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Importance of plant species for nitrogen removal using constructed floating wetlands in a cold climate
Ecological Engineering 138 (2019) 126–132
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Short communication
Importance of plant species for nitrogen removal using constructed floating wetlands in a cold climate
T
Maidul I. Choudhurya, , Joel Segerstena, Maria Hellmanb, Brendan G. Mckiea, Sara Hallinb, Frauke Eckea,c ⁎
a
Swedish University of Agricultural Sciences, Department of Aquatic Sciences and Assessment, Box 7050, SE-750 07 Uppsala, Sweden Swedish University of Agricultural Sciences, Department of Forest Mycology and Plant Pathology, Box 7026, 750 07 Uppsala, Sweden c Swedish University of Agricultural Sciences, Department of Wildlife, Fish, and Environmental Studies, 901 83 Umeå, Sweden b
ARTICLE INFO
ABSTRACT
Keywords: Denitrification Macrophytes Mining Nitrate Nitrogen uptake Nitrogen removal Sub-arctic
Constructed floating wetlands (CFWs) have been tested in different climatic regions and aquatic habitat types for nitrogen (N) removal from surface water, but there is limited knowledge about their applicability for N removal in cold climate regions. Most CFWs studies are conducted at the micro- or mesocosm scale, while the application of CFWs at in situ is rare. Moreover, most CFWs studies have focused on plant N accumulation without considering macrophyte root-associated denitrification as a possible N removal pathway. Here, we study the N removal potential of CFWs through N accumulation by macrophytes and potential denitrification activity (PDA) associated with plants. At a mining area in the sub-arctic region of Sweden receiving N-rich mine effluents, we tested the concept of CFWs and evaluated the performance of six native, emerging macrophyte species planted in CFWs. The CFWs were deployed in two types of systems: in situ in the recipient lake, subjected to ambient N concentrations, and CFWs placed in water-side “eco-tanks”, subjected to higher N concentrations. We showed that macrophyte establishment in CFWs is feasible under cold climatic conditions, both in situ and eco-tanks. The standing biomass of macrophytes, bulk N accumulation in plant biomass and PDA in mesocosms were 0.54–2.25 kg m−2, 7.56–24.75 mg N m−2 d−1 and 31.82–2250.77 mg N2O-N m−2 d−1, respectively. In the recipient, the variation was larger and the values were higher (standing biomass, 0.37–6.74 kg m−2; bulk N accumulation, 8.09–106.93 mg N m−2 d−1; PDA, 11.89–8446.15 mg N m−2 d−1). Macrophyte root-associated denitrification was the main N removal pathway in the CFWs. Given the demonstrated applicability of CFWs and the high denitrification rates that can be obtained, future studies should focus on designing CFWs to enhance denitrification as this process leads to permanent removal of N from the water phase.
1. Introduction Nitrogen (N) released to freshwaters from undetonated ammoniumnitrate based explosives that are used in blasting operations has become an emerging environmental issue, especially in cold climate regions where anthropogenic pressures associated with mining are increasing (Herbert et al., 2014; Bailey et al., 2013). Constructed wetlands (CWs) offer a possibility for remediation by promoting nutrient transformation processes, representing key ecosystem processes (Vymazal, 2007; Verhoeven et al., 2006; Thullen et al., 2005). Denitrification, i.e. microbial reduction of nitrate (NO3−) to N2 under oxygen limiting conditions, and N assimilation by aquatic plants (macrophytes) are the main N removal processes in wetlands. Although sediment is an important wetland habitat for denitrification (Kjellin et al., 2007; Whitmire and
⁎
Hamilton, 2005), denitrification rates are especially high in the rhizosphere or periphyton of macrophytes compared to bare sediments (Kofoed et al., 2012; Ruiz-Rueda et al., 2009) and periphytic communities can significantly contribute to N removal through denitrification (Hallin et al., 2015; Eriksson and Weisner, 1997). In addition to the sediment-water interface, denitrification can potentially be facilitated in the water column of wetlands by hydroponic cultivation of macrophytes, but there is limited knowledge on its potential at low temperature, and which macrophyte species that would be most suitable under those conditions. To increase the contact between NO3− rich water and macrophyte root systems, constructed floating wetlands (CFWs) planted with emerging macrophytes have been used in different freshwater systems including storm water reservoirs, ponds and lakes (Song et al., 2014;
Corresponding author. E-mail address: [email protected] (M.I. Choudhury).
https://doi.org/10.1016/j.ecoleng.2019.07.012 Received 4 December 2018; Received in revised form 7 July 2019; Accepted 15 July 2019 0925-8574/ © 2019 Elsevier B.V. All rights reserved.
