research 46 (2012) 5102e5114

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Halophyte filter beds for treatment of saline from

J.M. Webb a, R. Quinta˜ a, S. Papadimitriou a, L. Norman a, M. Rigby b, D.N. Thomas a,c, L. Le Vay a,* a School of Sciences, College of Natural Sciences, Bangor University, Wales, LL59 5AB, UK b Llyn Aquaculture Ltd, Afonwen Farm, Chwilog, Pwllheli, Gwynedd, Wales, LL53 6TX, UK c Finnish Environment Institute, Marine Research Center, Helsinki, Finland article info abstract

Article history: The expansion of aquaculture and the recent development of more intensive land-based Received 22 December 2011 marine farms require efficient and cost-effective systems for treatment of highly Received in revised form nutrient-rich saline wastewater. Constructed with halophytic offer the 11 June 2012 potential for waste-stream treatment combined with production of valuable secondary Accepted 20 June 2012 crops. Pilot filter beds, constructed in triplicate and planted with the - Available online 28 June 2012 marsh plant europaea, were evaluated over 88 days under commercial operating conditions on a marine fish and shrimp farm. waste was primarily in the form of Keywords: dissolved inorganic nitrogen (TDIN) and was removed by 98.2 2.2% under ambient 1 Æ Salicornia loadings of 109e383 mmol lÀ . There was a linear relationship between TDIN uptake and 1 Saltmarsh loading over the range of inputs tested. At peak loadings of up to 8185 590 mmol lÀ 2 1 Æ (equivalent to 600 mmol N mÀ dÀ ), the filter beds removed between 30 and 58% 2 1 Filter bed (250 mmol N mÀ dÀ ) of influent TDIN. Influent dissolved inorganic phosphorus levels 1 Aquaculture ranged from 34 to 90 mmol lÀ , with 36e89% reduction under routine operations. Dissolved 1 Waste water organic nitrogen (DON) loadings were lower (11e144 mmol lÀ ), and between 23 and 69% of influent DON was removed during routine operation, with no significant removal of DON under high TDIN loading. Over the 88-day study, cumulative nitrogen removal was 2 2 1.28 mol mÀ , of which 1.09 mol mÀ was retained in plant tissue, with plant uptake 1 1 ranging from 2.4 to 27.0 mmol N gÀ dry weight dÀ . The results demonstrate the effec- tiveness of N and P removal from wastewater from land-based intensive marine aqua- culture farms by constructed wetlands planted with S. europaea. ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction Marine and coastal aquaculture represent a significant component of seafood production and current expansion of Recent estimates show that aquaculture provides 47% (51 this sector is largely the result of the intensification of marine million MT) of global human fish consumption (FAO, 2009). In farming of carnivorous fish and shrimp species in coastal order to keep up with population growth and increasing per cages and ponds. In the main, these are ‘flow through’ or ‘open capita fish consumption, aquaculture output is set to increase systems’ which can discharge high volumes of wastewater by a further 60e100% over the next 20e30 years (FAO, 2009). containing suspended solids (excreta and food waste) and

* Corresponding author. Tel.: 44 1248 388115. þ E-mail address: [email protected] (L. Le Vay). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2012.06.034 water research 46 (2012) 5102e5114 5103

