WETLAND SYSTEMS: ECOLOGY, FUNCTIONS AND MANAGEMENT

Ornamental plants for micropollutant removal in wetland systems

Cristina Macci & Eleonora Peruzzi & Serena Doni &Renato Iannelli & Grazia Masciandaro

Received: 13 December 2013 /Accepted: 21 April 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The objective of this paper was to evaluate theefficiency of micropollutant removal, such as Cu, Zn, car-bamazepine, and linear alkylbenzene sulfonates (LAS),through the use of a subsurface vertical flow constructedwetland system with ornamental plants. Zantedeschiaaethiopica, Canna indica, Carex hirta, Miscanthussinensis, and Phragmites australis were selected andplanted in lysimeters filled up with gravel. The lysimeterswere completely saturated with synthetic wastewater (N280 mg L−1, P 30 mg L−1, Cu 3.6 mg L−1, Zn 9 mg L−1,carbamazepine 5 μg L−1, linear alkylbenzene sulfonates14 mg L−1), and the leaching water was collected for anal-ysis after 15, 30, and 60 days in winter-spring and spring-summer periods. Nutrients (N and P) and heavy metalsdecreased greatly due to both plant activity and adsorption.C. indica and P. australis showed the highest metal contentin their tissues and also the greatest carbamazepine andLAS removal. In these plants, the adsorption/degradationprocesses led to particularly high oxidative stress, as evi-denced by the significantly high levels of ascorbate perox-idase activity detected. Conversely, Z. aethiopica was theless efficient plant in metal and organic compound removaland was also less stressed in terms of ascorbate peroxidaseactivity.

Keywords Constructed wetlands . Ornamental plants .

Pharmaceuticals and personal care products (PPCP) . Heavymetals . Carbamazepine . Linear alkylbenzene sulfonates(LAS)

Introduction

Cost-effective and environmentally friendly technologies forthe remediation of wastewater polluted by toxic substances areemerging as a topic of global interest (Amjad et al. 2013).Wastewater treatment by constructed wetlands (CWs) hasgained popularity in the last four decades as an alternative toconventional treatments. In addition to decontamination, theselow-cost systems provide further multiple benefits, such asCO2 reduction, creation of habitat for plants and animals,recreational and educational exploitability, and aesthetic/amenity value (Smith 2007).

Planted vegetative filters are well known to be extremelyeffective in removing nutrients and pathogens fromwastewater(Ghermandi et al. 2007; Vymazal 2007; Snow et al. 2008;Cooper 2009; Kadlec 2009). The effectiveness of constructedwetland systems has been demonstrated for a variety of inor-ganic contaminants including sulfur, metals, and metalloids(Batty et al. 2008; Kadlec and Wallace 2009). These systemshave also shown the capacity for removing several organicpollutants such as petroleum hydrocarbons, chlorinated hydro-carbons, pesticides, and fertilizers (Huang et al. 2004; Kadlecand Wallace 2009; Vymazal 2011a). However, whereas nu-merous studies are focused on organic contaminant removalfrom agricultural or urban runoff (Schulz et al. 2003), only afew studies are directed to assess the capacity of CWs toremove pharmaceuticals and personal care products (PPCPs)(Gross et al. 2004). PPCPs are a class of compounds (emergingpollutants) that have raised great concern in recent years(Daugthon 2004). They have been detected in several aquatic

Responsible editor: Philippe Garrigues

C. Macci (*) : E. Peruzzi : S. Doni :G. MasciandaroNational Research Council – Institute of Ecosystem Study(CNR-ISE), 56124 Pisa, Italye-mail: cristina.macci@ise.cnr.it

R. IannelliDepartment of Engineering of Energy, Systems, Territory andConstructions (UNIPI-DESTEC), University of Pisa, Pisa, Italye-mail: r.iannelli@ing.unipi.it

Environ Sci Pollut ResDOI 10.1007/s11356-014-2949-x

environments (Heberer 2002) and effluents from wastewatertreatment plants (Ternes et al. 2004) in concentrations rangingfrom nanograms per liter to micrograms per liter. In a study onorganic micropollutant removal in a full-scale constructedwetland fed with secondary effluent, two PPCPs—carbamaz-epine (drug used in the treatment of epilepsy) and clofibric acid(the main metabolite of clofibrate, a lipid-lowering agent usedfor controlling cholesterol and triacylglycerol levels in theblood)—were removed at about 40 % (Matamoros et al.2008a). This removal rate is significantly higher than the valueof less than 30 % exhibited by most conventional wastewatertreatments (Gómez et al. 2007; Kosjek et al. 2007).Conversely, in another microcosm-scale CWexperiment, onlyminimal quantities of carbamazepine (about 5 %) were re-moved (Matamoros et al. 2008b). Since the removal of recal-citrant xenobiotics by natural treatments has been only partiallyexplored so far, further studies are needed to fully understandthe processes taking place in CWs for PPCP removal.