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Wang and Sample, 2014). Nitrogen is released to freshwaters from different point and non-point sources (Carpenter et al., 1998) and onsite treatment of nutrients by using CFWs have been used for nutrient removal in for example, intensive recirculating aquaculture systems (Piedrahita, 2003; van Rijn, 1996), meat processing industry (van Oostrom, 1995) and chemical refinery plants (Li et al., 2012). CFWs generally consist of a mat or structure floating in the waterbody and planted with emerging macrophytes that develop extensive root systems in the water column. Although CFWs have been tested in different geographic regions and systems, most studies have either been conducted in closed systems in micro- or mesocosm experiments (White and Cousins, 2013; Wu et al., 2011; Yang et al., 2008), whereas in situ applications of CFWs for nutrient removal remain limited, especially in a cold climate (reviewed by Pavlineri et al., 2017). This study is a proof of concept for applying CFWs for N removal in a cold climate, more specifically, a sub-arctic area subjected to high nitrate loading from mining exploration. The objectives were to evaluate species-specific performance i.e. macrophyte establishment and survival at the site, biomass production and determine the relative importance of N accumulation in plant biomass and denitrification associated with plant roots for N removal. We evaluated the N removal performance of six native sub-arctic macrophyte species established in CFWs placed in situ in a recipient lake and in CFWs subjected to much higher N-loadings in controlled water-side eco-tanks (Fig. 1). The ecotanks were included in the study to evaluate if floating wetlands
a)
deployed in a land-based system can also facilitate N removal, which might be useful in case where the area of wetland is insufficient for deployment of a floating wetland in situ (e.g. in intensive recirculating aquaculture systems). In accordance with previous studies (Choudhury et al., 2018; Hallin et al., 2015), we expected that N accumulation in plant biomass and denitrification associated with plant roots differ among the studied species. 2. Materials and methods 2.1. Study area The study was conducted in a mining area situated in Kiruna, Northern Sweden, located 145 km north of the Arctic Circle (67°51′N 20°13′E). The mean annual temperature in this area is −2 °C, with maximum-minimum temperature range in summer of 25.9 to −2.1 °C (mean 10 °C), while in winter the range is 5.9 °C down to −37 °C (SMHI, 2016). The active growing period for plants in this area is approximately 120 days (Raab and Vedin, 1995). We deployed our CFWs in two systems: in situ in the natural recipient of the mining effluent (Lake Mettä-Rakkurijärvi; area: 0.6 km2) and in two outdoor plastic eco-tanks (height 1.2 m; diameter 2 m and volume 3600 L; Strandvik Plast AS, Bergen, Norway). Both systems received water from the clarification pond at the mining site. Nitrate is the main N species in the effluent from the clarification pond (Herbert et al., 2014) and
Eco-tank Inlet
Inlet CFWs Outlet
Outlet
3600 L
b)
3600 L
Recipient (in situ)
CFWs
Fig. 1. Schematic diagram of constructed floating wetlands (CFWs) planted with emerging macrophytes established in the a) eco-tanks and, b) recipient (in situ) at the mining area. 127
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Table 1 Water chemistry in the eco-tanks and recipient containing constructed floating wetlands (CFWs; mean ± SD). System
NO3-N (mg l−1)
PO4-P (mg l−1)
pH
TOC (mg l−1)
Conductivity (µS m−1)
Eco-tank Recipient
21.26 (1.35) 9.85 (4.45)
< 0.005 < 0.005
7.7 (0.44) 7.4 (0.77)
6.22 (3.14) 3.05 (1.90)
267.83 (0.75) 162.50 (45.96)
phosphate is very limited in this system (Table 1). The annual water discharge from tailing pond to clarification pond is 51.8 × 106 m3 and overall annual water residence time is approximately 14 days. The ecotanks were directly fed with water from the clarification pond using a pump with mean residence time of approximately 14 days (Fig. 1a). Continuous water flow was maintained during summer (June – August) from 2014 to 2016 but was stopped during winter (October – May). The water chemistry (i.e. NO3-N, PO4-P, pH, total organic carbon and conductivity) of eco-tanks and recipient is given in Table 1.
2.4. Potential denitrification activity (PDA) The acetylene inhibition technique without chloramphenicol (Pell et al., 1996) was used to determine potential denitrification activity (PDA) of the microbial communities associated with the fresh roots of macrophytes sampled at the end of the experiment. After harvesting, root samples were stored at refrigerator maximum for 3–6 days at 4 °C until analysis. The plant roots were cut in 2–3 cm pieces and slurries of 3–7 g of fresh plant roots were put in gas-tight 100 ml flasks. Then 30 ml sterile, deionized water was added to each flask. To provide anoxic conditions, the headspace was exchanged to 1 atm N2 by evacuating and filling the flask 5 times. The root slurries were pre-incubated for 20 min at 25 °C with constant agitation (175 rpm) prior to injection of acetylene (10% v/v) and a substrate solution consisting of a mixture of electron donors and KNO3 was added through the septa with a syringe, resulting in a final concentration of 1 mM glucose, 3 mM sodium acetate, 1.5 mM sodium succinate and 3 mM KNO3. The slurries were incubated 8 h at 25 °C with constant agitation (175 rpm) and 1 ml gas samples were withdrawn after 2, 4, 6 and 8 h, respectively, and analysed for N2O on a gas chromatograph equipped with an electron capture detector (Clarus 500 GC, Perkin Elmer, CT, USA). The denitrification rate was calculated from non-linear regression of the N2O produced during incubation. It was not possible to harvest the entire root biomass of macrophytes since the roots also developed within the pore spaces in the construction fibres of CFWs. Therefore, we calculated belowground biomass of macrophytes by multiplying aboveground biomass with 2.1 (for C. rostrata see Aerts et al., 1992). Belowground biomass was then used to calculate bulk PDA (Eq. (2)).
2.2. Constructed floating wetlands (CFWs) and experimental design Commercially available 3.25 m2 (W × L × H:154 × 243 × 20 cm) PET-plastic floating wetlands (Veg Tech, Sweden; article no. 9-15055) with 78 evenly distributed wells (53 cm2) for planting macrophytes were used in the recipient lake. CFWs in the eco-tanks were constructed of the same material, but reduced in size to fit the water tanks and had 52 wells, each. We placed one CFW in each of the two eco-tanks and two CFWs in the recipient. We collected six native and common wetland macrophyte species near the recipient: Comarum palustre L. (purple marshlocks), Equisetum fluviatile L. (water horsetail), Carex rostrata Stokes (bottle sedge), Eriophorum angustifolium Honck. (common cottonsedge), Filipendula ulmaria (L.) Maxim. (meadowsweet) and Menyanthes trifoliata L. (bog bean). Every macrophyte species was represented in every CFW (Fig. 1). CFWs contained a total of 5–10 (eco-tanks) and 6–10 (recipient) replicates per species (see Table S1). Macrophytes were planted in the CFWs in June 2014 and cultivated until August 2016. The CFWs in the recipient were anchored with iron chains attached to concrete weights.
Bulk PDA =
2.3. N Accumulation in plant biomass
(
Biomass area
) × N conc. Gp
(2)
where Biomass = aboveground biomass (kg dry weight); area = area of planting well (0.0053414 m2); PDA = potential denitrification activity (mg N2O-N kg dry weight−1 day−1).
We harvested individual macrophytes in each well in August 2016. In the eco-tanks, 1–3 specimens per species and CFW were harvested resulting in a total of 3–6 specimens per species and in the recipient experiment, 1–5 specimens of each species per CFW resulting in total 4–7 specimens per species (see Table S1). Aboveground biomass was separated from root biomass. Aboveground or photosynthetic biomass (in case of C. palustre and M. trifoliata that have roots attached with stems) was dried at 50 °C for seven days to determine dry weight (DW) of each specimen. The N content in the plant biomass was analysed at the Forest Research Lab, Farnham, UK, according to ISO 10,694 & 13878. We calculated standing plant biomass based on well area and used the N content in plant biomass to calculate bulk N accumulation (mg N m−2 day−1) by aboveground (or photosynthetic) biomass, assuming an active growing period of 120 days (Raab and Vedin, 1995) (Eq. (1)).