dissolved metabolites in the form of organic matter and value as an oil seed crop, a seasonal vegetable and also for inorganic nitrogen and phosphorus. With fish farms being potential use in the health, beauty and nutraceutical indus- subject to best practice codes of conduct (Boyd, 2003), and tries (Glenn et al., 1991; Rhee et al., 2009). The present study set legislation imposing financial penalties on the polluter (e.g. EU out to test the effectiveness of a CW planted with Salicornia Water Framework Directive 2000/60/CE), development of europaea agg. (L) for treating effluent water from a commer- land-based intensive marine recirculating aquaculture cially-operating marine fish and shrimp RAS. systems (RAS) offers significant potential to treat reduced volumes of wastewater prior to discharge (Tal et al., 2009). In conventional aquaculture 2. Materials and methods systems, solids are removed by gravitational and/or mechanical methods. Settlement is used to remove the denser 2.1. Filter design and operation solids, while filtration (commonly screen filtration, expand- able granular biofilters (EGBs), and foam fractionation) is used Triplicate pilot water treatment filter beds were installed in for removing suspended and fine solids (Cripps and Bergheim, a single span poly-tunnel, 5 m 20 m (W L) on an intensive   2000; Piedrahita, 2003). Removal of dissolved metabolites marine fish farm in Pwllheli, North Wales, UK. Each bed had requires oxidation of organic matter and steps to promote 14.5 m2 surface area and 4.35 m3 volume (1 m 14.5 m 0.3 m,   nitrification or denitrification. Ion-exchange and carbon filters W L H ). The filter beds were constructed of timber frames   are quickly biofouled and ion-exchange filters are rendered on a sand base, with butyl rubber liners, and filled to 200 mm inactive in ion-rich and these methods, although depth with 40 mm clean, graded, single size smooth limestone effective, are costly both in terms of capital investment, (Cefn Graianog Quarry, Chwilog, UK), to allow subsurface flow. energy consumption and maintenance requirements. This was overlaid with a 100 mm layer of 6 mm, mixed M  Constructed wetlands (CW) are a well-established, cost- grade quarry to a depth of 100 mm (Fig. 1). The two layers were effective, method for treating wastewater, such as municipal separated by a sheet of plastic mesh (2 mm2 pore size; www. or domestic , industrial and agricultural wastewater, boddingtons-ltd.com insect mesh, ref. 47000) overlaid with 2 landfill , and runoff. A constructed 17 g mÀ geotextile frost protection fleece (http://www. wetland is a system engineered to recreate the vegetation, lbsgardenwarehouse.co.uk, ref. R-F2050). This combination sediment, and microbial assemblages found within its natu- was designed to provide a semi-permeable barrier, preventing rally occurring counterparts, whilst utilizing the biogeo- sand from falling into the pore spaces between the larger chemical processes occurring therein (Vymazal, 2005). The stones below, whilst allowing root growth into the lower stone role of the higher plants is crucial in establishing a successful layer. At each end, a perforated 300 mm diameter PVC pipe CW since they maintain the hydraulic conductivity of the was fitted vertically through the gravel and sand layers, substrate, increase microbial assemblages in the root zone serving as both filling and sampling points as well as water and participate in nutrient uptake. CW design usually consists level indicators. of a lined bed filled with porous media (generally rock or Salicornia europaea agg (L) were grown from seeds taken gravel) and planted with emergent hydrophytes and from 2nd generation cultivated plants. Germination took commonly employed designs incorporate horizontal sub- place in a controlled environment greenhouse (photoperiod of surface flow (HF), where pre-treated waste water enters the 16 light:8 dark and of 18 C). Seeds were sown CW at the inlet and travels slowly down the length of the bed, onto the surface of P576 plug trays filled with John Innes No.1 passing through the filtration media under the surface of the and irrigated with . After two weeks, bed until it reaches the outlet/discharge point. Remediation seedlings emerged and the trays were thinned out to 1 plant relies on a combination of physical, chemical and biological per plug. was increased to 10 psu with processes, sedimentation, precipitation, volatilization, TROPICMARINä artificial salt and seedlings received adsorption to soil particles, plant uptake and microbial Phostrogen soluble plant feed (N:P:K 14:10:27 trace þ conversion. Several pilot scale and field studies have been elements; Bayer CropScience Ltd, Cambridge, UK). Two- carried out and demonstrated the viability of using CWs to month old plants were transplanted into the filter beds at 2 treat aquaculture wastewater, mainly in freshwater and some a density of 90 mÀ , so that each bed contained approximately in systems (e.g. Lin et al., 2005), however, to 1250 plants. date there has been limited work using CWs with marine The pilot filter beds processed waste water from systems (Lymbery et al., 2006). a commercially-operating intensive recirculating marine Freshwater RASs are ideally suited to , where aquaculture facility (Llyn Aquaculture Ltd) producing shrimp, plants are grown in an inert medium (e.g. perlite, sand, gravel, sole and turbot (Fig. 2). They were operated on a batch- rockwool) supplied with wastewater from the RAS, with the treatment, flood and drain system, as standard water aim of reducing overall organic and inorganic nutrient load management for the facility requires once-daily discharge of while producing a crop (Lennard and Leonard, 2006). To apply batches of waste water flushed from sediment traps, and a similar approach to marine systems would require weekly batches of waste water used to backflush biofilter and a commercially-valuable halophytic plant that can thrive in bead filter units. All wastewater drained to an outdoor, saline wastewater (Calheiros et al., 2012). One of the few uncovered settlement pond (10 4 0.5 m), from which it was   candidates is samphire (Salicornia spp.), an edible succulent pumped (400 W 240 V automatic dirty water pump; www. leafless genus that grows in saltmarshes and saline environ- screwfix.com) into a covered and lined 12 m3 header tank ments (Davy et al., 2001). Salicornia spp. have a commercial via a 300 L vortex separation tank containing plastic biofilter 5104 water research 46 (2012) 5102e5114

Fig. 1 e Cross-section of pilot filter bed (a) Lengthwise and (b) width wise.

media removing suspended particulate matter. The header HI 98127 pH meter. Salinity was measured with a WTW sali- tank wastewater was pumped to each of the three filter beds nometer. Water temperature was measured using a Tinytag via a 25 mm flexible pipe and a Kent V110 25 mm volumetric Aquatic (TG-3100) data logger, while ambient air temperature flow meter. The filter beds were filled to just below the surface and humidity were measured on a Tinytag View 2 Int Temp/ of the upper sand layer to prevent algal fouling of the filter RH (TV-4500) data logger (http://www.geminidataloggers. surface and each was fitted with a moveable standpipe which com). Prior to filling, filter beds were flushed to remove could be lowered to allow complete draining of the systems. water remaining from the previous flooding. Immediately The interval between each flood and drain procedure was 24 h. after re-filling, triplicate 20 ml water samples were taken from Sub-surface flow through the gravel layer was continuously each filter bed using a 20 ml disposable sterile plastic syringe maintained by a submersible 18 W pump (Hozelock Cascade fitted with a 30 cm length of acid-washed Teflon tubing. 700) located in the sump at one end of each filter bed and Sample water was flushed through the syringe and tubing discharging via a 15 mm flexible hose to the sump at the twice before the sample was then passed through a Whatman distant end (Fig. 1b). GD/X syringe filter (25 mm, nominal pore size of 0.45 mm) into an acid washed 20 ml plastic bottle. A fourth 20 ml water 2.2. Simulated high nitrogen load sample was taken from each bed, and was filtered similarly into 2 5 ml glass vials and fixed with phosphoric acid for  The pilot operation of the filter beds ran for a total of 88 days. subsequent dissolved organic carbon (DOC) analyses. All

After the first 58 days, nitrogen loading was increased by samples were frozen ( 20 C) until analyses. Following each À dosing the header tank with ammonium nitrate agricultural fill, the content, pH and the salinity of the water in grade fertiliser, to increase nitrogen concentrations to those each filter bed were measured. Evaporation and transpira- known to occur at peak discharge levels for this facility. Apart tional water loss was quantified by topping up each filter bed from this, water management, filter bed operation and to original fill levels after 24 h. sampling remained unchanged until the end of the test period. 2.4. Plant growth

2.3. Water sampling The plants in each filter were cropped every three weeks, with all growth 10 cm above the sediment surface removed. The Water samples were taken at 0 and 24 h after filling, three total plant biomass removed from each filter bed at each times a week from each of the 3 filter beds over the 88 days. cropping is termed plant yield. Prior to each cropping, four Water pH within each filter bed was measured using a Hanna randomly selected plants were removed completely from Fig. 2 e Schematic overview of water flow from seawater intake, through aquaculture production units to wastewater treatment. 5106 water research 46 (2012) 5102e5114 each filter bed. The roots were rinsed carefully to remove 2.7. Statistical analysis sand, and plants were separated into below-ground (root) and above-ground biomass, which were oven-dried at 50 C and Analysis of water samples produced seven data sets of NH4þ weighed, Subsamples were then taken and were finely ground NO2À,NO3À, DIP, DOC, DON, and DOC:DON for both influent and for biochemical analysis. effluent, as well as for ambient and high nutrient loading. Each nutrient data set was analyzed independently, to deter- mine significant differences between influent and effluent 2.5. Water analysis during the periods of ambient and high nutrient loading, and between replicate filter beds. All statistical analyses were