In CWs, contaminant removal is accomplished by diversemechanisms which include sedimentation/coagulation, filtra-tion, plant uptake/removal, adsorption (binding to sand parti-cles and roots), formation of solid compounds, cation ex-change, and microbial-mediated reactions, especially oxida-tion (Ganjo and Khwakaram 2010). The removal efficiencydepends on several factors: CW type (e.g., surface, subsur-face, vertical or horizontal flow; possible recirculation),wastewater loading quality and quantity (Sklarz et al. 2009),plant species and their combinations (Brisson and Chazarenc2009), climate, medium type, and plant management policies,such as harvesting regime.

By far, the most frequently used plant in CWs isPhragmites australis (common reed) (Vymazal 2011b).Species of the genera Typha (Typha latifolia, Typhaangustifolia, Typha domingensis, Typha orientalis, andTypha glauca) and Scirpus (e.g., Scirpus lacustris, Scirpusvalidus, Scirpus californicus, and Scirpus acutus) are alsocommonly used (Vymazal 2011b). Although these plant spe-cies have been extensively studied in CWs for wastewatertreatment, relatively little is known about the employ of orna-mental plants (Belmont and Metcalfe 2003; Zurita et al. 2006;Chen et al. 2009). In recent years, more attractive plants inseveral environmental applications have been increasinglyimplemented (Chen et al. 2009; Doni et al. 2013; Macciet al. 2013). A wider adoption of ornamental plants as analternative to macrophytes becomes possible when CWs arearranged in such a way as to avoid perennial medium satura-tion, such as in vertical flow constructed wetlands (Giraldi andIannelli 2009). Chen et al. (2009) analyzed the nutrient re-moval abilities of herbaceous perennial ornamentals (Cannaindica, Iris, Zantedeschia aethiopica, and Cyperus papyrusL.) in comparison to those of wetland plants (Peltandra,Pontederia L., and Sagittaria lancifolia L.). The resultsshowed that systems planted with canna removed more

nitrogen and phosphorous (98.7 % N and 91.8 % P) thanthose planted with iris and arrow arum (31.6 and 31.5 % N;38.5 % and 26.3 % P, respectively), and canna/pickerelweed-planted systems removed more nutrients than calla lily.

The aim of the present paper was to evaluate the removalefficiency of nutrients (N and P), heavy metals (Cu and Zn),carbamazepine, and linear alkylbenzene sulfonates (LAS),through the use of a subsurface vertical flow constructedwetland system. The following ornamental vegetal specieswere chosen as test plants: C. indica, Carex hirta,Miscanthus sinensis, and Z. aethiopica (calla). Besides theirpotential of contaminant removal, they were also selectedbecause they are aesthetically attractive and noninvasive. Inaddition, P. australis was chosen as reference species becauseof its universal use in wastewater treatment.

Materials and Methods

Experimental layout

The experiment consisted of 12 lysimeters (75 cm height and31 cm external diameter) located outdoors in the premises ofthe National Research Council, Institute of Ecosystem Studyin Pisa (Italy). Ten lysimeters (Fig. 1) were planted with thefollowing plants (two lysimeters per species): C. indica,

Plant species

Gravel (1 - 3 cm)

Water effluent

55

02

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75 Gravel (6 - 8 mm)

Fig. 1 Scheme of one lysimeter used in the experiment

Environ Sci Pollut Res

C. hirta,M. sinensis, P. australis, and Z. aethiopica, while twolysimeters were left without plant and used as control. Theplants were put in such a number to completely cover thelysimeter surface. The lysimeters were filled up with gravel(bottom layer 20 cm, 1–3 cm size, and top layer 55 cm, 6–8 mm size) and equipped with a valved drainage hose at thebottom for water collection.

