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Marine Pollution Bulletin 50 (2005) 1386–1397

Inorganic nitrogen control in wastewater treatment pondsfrom a fish farm (Orbetello, Italy): Denitrification

versus Ulva uptake

Marco Bartoli a,*, Daniele Nizzoli a, Mariachiara Naldi a, Luigi Vezzulli b,Salvatore Porrello c, Mauro Lenzi d, Pierluigi Viaroli a

a DSA, Universita di Parma, 43100 PR, Italyb DIPTERIS, Universita di Genova, 16142 GE, Italy

c ICRAM, 300-00166 Rome, Italyd LEALab, OPL s.r.l., 58015 Orbetello, GR, Italy

Abstract

The aim of this study was to quantify the N removal efficiency of an Ulva-based phytotreatment system receiving wastewatersfrom a land-based fish farm (Orbetello, Italy), to identify the main biogeochemical pathways involved and to provide basic guide-lines for treatment implementation and management. Fluxes of O2 and nutrients in bare and in Ulva colonised sediments wereassessed by light/dark core incubations; denitrification by the isotope pairing technique and Ulva growth by in situ incubation ofmacroalgal disks in cages. O2 and nutrient budgets were estimated as sum of individual processes and further verified by 24-h inves-tigations of overall inlet and outlet loads. Ulva uptake (up to 7.8 mmol N m�2 h�1) represented a net sink for water column andregenerated NHþ

4 whilst N removal via denitrification (10–170 lmol N m�2 h�1) accounted for a small percentage of inorganic nitro-gen load (<5%). Laboratory experiments demonstrated a high potential for denitrification (over 800 lMNm�2 h�1) indicating thatN loss could be enhanced. The control of Ulva standing stocks by optimised harvesting of surplus biomass may represent an effectivestrategy to maximise DIN removal and could result in the assimilation of �50% of produced inorganic nitrogen.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Phytotreatment ponds; Ulva; Nutrient assimilation; Denitrification; Inorganic nitrogen budget; Biomass harvest

1. Introduction

Aquaculture practices are increasing all over theworld due to progressive impoverishment of natural fishstock populations and world-wide increasing demandfor fish-associated proteins (FAO, 2000). Paradoxically,farming activities have a strong negative feedback onnatural fish populations as a consequence of two mainreasons: (a) the enormous need of fish (from naturalstocks) to be converted in farmed fish feed (Nayloret al., 2000 and references therein) and (b) the deteriora-

0025-326X/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2005.06.011

* Corresponding author. Fax: +39 0521 905402.E-mail address: bartoli@dsa.unipr.it (M. Bartoli).

tion of coastal areas (water, surface sediment and plantcommunities) due to high loads of organic matter andnutrients introduced by fish farms (Hall et al., 1990,1992; Holby and Hall, 1991; Holmer and Kristensen,1992; Karakassis et al., 1998; Christensen et al., 2000).For example, land-based fish farms require a huge inputof energy in order to sustain cultivated biomass in termsof water demand (hundreds of litres per second), oxygensupply, food, antibiotics and electricity. Furthermore,fish ponds require daily management due to in-pondrapid growth of various types of primary producers(mainly filamentous microalgae) that must be removedto allow water circulation and avoid night oxygenconsumption.

Orbetello

Lagoons

ThyrrenianSea

Nassafish farm

1

2

3

4

5

A

B

C

D

to thesea-lagoon

Orbetello

Lagoons

ThyrrenianSea

Nassafish farm

1

2

3

4

5

A

B

C

D

to thesea-lagoon

M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397 1387

The enormous mass of fish stocked in modern fishponds (up to 30/40 kg m�3) determines a rapid con-sumption of dissolved O2 and a considerable increaseof dissolved nitrogenous compounds (mostly NHþ

4 )due to direct fish excretion. Part of the not ingestedfood, together with fish faeces and dissolved excretion,are exported from the plant by outflowing water andpollute the coastal environment. During the 90s a num-ber of research projects aiming at the comprehensiveand management of coastal eutrophication and greentides were financed by the European Community (4thand 5th Research Frameworks, European Land OceanInteraction Studies); more recently seaweed cultivationhas been indicated as a possible tool for water qualityregulation in fish farms even if this approach seemsparadoxical (Neori et al., 1996; Schuenhoff et al., 2003;Chirapart and Lewmanmont, 2004; Fei, 2004). In Italymost of the land based fish farms are generally con-structed in the coastal area due to prevalent farmingof high value brackish or marine species (mostly seabass,seabream and eel) (Marino et al., 1999; Italian Ministryof Agricultural Activities, 2000). The final receptors foroutlet waters are the open sea or coastal lagoons.The Orbetello basin (Thyrrhenian coast, central Italy)