Dissolved ammonium (NH4þ) was determined with the fluori- conducted using MINITAB. The DON and DOC:DON data taken metric method of Holmes et al. (1999) using a HITACHI F2000 during ambient nutrient loading were normally distributed

fluorescence spectrophotometer. The major dissolved inor- with homogeneity of variance, but the NH4þ, DIP and DOC data ganic nutrients, nitrate, nitrite, dissolved inorganic phos- taken at this time required log transformation to meet the phorus (DIP) and silicic acid [Si(OH)4], were determined assumptions for normality and homogeneity of variance. All using standard colourimetric methodology (Grasshoff et al., then underwent ANOVA to compare the replicates and paired 1983) as adapted for flow injection analysis (FIA) on t-test to compare influent and effluent means. Log trans- a5echannel LACHAT Instruments Quick-Chem 8000 auto- formation had little effect on the normality and homogeneity analyzer (Hales et al., 2004). These analyses were monitored of variance of the pre-enrichment NO2À and NO3À and DOC daily using certified reference oceanic water (batch 54, Scripps data, and the influent and effluent medians were compared Institute of , University of California, San Diego) using the Wilcoxon signed rank test, and differences between as an external standard and riverine water collected from the the replicates were tested using the KruskaleWallis test. The

River Clwyd, UK, as an internal standard. The external stan- post-enrichment NH4þ,NO3À, DON and DOC data were nor- 1 dard yielded: [NO2À] 0.02 ( 0.02) mmol kgÀ (n 34; reported mally distributed with homogeneity of variance and a one- ¼1 Æ ¼ 1 [NO2À] 0.00 mmol kgÀ ), [NO3À] 1.27 ( 0.15) mmol kgÀ (n 31; way ANOVA was used to compare replicates and paired ¼ ¼1 Æ ¼ 1 reported [NO3À] 1.25 mmol kgÀ ), [DIP] 0.34 ( 0.03) mmol kgÀ t-tests to compare influent and effluent means. The post- ¼ ¼ 1Æ (n 27; reported [DIP] 0.39 mmol kgÀ ). The internal enrichment DIP and NO2À data were log transformed, to ach- ¼ ¼ 1 standard yielded, [NO2À] 1.70 ( 0.12) mmol kgÀ ieve normality and homogeneity of variance, prior to ANOVA ¼ Æ 1 (n 12), [NO3À] 117 ( 3) mmol kgÀ (n 17), to compare the replicates and paired t-tests to compare ¼ ¼ Æ 1 ¼ [DIP] 2.60 ( 0.18) mmol kgÀ (n 11), and influent and effluent means. Log transformation had little ¼ Æ 1 ¼ [Si(OH) ] 17 ( 1) mmol kgÀ (n 13). effect on the normality and homogeneity of variance of the 4 ¼ Æ ¼ Dissolved organic nitrogen (DON) was determined by post-enrichment DOC:DON data and the influent and effluent subtraction of NO3À,NO2À and NH4þ from the total dissolved medians were compared using the Wilcoxon signed rank test, nitrogen (TDN) analyzed by FIA on the LACHAT autoanalyzer and differences between the replicates were tested using the using on-line peroxodisulphate oxidation coupled with KruskaleWallis test. ultraviolet radiation at pH 9.0 and 100 C(Kroon, 1993), with The effluent and nutrient concentrations and DOC:DON 1 1 a precision of 3 mmol kgÀ at 133 mmol TDN kgÀ and were (with the exception of DON during the period of high 1 1 1 mmol kgÀ at 9 mmol DON kgÀ based on the internal riverine nutrient loading) in all cases significantly lower at p 0.001  water material described above. Dissolved organic carbon than the measured influent levels, and in all cases there was (DOC) was determined by high temperature combustion on an no significant difference at p 0.05 between measurements  MQ 1001 TOC analyzer calibrated daily with potassium taken across the replicate filter beds. Therefore all data, phthalate (Qian and Mopper, 1996). The performance of the unless otherwise stated, will be quoted as the mean 1 analyzer was tested daily on the certified reference material of concentration standard deviation (mmol lÀ ). Æ deep (700 m) Florida Strait seawater (Hansell Laboratory, 1 University of Miami, RSMAS; batch 5 FS: 47e48 mmol lÀ ), the 1 method yielded 46 ( 3) mmol lÀ (n 74). Deep oceanic water, Æ ¼ collected on 19/11/04 at 1000 m depth in the 3. Results and used throughout as an internal standard, yielded a similar 1 precision at a low DOC concentration (59, 3 mmol lÀ , n 35). Air temperature and humidity over the duration of pilot Æ ¼ operation are shown in Fig. 3, and influent and effluent water temperature, salinity and pH are shown in Fig. 4. Air 2.6. Plant tissue analysis within the poly-tunnel ranged from 8.2 to

55.0 C (mean 1s: 23.1, 10.8 C). Humidity ranged from 19 to Æ Æ Plant nitrogen content was determined using a CARLO ERBA 100% RH (mean 1s:78 25%). In all cases there was no Æ Æ NA 1500 Elemental CHN Analyzer with finely ground plant significant difference ( p 0.05) between  material following weighing into pre-combusted (500 C, 3 h) measurements taken across the replicate filter beds. Influent silver boats, acidification with 10% (w/v) HCl, and oven-drying water temperature across the three replicate filter beds ranged at 40 C. Plant phosphorus was determined on ground plant from 16.5 to 26.1 C (mean 20.2 1.7 C), and effluent water Æ subsamples the methods of Solo´ rzano and Sharp (1980) as temperature ranged from 17.4 to 25.7 C (mean 1s: Æ modified by Fourqueran and Zieman (1992) for particulate P 21.5 1.8). Influent salinity ranged from 10 to 29 psu Æ determination. (mean 1s:20 5 psu), while effluent salinity ranged from 14 Æ Æ water research 46 (2012) 5102e5114 5107