The plants were irrigated with tap water for 2 weeks topromote rooting and adaptation. After this period, the lysim-eters were completely saturated with synthetic wastewater(28–33 L). The water was manually removed and replacedwith new synthetic water after 15, 30, and 60 days in winter-spring (W15d, W30d, W60d) (5 January to 20 April, 2012)and spring-summer (S15d, S30d, S60d) (21 April to 6 August,2012 ) treatment periods.

The wastewater collected from each of the two lysimetersper species was analyzed in the laboratory in triplicate, and theresults were reported as averages among triplicates and lysim-eters. The average daily temperature was 10.5 °C in winter-summer and 23.5 °C in spring-summer.

The synthetic wastewater was prepared using NH4NO3,(NH4)2HPO4, ZnSO4·7H2O, CuSO4·5H2O, carbamazepine,and LAS, with final concentrations of 280 mg N L−1, 30 mg PL−1, 3.6 mg Cu L−1, 9 mg Zn L−1, carbamazepine 5 μg L−1,and LAS 14 mg L−1.

At the end of the experiment, the plants were removed, androots and aerial parts (leaves and stems) were divided and driedseparately at 45 °C until constant weight was reached. Heavymetals were measured in dried roots and aerial parts, whileascorbate peroxidase activity was determined in fresh leaves.

Plants (two specimens per species) grown in pots filledwith 40 kg of a metal-free standard agronomic substrate wereused as controls. These control pots were kept in the sameenvironmental condition as that of the treated plants for thesame period.

Water effluent analysis

The following parameters were measured in the water sam-ples: pH by using a Titroprocessor 672 electrode (Metrohm;Herisau, Switzerland), N by using selective electrodes(SevenMulti, Mettler Toledo, Greifensee, Switzerland), P byusing a colorimetric method reported by Murphy and Riley(1962), and heavy metals (Cu and Zn) by inductively coupledplasma (ICP) (Liberty AX, Sequential ICP-OES; Varian, PaloAlto, CA, USA) after acidification with nitric acid.

LAS were detected with a high-performance liquid chro-matograph (HPLC) (Agilent 1,100 series, Santa Clara, CA,USA) with an ultraviolet diode array (λex=226 nm) andfluorescence detectors (λex=226 nm; λem=301 nm) (Santoset al. 2007).

To determine carbamazepine, water samples were acidifiedwith sodium formate-formic acid buffer to pH 3–3.5 to

enhance trapping of the acidic compounds on the solid-phase extraction (SPE) sorbent. Solid-phase extraction andgas chromatography were performed following the methodof Kosma et al. (2007), using Supelco (Bellefonte, PA) ENVI18 cartridge and GC-MS TRACE DSQ (Thermo FisherScientific, Austin, TX, USA).

Plant analysis

Plant tissues (roots and aerial parts) were analyzed for heavymetals (Cu and Zn) after they were dried at 45 °C untilconstant weight was reached. Samples were digested withhydrogen peroxide-nitric acid (H2O2/HNO3 1:3) in micro-wave, and the total heavy metals were determined by ICP(Liberty AX, Sequential ICP-OES; Varian).

Cu and Zn translocations from root to shoot were measuredand expressed by translocation factor (TF) defined as follows:

TF ¼ Cshoot Croot

where Cshoot and Croot are the metal concentrations in plantshoots (mg kg−1) and roots (mg kg−1), respectively.

Ascorbate peroxidase (APX) activity was assayed accord-ing to the method of Israr et al. (2006). The enzyme wasextracted from the leaves, after homogenizing the sample(1.0 g) in a prechilled mortar and pestle under ice-cold condi-tions in 5.0 mL of extraction buffer, containing 50 mM phos-phate buffer (pH 7.5), 1 % polyvinylpyrrolidone (PVP), 0.5 %Triton X-100, and 1 mM EDTA. The homogenate was centri-fuged at 12,000×g for 20 min at 4 °C. The supernatant wasadded to a reaction mixture (1 mL) containing 100 mM po-tassium phosphate buffer (pH 7.0), 0.5 mM ascorbate, and0.3 mM H2O2. The oxidation of ascorbic acid was measuredby absorbance decrease at 290 nm for 3 min. The enzymeactivity was calculated using the extinction coefficient2.8 mM−1 cm−1 and expressed in enzyme units (mg protein)−1.One unit of enzyme is the amount necessary to decompose1 μmol substrate min−1 at 25 °C.

