Chemosphere 72 (2008) 1823–1828

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Chemosphere

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Technical Note

Interactive effects of nitrogen and phosphorus loadings on nutrient removalfrom simulated wastewater using Schoenoplectus validus in wetland microcosms

Zhenhua Zhang a, Zed Rengel a,*, Kathy Meney b

a Soil Science and Plant Nutrition, School of Earth and Geographic Sciences, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australiab Syrinx Environmental Pty Ltd, 12 Monger Street, Perth WA 6000, Australia

a r t i c l e i n f o

Article history:Received 31 October 2007Received in revised form 6 May 2008Accepted 6 May 2008Available online 17 June 2008

Keywords:Constructed wetlandEutrophicationLoading rateN and P interactionSchoenoplectus validusWastewater

0045-6535/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2008.05.014

* Corresponding author. Tel.: +61 8 6488 2557; faxE-mail address: zed.rengel@uwa.edu.au (Z. Rengel

a b s t r a c t

The concentrations of nutrients (N and P) in the wastewater and loading rate to the constructed wetlandsmay influence the nutrient removal from the secondary-treated municipal wastewater using wetlandplants. Three loading rates of N (low 5.7, medium 34.3 and high 103 mg N d�1) and two of P (low 3.4and high 17.1 mg P d�1) were studied in simulated secondary-treated municipal wastewater usingSchoenoplectus validus (Vahl) A. Löve & D. Löve in the vertical free surface-flow wetland microcosms. After70-d growth, there were significant interactive effects of N and P on the total, above-ground and root bio-mass. The below-ground biomass (rhizome and root) was negatively affected by the high N treatment.The tissue concentrations of N increased with an increase in N additions and decreased with an increasein P applications, whereas the tissue concentrations of P increased with an increase in P additions anddecreased with an increase in N applications at the low P treatment, but increased at the high P treat-ment. Significant interactive effects of N and P loadings were found for the removal efficiencies of NH4

and P, but not that of NOx. The plant uptake, substrate storage and other losses (e.g. denitrification andformation of organic film) had similar contribution to N removal when N loading was relatively low.The P storage by substrate was the main contribution to P removal when P loading was high, but plantuptake was the major factor responsible for P removal when P loading was low and N loading was high.The high nutrient availability and optimum ratio of N:P are required to stimulate growth of S. validus,resulting in preferential allocation of resources to the above-ground tissues and enhancing the nutrientremoval efficiencies, but the high N concentration in wastewater may hamper the growth of S. validus inconstructed wetlands.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

High nutrient inputs and eutrophication continue to be one ofthe most important water quality problems in many water bodies.N and P, the primary nutrients implicated in eutrophication, enterwater bodies via a variety of pathways. Sewage pollution of coastalrivers, estuaries, and near-shore waters has been implicated ineutrophication around the world, e.g. in Australia (Davis and Koop,2006) because little sewage effluent is reclaimed and reused; in-stead, secondary-treated effluent is discharged into rivers, estuar-ies or oceans (Greenway, 2005).

The use of constructed wetlands for purification of secondary-treated wastewater has expanded since the 1990s (Kadlec andKnight, 1996). However, a wide range of nutrient removal efficien-cies in constructed wetlands was reported, with many wetlandsfailing to meet relevant government standards, especially for totalN and P concentrations in discharged wastewater (Fraser et al.,

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: +61 8 6488 1050.).

2004; Greenway, 2005). Variation among studies may be influ-enced by differences in vegetation type and density, media, reten-tion times, loading rates, temperature, design and size of thesystems.

Macrophytes are assumed to be the main biological componentof wetlands. They not only take up nutrients from wastewater andsubstrates directly, but also act as catalysts for purification reac-tions by increasing the environmental diversity in the rhizosphere,and promoting a variety of chemical and biological reactions thatenhance purification in constructed wetlands (Jenssen et al.,1993). N and P are key nutrients in the life cycles of wetland plants.However, the concentrations of N and P in the wastewater andloading rates to the constructed wetlands vary depending on thesource of wastewater, time of year and the type of wastewatertreatment facility. These changes to nutrient availability could af-fect plant growth responses and resource allocation, and possiblyinfluence nutrient removal efficiency in constructed wetlands(Tanner, 2001; Zhang et al., 2007c).

