the columns fed at the frequency of 4 pulses/d, this is, the PhC-4P-noPlant (68.3%) and PhC-4PPlant (47.3%) columns. The results showed that the effect of the feeding frequency manifested only in the planted columns suggesting that the plants amplify this effect. This also indicates that the knowledge regarding unplanted systems (such as intermittent sand filters) cannot be directly transferred to constructed wetlands. Interestingly, Ávila et al. (2014b) also observed that the DCF was more efficiently removed at lower loading frequency (as the only substance out of eight studied) in the VF-CW planted with Phragmites australis attributing this to lower oxidation of the system. The other compounds were not affected by the loading frequency or showed opposite trend. The behavior of SMX was not studied in this regard. For both SMX and DCF, the removal efficiency was deteriorated at the higher loading frequency. Thus, the adjustment of the loading frequency (between 1 or 4 pulses daily) in planted systems appears to be of major importance for improving the removal of DCF and SMX as model PhCs. This observation is especially relevant for the CW systems, which inherently are planted systems (Vymazal, 2011). The results for the removal of N-NH4 (Table 2) showed that the lower loading frequency resulted in much lower removal of this N form. This may create a potential conflict when considering the treatment goals, especially taking into account the anticipated regulations for the concentrations of organic micropollutans in the effluents of wastewater treatment systems. The effect of the presence of plants on the removal of PhCs was negative (it decreased the removal) and more pronounced at the higher

3.3. Transformation products of DCF and SMX in the effluents The tentative transformation products of SMX (TPs-SMX) and DCF (TPs-DCF) are presented in Table 3. The chemical structures of the TPs and the parent compounds are presented in Table S5 (SI) and supplemented with the results given in Table 3 for the convenience of the reader. Marvin software was used for drawing and displaying chemical structures (ChemAxon, 2017). The chemical structures of the identified tentative TPs-SMX suggest that five transformation reactions affected the parent compound during the treatment in the CWs. These reactions were: hydroxylation, deamination, conjugation with glutathione, demethylation and N-acetylation. As shown in Table 3 six of nine TPs-SMX contained single hydroxy group (OH) or multiple OHs. Thus based on the number of TPs-SMX, 191

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Table 3 Occurrence of TPs-SMX and TPs-DCF in the effluents (“-” not detected; “+” detected; the detection threshold for TPs was signal-to-noise-ratio (S/N) for non-target analyses ≥ 5.0 and the signal intensity level for both quadrupoles (Q1 and Q3) ≥ 1000 AU).

Transformation product (TPs) hydroxy-SMX dihydroxy-SMX trihydroxy-SMX glutathionyl-hydroxy-SMX glutathionyl-dihydroxy-SMX glutathionyl-desamino-hydroxy-SMX desamino-SMX desmethyl-SMX N-acetyl-SMX trihydroxy-DCF

PhCs-1P-Plant + + + + + + + + +

Effluents (column symbols) PhCs-1P-noPlant PhCs-4P-Plant + + + + + + + + + + + + + + + + +

PhCs-4P-noPlant + + + + + + + +

Osorio et al., 2016; Peng et al., 2017) but it was also observed under denitrifying conditions (Rodríguez-Escales and Sanchez-Vila, 2016). In the present study, desamino-SMX was detected in all the effluents of the PhC columns, therefore no qualitative effect of plants and feeding frequency can be distinguished. Desamino-SMX was found to be more toxic (based on acute toxicity test with Aliivibrio fischeri) than the parent compound (Osorio et al., 2016). Another tentatively identified TPs-SMX was desmethyl-SMX as the product of demethylation. This is reaction in which methyl group (–CH3) is removed from a molecule. Demethylation was previously observed for other organic micropollutants under both anaerobic conditions (e.g. in soil microcosms by Morrica et al. (2001)) and aerobic conditions in an activated sludge system (Jewell et al., 2016). In this study, similarly as desamino-SMX, desmethyl was detected in all the effluent of the PhCs. N-acetyl-SMX, which was detected in the effluent of the columns fed with 1 pulse/d, is a compound produced by the N-acetylation reaction. In real municipal wastewater this compound occurs as a metabolite of SMX formed during human metabolism, which is excreted in the faeces. It was also reported to be a product of bacterial metabolism of ammonia oxidizing bacteria in nitrifying activated sludge wastewater treatment system (Kassotaki et al., 2016). N-acetyl-SMX is also one of the main metabolite of SMX in soil environment (Andriamalala et al., 2018). In wastewater treatment systems or even in the sewer it can be retransformed to the parent compound (Göbel et al., 2005). In this study, due to the lack of detectable of amounts of any TPs-SMX in the feed, it was possible to confirm formation of this compound during wastewater treatment. N-acetyl-SMX occurred only in the effluent of the columns fed at the lower frequency, which were assumed to provide lower retention time (and thus shorter contact between the pollutants and the media) than the columns with higher feeding frequency (Ávila et al., 2014b; Molle et al., 2006; Torrens et al., 2009). This can be elucidated by two phenomena. One is that N-acetyl-SMX was formed only in the columns fed with 1 pulse/d because of specific conditions supportive of this metabolic pathway in the microorganisms. The other could have been its initial production and subsequent retransformation in the columns with longer retention time (fed by 4 pulses/d) leading to undetectable amount in the effluent. Some of the detected TPS-SMX can exert residual antibiotic activity (Majewsky et al., 2014; Achermann et al., 2018) or can be retransformed to the parent compound (Göbel et al., 2005), causing potential threat to the environment exposed to the effluents of wastewater treatment plants. The potential transformation pathways for SMX in the present experiment are shown in Fig. S4 (SI). The only detected tentative TP-DCF was trihydroxy-DCF. It was