Gravel analysis

The gravel was digested with hydrogen peroxide-nitric acid(H2O2/HNO3 1:3) in microwave, and Cu and Zn were deter-mined by ICP (Liberty AX, Sequential ICP-OES; Varian).

Statistical analysis

STATISTICA 7.0 software (StatSoft Inc., Tulsa, OK, USA)was used for data analysis. All results are means of twolysimeters; wastewater from each lysimeter was analyzed inthe laboratory in triplicate, and the results were reported asaverages among triplicates and lysimeters.

Environ Sci Pollut Res

Differences among sampling times (time effect), plants(species effect), and their interactions (time×species) weretested by analysis of variance (ANOVA). Significantly differ-ent values within time and species effects were reported intables as lower- and uppercase letters, respectively; the inter-action effect was listed at the end of each table. For plantanalysis, significant differences between each treated plantand the corresponding control plant (treatment effect) andbetween different plant species (species effect) were reportedin Fig. 4 as lower- and uppercase letters, respectively. Meanswere compared using honestly significant difference (HSD)Tukey’s test (P<0.05).

Moreover, a correlation matrix of the data was also calcu-lated in order to determine the relationship between the pa-rameters (table not reported).

Results and discussion

Water effluent quality

Nutrients

Water effluent showed a similar trend for nutrient concentra-tions (N and P), with significant differences between the twoexperimental seasons (Table 1). In the winter-spring period, aslight variability in nutrient concentration was observed indifferent incubation times (15, 30, and 60 days), while a cleardecrease in N and P content with the increase of incubationtime was found in the spring-summer cycle (Table 1). Totalremoval of N in 60 incubation days was observed in all planttreatments with the only exception of M. sinensis.

As expected, in agreement with previous papers (Picardet al. 2005; Salvato and Borin 2010), the highest nutrientremoval rates were observed during the spring-summer grow-ing season (Table 1). Moreover, the greater reduction in N inthe last two sampling times suggested a positive correlationbetween N removal and temperature. Similar trends have beensuggested by Picard et al. (2005), Akratos and Tsihrintzis(2007), and Hijosa-Valsero et al. (2011).

Low values of P and N were detected after 15 days, whichwere lower than those measured at 30 days and could beattributed to the initial nutrient deficiency period (water-onlyperiod), which stimulated a rapid N adsorption by the plants.Among the tested plants, M. sinensis and Z. aethiopica wereparticularly efficient in P reduction, showing a removal of74 % in the whole monitored period (average data of twoseasons). In the same period, P reduction exhibited by theother plant systems ranged between 63 and 69 % (Table 2).Regarding N removal, a similar efficiency (around 65–67 %)was found for all plants, expect M. sinensis, which wasslightly less effective (62 %).

On the other hand, the highest concentrations of N and Pwere found, as expected, in the discharge water of the controlsystem at all sampling times (Table 1). At the end of theexperiment, a N reduction of about 33 % was found in theunplanted controls, in agreement with Salvato and Borin(2010) who found an abatement of 24%. According to severalauthors (Kadlec and Wallace 2009; Bastviken et al. 2009;Kantawanichkul et al. 2009; Salvato et al. 2012), this resultdemonstrates the importance of vegetation in removing Nfrom wastewater since, considering the total period, theplanted treatments removed about two times (66 %) more Nthan the unplanted control (33 %).

Adsorption and precipitation phenomena associated withthe gravel substrate should also be taken into account, inparticular for P, which, as phosphate, is able to bind calciumcarbonate derived from the gravel medium (calcareous ori-gin). The results of the first sampling times (W15d andW30d)suggested that the greatest reduction in P was due to the gravelaction, the concentration in the control being similar to thatfound in the planted systems. Moreover, the decrease in waterP content in the last two sampling times could be due to Pprecipitation with pH reduction (from 8 to 7), as alreadyobserved (Zanini et al. 1998; Song et al. 2002; Serrano et al.2003; Peng et al. 2007).