Bulk nitrogen accumulation =
(Biomass × 2.1) × PDA area
2.5. Potential N removal pathways The ratio between potential denitrification activity and N accumulation in plant biomass was calculated according to Hallin et al. (2015) to determine the relative importance of denitrification and plant accumulation of N, for nitrogen removal associated with different macrophyte species (Eq. (3)).
Nitrogen removal pathway =
Bulk PDA Bulk N accumulation
(3)
A value above 1 indicates denitrification as the main N removal pathway, whereas a ratio below 1 indicates plant accumulation as the main N removal pathway (for calculation of bulk N accumulation and bulk PDA see Eqs. (1) and (2), respectively).
(1)
2.6. Data analyses
where Biomass = aboveground biomass (kg dry weight); area = area of planting well (0.0053414 m2); N conc. = nitrogen concentration in aboveground biomass (mgN kg dry weight−1); Gp = active growing period (120 days).
One-way ANOVA was performed to test for differences among macrophyte species in standing biomass, bulk plant N accumulation, bulk denitrification and potential N removal pathways, with separate ANOVAs conducted for the eco-tanks and recipient. To meet the assumptions of parametric tests, data was log transformed for standing 128
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biomass, bulk plant N accumulation and bulk denitrification, and square root transformed for potential N removal pathway. Adjustment for multiple comparisons was made by Tukey’s HSD. Statistical analyses were performed using the software IBM SPSS Statistics for Macintosh, Version 22.0 (Armonk, NY: IBM Corp).
et al., 2015; Aerts et al., 1992). In the eco-tanks, F. ulmaria showed higher bulk N accumulation compared to E. fluviatile and M. trifoliata (Fig. 2b, Table S2). Similarly, F. ulmaria had higher bulk accumulation compared to E. fluviatile, M. trifoliata and E. angustifolium in the recipient whereas C. palustre had higher bulk N accumulation compared to E. fluviatile and M. trifoliata (Fig. 2f, Table S3). Overall, bulk N accumulation in macrophytes was generally lower than that of other species planted in CFWs in warmer climatic regions, i.e. tropical, subtropical, temperate and continental climates (Table 3). Low summer temperature and a short growing season are the main controlling factors for biomass production of macrophytes in the sub-arctic region (Solander, 1983), which likely explains the lower bulk N accumulation by macrophytes when compared to temperate or tropical regions. Even though our study was conducted in a cold climate, the observed N accumulation by C. palustre and F. ulmaria in particular is comparable with that by commonly used macrophyte species in constructed wetlands, e.g. Typha spp. and Phragmites australis (Table 3).
3. Results and discussion 3.1. Establishment and standing biomass of macrophytes Our study demonstrates that CFWs, frequently applied in tropical and temperate climates (Pavlineri et al., 2017), also effectively remove nitrate from freshwater systems under cold climatic conditions. The successful establishment and survival of all six macrophyte species is partly likely to reflect the use of local-sourced plant specimens and species, rather than the more usual practice of purchasing plants from commercial cultures for deployment in CFWs. Standing biomass of macrophytes differed significantly among species, both in eco-tanks and the recipient (Table 2). In the eco-tanks, C. rostrata and F. ulmaria showed a higher standing biomass compared to E. fluviatile and M. trifoliata, whereas C. palustre showed higher standing biomass than E. fluviatile. No differences in standing biomass were observed between other species (Table S2). In the recipient, M. trifoliata had the lowest biomass production compared to all other species except E. fluviatile (Table S3). E. fluviatile had lower biomass production compared to C. rostrata, C. palustre and F. ulmaria (Table S3). In line with this, E. fluviatile in general has low aboveground biomass compared to belowground biomass (Grime et al., 2007) and M. trifoliata usually occurs in oligotrophic habitats (Fitter and Peat, 1994). Further, F. ulmaria and C. rostrata prefer eutrophic and mesotrophic habitats, respectively (Grime et al., 2007; Fitter and Peat, 1994), whereas F. ulmaria is a competitive species (Grime et al., 2007). The standing biomass of C. rostrata (2.2 kgDW m−2) in the recipient and eco-tanks was greater than that reported by Aerts et al. (1992) after growing this species in situ for 2 years (1.2 kg-DW m−2), and also exceeds the biomass observed for this species in a boreal fen (25.2 g-DW m−2) (Saarinen, 1996). This is most likely to reflect the very high nitrate levels in our study. Overall, our results suggest that the ecology and known life strategies of the studied macrophytes determined their biomass development in the CFWs.
3.3. Potential denitrification activity (PDA) and dominant N removal pathway In the eco-tanks, PDA was higher for C. palustre compared to E. fluviatile, whereas there was no difference among the remaining species (Fig. 2c, Table S2). In the recipient, C. palustre and F. ulmaria had higher PDA compared to the other species (Fig. 2g, Table S3), with PDA being negligible for M. trifoliata in both systems (Fig. 2c, g, Table S2 and S3). M. trifoliata is known to have a high allelopathic potential (Grutters et al., 2017) that might inhibit bacterial activity in close vicinity of roots. In our study, potential N removal rates through denitrification in the eco-tanks ranged between 32 and 2250 mg N2O-N m−2 day−1. These high rates are likely to underpin the complete removal of the NO3− loading (1528 mg NO3-N m−2 day−1) in the clarification pond at the studied mining area. All macrophyte species in the CFWs showed denitrification as the main N removal pathway (Fig. 2d, h). This result is consistent with previous studies in constructed wetlands (Hallin et al., 2015; Van Cleemput et al., 2007). The root-associated denitrification activity differed among macrophyte species in our study, but was not related to plant N accumulation. This is in contrast to previous work showing that macrophytes that accumulate more N have lower root associated denitrification activity and vice versa (Hallin et al., 2015). The nitrate supply around the roots in hydroponically cultivated species is likely to be greater than for species growing in wetlands that rely more on nitrate in the sediment. This might reduce the importance of competition for nitrate between the denitrifiers and the plants as a control of denitrification activity, potentially explaining the decoupling of root-associated denitrification and plant N accumulation observed in CFWs in the current study. Our study highlights the importance of considering denitrification activity associated with macrophyte roots in CFWs, which has surprisingly rarely been reported in previous studies (Table 3). Denitrification potential in CFWs has been measured in biofilms on rice straw and plastic materials (Zhang et al., 2018, and the references therein). However, the periphyton of macrophyte roots and submerged shoots can significantly contribute to denitrification in wetlands (Hallin et al., 2015; Eriksson and Weisner, 1997) and more efforts are needed to estimate the quantitative importance of denitrification for nitrate removal in CFWs.