and the surrounding coastal area receive currently about200,000 m�3 d�1 of low quality water discharged from anumber of land-based fish farms for an estimated N andP daily load of �362 and �18 kg respectively (Lenziet al., 2003). These loads have been considered responsi-ble for the deterioration of the whole lagoon system andin particular for dystrophic events associated withmacro-algal growth and collapse (Lenzi, 1992). Local policies inthe Orbetello area have recently imposed the construc-tion of phytotreatment ponds between the fish farmsand the sea-lagoon channel in order to abate organicand nitrogen loads. These phytotreatment ponds basetheir buffer function on spontaneous growth of primaryproducers (and thus on the uptake of nutrients by grow-ing plants), on sedimentation of particles and on benthicprocesses (P adsorption/precipitation, denitrification,burial in deep sediments) (Porrello et al., 2003a,b,c).In this study we evaluated the N removal efficiency in

a series of 4 phytotreatment ponds receiving wastewa-ters from a land-based fish farm. In particular, we com-pared the relative importance of bacterial denitrificationand macroalgal uptake for overall N removal. Simpleguidelines for optimal management strategies of phyto-treatment ponds are finally proposed.

channelchannel

Fig. 1. Schematic representation of the Nassa fish farm. Squaresrepresent phytotreatment ponds A–D; numbers indicate inlet andoutlet sampling sites. Ulva was growing in ponds B–D in May 2002and 2003 whilst in September 2002 all ponds were devoid ofmacroalgae. A dense population of mullets was present in pond A.

2. Materials and methods

2.1. Study area and sampling

The studywas carried out at theNassa fish farm (Orbe-tello, Italy), that produces annually about 100 tonnes of

seabream (Sparus aurata) and seabass (Dicentrarchus lab-rax). Fish are farmed in a series of parallel tanks suppliedwith water pumped from the sea; outflowing waters aredriven into a phytotreatment plant. A detailed descriptionof the fish farm and phytotreatment ponds is given in Por-rello et al., 2003a. Total water volume is 9000 m3 and7000 m3 in fish and phytotreatment ponds, respectively,andwater flow is 140 l s�1. The phytotreatment plant con-sists of four ponds arranged in series (Fig. 1) with a sur-face of �2600 m2 each and a mean depth of �0.7 m.The seaweed Ulva rigida grows spontaneously in theponds attaining in late spring critical biomass values (asdry weight, dw > 300�400 gdw m�2) and is not managedresulting in summer collapse of the production. Fromthe phytotreatment ponds nutrient-rich water and partof the Ulva biomass are driven into the channel that con-nects the Orbetello lagoon with the open sea.Sampling was carried out during three field surveys,

in May and September 2002 and in May 2003. Duringthe study periods measurements were performed atponds A and D. Pond A received water directly fromthe fish farm, and was devoid of macroalgae probablydue to massive presence of mullets. In contrast, Ulvastands were present at ponds B, C and D with biomassof approximately 400–500 gdw m

�2 and 200–300gdw m

�2 in May 2002 and May 2003, respectively. InSeptember 2002, macroalgae were absent in all pondsdue to a collapse event which occurred in summer.

2.2. Oxygen, dissolved inorganic carbonand nitrogen net daily balance

On 16/05/02, 26/09/02 and 20/05/03 water samples(1 l) were withdrawn approximately every 3 h for 24 h

1388 M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397

from stations 1, 2, 4 and 5 (Fig. 1); simultaneously, tem-perature, pH, salinity and dissolved oxygen concentra-tions were measured in situ with a YSI 556 multipleprobe. Water samples (�200 ml) were filtered withGF/F Whatman filters within 1 h from collection andimmediately frozen for later analyses. Ten milliliters ofunfiltered water were analysed for total dissolved in-organic carbon (DIC). Filtered water was analysed forammonium, nitrite and nitrate (DIN ¼ NHþ

4 þNO�x );

filters were analysed for total particulate nitrogen(PN). At each site O2, DIC, DIN and PN concentra-tions, once calculated, were multiplied by water flow(140 l s�1) and integrated over 24 h. Net balances ofO2, DIC, DIN and PN, expressed as moles producedor consumed per day, were finally obtained for pondsA and D and for the whole phytotreatment system bysubtracting inlet from outlet values.