Inorganic contaminants

Avery low amount of heavy metals (Cu and Zn), in particularCu, was measured in the discharged water of all treatmentsand sampling times, including the control (Table 1). Thisfinding suggests that gravel adsorption is probably the maincause of the great reduction of heavy metal concentrationsobserved in the experimental systems. Considering theamount of metals detected in the wastewater (Table 1) andthat adsorbed by the plants (Fig. 4), it could be assumed thatgravel contributed to metal removal for about 96–99 %. Somestudies have already hypothesized the capability of mineralsubstrates (gravel and sand) in heavy metal adsorption (Genç-Fuhrman et al. 2007; Iannelli et al. 2011; Oluyemi 2013;Norris et al. 2013); however, further studies are needed tobetter understand the gravel absorption mechanisms takingplace in these plant-gravel systems. A preliminary attempt tocorroborate the hypothesis of gravel absorption was made inthis study by measuring the concentration of metals in thegravel at the end of the experimental period. Not surprisingly,mean concentrations of 13 and 30 mg kg−1 were found for Cuand Zn, respectively.

However, although the total metal reduction was verysimilar in all planted systems, C. indica seemed to be the mosteffective in removing both metals, followed by C. hirta,M. sinensis, and P. australis. Z. aethiopica was the leastefficient in the reduction of both metals.

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Organic contaminants

The results related to the organic pollutants are more interest-ing, since only in the most recent years has the capacity ofCWs to remove PPCPs been investigated, and very few stud-ies have been carried out using ornamental plants of highaesthetical value.

In this study, all the species were able to partially removecarbamazepine, showing a percentage of reduction rangingfrom 25 to 61% (Table 2). These results are in agreement withthose ofMatamoros et al. (2008a), who reported a reduction of30–47 %. However, in this study, the authors classified car-bamazepine as a poorly removed compound, its eliminationrate being lower than 50 %. In addition, when comparing

Table 1 Nutrient and inorganic contaminant concentrations in water effluents of test species and various sampling times