3.2. N accumulation in plant biomass Plant bulk N accumulation differed significantly among species in all CFWs (Table 2) and largely coincide with plant biomass development. This indicates that certain macrophyte species are better than others for N removal by plant assimilation (Fig. 2b, f; Table S2 and S3), which agrees with previous studies (Choudhury et al., 2018; Hallin Table 2 One-way ANOVA testing the effect of macrophyte species on standing plant biomass (g-DW m−2), N accumulation by plant biomass (mg N m−2 day−1), potential denitrification activity (PDA) (mg N2O-N m−2 day−1) on roots of macrophytes and main N removal pathways, i.e. ratio between PDA and N accumulation in constructed floating wetlands (CFWs) investigated in cold climate. System
Response variable
df
Eco-tank
Standing biomass Plant N accumulation PDA PDA: N accumulation
5, 5, 5, 5,
Recipient (or in situ)
Standing biomass Plant N accumulation PDA PDA: N accumulation
5, 5, 5, 5,
F
p
19 19 19 19
5.53 5.94 63.94 116.77
** ** *** ***
29 29 29 29
13.46 11.32 41.61 279.90
*** *** *** ***
4. Final remarks We showed that CFWs planted with macrophytes could be used for treatment of nitrate contaminated mine water under cold climatic conditions. Macrophyte-mediated denitrification, resulting in removal of NO3- from the water, was the main N removal pathway in the CFWs. However, the choice of macrophyte species will determine the overall nitrate removal by plant bulk N accumulation and denitrification, and
*** < 0.001, ** < 01, * < 0.05, ns = not significant. 129
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Fig. 2. Operation of constructed floating wetlands in eco-tanks and the recipient (in situ) for nitrogen (N) removal from surface water by six macrophyte species (Comarum palustre, Equisetum fluviatile, Carex rostrata, Eriophorum angustifolium, Filipendula ulmaria and Menyanthes trifoliata), a, e) standing plant biomass, b, f) N accumulation in plant biomass, c, g) potential denitrification activity (PDA) associated with macrophytes roots, d, h) ratio between PDA and plant accumulation of N (a ratio value > 1 indicates denitrification as main N removal pathway while a value < 1 indicates plant accumulation as main N removal pathway). Different numbers above the bars indicate significant differences between plant species (p < 0.05).
successful application of CFWs will rely on the correct choice of macrophyte species. Given the demonstrated utility of both land-based CFWs and those in water bodies, future studies should focus on
designing CFWs that support enhanced denitrification as this process outcompetes that of plant bulk N accumulation and also leads to permanent removal of nitrate from water. 130
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Table 3 Nitrogen (N) accumulation and potential denitrification activity (PDA) associated with macrophyte roots in constructed floating wetlands applied in different climatic regions and systems. Plant species
N accumulation (mg N m−2 day−1)a
PDA (mg N m−2 day−1)
System
Climatic region
References
Typha angustifolia Iris pseudacorus Iris pseudacorus Typha orientalis Phragmites australis Phragmites australis Phragmites australis Juncus effusus Juncus effusus Canna flaccida Scirpus validus Salix babylonica Alisma subcordatum Carex stricta Iris versicolor Pontederia cordata
13.2 204.4 161.3 44 27.1 – 65 71 390 220 95 34.4 12.2 168.1 62.1 85.3
– – – – – 947.3 – – – – – – – – – –
CFW-mesocosm CFW-mesocosm CFW-mesocosm CFW- mesocosm CFW- mesocosm CFW- microcosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- in situ CFW- in situ CFW- in situ CFW- in situ
n.m n.m. n.m sub-humid continental sub-humid continental n.m n.m n.m Humid sub tropical Humid sub tropical sub-humid continental Temperate Humid subtropical Humid subtropical Humid subtropical Humid subtropical
Keizer-Vlek et al. (2014) Keizer-Vlek et al. (2014) Gao et al. (2018) Wu et al. (2011) Wu et al. (2011) Prajapati et al. (2017) Saad et al. (2016) Saad et al. (2016) White and Cousins (2013) White and Cousins (2013) Wu et al. (2011) Zhu et al. (2011) McAndrew et al. (2016) McAndrew et al. (2016) McAndrew et al. (2016) McAndrew et al. (2016)
a
Mass uptake was given as g/m2 and was divided by tissue sampling interval (days); n.m. = not mentioned.
Declaration of Competing Interest
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The authors declare that they have no conflict of interest. Acknowledgements This study was financed by VINNOVA (The Swedish Innovation Agency), LKAB and Boliden Minerals AB through the project ′′miNing′′ (grant numbers: 2013-03325 and 2014-01134). We thank two anonymous reviewers for their valuable comments. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoleng.2019.07.012. References Aerts, R., Decaluwe, H., Konings, H., 1992. Seasonal allocation of biomass and nitrogen in 4 carex species from mesotrophic and eutrophic fens as affected by nitrogen supply. J. Ecol. 80 (4), 653–664. Bailey, B.L., Smith, L.J.D., Blowes, D.W., Ptacek, C.J., Smith, L., Sego, D.C., 2013. The Diavik waste rock project: persistence of contaminants from blasting agents in waste rock effluent. Appl. Geochem. 36, 256–270. Carpenter, S.R., Caraco, N.F., Correll, D.L., Howarth, R.W., Sharpley, A.N., Smith, V.H., 1998. Nonpoint pollution of surface waters with phosphorus and nitrogen. Ecol. Appl. 8 (3), 559–568. Choudhury, M.I., McKie, B.G., Hallin, S., Ecke, F., 2018. Mixtures of macrophyte growth forms promote nitrogen cycling in wetlands. Sci. Total Environ. 635, 1436–1443. Eriksson, P.G., Weisner, S.E.B., 1997. Nitrogen removal in a wastewater reservoir: the importance of denitrification by epiphytic biofilms on submersed vegetation. J. Environ. Qual. 26 (3), 905–910. Fitter, A.H., Peat, H.J., 1994. The ecological flora database. J. Ecol. 82 (2), 415–425. http://www.ecoflora.co.uk. Gao, L., Zhou, W., Wu, S., He, S., Huang, J., Zhang, X., 2018. Nitrogen removal by thiosulfate-driven denitrification and plant uptake in enhanced floating treatment wetland. Sci. Total Environ. 621, 1550–1558. https://doi.org/10.1016/j.scitotenv. 2017.10.073. Grime, J.P., Hodgson, J.G., Hunt, R., 2007. Comparative Plant Ecology: A Functional Approach to Common British species, 2nd ed. Castlepoint Press, Colvend. Grutters, B.M.C., Saccomanno, B., Gross, E.M., Van de Waal, D.B., van Donk, E., Bakker, E.S., 2017. Growth strategy, phylogeny and stoichiometry determine the allelopathic potential of native and non-native plants. Oikos 126 (12), 1770–1779. Hallin, S., Hellman, M., Choudhury, M.I., Ecke, F., 2015. Relative importance of plant uptake and plant associated denitrification for removal of nitrogen from mine drainage in sub-arctic wetlands. Water Res. 85, 377–383. Herbert, R.B., Winbjork, H., Hellman, M., Hallin, S., 2014. Nitrogen removal and spatial distribution of denitrifier and anammox communities in a bioreactor for mine drainage treatment. Water Res. 66, 350–360.