Fig. 3 e The mean daily air temperature (a) and humidity (b) with associated minima and maxima measured in the experimental polytunnel (days 179-245).

to 31 psu (mean 1s:22 4 psu) before adjustment for Æ Æ evaporation. Influent pH across the three replicate filter beds ranged from 6.8 to 8.8 while effluent pH ranged from 6.4 to 7.4. The mean pH of influent and effluent was 7.7 0.4 and Æ 6.9 0.2, respectively. Æ

3.1. Inorganic nutrients Fig. 4 e The mean salinity (a), pH (b) and temperature (c) of waste-water entering (influent) and exiting (effluent) 3.1.1. Ammonium (NH4þ) triplicate pilot filter beds. Influent NH4þ concentrations at ambient nutrient loading over the first 58 days of operation varied between 85.2 15.9 and 1 Æ 1 344.4 4.8 mmol lÀ (overall mean: 191.2 81.9 mmol lÀ ). The Æ Æ filter beds removed between 91 12% and w100% of influent Æ NH4þ, reducing its concentration in the effluent to between 1 0.7 0.3 and 16.5 20.9 mmol lÀ (Fig. 5a). 3.1.2. Nitrite (NOÀ) Æ Æ 2 As intended, the addition of the fertilizer increased the The influent NO2À concentrations at ambient nutrient loading

NH4þ concentration in the influent to a maximum of over the first 58 days of operation fluctuated between 4.2 0.4 1 1 1 Æ 4349 553 mmol lÀ . The NH4þ concentration then declined and 35.0 1.5 mmol lÀ (overall mean: 17.2 9.1 mmol lÀ ). The Æ 1 Æ Æ steadily to 1514 164 mmol lÀ over the following 7 days. The filter beds removed 90 9% to w100% (overall mean: 97 3%) Æ Æ Æ filter beds removed from 23 12% to 50 6% of influent NH4þ, of influent NO2À, reducing its concentration in the effluent to Æ Æ 1 resulting in effluent NH4þ concentration between 812 121 between 0.04 and 1.7 2.0 mmol lÀ (overall mean: 1 Æ 1 Æ and 3464 79 mmol lÀ (Fig. 6a). 0.4 0.5 mmol lÀ )(Fig. 5b). Æ Æ 5108 water research 46 (2012) 5102e5114

During the period of high nutrient loading, influent NO2À a 1 Ambient nutrient loading concentrations increased steadily from 27.9 30.8 mmol lÀ Æ 1 on day 1 to a maximum of 220.2 24.5 mmol lÀ on day 7 400 1Æ (overall mean: 135.3 80.8 mmol lÀ ). The filter beds removed 350 Æ 300 between 66 34% and 85 4% (overall mean: 75 8%) of ) Æ Æ Æ 250 influent NO2À, resulting in effluent concentrations ranging 1 200 from 3.5 0.7 to 59.8 8.9 mmol lÀ (overall mean: (µmol l (µmol ] Æ 1 Æ 150 29.1 23.3 mmol lÀ )(Fig. 6b).

[NH 100 Æ 50 3.1.3. Nitrate (NO3À) 0 The influent NOÀ concentrations at ambient nutrient loading -50 157 161 166 171 174 179 185 189 194 200 203 211 3 Day of the Year over the first 58 days of operation fluctuated between 4.0 0.2 1 1 Æ and 114 5 mmol lÀ (overall mean: 38.1 30.8 mmol lÀ ). The b Æ Æ 40 filter beds removed 91 4e100% (overall mean: 98 3%) of Æ Æ 35 influent NO3À, reducing its concentration in the effluent from 1 30 the filter beds to between 0.0 and 2.9 2.8 mmol lÀ (overall ) 25 1 Æ mean: 0.4 0.7 mmol lÀ )(Fig. 5c). 20 Æ

(µmol l (µmol During the 7 day period of high nutrient loading, the ] 15 concentrations of NO3À in the influent reached a maximum of [NO 10 1 3808 111 mmol lÀ on day 218 and then declined to 5 Æ 1 1 723 46 mmol lÀ (overall mean: 2023 1370 mmol lÀ ) over the 0 Æ Æ -5 157 161 166 171 174 179 185 189 194 200 203 211 subsequent 7 days before returning to ambient levels shortly afterwards. The filter beds removed from 33 7e83 2% Day of the Year Æ Æ (overall mean: 54 21%) of influent NOÀ, resulting in effluent c Æ 3 140 NO3À concentrations between 135.3 23.0 and 1 Æ 1 120 1824 274 mmol lÀ (overall mean: 1032 718 mmol lÀ )(Fig. 6c). Æ Æ 100 ) 80 3.1.4. Total dissolved inorganic nitrogen (TDIN) (µmol l (µmol

] 60 The TDIN concentration in the influent, at ambient nutrient 40 loading during the first 58 days of operation, fluctuated between [NO 1 20 108.9 19.5 and 383.0 6.9 mmol lÀ (overall mean: Æ 1 Æ 0 246.5 68.3 mmol lÀ ). The filter beds removed 91 9% to 157 161 166 171 174 179 185 189 194 200 203 211 Æ Æ -20 w100 0.1% (overall mean: 98 2%) of influent TDIN, thus Æ Æ Day of the Year reducing the TDIN concentration in the effluent to between 1 1 d 0.9 0.2 and 7.7 5.1 mmol lÀ (overall mean: 2.8 1.9 mmol lÀ ) Æ Æ Æ 450 (Fig. 5d). 400 During the 7 day period of high nutrient loading, the TDIN 350