Can Car Mis Phr Zan Con

pH

W15d 7.30b C 7.21ab C 7.67a B 7.29b C 7.21b C 8.18a A

W30d 7.35ab B 7.19b B 7.40b B 7.50a B 7.39ab B 8.13a A

W60d 7.51a BC 7.42a C 7.74a B 7.34ab C 7.45a C 8.12a A

S15d 7.51a B 7.16b C 7.60ab B 7.35ab C 7.46a B 8.10a A

S30d 7.52a B 7.34a B 7.52ab B 7.57a B 7.55a B 7.85b A

S60d 7.34ab A 7.34a A 7.44b A 7.30b A 7.46a A 7.35c A

N mg L−1

W15d 147a B 105c C 106c C 100b C 105c C 196b A

W30d 150a B 152b B 166b B 131a B 141b B 217b A

W60d 84.6b D 141b CB 148b B 176a B 136b C 226ab A

S15d 150a C 194a B 232a A 155a C 200a B 254a A

S30d 61.4c C 102c B 68.8d C 86.8b CB 61.1d C 186b A

S60d 0.00d B 0.00d B 15.5e B 0.00c B 0.00e B 153b A

P mg L−1

W15d 14.3b A 12.7b AB 9.90b B 13.7b A 9.80b B 12.1c AB

W30d 19.9a A 18.9a A 16.3a B 19.7a A 15.7a B 18.8a A

W60d 14.1b B 10.4c C 9.20b CD 19.0a A 7.90b D 18.4a A

S15d 14.7b A 12.9b AB 12.4b B 15.9ab A 9.3bc C 14.6b A

S30d 8.27c AB 9.17c A 4.99c C 8.01c AB 6.80c B 10.6c A

S60d 0.00d C 4.17d B 1.00d C 2.27d BC 1.22d C 7.55d A

Zn mg L−1

W15d 0.072b C 0.061b CD 0.026d D 0.241a B 0.207d B 0.955a A

W30d 0.182a B 0.022c C 0.000d C 0.226a A 0.181b B 0.169c B

W60d 0.010c A 0.000c A 0.007d A 0.000d A 0.000e A 0.010f B

S15d 0.070b AB 0.083b A 0.066c B 0.044c C 0.093d A 0.047e C

S30d 0.092b D 0.173a B 0.137b C 0.095b D 0.248a A 0.073d D

S60d 0.026c E 0.085b D 0.305a B 0.049c D 0.147c C 0.772b A

Cu mg L−1

W15d 0.023bc B 0.014d B 0.022c B 0.028c B 0.023d B 0.046c A

W30d 0.019c D 0.001e E 0.000d E 0.105a A 0.089b B 0.052c C

W60d 0.000d B 0.000e B 0.000d B 0.000d B 0.000d B 0.021d A

S15d 0.065a D 0.042c E 0.091a C 0.029c E 0.127a B 0.141a A

S30d 0.034b D 0.160a A 0.062b C 0.026c D 0.061c C 0.093b B

S60d 0.012c E 0.061b D 0.092a B 0.046b D 0.082b B 0.106b A

Different lowercase letters indicate statistically different values for each plant species within time according to HSD Tukey’s test (P<0.05). Differentuppercase letters indicate statistically different values for each time within plant species according to HSD Tukey’s test (P<0.05). The interaction effect(species×time) is significant (P<0.001) for each parameterCan, Canna indica; Car, Carex hirta; Mis, Miscanthus sinensis; Phr, Phragmites australis; Zan, Zantedeschia aethiopica; W, winter-spring period, S,spring-summer period

Environ Sci Pollut Res

planted systems with the unplanted control, it is reasonable toassume that the presence of plants was essential in carbamaz-epine degradation (Dordio et al. 2010), since the controlexhibited a total reduction of only 8 % (Table 2).

Plants can contribute directly or indirectly to pollutantremoval in CWs. Direct processes include uptake (whichcan lead to accumulation, degradation, or phytovolatilization),adsorption, and release of plant exudates at the rhizospherelevel. Indirect processes are related to biofilm growth aroundroots and to oxygen pumping toward the rhizosphere, thuschanging physicochemical parameters inside the constructedwetland.

Among the test plants, P. australis and C. indica have beenable to better degrade carbamazepine, in particular in thewinter-spring period, while the other plants showed an appre-ciable degradation only in the longest incubation time (W60d)(Fig. 2). These results suggested a greater activity ofP. australis and C. indica (root and associated microorgan-isms) than the other species in the quiescent season. Indeed,considering the whole monitored period, the removal efficien-cy was 51 % for C. indica and 61 % for P. australis. Z.aethiopica, which showed one of the best performances innutrient removal, was the plant with the lowest carbamazepineabatement, only 25 %, less than the values for P. australis andC. indica (Table 2).

In addition, as also found by Matamoros et al. (2008a) andDordio et al. (2010), a seasonal trend of carbamazepine re-moval was observed, with a higher reduction in summer(46 %) than in winter (40 %). Also, the retention time was afactor of great importance in carbamazepine reduction; ahigher degradation was, in fact, observed for each plant after60 days in both seasons (Fig. 2).

The phenomenon of gravel adsorption seemed not to affectcarbamazepine since its concentrations in water dischargedfrom the control at all sampling times were coherent with theadded amounts. Also, for LAS degradation, P. australis turnedout to be the most effective species, reaching a total removalof 78 %, followed by C. hirta, C. indica, andM. sinensis witha reduction of 72–73 % (Table 2). For this organic pollutant,

the worst performance was again observed in Z. aethiopica(60 %). The present findings were similar to the work ofHuang et al. (2004) who found a biodegradation of LAS ofup to 71 %.

A greater reduction was already observed after 30 days forLAS than for carbamazepine although with few significantdifferences between 30 and 60 days, in particular in winter(Fig. 3). This result was probably due to the easier degrad-ability of these compounds, in comparison to the more recal-citrant carbamazepine. Gravel absorption seemed not to par-ticipate in LAS removal (similar to carbamazepine), as shownby the high concentration measured in water discharged by thecontrol at all sampling times.

Plant adaptation and action

All test plants used in the experimentation were successfullyadapted to the treatment, as evidenced by the regular growthand good healthy state (e.g., C. indica also flowered). At theend of the experimental period, plants were removed andanalyzed for metal content in their roots and aerial parts(leaves and stems). The concentrations were compared tothose measured in the control plants (Fig. 4).

Cu concentration in the roots was significantly higher intreated plants than in control plants, with the only exception ofZ. aethiopica, thus suggesting a certain efficiency of all butthis species in accumulating Cu. In Z. aethiopica, the concen-trations in the experimental and control plants were not sig-nificantly different. Among all plants, only M. sinensis andP. australis were able to translocate Cu to their aerial parts,even though the translocation factors were only 0.11 and 0.08,respectively (data not shown).