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Whitmire, S.L., Hamilton, S.K., 2005. Rapid removal of nitrate and sulfate in freshwater wetland sediments. J. Environ. Qual. 34 (6), 2062–2071. Wu, H.M., Zhang, J.A., Li, P.Z., Zhang, J.Y., Xie, H.J., Zhang, B., 2011. Nutrient removal in constructed microcosm wetlands for treating polluted river water in northern China. Ecol. Eng. 37 (4), 560–568. Yang, Z.F., Zheng, S.K., Chen, J.J., Sun, M., 2008. Purification of nitrate-rich agricultural runoff by a hydroponic system. Bioresour. Technol. 99 (17), 8049–8053. Zhang, L.L., Sun, Z.Z., Xie, J., Wu, J., Cheng, S.P., 2018. Nutrient removal, biomass accumulation and nitrogen-transformation functional gene response to different nitrogen forms in enhanced floating treatment wetlands. Ecol. Eng. 112, 21–25.
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Short communication
Importance of plant species for nitrogen removal using constructed floating wetlands in a cold climate
T
Maidul I. Choudhurya, , Joel Segerstena, Maria Hellmanb, Brendan G. Mckiea, Sara Hallinb, Frauke Eckea,c ⁎
a
Swedish University of Agricultural Sciences, Department of Aquatic Sciences and Assessment, Box 7050, SE-750 07 Uppsala, Sweden Swedish University of Agricultural Sciences, Department of Forest Mycology and Plant Pathology, Box 7026, 750 07 Uppsala, Sweden c Swedish University of Agricultural Sciences, Department of Wildlife, Fish, and Environmental Studies, 901 83 Umeå, Sweden b
ARTICLE INFO
ABSTRACT
Keywords: Denitrification Macrophytes Mining Nitrate Nitrogen uptake Nitrogen removal Sub-arctic
Constructed floating wetlands (CFWs) have been tested in different climatic regions and aquatic habitat types for nitrogen (N) removal from surface water, but there is limited knowledge about their applicability for N removal in cold climate regions. Most CFWs studies are conducted at the micro- or mesocosm scale, while the application of CFWs at in situ is rare. Moreover, most CFWs studies have focused on plant N accumulation without considering macrophyte root-associated denitrification as a possible N removal pathway. Here, we study the N removal potential of CFWs through N accumulation by macrophytes and potential denitrification activity (PDA) associated with plants. At a mining area in the sub-arctic region of Sweden receiving N-rich mine effluents, we tested the concept of CFWs and evaluated the performance of six native, emerging macrophyte species planted in CFWs. The CFWs were deployed in two types of systems: in situ in the recipient lake, subjected to ambient N concentrations, and CFWs placed in water-side “eco-tanks”, subjected to higher N concentrations. We showed that macrophyte establishment in CFWs is feasible under cold climatic conditions, both in situ and eco-tanks. The standing biomass of macrophytes, bulk N accumulation in plant biomass and PDA in mesocosms were 0.54–2.25 kg m−2, 7.56–24.75 mg N m−2 d−1 and 31.82–2250.77 mg N2O-N m−2 d−1, respectively. In the recipient, the variation was larger and the values were higher (standing biomass, 0.37–6.74 kg m−2; bulk N accumulation, 8.09–106.93 mg N m−2 d−1; PDA, 11.89–8446.15 mg N m−2 d−1). Macrophyte root-associated denitrification was the main N removal pathway in the CFWs. Given the demonstrated applicability of CFWs and the high denitrification rates that can be obtained, future studies should focus on designing CFWs to enhance denitrification as this process leads to permanent removal of N from the water phase.
1. Introduction Nitrogen (N) released to freshwaters from undetonated ammoniumnitrate based explosives that are used in blasting operations has become an emerging environmental issue, especially in cold climate regions where anthropogenic pressures associated with mining are increasing (Herbert et al., 2014; Bailey et al., 2013). Constructed wetlands (CWs) offer a possibility for remediation by promoting nutrient transformation processes, representing key ecosystem processes (Vymazal, 2007; Verhoeven et al., 2006; Thullen et al., 2005). Denitrification, i.e. microbial reduction of nitrate (NO3−) to N2 under oxygen limiting conditions, and N assimilation by aquatic plants (macrophytes) are the main N removal processes in wetlands. Although sediment is an important wetland habitat for denitrification (Kjellin et al., 2007; Whitmire and
⁎
Hamilton, 2005), denitrification rates are especially high in the rhizosphere or periphyton of macrophytes compared to bare sediments (Kofoed et al., 2012; Ruiz-Rueda et al., 2009) and periphytic communities can significantly contribute to N removal through denitrification (Hallin et al., 2015; Eriksson and Weisner, 1997). In addition to the sediment-water interface, denitrification can potentially be facilitated in the water column of wetlands by hydroponic cultivation of macrophytes, but there is limited knowledge on its potential at low temperature, and which macrophyte species that would be most suitable under those conditions. To increase the contact between NO3− rich water and macrophyte root systems, constructed floating wetlands (CFWs) planted with emerging macrophytes have been used in different freshwater systems including storm water reservoirs, ponds and lakes (Song et al., 2014;
Corresponding author. E-mail address: [email protected] (M.I. Choudhury).
https://doi.org/10.1016/j.ecoleng.2019.07.012 Received 4 December 2018; Received in revised form 7 July 2019; Accepted 15 July 2019 0925-8574/ © 2019 Elsevier B.V. All rights reserved.