hydroxylation was the predominant transformation of SMX. Hydroxylation was observed to be a sole transformation but it also co-occurred with other reactions (deamination and glutathione conjugation), which was reflected in the structure of the TPs-SMX. For hydroxylation as a single transformation, one OH group (monohydroxylation), two OH groups (dihydroxylation) or three OH groups (trihydroxylation) were attached to the parent compound. Monohydroxylated and dihydroxylated TPs-SMX were also detected to be conjugated with glutathione (glutathionyl-hydroxy-SMX and glutathionyl-dihydroxy-SMX, respectively) and also to co-occur with detachment of the amino group (–NH2; deamination) from the parent compound (glutathionyl-desamino-hydroxy-SMX). Glutathione is a marker of oxidative stress in plants and in bacteria, however, the lack of excretory pathways in plants, suggests that the glutathione conjugates detected in the effluents were of bacterial origin (Nowrotek et al., 2017) or originated from detrital plant biomass. Monohydroxylated TP-SMX (hydroxy-SMX) was detected only in the effluents of the unplanted columns, whereas dihydroxy-SMX and trihydroxy-SMX were detected in all the effluents. However, the presence of glutathionyl-hydroxy-SMX in all the effluents, suggests that hydroxyl-SMX temporarily occurred also in the planted columns, but was subsequently transformed. One of the possible transformation reactions was conjugation with glutathione, which is expected to occur after hydroxylation of a compound (Josephy et al., 2005), which could have rendered undetectable amounts of hydroxylSMX. Another explanation can be that hydroxy-SMX gained additional OH group(s) and transformed into di- or trihydroxy-SMX. The presence of glutathionyl-desamino-hydroxy-SMX, which was noted only in the planted columns, can suggest that plants provided higher diversity of conditions in the system that enabled co-occurrence of various transformation reactions. The hydroxylated TPs-SMX and conjugates with glutathione are regarded as less toxic than the parent compound (Majewsky et al., 2014; Achermann et al., 2018). The hydroxylation of benzene ring in SMX is the first step of possible degradation pathways of SMX, next step is oxidation of the amine group at benzene ring, ring opening and S-N bond cleavage (Zhang et al., 2017). Hydroxylation and deamination were observed for the identified TPs-SMX, but further steps of transformation indicated by Zhang et al. (2017) cannot be proved in the studied CW system. Further degradation steps could have been prevented by unconducive process conditions, such as insufficiently long retention time, but also lack of specific redox conditions and bacterial groups, or could have been undetected in the analysis. The products of deamination reaction were desamino-SMX and glutathionyl-desamino-hydroxy-SMX. In the literature, the formation of deaminated TPs-SMX was attributed to the activity of ammonia oxidizing bacteria (Hijosa-Valsero et al., 2011; Kassotaki et al., 2016; 192