) concentration in the influent reached a maximum of 300 1 250 8185 590 mmol lÀ on day 218 and then steadily decreased to Æ 1 1 (µmol l (µmol 2391 248 mmol lÀ (overall mean: 4914 2557 mmol lÀ ) over 200 Æ Æ 150 the subsequent 7 days before returning to ambient levels [TDIN] 100 shortly afterwards (Fig. 6d). The filter beds removed 50 30 34e58 4% (overall mean: 40 12%) of influent TDIN, 0 Æ Æ Æ resulting in TDIN concentrations in the effluent fluctuating -50 157 161 166 171 174 179 185 189 194 200 203 211 1 Day of the Year between 1007 144 and 5291 208 mmol lÀ (overall mean: Æ 1 Æ 3081 1812 mmol lÀ ). Although the percentage of TDIN e Æ 100 removed is greatly reduced in these experimental conditions 90 when compared to ambient nutrient loading, the removal was 80 ) 70 still high when considering the reduction in the TDIN 1 60 concentration by 2894 408 mmol lÀ in the first 24 h (µmol l (µmol 50 Æ 40 period, followed by reduction between 1037 30 and [DIP] 30 1 Æ 2017 36 mmol lÀ over each of the following periods of six day 20 Æ 10 each. Overall, the relationship between daily loading and 0 removal of nitrogen was linear over the range tested, and 157 161 166 171 174 179 185 189 194 200 203 211 higher rates of N removal may be achievable (Fig. 9). Day of the Year

Influent

Fig. 5 e The mean concentration (mmol lL1) (±SD) of dissolved inorganic ammonium (a), nitrate (b), nitrite (c), exiting (effluent) triplicate Salicornia planted filter beds TDIN (d) and DIP (e) in waste-water entering (influent) and during a period of ambient nutrient loading. water research 46 (2012) 5102e5114 5109

a High nutrient loading 3.1.5. Dissolved inorganic phosphate (DIP) 6000 Influent DIP concentrations at ambient nutrient loading over

5000 the first 58 days of operation fluctuated between 34.1 0.9 and 1 1 Æ ) 89.9 0.8 mmol lÀ (overall mean: 59.1 18.3 mmol lÀ ). The filter 4000 Æ Æ beds removed 36 6e89 2% (overall mean: 67 14%) of 3000 Æ Æ Æ (µmol l (µmol

] influent DIP, resulting in an effluent DIP concentration range of 2000 1 1 e À À

[NH 10.5 2.1 44.4 1.3 mmol l (overall mean: 19.7 10.0 mmol l ) Æ Æ Æ 1000 (Fig. 5e). During the period of high nutrient loading, influent DIP 1 0 concentrations reached a maximum of 354.1 21.7 mmol lÀ on 218 222 223 225 229 231 232 237 239 244 245 Æ -1000 day 218. The DIP concentrations declined steadily to Day of the Year 1 215.6 13.2 mmol lÀ over the following 7 days (overall mean: Æ 1 282.7 57.1 mmol lÀ )(Fig. 6e). The filter beds removed b Æ 300 19 3e40 27% (overall mean: 31 9%) of influent DIP, Æ Æ Æ 250 resulting in effluent DIP concentrations between 187.3 6.4 and 1 Æ1 ) 200 227.2 10.4 mmol lÀ (overall mean: 201.3 2.5 mmol lÀ ). Æ Æ 150 (µmol l (µmol ] 100 3.1.6. Nutrient ratios

[NO The TDIN in the settled fish farm wastewater used to irrigate the 50 pilot filter beds was proportionately higher in NH4þ than NO3À or 0 222 223 225 229 231 232 237 239 244 245 NO2À, with mean molar ratio of 10:2:1, and had a relatively -50 constant molar TDIN:DIP of 4:1. Over the first 58 days of oper- Day of the Year c ation, influent TDIN and DIP concentrations fluctuated as 4500 a result of varying fish stock, feed loading and intervals between 4000 biofilter flushing (Fig. 5d and e). When operating under ambient 3500

) nutrient loading, the relative composition of TDIN in the 3000 2500 effluent from the filter beds was similar to that of the influent (µmol l (µmol

] 2000 wastewater, with a mean NH4þ:NO3À:NO2À 14:2:1. However, the 1500 ¼

[NO mean TDIN:P in the effluent declined to approximately 1:5, 1000 reflecting the more efficient removal of TDIN in the filters beds. 500 0 For the first seven days after the addition of ammonium -500 218 222 223 225 229 231 232 237 239 244 245 nitrate fertiliser, the TDIN composition of the irrigation water Day of the Year (influent) changed, with a mean NHþ:NOÀ:NOÀ 24:17:1 and 4 3 2 ¼ d a mean TDIN:DIP 17:1. 10000 ¼ 9000 8000 ) 7000 3.2. Organic nutrients 6000

(µmol l (µmol 5000 4000 3.2.1. Dissolved organic carbon (DOC)

[TDIN] 3000 Influent DOC levels, at ambient nutrient loading, over the first 2000 58 days of operation (Fig. 7a) fluctuated between 335.7 96.8 and 1000 1 Æ 1360 11.3 mmol lÀ . The filter beds removed 3 65 to 59 7% of 0 Æ Æ Æ -1000 218 222 223 225 229 231 232 237 239 244 245 inlet DOC, significantly reducing the concentration in effluent Day of the Year 1 to between 295.7 112.2 and 823.7 66.5 mmol lÀ . Æ Æ e Following the addition of fertilizer, influent DOC levels 400 increased (Fig. 8a) ranging between 1382 215 and 350 1 Æ 1693 61 mmol lÀ almost double that observed at ambient

) Æ 300 levels. During the seven days of high nutrient loading, the 250 filter beds ability to remove DOC deteriorated slightly, with (µmol l (µmol 200 150 2 13 to 37 7% of influent DOC being removed, significantly [DIP] Æ Æ 100 reducing effluent concentrations to between 807 61 and 1 Æ 50 1442 1467 mmol lÀ . Æ 0 218 222 223 225 229 231 232 237 239 244 245 3.2.2. Dissolved organic nitrogen (DON) Day of the Year Influent DON levels, at ambient nutrient loading, over the first Influent Effluent 58 days of operation fluctuated between 45.3 21.8 and 1 Æ Fig. 6 e The mean concentration (±SD) of dissolved 144.1 6.2 mmol lÀ , with concentrations in effluents fluctu- Æ 1 inorganic ammonium (a), nitrate (b), nitrite (c), TDIN (d) and ating between 28.9 0.6 and 66.4 16.8 mmol lÀ (Fig. 7b). Æ Æ DIP (e) in waste-water entering (influent) and exiting During the first 7 days of operation (days 157e164), DON (effluent) triplicate Salicornia planted filter beds during concentrations in filter effluents were not significantly a period of high nutrient loading. reduced compared to influent values ( p > 0.05). For the 5110 water research 46 (2012) 5102e5114