The highest Cu content was found in the tissues ofC. hirta,followed by P. australis andC. indica, showing a trend similarto that found in the discharged water, even though, as reportedabove, few and often nonsignificant differences were mea-sured in the effluent.

The more evident result was the inefficiency ofZ. aethiopica in the absorption of heavy metals, since alsothe Zn concentration in the roots and aerial parts of theexperimental plant was not significantly different from thecorresponding control. This result was in agreement with thehigh concentrations of metals generally found in the waterdischarged by Z. aethiopica (Table 1).

P. australis and C. hirta showed also the highest concen-trations of Zn in their roots, 19- and 11-fold than in the controlplants, while C. indica and M. sinensis tended to translocateZn to the aerial part, as suggested by the very high transloca-tion factors of 0.9 and 0.6, respectively (data not shown). Atranslocation factor of 0.6 was also observed for P. australisthat, in agreement with Cu results, can be surely considered aneffective plant in heavy metal translocation.

Table 2 Total percentage of reduction of nutrients and contaminants inthe treatments

N (%) P (%) Zn (%) Cu (%) LAS Carbamazepine

Can 67.4 63.5 99.2 99.3 71.6 51.0

Car 66.5 69.0 99.3 98.9 73.5 41.2

Mis 61.7 73.7 99.1 98.9 71.5 37.3

Phr 67.3 63.1 98.4 99.1 78.3 61.4

Zan 65.1 74.4 98.8 98.5 60.3 24.9

Cont 32.7 58.0 96.6 98.1 0.00 8.29

Can, Canna indica; Car, Carex hirta; Mis, Miscanthus sinensis; Phr,Phragmites australis; Zan, Zantedeschia aethiopica; W, winter-springperiod, S, spring-summer period

Environ Sci Pollut Res

However, the total amount of metals in the plants (rootsplus aerial parts) was quite insignificant (ranging from 1.6 to9.6 mg for Cu and from 4 to 14.7 mg for Zn) when comparedto the total amount added during the whole experimentalperiod (an average of 628 mg of Cu and 1,569 mg of Zn).P. australis and C. hirta showed the highest content of boththe investigated metals followed by C. indica. Therefore, thecontribution of the plants to metal removal was scarce in alltreatments (0.02–1.50 %), thus confirming the already de-scribed finding that the main role in metal removal was playedby the gravel substrate.

As described before, all plants were in healthy conditionsduring the whole experimental period. However, to investigatemore deeply the influence of contaminant load on the physiology

and biochemistry of plants, ascorbate peroxidase test was carriedout on the leaves of experimental and control plants.

Pollutants induce the formation of an excess of reactiveoxygen intermediates (ROIs) which have the capacity to dam-age biomolecules (lipids, proteins, DNA) and can ultimatelycause cell death (Mittler 2002). Plant tolerance to pollutants isrelated to their capacity to cope with ROI production by theactivation of enzymatic systems capable of scavenging andeliminating them. Among these, the widely studied ascorbateperoxidases (Mittler 2002; Masood et al. 2012) catalyze thereduction of hydrogen peroxide into water using ascorbic acidas a donor of electrons.

This enzyme activity increased in all treated plants whencompared to the control (Fig. 4), with the exception of

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Fig. 2 Concentration of carbamazepine in water effluents of the differentplant systems and sampling times. Different lowercase letters indicatestatistically different values for each plant species within time accordingto HSD Tukey’s test (P<0.05). Different uppercase letters indicate sta-tistically different values for each time within plant species according to

HSD Tukey’s test (P<0.05). The interaction effect (species×time) issignificant (P<0.001). Can, Canna indica; Car, Carex hirta; Mis,Miscanthus sinensis; Phr, Phragmites australis; Zan, Zantedeschiaaethiopica; W winter-spring period, S spring-summer period

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Fig. 3 Concentration of LAS in water effluents of the different plantsystems and sampling times. Different lowercase letters indicate statisti-cally different values for each plant species within time according to HSDTukey’s test (P<0.05). Different uppercase letters indicate statisticallydifferent values for each time within plant species according to HSD

Tukey’s test (P<0.05). The interaction effect (species×time) is significant(P<0.001). Can, Canna indica; Car, Carex hirta; Mis, Miscanthussinensis; Phr, Phragmites australis; Zan, Zantedeschia aethiopica; Wwinter-spring period, S spring-summer period