Although there is considerable information on plant produc-tivity, biomass and nutrient dynamics in natural and fertilized

1824 Z. Zhang et al. / Chemosphere 72 (2008) 1823–1828

wetlands, most studies on constructed wetlands receiving waste-waters have only addressed general aspects of plant growth andnutrient content (Tanner, 2001). There is a lack of knowledge onthe influence of various N and P loadings on the plant growthand nutrient removal efficiencies in constructed wetlands. Schoen-oplectus validus (Vahl) A. Löve & D. Löve and closely related speciesSchoenoplectus lacustris, Schoenoplectus acutus and Schoenoplectuscalifornicus have been used widely in constructed wetlands aroundthe world (Tanner, 2001). The objective of this study was to inves-tigate the interactive effects of N and P loadings on growth of S. val-idus and nutrient removal from simulated secondary-treatedmunicipal wastewater in the vertical free surface-flow (VFS) wet-land microcosms.

2. Materials and methods

2.1. Experimental setup

The experiment was established in a VFS wetland microcosm,and conducted in a phytotron chamber at the University of Wes-tern Australia (31�580 S, 115�490 E) with controlled day/night tem-peratures of 25/20 �C under natural light conditions from late Mayto July. The details of the microcosm design were described inZhang et al. (2007a,b).

In order to minimise variability in the experiment, a simulatednutrient solution was used in the microcosms, with the averagenutrient loadings mimicking the effluent from the secondary-trea-ted municipal wastewater at the wastewater treatment plants inPerth, Western Australia (Water Corporation of Western Australia,2000). Three loading rates of N (low 5.7, medium 34.3 and high103 mg N d�1 with N ratio of 1:1 NH4:NO3) and two of P (low3.4 and high 17.1 mg P d�1) were tested in the wetland micro-cosms with three replicates. Other macro- and micro-nutrients(mg L�1): 25 K, 10 Ca, 5 Mg, 7 S, 0.04 Zn, 0.01 Cu, 0.34 Fe, 0.05Mn, 0.26 B and 0.05 Mo were kept at the same level in all thetreatments. For potassium, part of this amount was provided bythe compounds used to supply P (as KH2PO4), and the rest by add-ing K2SO4.

Seedlings of S. validus were obtained from the local plant nurs-ery, and 6 healthy plants of similar size (3–4 shoots approximately0.1 m in length, dry weight 57 mg plant�1) were transplanted intoeach microcosm. Microcosms were filled with the treatment nutri-ent solution to achieve a water depth of 0.02 m above the sand sur-face; and the solution was renewed weekly. After 2 weekacclimation, microcosms were filled with 8 L nutrient solution ofeach treatment to achieve a water depth of about 0.05 m abovethe sand surface. Each microcosm was drained and refilled weekly.

Table 1Effects of N and P treatments on biomass (g m�2) of different parts of S. validus after70 days of growth in the wetland microcosms

Treatment Whole plant Above-ground Below-ground Rhizome Root

Low PLow N 108d 55c 52c 23b 30cMedium N 422bc 296b 126ab 76a 50bHigh N 345c 247b 98bc 58ab 40bc

High PLow N 112d 58c 53c 22b 31cMedium N 483ab 309b 175a 92a 82aHigh N 581a 455a 126ab 89a 37bc

Means with different letters within columns are significantly different based on LSD(P < 0.05).

2.2. Sampling and measurements

After nutrient treatments were imposed, volumes of influentand effluent solution were recorded, and samples were collectedonce per week. Influent samples were taken immediately beforethe microcosms were filled with simulated wastewater. Effluentsamples were taken immediately after draining the solution, andthe pH and dissolved oxygen (DO) were measured. Samples werefrozen before analysis of nutrients.

Plants were harvested after 10 week of nutrient treatments.Shoots were cut at the sand surface and their base washed to re-move any adhering sediments. Samples of sand were taken fromeach microcosm for nutrient analysis. Each microcosm was thenexcavated and plants were separated into above-ground (includingculms, leaves and inflorescences) and below-ground portions (rhi-zomes and roots). Roots were separated from sand under tap waterand collected onto a mesh. All plant samples were dried to a con-

stant weight at 70 �C for 5 d in a forced-air cabinet, weighed andground to pass a 0.75 mm mesh.