2.3. Benthic fluxes and denitrification

On 17/05/02 sediment–water fluxes of O2, DIC andDIN were measured via light and dark 1-h incubationsof intact sediment cores. Eight centimeters i.d. transpar-ent Plexiglas cores were used for pond A, withoutmacroalgae, whilst 20 cm i.d. cores were used for pondD, with Ulva; we incubated, simultaneously, cores con-taining bare sediments and cores containing sediment +Ulva thalli. During incubation the top opening of coreswas closed with floating transparent lids; water stirringinside cores was ensured by Teflon-coated magnetic barsrotating at 40 rpm (8 cm i.d. cores) and small aquariumpumps (flow �2.5 l min�1, 20 cm i.d. cores). For adetailed description of core preincubation and incuba-tion techniques see Dalsgaard et al. (2000).On 27/09/02, due to a collapse of Ulva production,

benthic fluxes were measured at both ponds only inthe dark with 8 cm i.d. cores according to the same pro-cedure described above.On 21/05/03 benthic fluxes were again measured in

the dark at both ponds; within the same incubationexperiment sediment metabolism was investigated dur-ing an oxic–anoxic transition. For this purpose the darkincubation was just prolonged (�50 h) with the coresclosed and rates were calculated in presence and absenceof oxygen.Denitrification rates were measured on 17/05/02 and

27/09/02 according to the method proposed by Nielsen(1992) using the same cores previously described forbenthic fluxes. The method allows measurements ofthe denitrification of nitrate diffusing to the sedimentfrom the water column (Dw) and denitrification ofnitrate produced within the sediment via nitrification(Dn). On 17/05/02, labelled NO

�3 was added to the water

phase of the cores (�30% enrichment) and incubationswere performed in the light and in the dark; at pondA we incubated bare sediments whilst at pond D we

incubated sediment ± Ulva thalli. Due to limited oxygenpenetration in the sediments (< 1 mm), incubations werestarted immediately after 15NO�

3 addition; 1 h later thecores were contaminated with 7 M ZnCl2, mixed andsubsamples were taken for labelled N2 analysis. On 27/09/02 incubations were carried out only in the dark onbare sediments from both ponds and increasingamounts of labelled nitrate (final concentration com-prised between 20 and 150 lM) were added to the waterphase of the cores for an evaluation of the sedimentdenitrification potential. The rates of denitrificationwere calculated according to the equations and assump-tions of Nielsen (1992):

D15 = p(15N14N) + 2p(15N15N) and D14 = p(15N14N) +2p(14N14N), where D15 and D14 equal rates of denitrifi-cation based on 15NO�

3 and 14NO�3 , respectively; and

p(14N14N), p(15N14N) and p(15N15N) equal rates of pro-duction of labelled and unlabelled N2 species. Estima-tion of D14 was obtained from: D14 = D15 · p(15N14N)/2p(15N15N). The proportion of D14 supported by unla-belled NO�

3 from the water column (Dw) was calculatedfrom: Dw = D15 · f/(1 � f), where f = mole fraction of14NO�

3 in the water column. The coupled nitrification-denitrification (Dn) was calculated by difference as:Dn = D14 � Dw.

2.4. Ulva elemental composition and in situ growth rates

On 15/05/02 Ulva samples were collected from pondD and gently washed to remove epiphytic material. Partof the collected thalli was dried and powdered for laterCHN analyses. Fresh thalli were cut into �300 discs(diameter = 5 cm); 20 discs were individually weighedfresh and after desiccation in an oven at 70 �C whilst 4series of 50 discs were suspended in 4 cylindrical cages(i.d. 20 cm, height 40 cm) positioned within the Ulvamats of pond D; three days later the cages were removedand all discs were gently washed, dried and weighed forthe determination of growth rates.On 19/05/03 Ulva growth rate was again evaluated

in situ but with a more complex setup. Four cylindricalcages (i.d. 40 cm, height 80 cm), each divided by net sep-tum in four compartments, allowed suspension of Ulvadiscs at different depths (�20, �40, �60, �80 cm fromthe surface). The four cages were filled with increasingamounts of Ulva thalli, equally distributed among thefour compartments in each cage, in order to obtain bio-mass values equivalent to 250 (in situ estimated value),500, 750 and 1000 gdw m

�2. Ten Ulva discs (diame-ter = 5 cm) were then positioned inside each compart-ment and allowed to grow for three days at differentdepths and within a more or less packed Ulva bed.Dry weight of Ulva discs was determined on 20 repli-cates at the beginning of the experiment and on discsfrom all compartments at the end of the experiment.