Environ Sci Pollut Res

Z. aethiopica, indicating that the contaminant load induced anoxidative stress (Juszczuk et al. 2001; Malusà et al. 2002)through higher concentrations of H2O2 in the tissues, leadingto an increase in enzyme activity in order to diminish theintracellular level of H2O2 (Dordio et al. 2009). Themaximum increase in ascorbate peroxidase activity was ob-served for C. indica (18-fold), followed by M. sinensis,C. hirta, and P. australis (4–7-fold), while Z. aethiopicaseemed not to be stressed by the pollutant, as it showedinsignificant differences among the experimental plants andcontrols.

A great positive correlation was found between this en-zyme activity and the concentrations of metals in the aerialparts of all tested plants (R2=0.7642). This finding suggests agreat sensibility of these plants to the metal concentration, asconfirmed by the fact that Z. aethiopicawas the only plant thatshowed no adsorption of metals in its tissue (Fig. 1). Also,Zurita et al. (2009) observed the capability of this plant toadapt well to negative environmental conditions due to the

presence of contaminants, not showing any stress. Also, thedegradation of organic compounds such as carbamazepineseemed to induce stress in all the experiments. In fact, aninteresting correlation was found between the ascorbate per-oxidase enzyme and the quantity of carbamazepine degraded(total mg added minus total mg in discharged waters)(R2=0.753). This finding indicates that all the tested plants(with the exception of Z. aethiopica) are effective in carba-mazepine removal, even though inducing a stress condition asshown by the synthesis of the ascorbate peroxidase enzyme(Mittler 2002; Masood et al. 2012).

Conclusions

The present study provides evidence that constructed wetlandswith different high-value ornamental plants can be used effec-tively for removal of nutrients (N and P), heavy metals (Cuand Zn), carbamazepine, and LAS in wastewater. N and P

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Fig. 4 Concentration of metals (root and aerial parts) and ascorbateperoxidases (leaves) in the different experimental and control plants.Different lowercase letters indicate statistically different values betweeneach treated plant and its control plant (treatment effect) according toHSD Tukey’s test (P<0.05). Different uppercase letters indicate

statistically different values within plant species (species effect) accordingtoHSDTukey’s test (P<0.05).Can,Canna indica;Car,Carex hirta;Mis,Miscanthus sinensis; Phr, Phragmites australis; Zan, Zantedeschiaaethiopica

Environ Sci Pollut Res

decreased greatly in all vegetated systems both for plantactivity and gravel adsorption, with the highest removal ratesin the spring-summer growing season.

The heavy metals were almost completely removed by allthe experimental systems, irrespective of the presence/absenceand type of plants. Among the plants, C. hirta, C. indica, andPhragmites seemed to be the most efficient in removing bothmetals, as suggested by the highest metal content in theirtissues, while the Z. aethiopica system was clearly the leastefficient.

However, the results suggested that the main removalaction was due to a phenomenon of gravel adsorption and thatfurther in-depth studies are needed to fully understand thisprocess. Different than for inorganic compounds, the organiccontaminant removal in the discharged water was primarilydue to plant activities. C. indica and P. australis had thehighest reduction efficiency for both carbamazepine andLAS, while Z. aethiopica was still the least effective plant.

In addition, the warmer season (spring-summer period) andthe longer incubation time (60 days) clearly favored nutrientand contaminant removal. Nevertheless, the capability of the-se plants to cope with the contaminants involved an expendi-ture of energy which led to the synthesis of ascorbate perox-idase which is an enzyme produced in situations of oxidativestress. The greatest enzyme activity was, in fact, measured inthe most active plant in pollutant removal. Conversely,Z. aethiopica, the least efficient plant in metal and organiccompound removal, turned out to be the least stressed plant.

In conclusion, in terms of removal efficiency of the ex-plored contaminants, all tested plants, with the exception ofZ. aethiopica, could replace P. australis in constructed wet-land systems, reaching the same or better depuration efficien-cy and having a higher aesthetical value.

Acknowledgements The study was carried out within the frameworkof a project financed by San Giuliano Terme Municipality, Pisa (Italy).The authors would like to thank the graduating student Guido Cioli andthe technician Virginia Giansoldati for their work in the execution of theexperiments.

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