The details of the methods for pH and DO measurements inwater, N and P determinations in water, plant and sand, and thenutrient removal efficiency calculation were described in Zhanget al. (2007a,b). PO4�P in water was determined using the ascorbicacid method (APHA, 1998).

The mass balance approach was used to partition added N andP. The factors considered were (1) amounts added to and exportedfrom the microcosms; (2) amounts accumulated by the macro-phytes and stored (e.g. adsorbed, precipitated) in the substrate(the difference in amounts determined in plants or substrate atthe beginning and the end of treatments); and (3) unaccountedfor (e.g. losses due to denitrification and periphyton growth).

2.3. Statistics

Statistical tests were performed using statistical package for thesocial sciences (SPSS) version 15. Percentage nutrient removal val-ues were normalized using an arcsine transformation. Measure-ments of each parameter in water samples at different samplingdate were not independent; therefore, difference in pH, concentra-tion of DO, and nutrient removal efficiency were tested using a re-peated measures two-way analysis of variance (ANOVA). Thesampling date was set as the repeated measure. Two-way ANOVAwas used to determine significance of nutrient effects on biomassand total contents of N and P in various components of the micro-cosms. Least significant difference (LSD) was applied to test for sig-nificance between treatment means.

3. Results

3.1. Biomass

The total, above-ground and root biomass was significantly af-fected by interactions of N and P treatments, whereas the below-ground and rhizome biomass was significantly affected by N treat-ments only, with no significant interaction detected (Table 1). Thehighest total biomass (581 g m�2) was in the high N-high P treat-ment and the lowest (108 g m�2) in the low N-low P treatment.The biomass of below-ground tissues (rhizome and root) was neg-atively influenced by the high N treatment. The above-ground bio-mass was lower in the high N-low P (345 g m�2) than medium N-low P treatment (422 g m�2).

3.2. Concentrations of N and P in plant tissues

There was a significant interaction between N and P treatmentsfor the tissue concentrations of N and P. In general, the highestconcentrations of N and P were in shoots. The highest concentra-

Table 3Nutrient removal efficiency (%) influenced by N and P treatments in wetlandmicrocosms planted with S. validus

Treatment Nutrient removal efficiency (%)

NH4 NOx PO4�P

Low PLow N 97 ± 1ab 97 ± 1a 74 ± 3aMedium N 92 ± 3b 66 ± 7b 80 ± 4aHigh N 59 ± 3d 21 ± 6c 77 ± 4a

High PLow N 99 ± 0ab 99 ± 1a 48 ± 2bMedium N 99 ± 1a 86 ± 8a 71 ± 2aHigh N 72 ± 2c 31 ± 3c 76 ± 3a

Means (±SE, n = 24) with different letters within columns are significantly differentbased on LSD (P < 0.05).

-40

-20

0

20

40

60

80

100

NO

x rem

oval

effi

ency

(%)

0

20

40

60

80

100

NH

4 rem

oval

effi

cien

cy(%

)Low N-low P Medium N-low P High N-low PLow N-high P Medium N-high P High N-high P

0

20

40

60

80

100

10

Week after planting

PO4-

P re

mov

al e

ffici

ency

(%)

2 3 4 5 6 7 8 9 10

Fig. 1. Removal efficiencies of NH4, NOx and PO4�P influenced by duration and typeof treatments in wetland microcosms planted with S. validus.

Z. Zhang et al. / Chemosphere 72 (2008) 1823–1828 1825

tion of N occurred in the high N-low P treatment, whereas thehighest concentration of P occurred in the high N-high P treatment.The N/P ratios increased significantly with the increasing N and de-creased with the increasing P supply (Table 2).