Ambient nutrient loading High nutrient loading a a 1600 2000 1400 1800 1600 ) )

-1 1200 -1 1400 1000 1200 800 1000 600 800 [DOC] (µmol l [DOC] (µmol [DOC] (µmol l [DOC] (µmol 600 400 400 200 200 0 0 157 161 166 171 174 179 185 189 194 200 203 211 218 222 223 225 229 231 232 236 237 239 244 b Day of the Ye ar Day of the Ye ar b

180 900 160 800 700 )

) 140 -1 -1 600 120 500 100 400 80 300 200 [DON] (µmol l [DON](µmol [DON] (µmol l [DON] (µmol 60 100 40 0 20 218 222 223 225 229 231 232 237 239 244 0 Day of the Ye ar 157 164 168 174 179 185 189 194 200 203 211 c Day of the Ye ar c

Fig. 8 e The mean concentration (±SD) of dissolved organic carbon, DOC (a) and nitrogen, DON (b) and the DOC:DON Fig. 7 e The mean concentration (±SD) of dissolved organic ratio (c) in waste-water entering (influent) and exiting carbon, DOC (a) and nitrogen, DON (b) and the DOC:DON (effluent) triplicate Salicornia planted filter beds during ratio (c) in waste-water entering (influent) and exiting a period of high nutrient loading. (effluent) triplicate Salicornia planted filter beds during a period of ambient nutrient loading.

influent values. With the return to ambient loading (days 229e244) effluent values were significantly lower than those remainder of the ambient loading period (days 166e215) observed in the influent with filter beds removing 23 6 to Æ effluent values were significantly lower than those observed 63 9% of influent DON. Æ in the influent with filter beds removing 12 44 to 68 3% of Æ Æ influent DON with uptake gradually increasing with time. 3.2.3. DOC:DON In the seven days following addition of fertiliser, influent The observed increases and decreases in DOC and DON are DON concentrations (Fig. 8b) were approximately six times reflected in the organic DOC:DON ratios, with a changing higher than that seen during ambient nutrient loading pattern during the course of the experiment. Initially (days 1 ranging between 444.7 133.3 and 542.8 117.8 mmol lÀ , with 157e166), influent DOC:DON ratio values were significantly Æ Æ concentrations in effluents fluctuating between 438.1 107.4 higher than those in the effluent, indicative of the apparent 1 Æ and 667.0 161.9 mmol lÀ (days 218e229) before returning to greater removal of DON by the CW (Fig. 7c). By day 168 there Æ ambient levels shortly afterwards (days 231e245). During the 7 was no significant difference ( p > 0.05) between the influent days of high loading (days 218e225), DON concentrations in and effluent DOC:DON ratios, and between day 174e215, filter effluents were not significantly reduced compared to DOC:DON ratios were consistently higher in the effluent than water research 46 (2012) 5102e5114 5111

(40e42%). It is possible to calculate nitrogen removal efficiency with increasing N loading during the period before TDIN was depleted in the effluent water. Fig. 9 shows the relationship between the daily load and removal of TDIN from the pilot CW during the period of high nutrient loading. This shows that daily nitrogen removal increased linearly with increased loading, yielding a TDIN removal rate of 2 1 21e239 mmol N mÀ dÀ . However, the efficiency in terms of residual TDIN concentration in the effluent declined along the same nutrient loading gradient. Overall the time-integrated (cumulative) apparent nitrogen removal by the filters beds 2 was 1.28 0.05 mol N mÀ . Over the same period, total N Æ accumulation in plant tissue (estimated from N content and growth of plant root and shoot tissues and harvested biomass) 2 was 1.09 mol N mÀ , indicating that up to 85% of nitrogen e Fig. 9 Relationship between total inorganic nitrogen removed from waste water was retained in plant tissue (TDIN) daily load and TDIN removal from triplicate (Fig. 10). During the high nutrient loading period, S. europaea Salicornia planted filter beds during the period of high plant biomass in the filter beds did not vary significantly, with L2 L1 nutrient loading. Units are mmol N m d , equation of 2 a mean of 8.85 2.32 g mÀ . This suggests that under high [ D 2 [ Æ fitted curve is y 0.34x 19.68, R 0.969. Mean nutrient loading conditions, the plants were able to take up Salicornia europaea plant biomass during this period was 1 1 2.4e27.0 mmol N dÀ gÀ dry weight. L2 8.85 ± 2.32 g m . At ambient nutrient loading, the DIP removal efficiency averaged 67 14%, dropping to 31 9% during the period of Æ Æ high nutrient loading. However, the mean daily DIP removal at 2 1 the influent water reflecting the significant removal ( p < 0.05) this time increased from 2.81 1.24 mmol mÀ dÀ to 2 1 Æ of DON from the influent. In the 7 days following the addition 6.60 3.05 mmol mÀ dÀ . Overall, the apparent cumulative Æ 2 DIP removal by the filters beds was 0.11 0.01 mol mÀ . Over of ammonium nitrate fertiliser, there was no significant Æ difference ( p > 0.05) between the influent and effluent DOC:- the same period, total P accumulation in plant tissue, esti- 2 DON ratios (Fig. 8c), but on the return to ambient influent mated as outlined for nitrogen above, was 0.08 mol mÀ , levels by day 229, the previous pattern returns with DOC:DON indicating that up to 73% of DIP removed from waste water in the effluent significantly higher than the influent. was retained in plant tissue (Fig. 10).