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Wang and Sample, 2014). Nitrogen is released to freshwaters from different point and non-point sources (Carpenter et al., 1998) and onsite treatment of nutrients by using CFWs have been used for nutrient removal in for example, intensive recirculating aquaculture systems (Piedrahita, 2003; van Rijn, 1996), meat processing industry (van Oostrom, 1995) and chemical refinery plants (Li et al., 2012). CFWs generally consist of a mat or structure floating in the waterbody and planted with emerging macrophytes that develop extensive root systems in the water column. Although CFWs have been tested in different geographic regions and systems, most studies have either been conducted in closed systems in micro- or mesocosm experiments (White and Cousins, 2013; Wu et al., 2011; Yang et al., 2008), whereas in situ applications of CFWs for nutrient removal remain limited, especially in a cold climate (reviewed by Pavlineri et al., 2017). This study is a proof of concept for applying CFWs for N removal in a cold climate, more specifically, a sub-arctic area subjected to high nitrate loading from mining exploration. The objectives were to evaluate species-specific performance i.e. macrophyte establishment and survival at the site, biomass production and determine the relative importance of N accumulation in plant biomass and denitrification associated with plant roots for N removal. We evaluated the N removal performance of six native sub-arctic macrophyte species established in CFWs placed in situ in a recipient lake and in CFWs subjected to much higher N-loadings in controlled water-side eco-tanks (Fig. 1). The ecotanks were included in the study to evaluate if floating wetlands
a)
deployed in a land-based system can also facilitate N removal, which might be useful in case where the area of wetland is insufficient for deployment of a floating wetland in situ (e.g. in intensive recirculating aquaculture systems). In accordance with previous studies (Choudhury et al., 2018; Hallin et al., 2015), we expected that N accumulation in plant biomass and denitrification associated with plant roots differ among the studied species. 2. Materials and methods 2.1. Study area The study was conducted in a mining area situated in Kiruna, Northern Sweden, located 145 km north of the Arctic Circle (67°51′N 20°13′E). The mean annual temperature in this area is −2 °C, with maximum-minimum temperature range in summer of 25.9 to −2.1 °C (mean 10 °C), while in winter the range is 5.9 °C down to −37 °C (SMHI, 2016). The active growing period for plants in this area is approximately 120 days (Raab and Vedin, 1995). We deployed our CFWs in two systems: in situ in the natural recipient of the mining effluent (Lake Mettä-Rakkurijärvi; area: 0.6 km2) and in two outdoor plastic eco-tanks (height 1.2 m; diameter 2 m and volume 3600 L; Strandvik Plast AS, Bergen, Norway). Both systems received water from the clarification pond at the mining site. Nitrate is the main N species in the effluent from the clarification pond (Herbert et al., 2014) and
Eco-tank Inlet
Inlet CFWs Outlet
Outlet
3600 L
b)
3600 L
Recipient (in situ)
CFWs
Fig. 1. Schematic diagram of constructed floating wetlands (CFWs) planted with emerging macrophytes established in the a) eco-tanks and, b) recipient (in situ) at the mining area. 127
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Table 1 Water chemistry in the eco-tanks and recipient containing constructed floating wetlands (CFWs; mean ± SD). System
NO3-N (mg l−1)
PO4-P (mg l−1)
pH
TOC (mg l−1)
Conductivity (µS m−1)
Eco-tank Recipient
21.26 (1.35) 9.85 (4.45)
< 0.005 < 0.005
7.7 (0.44) 7.4 (0.77)
6.22 (3.14) 3.05 (1.90)
267.83 (0.75) 162.50 (45.96)
phosphate is very limited in this system (Table 1). The annual water discharge from tailing pond to clarification pond is 51.8 × 106 m3 and overall annual water residence time is approximately 14 days. The ecotanks were directly fed with water from the clarification pond using a pump with mean residence time of approximately 14 days (Fig. 1a). Continuous water flow was maintained during summer (June – August) from 2014 to 2016 but was stopped during winter (October – May). The water chemistry (i.e. NO3-N, PO4-P, pH, total organic carbon and conductivity) of eco-tanks and recipient is given in Table 1.
2.4. Potential denitrification activity (PDA) The acetylene inhibition technique without chloramphenicol (Pell et al., 1996) was used to determine potential denitrification activity (PDA) of the microbial communities associated with the fresh roots of macrophytes sampled at the end of the experiment. After harvesting, root samples were stored at refrigerator maximum for 3–6 days at 4 °C until analysis. The plant roots were cut in 2–3 cm pieces and slurries of 3–7 g of fresh plant roots were put in gas-tight 100 ml flasks. Then 30 ml sterile, deionized water was added to each flask. To provide anoxic conditions, the headspace was exchanged to 1 atm N2 by evacuating and filling the flask 5 times. The root slurries were pre-incubated for 20 min at 25 °C with constant agitation (175 rpm) prior to injection of acetylene (10% v/v) and a substrate solution consisting of a mixture of electron donors and KNO3 was added through the septa with a syringe, resulting in a final concentration of 1 mM glucose, 3 mM sodium acetate, 1.5 mM sodium succinate and 3 mM KNO3. The slurries were incubated 8 h at 25 °C with constant agitation (175 rpm) and 1 ml gas samples were withdrawn after 2, 4, 6 and 8 h, respectively, and analysed for N2O on a gas chromatograph equipped with an electron capture detector (Clarus 500 GC, Perkin Elmer, CT, USA). The denitrification rate was calculated from non-linear regression of the N2O produced during incubation. It was not possible to harvest the entire root biomass of macrophytes since the roots also developed within the pore spaces in the construction fibres of CFWs. Therefore, we calculated belowground biomass of macrophytes by multiplying aboveground biomass with 2.1 (for C. rostrata see Aerts et al., 1992). Belowground biomass was then used to calculate bulk PDA (Eq. (2)).