3.3. Nutrient removal efficiency

There was a significant interactive effect of N and P loadings onthe removal efficiencies for NH4 and PO4�P, but not for NOx (Table3). The lowest removal efficiency for NH4 (59%) and NOx (21%) wasin the high N-low P treatment, and for P (48%) in the low N-high Ptreatment. The removal efficiency was relatively higher for NH4

than NOx, except in the low N treatments. Negative removal ofNOx was observed in the high N-low P treatment at the last sam-pling (week 10) (Fig. 1). The lowest PO4�P removal efficiency wason week 7 (except in high N-high P) (Fig. 1). The significant(P < 0.001) differences over time indicated the removal efficiencieswere influenced by plant growth and/or other factors in themicrocosms.

3.4. pH and DO in effluents

The significant interactive effects of N and P loadings werefound for DO concentration in the effluents, but not for pH (Table4). In general, pH and DO concentrations decreased with an in-crease in N additions. The highest effluent pH (6.0) and lowestDO (2.6 mg L�1) were in the medium N-high P treatment, whereasthe lowest pH (3.8) was in the high N-high P and the highest DO(4.7 mg L�1) in the low N-low P treatment.

3.5. Nutrient partitioning in the water-substrate-plant continuum

3.5.1. N and P in the effluentsThe recovery percentages of N in the effluents increased with an

increase in N applications and decreased with P additions, whereasthat of P decreased with an increase in N applications and in-creased with an increase in P additions (except in the mediumN-low P treatment). The recovery of N in the effluent was relativelyhigh (47%) in the high N-low P treatment, whereas that of P wasrelatively high (30%) in the low N-high P treatment (Table 5).

3.5.2. N and P uptake by plantsThe greatest N (42%) and P (50%) removal by plants was in the

medium N-low P treatment, whereas the lowest plant removals ofN (30%) and P (4%) were in the high N-high P and low N-high Ptreatments, respectively (Table 5).

3.5.3. N and P in the substrateSignificant differences in the substrate content were found for

total P but not N among treatments. Total P in the substrate was

Table 2The concentrations (g kg�1) of N and P, and N/P ratios in the various plant tissues of S. validus influenced by N and P treatments after 70 days of growth in the wetland microcosms

Element Tissue Low P High P

Low N Medium N High N Low N Medium N High N

N Shoot 12.4e 21.7c 32.2a 10.9e 15.5d 29.2bRhizome 6.9d 16.0b 29.6a 6.4d 10.1c 27.0aRoot 7.3d 13.7b 21.5a 7.1d 10.0c 22.1a

P Shoot 3.2b 2.3c 2.1c 3.9b 3.6b 4.5aRhizome 2.9c 2.2d 2.1d 3.4bc 3.7ab 4.3aRoot 1.6c 1.6c 1.7c 2.1c 2.9b 3.5a

N/P Shoot 3.8d 9.4b 15.5a 2.8e 4.3d 6.5cRhizome 2.4d 7.4b 14.2a 1.9d 2.7d 6.3cRoot 4.6d 8.6b 12.9a 3.5e 3.4e 6.3c

Means with different letters within rows are significantly different based on LSD (P < 0.05).

Table 4The pH and concentration of dissolved oxygen (DO) (mg L�1) in the effluentsinfluenced by N and P treatments in wetland microcosms planted with S. validus

Treatment pH DO

Low PLow N 5.7ab 4.7aMedium N 5.5b 4.1aHigh N 3.9c 3.6b

High PLow N 5.9a 4.2aMedium N 6.0a 2.6cHigh N 3.8c 3.9b

Values with different letters within columns are significantly different based on LSD(P < 0.05).

Table 5Nitrogen and P mass balance (% of input) influenced by N and P treatments in wetlandmicrocosms planted with S. validus

Treatment Effluent Plant uptake Soil storage Other losses

N P N P N P N P

Low PLow N 3 20 35 16 30 54 32 10Medium N 15 15 42 50 4 25 39 10High N 47 17 19 39 1 39 33 6

High PLow N 1 39 32 4 31 53 35 4Medium N 3 22 34 19 4 54 59 5High N 38 18 30 28 1 45 31 9

1826 Z. Zhang et al. / Chemosphere 72 (2008) 1823–1828

significantly higher in the high P (0.0458 g kg�1) than in the low P(0.0316 g kg�1) treatments. P storage by substrate (25–54%) wasrelatively high compared with N (1–31%). P storage by substratewas higher than P uptake by plants in the low N-low P and all highP treatments (Table 5).