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columns as compared with the influent values (p < 0.05) (Table S6, SI). It was observed that the effluent toxicity is lower (by 5–10%) in the unplanted columns (“noPlant” columns) than in the counterpart planted columns (“Plant” columns). This trend was consistently observed for all the four corresponding pairs of planted and unplanted columns, regardless of the presence of PhCs and the loading frequency. The LI was observed to be positively correlated with the DOC concentrations in the effluent consistently for all the four pairs of column types. The DOC concentrations were univocally higher in the planted columns in respect to their unplanted counterparts (Table 2), which could be attributed to lower removal of the influent DOC, but also to the presence of plant related dissolved compounds such as detrital carbon or plant exudates. The toxic effect can be especially attributed to the plant exudates, which can also include allelopathic chemical substances and antibacterial agents, which was observed in wetland plants (Batty, 2003; Vymazal, 2011). Also a common trend was noted, that the toxicity of the effluents from the columns fed with 1 pulse/d is lower than in the counterpart columns fed at the frequency of 4 pulses/d, however this difference was significant (p < 0.05) only in the case of the unplanted PhCs columns (Table S6, SI). Additionally, it was observed that the LI values were not significantly different (p ≥ 0.05) between the counterpart PhCs and CTRL columns. The only exception was the significantly (p < 0.05) lower LI value (by approx. 2%) in the PhC-1P-noPlant columns as compared with the CTRL-1P-noPlant columns.

detected in the effluents from all the types of columns. Indeed, degradation of DCF in aerobic biological wastewater treatment systems occurs mainly via hydroxylation reaction as the initial step of transformation (Bouju et al., 2016). According to Ghattas et al. (2017), oxygenation of the chlorinated benzene ring (by hydroxylation) enables further transformation of DCF, which is dechlorination. Whereas dechlorination of DCF occurs under anaerobic conditions, the hydroxylation reaction is much more likely under predominantly aerobic conditions (Ghattas et al., 2017). In wastewater treatment systems, the frequently reported product of DCF hydroxylation was 4′-hydroxy-DCF. It was identified as the main TPs-DCF in a horizontal-flow CW (Ávila et al., 2013) and in various surface-flow and subsurface-flow mesocosm CW treating urban wastewater (Hijosa-Valsero et al., 2016). 4′-hydroxyDCF was also found to be a metabolite of DCF in plants (Huber et al., 2012), but the lack of excretory pathways in plants precludes the presence of this TP of plant origin in the effluent. Triple hydroxylation of DCF was, however, not thus far reported as a possible transformation of DCF in CWs. It cannot be proved that trihydroxy-DCF underwent any further transformations, such as, for example, dechlorination mentioned above. The dechlorination reaction of DCF in the studied system was probably inhibited by the lack of reducing conditions. The potential transformation pathways for SMX in the present experiment are shown in Fig. S5 (SI). 3.4. Ecotoxicological assessment – microtox bioassay The toxicity of the influents and effluents measured as luminescence inhibition (LI) in the Aliivibrio fischeri test is shown in Fig. 1. The presence of PhCs in the influent did not cause significantly higher (p ≥ 0.05) LI in comparison with the control influent (Table S6, SI). The LI was markedly reduced during treatment in all the types of

3.5. Bacterial community structure The analysis (based on the next generation sequencing) of the bacterial community in the 10-cm uppermost layer of the PhC-4P columns revealed that the dominant phyla in all substrate and roots and

Fig. 1. Influents and effluents ecotoxicity (central point indicates arithmetic average; whiskers show min and max non-outlying values), n = 7. 193

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CTRL-4P-Plant; roots and rhizomes CTRL-4P-Plant; substrate CTRL-4P-noPlant; substrate PhCs-4P-Plant; roots and rhizomes PhCs-4P-Plant; substrate PhCs-4P-noPlant; substrate 0%

20%

40%

60%

80%

Proteobacteria

Chloroflexi

Bacterioidetes

Actinobacteria

Firmicutes

Planctomycetes

Cyanobacteria

others

100%

Fig. 2. The composition of the microbial community in the 10-cm uppermost layer of the constructed wetland columns at phylum level.