2.2. Constructed floating wetlands (CFWs) and experimental design Commercially available 3.25 m2 (W × L × H:154 × 243 × 20 cm) PET-plastic floating wetlands (Veg Tech, Sweden; article no. 9-15055) with 78 evenly distributed wells (53 cm2) for planting macrophytes were used in the recipient lake. CFWs in the eco-tanks were constructed of the same material, but reduced in size to fit the water tanks and had 52 wells, each. We placed one CFW in each of the two eco-tanks and two CFWs in the recipient. We collected six native and common wetland macrophyte species near the recipient: Comarum palustre L. (purple marshlocks), Equisetum fluviatile L. (water horsetail), Carex rostrata Stokes (bottle sedge), Eriophorum angustifolium Honck. (common cottonsedge), Filipendula ulmaria (L.) Maxim. (meadowsweet) and Menyanthes trifoliata L. (bog bean). Every macrophyte species was represented in every CFW (Fig. 1). CFWs contained a total of 5–10 (eco-tanks) and 6–10 (recipient) replicates per species (see Table S1). Macrophytes were planted in the CFWs in June 2014 and cultivated until August 2016. The CFWs in the recipient were anchored with iron chains attached to concrete weights.
Bulk PDA =
2.3. N Accumulation in plant biomass
(
Biomass area
) × N conc. Gp
(2)
where Biomass = aboveground biomass (kg dry weight); area = area of planting well (0.0053414 m2); PDA = potential denitrification activity (mg N2O-N kg dry weight−1 day−1).
We harvested individual macrophytes in each well in August 2016. In the eco-tanks, 1–3 specimens per species and CFW were harvested resulting in a total of 3–6 specimens per species and in the recipient experiment, 1–5 specimens of each species per CFW resulting in total 4–7 specimens per species (see Table S1). Aboveground biomass was separated from root biomass. Aboveground or photosynthetic biomass (in case of C. palustre and M. trifoliata that have roots attached with stems) was dried at 50 °C for seven days to determine dry weight (DW) of each specimen. The N content in the plant biomass was analysed at the Forest Research Lab, Farnham, UK, according to ISO 10,694 & 13878. We calculated standing plant biomass based on well area and used the N content in plant biomass to calculate bulk N accumulation (mg N m−2 day−1) by aboveground (or photosynthetic) biomass, assuming an active growing period of 120 days (Raab and Vedin, 1995) (Eq. (1)).
Bulk nitrogen accumulation =
(Biomass × 2.1) × PDA area
2.5. Potential N removal pathways The ratio between potential denitrification activity and N accumulation in plant biomass was calculated according to Hallin et al. (2015) to determine the relative importance of denitrification and plant accumulation of N, for nitrogen removal associated with different macrophyte species (Eq. (3)).
Nitrogen removal pathway =
Bulk PDA Bulk N accumulation
(3)
A value above 1 indicates denitrification as the main N removal pathway, whereas a ratio below 1 indicates plant accumulation as the main N removal pathway (for calculation of bulk N accumulation and bulk PDA see Eqs. (1) and (2), respectively).
(1)
2.6. Data analyses
where Biomass = aboveground biomass (kg dry weight); area = area of planting well (0.0053414 m2); N conc. = nitrogen concentration in aboveground biomass (mgN kg dry weight−1); Gp = active growing period (120 days).
One-way ANOVA was performed to test for differences among macrophyte species in standing biomass, bulk plant N accumulation, bulk denitrification and potential N removal pathways, with separate ANOVAs conducted for the eco-tanks and recipient. To meet the assumptions of parametric tests, data was log transformed for standing 128
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biomass, bulk plant N accumulation and bulk denitrification, and square root transformed for potential N removal pathway. Adjustment for multiple comparisons was made by Tukey’s HSD. Statistical analyses were performed using the software IBM SPSS Statistics for Macintosh, Version 22.0 (Armonk, NY: IBM Corp).
et al., 2015; Aerts et al., 1992). In the eco-tanks, F. ulmaria showed higher bulk N accumulation compared to E. fluviatile and M. trifoliata (Fig. 2b, Table S2). Similarly, F. ulmaria had higher bulk accumulation compared to E. fluviatile, M. trifoliata and E. angustifolium in the recipient whereas C. palustre had higher bulk N accumulation compared to E. fluviatile and M. trifoliata (Fig. 2f, Table S3). Overall, bulk N accumulation in macrophytes was generally lower than that of other species planted in CFWs in warmer climatic regions, i.e. tropical, subtropical, temperate and continental climates (Table 3). Low summer temperature and a short growing season are the main controlling factors for biomass production of macrophytes in the sub-arctic region (Solander, 1983), which likely explains the lower bulk N accumulation by macrophytes when compared to temperate or tropical regions. Even though our study was conducted in a cold climate, the observed N accumulation by C. palustre and F. ulmaria in particular is comparable with that by commonly used macrophyte species in constructed wetlands, e.g. Typha spp. and Phragmites australis (Table 3).
3. Results and discussion 3.1. Establishment and standing biomass of macrophytes Our study demonstrates that CFWs, frequently applied in tropical and temperate climates (Pavlineri et al., 2017), also effectively remove nitrate from freshwater systems under cold climatic conditions. The successful establishment and survival of all six macrophyte species is partly likely to reflect the use of local-sourced plant specimens and species, rather than the more usual practice of purchasing plants from commercial cultures for deployment in CFWs. Standing biomass of macrophytes differed significantly among species, both in eco-tanks and the recipient (Table 2). In the eco-tanks, C. rostrata and F. ulmaria showed a higher standing biomass compared to E. fluviatile and M. trifoliata, whereas C. palustre showed higher standing biomass than E. fluviatile. No differences in standing biomass were observed between other species (Table S2). In the recipient, M. trifoliata had the lowest biomass production compared to all other species except E. fluviatile (Table S3). E. fluviatile had lower biomass production compared to C. rostrata, C. palustre and F. ulmaria (Table S3). In line with this, E. fluviatile in general has low aboveground biomass compared to belowground biomass (Grime et al., 2007) and M. trifoliata usually occurs in oligotrophic habitats (Fitter and Peat, 1994). Further, F. ulmaria and C. rostrata prefer eutrophic and mesotrophic habitats, respectively (Grime et al., 2007; Fitter and Peat, 1994), whereas F. ulmaria is a competitive species (Grime et al., 2007). The standing biomass of C. rostrata (2.2 kgDW m−2) in the recipient and eco-tanks was greater than that reported by Aerts et al. (1992) after growing this species in situ for 2 years (1.2 kg-DW m−2), and also exceeds the biomass observed for this species in a boreal fen (25.2 g-DW m−2) (Saarinen, 1996). This is most likely to reflect the very high nitrate levels in our study. Overall, our results suggest that the ecology and known life strategies of the studied macrophytes determined their biomass development in the CFWs.