4. Discussion

4.1. Plant growth

The significantly interactive effects of N and P loadings on plantgrowth were observed in the present study (Table 1). Similarly, theinteractive effects of N and P on growth of Canna indica have beenobserved (Zhang et al., 2008). The interaction between N (NO3) andP supply strongly affected growth of Typha latifolia, Typha angusti-folia, Sparganium eurycarpum and Phragmites australis in the sandculture experiments (Ulrich and Burton, 1988). However, thatinteractive effect between N and P supply was not observed forP. australis (Romero et al., 1999) and Typha orientalis (Cary andWeerts, 1984) in the hydroponic culture experiments. The differ-ences may caused by different nutritional conditions such as thetotal amount of added nutrients, the N/P ratios, the proportionsof NH4 versus NO3, and the presence or absence of organic matterin the substrate. The simple comparison of growth reactions ofwetland plants under nutrient treatments in different studies isdifficult (Vojtíšková et al., 2004).

Although the significant difference was not observed betweenmedium N and high N treatments (except for root biomass in themedium N-high P and the high N-high P treatments), the below-ground (rhizome and root biomass) were slightly negatively influ-enced by the high N treatments. In addition, the above-ground bio-mass was negatively affected by the high N-low P treatment.

The lower pH in the solution (Table 4) could have influencedplant growth. Brix et al. (2002) observed that the growth of T. lat-ifolia almost completely stopped at pH 3.5 in the solution culture.

The growth inhibition at low pH was probably due to a reducednutrient uptake and a consequential limitation of growth by nutri-ent stress. In addition, S. validus in this study possibly sufferedammonia toxicity in the high N treatments. Ammonia can be toxicto a variety of plant species due to accumulation of NH4 in tissuesor a diminished cation (such as K+, Mg2+ or Ca2+) uptake (Britto andKronzucher, 2002). Even species whose tolerance to NH4 is pro-nounced can suffer toxicity symptoms, given a high enough appli-cation of ammonium (Britto and Kronzucher, 2002). For instance,growth of S. acutus var. acutus (Hill et al., 1997) and Schoenoplectustabernaemontani was decreased by ammonium (Clarke and Bald-win, 2002). However, it is worth noting that although the concen-trations of ammonium in the high N treatments in this study werethe similar to studies mentioned above, we had N ratio of 1:1NH4:NO3.

4.2. N and P uptake by plants

In general, the concentrations of N and P in the plant tissues in-creased with an increase in N and P supply (Table 2). The presentresults indicated that S. validus could accumulate high concentra-tions of N and P in above-ground tissues under conditions of abun-dant supply. This capacity in addition to its high growth rate isdesirable for aquatic plants used in constructed wetlands forwastewater treatment. The highest concentrations were29 g N kg�1 and 4.5 g P kg�1 in shoots, the values comparable tothose in the literature (e.g. Cronk and Fennessy, 2001). Over 60%of N and P taken up by S. validus were located in the above-groundtissues; hence, harvesting shoots would remove most of thesenutrients accumulated by plants in constructed wetlands forwastewater treatments.

4.3. Nutrient removal

In the present study, the removal efficiencies of N and P (Table3) were comparable to the reported studies. For example, about80–99% of N and P could be removed by 21 aquatic plant speciesin the planted VFS microcosms after 14-d retention time (Iamcha-turapatr et al., 2007). A mass reduction of 85% N and 21% P fromsecondary-treated municipal wastewater was found in surface-flow constructed wetland at Cairns, Australia (Greenway andWoolley, 2001).

In the present study, the removal efficiency for NH4 was rela-tively high compared with that for NOx in the medium and highN treatments. Higher NH4 removal could be caused by (1) S. validuspreferring NH4 over NOx for uptake; (2) nitrification; and (3) thesand substrate adsorbing NH4 to a certain degree. Vymazal(2007) pointed out that the potential to remove nitrate is verylow in vertical-flow wetlands.