3.3. Nutrient removal efficiency and retention in plants 4. Discussion During the first 58 days of the sampling period at ambient 1 nutrient loading, the filter beds removed 97e100% of the The concentrations of TDIN (93e439 mmol lÀ ) and DIP 1 influent TDIN. During the 7-day period of nutrient enrich- (34e90 mmol lÀ ) observed in fish farm wastewater in the ment, the filter beds exhibited lower TDIN removal efficiency present study were within the range, or even exceeded, those

1.2 0.12

1 0.10

0.8 0.08 )

0.6 0.06 (mol m mol m mol ] P [N] [

0.4 0.04

0.2 0.02

0 0.00 166 185 208 230 246 Day of the Year nitrogen phosphorus

Fig. 10 e The mean cumulative stored plant N and P calculated over five successive harvests from triplicate Salicornia planted filter beds. 5112 water research 46 (2012) 5102e5114

measured in the wastewater from other aquaculture RAS Salicornia virginica in salt marshes not only cause a shift in (Cripps and Bergheim, 2000; Porello et al., 2003; Lin et al., 2005). species composition in favour of S. virginica, reflecting an Overall, under the hydraulic regime applied during the 58 day ability to outcompete other plants for available N, period of ambient loading, the pilot CW acted as a highly but also produced significant increases in biomass (Covin and effective biofilter for the aquaculture effluent, removing Zedler, 1988; Boyer and Zedler, 1999). 91e99% of influent TDIN and 41e88% of influent DIP, Macrophytes play a key role in the performance of fresh- comparing favourably with those reported in small-scale water CW, facilitating the nitrification/denitrification process, experimental studies. Brown et al. (1999) achieved 98% maintaining the hydraulic conductivity of the substrate, and 94% removal total and inorganic nitrogen in the increasing microbial assemblages in the root zone and irrigation water and in the Lin et al. (2002a,b) experiment, participating in nutrient uptake (Brix, 1994; Haberl et al., 1995). the SSF section of their CW removed 95e98% of the Research into the role of plants in CWs tends to have focussed 1 1 0.15e21.5 mg TDIN lÀ (11e1537 mmol lÀ ) observed in the on freshwater systems with wetland plants such as Phragmites influent. These levels of nutrient removal efficiency are and Typha as essential components of the CW ecosystem, considerably greater than those reported by Lymbery et al. releasing oxygen from their root system into the rhizosphere (2006), where TDIN nutrient removal from RAS effluent and hence facilitating the establishment of the microbial using CWs was 69%. communities responsible for nitrogen removal (Faulwetter The addition of ammonium nitrate fertiliser to the fish et al., 2009). Like the rhizomes of macrophytes, the roots of farm effluent increased TDIN loading by an order of magni- salt marsh Salicornia sp., possess aerenchyma which allow tude above ambient levels. However, TDIN and DIP concen- exchange of gases between the shoot and the root and indi- trations remained high in the effluent, comparable to those rectly aerate the surrounding soil zone potentially resulting in reported in effluent from other aquaculture RAS systems increased nitrification/denitrification efficiency (Brix, 1994; (Pagand et al., 2000; Adler et al., 2000; Lin et al., 2005). Under Haberl et al., 1995; Faulwetter et al., 2009). The present CW was the hydraulic regime applied at high nutrient loading, the CW carefully designed to optimise plant and aerobic microbial operated at lower removal efficiency for TDIN (30e58%) and nutrient removal whilst promoting plant yield. The daily flood DIP (19e40%). However, overall assimilation of nitrogen by the and drain procedure, which simulated the tidal immersion bed was significantly higher under high loading conditions cycles experienced by saltmarsh plants, also allowed aeration and, as indicated by the linear relationship between loading at the root zone for several hours before re-submergence, thus and removal (Fig. 9), the limit of TDIN removal capacity was reducing the build-up of anaerobic conditions usually not reached over the range tested in the present study. The accountable for low nitrification (Vymazal, 2005). In addition, 2 1 highest removal rate observed (263 mmol mÀ dÀ ) greatly large pore spaces within the stone layer reduced clogging, exceeds those reported in previous studies in CWs. For whilst continuous subsurface circulation of water within each example, Lin et al. (2005) observed removal rates of bed improved nutrient flow across the root zone whilst 2 1 17 mmol N mÀ dÀ and Konnerup et al. (2011) reported reducing the build up of anoxic zones within the CW 2 1 a removal rate of 27 mmol mÀ dÀ . Several studies have substrate. investigated the efficiency of CWs under different HRT and In the present study, apparent DON removal depended on HLR, with a general trend of increasing efficiency with the total inorganic nitrogen loading to the filter beds. Under increasing HRT (eg Lin et al., 2005; Schulz et al., 2003) sug- ambient loading, TDIN was being almost completely removed gesting that during periods of high nitrogen loading, a HRT and DON was significantly reduced and there was a slightly greater than 24 h may increase N removal efficiency. Under higher DOC:DON ratio in effluent than influent water. By the hydraulic regime operated in the present study, if daily comparison, under experimentally-elevated TDIN loadings, 2 1 loading remained below 50 mmol mÀ dÀ , complete removal when there was excess TDIN in effluent water, there was no of TDIN in waste-water was achievable in 24 h. For higher apparent DON removal and the DOC:DON ratio increased as loading levels, the effectiveness of longer retention times may water passed through the filter beds. These results indicate that usefully be investigated. when TDIN was available in excess, DON removal was negli- The majority of nitrogen removal in wetlands is thought to gible suggesting that uptake of DON was by primary producers result from the microbial processes of nitrification and deni- (microbial community or plants). can uptake both trification and to a lesser degree, sedimentation, filtration, organic and inorganic N, and the DON may then be mineralized precipitation and volatization. Uptake by the plant is thought and made available to higher plants. Konnerup et al. (2011) to account for a minor fraction of the total reduction of observed apparent DON removal in a CW planted with Canna nutrients (Kadlec and Knight, 1996; Koottatep and Polprasert, x generalis, where 90% of TDN was present as DON and TDIN 1 1997; Lin et al., 2002b; Haddad et al., 2006). However there is concentrations were low (30e48 mmol N lÀ ). The ability of limited research available on the uptake capacity of Salicornia plants to uptake DON directly as free amino acids has been spp. and the highly effective removal of nitrogen by S. europaea widely demonstrated (see Na¨sholm et al. (2009) for a review), in the present study may reflect an adaptation in plants that and recently direct uptake of peptides has been measured have evolved to compete for and sequester nitrogen in what (Hill et al., 2011a, 2011b). Halophytic plants have been shown to are periodically nitrogen-limited environments. Nitrogen take up DON in quantities up to 20% of the DIN uptake, and DON availability in salt marshes not only varies throughout the uptake has been shown to be independent of DIN concentra- year but also spatially and peaks in available N tend to tion (Mozdzer et al., 2010). Direct uptake of amino acids and immediately precede a phase of vegetative growth (Jefferies, peptides by Salicornia plants has also been observed (R Quinta, 1977). In situ additions of inorganic nitrogen to stands of unpublished data), which would suggest that, as well as water research 46 (2012) 5102e5114 5113