3.2, the increased mobility of SMX (Banzhaf et al., 2012) would indicate bottom layers of a VF-CW system as the main SMX-biodegradation compartment and conversely as the compartment the most impacted by SMX and its TPs.

rhizomes samples is Proteobacteria (Fig. 2) with a relative abundance above 50% in all the sampling spots. It was observed that especially the bacteria belonging to the Chloroflexi phylum became noticeably more abundant on the roots and rhizomes in the PhCs-4P-Plant column and in the substrate of the PhCs-4P-noPlant column in comparison to counterpart samples from the CTRL columns (Fig. 2). The species dominant in all the samples was Paracoccus, belonging to Alphaproteobacteria is (Fig. S6, SI). Bacteria belonging to this genus are often present in different types of WWTPs (Wang et al., 2014) and they are also known to be able to grow in a biofilm (Kumar and Spiro, 2017). It could be suspected that the ability to grow in biofilm predispose these bacteria to be abundant in CWs columns, which support attached-growth mode. The genus Chloroflexus was especially abundant on the roots and rhizomes in the PhCs-4P-Plant column and in the substrate of the PhCs-4P-noPlant column (Fig. S6, SI) similarly as observed for the Chloroflexi phylum. It can be hypothesized that this genus played a role in the degradation of these compounds. Chloroflexus is a thermophilic filamentous anoxygenic phototrophic bacterium which is able to grow phototrophically under anaerobic conditions but also chemotrophically under aerobic conditions in the absence of light (Tang et al., 2011). The biodiversity of the bacterial communities calculated using Shannon Biodiversity Index (H) indicated the lowest biodiversity on the roots and rhizomes in the PhCs-4P-Plant column, while the roots and rhizomes in the CTRL-4P-Plant column supported the highest biodiversity (the H values of 1.19 and 2.56, respectively, Fig. S8). Similar observation was made for the H values describing the communities in the substrate of planted columns, but no difference was observed for the unplanted columns. These results support the hypothesis of bactericidal influence of SMX or its derivatives (however, the effect of DCF cannot be ruled out) on the bacterial community of CWs within the zone directly exposed to the influent wastewater (the upper 10 cm of the filtering bed), with particularly pronounced effect on the bacteria associated directly with the rhizosphere or occurring in its vicinity. The implications of bacterial diversity on the treatment efficiency of CWs have not so far been explicitly pointed out, yet high microbial diversity of CWs or any other eco-treatment system is regarded as their critical feature (Wu et al., 2018). It should be noted that the presented results describe the bacterial structure only in the uppermost layer of the substrate, which was assumed to host major bacterial activity within the columns (Tietz et al., 2007). Also, considerable amount of biomass could have been expected at the lower layers despite negligible activity. As mentioned in Section

4. Conclusions The following conclusions can be formulated based on the obtained results:

• The • • •

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removal efficiency of DCF and SMX in unsaturated intermittently-fed VF-CWs can vary substantially from moderate (approx. 50%) to high (approx. 75% for DCF) or very high (> 90% for SMX) depending on the presence of plants and the feeding frequency. Both DCF and SMX presented similar response to the studied effects: the presence of plants had detrimental effect on their removal efficiency, while the lower loading frequency enhanced their removal. The effect of the loading frequency was significant only in the planted columns, suggesting combined effect of these two variables. The plants did not participate in the detoxification of the wastewater. Conversely, the effluents from planted columns were slight more toxic towards Aliivibrio fischeri than effluents from the unplanted controls (also those without PhCs), which can be attributed to antimicrobial effect of plant exudates, among other factors. The only transformation reaction that was identified for DCF was multiple hydroxylation, regardless of the presence of plants and the loading frequency. The set of the identified TPs-SMX indicated increased diversity of the transformation reactions: (multiple) hydroxylation, demethylation, deamination, conjugation with glutathione and N-acetylation. The flower frequency stimulated N-acetylation, however it cannot be ruled out that this reaction was reversible at higher loading frequency. The presence of plants accelerated the transformation of hydroxyl-SMX to other TPs-SMX. DCF and SMX did not substantially affect the removal of DOC, TKN and N-NH4, and did not exert significant toxic effect towards test organism Aliivibrio fischeri at the influent concentrations. Also the final effluent with residual amounts of DCF and SMX, and their TPs had exerted the same toxic effect as control samples without PhCs. DCF and SMX affected the bacterial community structure in the uppermost layer (the only studied) of the CWs by changing the relative abundance of specific taxa. The PhCs decreased the biodiversity of the bacterial community in the planted columns, but not in the unplanted columns.

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In general, this study showed that vertical-flow constructed wetlands can be efficient in the removal of DCF and SMX at the elevated concentrations typical of domestic wastewater. Given that the constructed wetlands are inherently planted system, it is suggested that the loading frequency is the operational variable that could be adjusted to enhance the removal of these pharmaceuticals and potentially other pharmaceutical compounds.

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