3.3. Potential denitrification activity (PDA) and dominant N removal pathway In the eco-tanks, PDA was higher for C. palustre compared to E. fluviatile, whereas there was no difference among the remaining species (Fig. 2c, Table S2). In the recipient, C. palustre and F. ulmaria had higher PDA compared to the other species (Fig. 2g, Table S3), with PDA being negligible for M. trifoliata in both systems (Fig. 2c, g, Table S2 and S3). M. trifoliata is known to have a high allelopathic potential (Grutters et al., 2017) that might inhibit bacterial activity in close vicinity of roots. In our study, potential N removal rates through denitrification in the eco-tanks ranged between 32 and 2250 mg N2O-N m−2 day−1. These high rates are likely to underpin the complete removal of the NO3− loading (1528 mg NO3-N m−2 day−1) in the clarification pond at the studied mining area. All macrophyte species in the CFWs showed denitrification as the main N removal pathway (Fig. 2d, h). This result is consistent with previous studies in constructed wetlands (Hallin et al., 2015; Van Cleemput et al., 2007). The root-associated denitrification activity differed among macrophyte species in our study, but was not related to plant N accumulation. This is in contrast to previous work showing that macrophytes that accumulate more N have lower root associated denitrification activity and vice versa (Hallin et al., 2015). The nitrate supply around the roots in hydroponically cultivated species is likely to be greater than for species growing in wetlands that rely more on nitrate in the sediment. This might reduce the importance of competition for nitrate between the denitrifiers and the plants as a control of denitrification activity, potentially explaining the decoupling of root-associated denitrification and plant N accumulation observed in CFWs in the current study. Our study highlights the importance of considering denitrification activity associated with macrophyte roots in CFWs, which has surprisingly rarely been reported in previous studies (Table 3). Denitrification potential in CFWs has been measured in biofilms on rice straw and plastic materials (Zhang et al., 2018, and the references therein). However, the periphyton of macrophyte roots and submerged shoots can significantly contribute to denitrification in wetlands (Hallin et al., 2015; Eriksson and Weisner, 1997) and more efforts are needed to estimate the quantitative importance of denitrification for nitrate removal in CFWs.
3.2. N accumulation in plant biomass Plant bulk N accumulation differed significantly among species in all CFWs (Table 2) and largely coincide with plant biomass development. This indicates that certain macrophyte species are better than others for N removal by plant assimilation (Fig. 2b, f; Table S2 and S3), which agrees with previous studies (Choudhury et al., 2018; Hallin Table 2 One-way ANOVA testing the effect of macrophyte species on standing plant biomass (g-DW m−2), N accumulation by plant biomass (mg N m−2 day−1), potential denitrification activity (PDA) (mg N2O-N m−2 day−1) on roots of macrophytes and main N removal pathways, i.e. ratio between PDA and N accumulation in constructed floating wetlands (CFWs) investigated in cold climate. System
Response variable
df
Eco-tank
Standing biomass Plant N accumulation PDA PDA: N accumulation
5, 5, 5, 5,
Recipient (or in situ)
Standing biomass Plant N accumulation PDA PDA: N accumulation
5, 5, 5, 5,
F
p
19 19 19 19
5.53 5.94 63.94 116.77
** ** *** ***
29 29 29 29
13.46 11.32 41.61 279.90
*** *** *** ***
4. Final remarks We showed that CFWs planted with macrophytes could be used for treatment of nitrate contaminated mine water under cold climatic conditions. Macrophyte-mediated denitrification, resulting in removal of NO3- from the water, was the main N removal pathway in the CFWs. However, the choice of macrophyte species will determine the overall nitrate removal by plant bulk N accumulation and denitrification, and
*** < 0.001, ** < 01, * < 0.05, ns = not significant. 129
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Fig. 2. Operation of constructed floating wetlands in eco-tanks and the recipient (in situ) for nitrogen (N) removal from surface water by six macrophyte species (Comarum palustre, Equisetum fluviatile, Carex rostrata, Eriophorum angustifolium, Filipendula ulmaria and Menyanthes trifoliata), a, e) standing plant biomass, b, f) N accumulation in plant biomass, c, g) potential denitrification activity (PDA) associated with macrophytes roots, d, h) ratio between PDA and plant accumulation of N (a ratio value > 1 indicates denitrification as main N removal pathway while a value < 1 indicates plant accumulation as main N removal pathway). Different numbers above the bars indicate significant differences between plant species (p < 0.05).
successful application of CFWs will rely on the correct choice of macrophyte species. Given the demonstrated utility of both land-based CFWs and those in water bodies, future studies should focus on
designing CFWs that support enhanced denitrification as this process outcompetes that of plant bulk N accumulation and also leads to permanent removal of nitrate from water. 130
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Table 3 Nitrogen (N) accumulation and potential denitrification activity (PDA) associated with macrophyte roots in constructed floating wetlands applied in different climatic regions and systems. Plant species
N accumulation (mg N m−2 day−1)a
PDA (mg N m−2 day−1)
System
Climatic region
References
Typha angustifolia Iris pseudacorus Iris pseudacorus Typha orientalis Phragmites australis Phragmites australis Phragmites australis Juncus effusus Juncus effusus Canna flaccida Scirpus validus Salix babylonica Alisma subcordatum Carex stricta Iris versicolor Pontederia cordata
13.2 204.4 161.3 44 27.1 – 65 71 390 220 95 34.4 12.2 168.1 62.1 85.3
– – – – – 947.3 – – – – – – – – – –
CFW-mesocosm CFW-mesocosm CFW-mesocosm CFW- mesocosm CFW- mesocosm CFW- microcosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- mesocosm CFW- in situ CFW- in situ CFW- in situ CFW- in situ
n.m n.m. n.m sub-humid continental sub-humid continental n.m n.m n.m Humid sub tropical Humid sub tropical sub-humid continental Temperate Humid subtropical Humid subtropical Humid subtropical Humid subtropical
Keizer-Vlek et al. (2014) Keizer-Vlek et al. (2014) Gao et al. (2018) Wu et al. (2011) Wu et al. (2011) Prajapati et al. (2017) Saad et al. (2016) Saad et al. (2016) White and Cousins (2013) White and Cousins (2013) Wu et al. (2011) Zhu et al. (2011) McAndrew et al. (2016) McAndrew et al. (2016) McAndrew et al. (2016) McAndrew et al. (2016)
a
Mass uptake was given as g/m2 and was divided by tissue sampling interval (days); n.m. = not mentioned.
Declaration of Competing Interest
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