bacteria, the plants themselves may have directly contributed well as providing a high value plant crop and biomass for to DOC and DON uptake within the filter beds. composting. However, S. europaea is an annual species and Nonetheless, DOC and DON were selectively removed in seasonal productivity and cost-effectiveness will depend on the CW as indicated by the differences in DOC:DON in the latitude and cost of artificial lighting. Ongoing work is inves- influent and effluent. This selectivity was not uniform over tigating the extension of the production season through use of the period studied, as the difference between influent and mixed communities of saltmarsh plants, including additional effluent DOC:DON shifted with time. These shifts may be biennial and perennial species that also have value as crops, a result of alterations in the nutrient supply, as bioavailability such as such Sarcocornia perennis and Aster tripolium. of DON depends on its composition (Stepanauskas et al., 2000), together with the progressive establishment of a microbial community and consequent population shifts: e.g. it may take 5. Conclusions from a month to a year for the microbial community to establish in a new CW (Lin et al., 2002a). Nitrogen in aquaculture wastewater was primarily in the  The CW acted as an effective biofilter for DIP in aquaculture form of dissolved inorganic nitrogen (TDIN) and was wastewater, removing 41e88% from influent water during the removed by 98.2 2.2% under ambient loadings of 58 day period of ambient loading, which is similar to values 1 Æ 109e383 mmol lÀ . reported in other aquaculture wastewater treatment systems There was a linear relationship between TDIN uptake and  (Lin et al., 2002a). Adsorption to sediment is considered to be loading over the range of inputs tested. At peak loadings of key mechanism for P retention in CW (Kadlec and Knight, 1 up to 8185 590 mmol lÀ (equivalent to mmoles 600 2Æ 1 1996). However, in the present study, over 70% of the DIP mmol N mÀ dÀ ), the filter beds removed between 30 and 2 1 removed from influent water was retained in plant tissue, 58% (250 mmol N mÀ dÀ ) of influent TDIN. indicating that sediments play a less important role in this Influent dissolved inorganic phosphate levels ranged from case. The use of coarse sand with a low adsorption capacity  1 34 to 90 mmol lÀ , with 36e89% reduction under routine might suit plant growth and the associated N uptake but this operations. appears to have reduced the effectiveness of the substrate in P Dissolved organic nitrogen (DON) loadings were lower uptake. However, phosphate uptake by S. europaea is likely to  1 (11e144 mmol lÀ ), and between 23 and 69% of influent DON be dependent on N supply. Webb (2005) found that in Salicornia was removed during routine operation, with no significant dolichostachya, a growth response to increasing P was only removal of DON under high TDIN loading. observed with excess N supply, suggesting that it is not Over the 88-day study, cumulative nitrogen removal was  2 2 limiting to growth. Over the 7 day period of nitrogen enrich- 1.28 mol mÀ , of which 1.09 mol mÀ was retained in plant ment, although the mean DIP removal efficiency from the tissue, with plant uptake ranging from 2.4 to 1 1 influent water dropped to 27.4%, uptake rose from 27.0 mmol N gÀ dry weight dÀ . 1 1 39.5 mmol lÀ to 81.5 mmol lÀ , suggesting that plant P The results demonstrate the effectiveness of treatment of  requirements increased with increasing N supply. wastewater from land-based intensive marine aquaculture Overall the pilot CW tested here performed well compared farms by constructed wetlands planted with S. europaea. to established alternative treatment systems. In a compara- tive study of various hydroponic systems with comparable influent N and P, Lennard and Leonard (2006) found gravel beds growing Lactuca sativa removed 91% of N and 53% of P, Acknowledgements while Porello et al. (2003) investigated the effectiveness of a system for phytotreatment of RAS waste and This study was supported by a European Union FP6 CRAFT observed only a 15% reduction in DIN from influent concen- project, Envirophyte (COOP-CT-2006-032167). RQ was sup- 1 ported by Fundac¸a˜o para a Cieˆncia e a Tecnologia, Portugal, trations of 67e69 mmol lÀ . Using a system of high rate algal ponds for RAS effluent treatment and with DIN concentra- (SFRH/BD/43234/2008) 1 tions in pond influent at 700 mmol N lÀ , Pagand et al. (2000) observed 59% removal efficiency over a 14 month period. references Present results also compare favourably with traditional bio- filtration and mechanical filtration, which can achieve 74% and 44% reduction in NH4þ and NO3À, respectively, in waste- Adler, P.R., Harper, J.K., Takeda, F., Wade, E.M., Summerfelt, S.T., water from temperate marine fish farms (Borges et al., 2003). 2000. Economic evaluation of hydroponics and other The high removal efficiency of DIN under ambient nitrogen treatment options for phosphorus removal in aquaculture loading and the significant removal of DIP indicate that con- effluent. Horticultural Science 35 (6), 993e999. structed wetlands planted with Salicornia are well-suited to Borges, M.-T., Morais, A., Castro, P.M.L., 2003. Performance of the treatment of batches of highly concentrated wastewater outdoor seawater treatment systems for recirculation in an released during routine filter backwashing in marine RAS. The intensive turbot (Scophthalmus maximus) farm. Aquaculture e high nutrient concentrations support high plant growth rates International 11 (6), 557 570. Boyd, C.E., 2003. Guidelines for aquaculture effluent management and hence efficient N uptake rates. Under normal RAS fish at the farm-level. Aquaculture 226 (1e4), 101e112. farm nutrient loading, the present study demonstrates that Boyer, K.E., Zedler, J.B., 1999. 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