4.4. pH and DO

In the present study, the pH in the effluents was influenced by Nand P treatments. A rapid reduction of pH at an early stage of theexperiment was found in the planted VFS wetland microcosms(Iamchaturapatr et al., 2007). The underlying reason might be theformation of CO2 and H2CO3 in water by the degradation of organiccompounds (residual organic matter in substrate and plant-de-rived matter) resulting in a pH decline (Kyambadde et al., 2004).Plants take up significant amounts of sparingly soluble nutrientsfrom the rhizosphere by acidifying the rhizosphere (Rao et al.,2002) via excreting H+ in exchange for cations and exuding organicacids and CO2 (Hinsinger et al., 2003). Nitrification can also de-crease the rhizosphere pH (Bezbaruah and Zhang, 2004).

In the present study, the concentration of DO was interactivelyaffected by N and P loadings. There might be several reasons for

Z. Zhang et al. / Chemosphere 72 (2008) 1823–1828 1827

this phenomenon in VFS wetland microcosms (Iamchaturapatret al., 2007). For example, plant shading in the high nutrient treat-ments may inhibit the growth of suspended photosynthetic organ-isms resulting in severely decreased O2 production in the water;also, high plant above-ground biomass may limit contact betweenatmospheric and water phases, lowering O2 transfer from air towater.

4.5. Nutrient mass balance

The results of the present study (Table 5) showed that N and Premovals by plant uptake (19–42% and 4–39% of the influent N andP, respectively) were relatively low compared with some publishedreports. For example, Breen (1990) stated that 51 and 67% of theinfluent N and P, respectively, were removed by plants in micro-cosms. In the field, nutrient removal from simulated nursery runoffby P. australis in the subsurface flow wetlands accounted for 76 and86% of total input of N and P, respectively (Huett et al., 2005). How-ever, nutrient removal by plants in the study presented here wascomparable to that in some constructed wetlands. Nutrient remo-vals from secondary-treated municipal wastewater by plants ac-counted for 15–80% N and 24–80% P in surface-flow constructedwetland at Cairns, Australia (Greenway and Woolley, 2001). Thenutrient uptake by Baumea articulata accounted for 11% of N re-moval and 3% of P removal (Browning and Greenway, 2003). Differ-ent nutrient loading rates were responsible for different nutrientuptake by plants in various studies.

In addition to plant uptake, N removal can occur by (i) NH3 vol-atilisation favoured by high pH, (ii) nitrification under aerobic con-ditions, (iii) denitrification under anaerobic conditions (Sekirandaand Kiwanuka, 1998), and formation of organic films (Breen,1990). In the present study, N removal by volatilisation was unli-kely, due to pH in the solution being less than 7 (Table 4). Hence,the other losses of N in the present study (Table 5) were likelydue to denitrification and formation of organic film. Indeed, deni-trification is a major N removal mechanism in most constructedwetlands (Vymazal, 2007) and organic films accounted for 11% ofthe influent N (Breen, 1990).

Phosphorus removal from wastewater by vertical-flow wet-lands may occur through three parallel mechanisms: substratesorption, plant uptake and microbial assimilation (Luederitzet al., 2001). Given that the medium used in this study was riversand containing oxides of Fe, Al, and Ca, it could enhance the effectof P retention by chemical adsorption and precipitation (cf. Ariaset al., 2001). The P sorption capacity in different sands varied from0.13 to 0.29 g P kg�1 (Xu et al., 2006). In the present study, the low-est removal efficiency of PO4�P was observed at week 7 after treat-ments started (Fig. 1), suggesting that the adsorption of PO4�P bysand decreased, and plant uptake increased, over the 10 weekcourse of this study.

Losses of 4–10% of P were recorded (Table 5), possibly due toformation of organic films that were not quantified in the presentstudy. These losses are similar to previously reported ones (Zhanget al., 2007b). Breen (1990) observed that organic film representeda total of 10% of the influent P. In addition, plants, algae and micro-organisms all utilise P as an essential nutrient, even though tissue Pis relatively small compared with C and N (Silvan et al., 2004).

5. Conclusions

The significant interactive effects of N and P loadings on plantgrowth and removal efficiencies of NH4 and PO4�P were observedin the wetland microcosms. High N concentrations in suppliedwastewater might affect the growth of S. validus. Therefore, in or-der to enhance nutrient removal efficiency, the optimum nutrient

availability and the N:P ratio are required to stimulate growth ofS. validus in constructed wetlands.

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