M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397 1389

During the 3-day experiment, water samples were with-drawn from inside the cages at three depths (throughtubing placed within Ulva mats) around midday everyday and around 4:00 AM every night. On water samplesNHþ

4 , NO�2 and NO

�3 concentrations were determined.

2.5. Water analyses

O2 concentration was measured by Winkler titration(Strickland and Parsons, 1972) and by polarographicsensors; dissolved inorganic carbon (DIC) by titrationwith 0.1 M HCl (Anderson et al., 1986). NO�

3 was deter-mined after reduction to NO�

2 in the presence of cad-mium, NO�

2 was determined spectrophotometricallyusing sulphanilamide and N-(1-naphtyl)ethylendiamine(Golterman et al., 1978). NHþ

4 was determined spectro-photometrically using salicylate and hypochlorite in thepresence of sodium nitroprussiate (Bower and Holm-Hansen, 1980).

14N15N and 15N15N abundance in N2 was analysedby mass spectrometry at the National EnvironmentalResearch Agency, Silkeborg, Denmark.Particulate nitrogen and Ulva elemental composition

were determined by combustion using a CHNS-O EA1108 Elemental Analyzer (Carlo Erba).

2.6. Sediment analyses

Surface sediments were collected on May 2002 fromponds A and D via 5 cm i.d. plexiglass cores (n = 3 foreach pond). For the analysis of density, porosity, organ-ic matter quantity (loss on ignition) and biopolymericcarbon concentration each sediment core was sectionedinto three layers (0–2, 2–5 and 5–10 cm depth). Organiccarbon (POC) and nitrogen (PON) were analysed bycombustion using a CHNS-O EA 1108 Elemental Ana-lyzer (Carlo Erba) after removal of carbonates by HClvapour in a desiccator (Hedges and Stern, 1984; Tanoue,1985).Proteins (PRT) were determined according to Har-

tree (1972). The absorbance was evaluated at 650 nm.Bovine albumin solutions were used as standards. Car-bohydrates (CHO) were analysed according to Duboiset al. (1956). The absorbance was measured at 490 nm.D(+) glucose solutions were used as standards. Lipids(LIP) were extracted by direct elution with chloroform

Table 1Physico-chemical features of water outflowing from the fish farm and enteri

Temp (�C) Salinity (&) pH O2 (%sat) DIC (mM)

16/05/02 23.6 ± 1.2 41.9 ± 0.1 7.6 ± 0.1 72.4 ± 9.1 2.36 ± 0.0326/09/02 21.5 ± 0.9 39.2 ± 0.6 7.4 ± 0.2 74.9 ± 8.7 2.42 ± 0.0620/05/03 21.7 ± 1.4 39.5 ± 1.9 7.6 ± 0.2 73.3 ± 6.4 2.59 ± 0.04

Reported numbers are mean ± standard deviation calculated over 8 diffDIC = dissolved inorganic carbon; DIN = dissolved inorganic nitrogen; PN

and methanol according to Bligh and Dyer (1956) andmeasured following Marsh and Weinstein (1959).Absorbance was measured at 375 nm. Tripalmitine solu-tions were used as standards. Protein, carbohydrate andlipid concentrations were converted to carbon equiva-lents by using the following conversion factors: 0.49,0.40 and 0.75 lg of C lg�1, respectively (Fabiano andDanovaro, 1994).Biopolymeric carbon concentration (BPC) was calcu-

lated according to Fabiano and Danovaro (1994) as thesum of lipid, protein and carbohydrate carbon.

2.7. Statistical analyses

Differences between light and dark fluxes and denitri-fication rates and between measurements carried out atdifferent ponds were tested with a one way ANOVA.Differences between initial and final weights of Ulva

discs in the growth experiments were tested witha one-way ANOVA in 2002, and with a two-wayANOVA, with algal density and water depth as factors,in 2003.

3. Results

3.1. O2, DIC, DIN and PN daily balance

Physico-chemical features of water outflowing fromthe fish farm and entering the phytotreatment pondsat station 1 were very similar within the day–night cyclesand among the three surveys (Table 1). O2 was alwaysbelow saturation (�73%) and its concentration (152 ±17 lM) was constant during the day; NHþ

4 was the dom-inant form of DIN (�90%) with concentrations up to72 lM. Values reported in Table 1, corrected for waterflow (140 l s�1), give an estimate of DIN and PN dailyloads produced by the fish farm of �780 and�270 mol N d�1, respectively. At station 2 outlet waterwas generally poorer in O2 and richer in DIC andDIN, indicating that at pond A respiratory processeswere dominating and that the system was regeneratingdissolved nutrients.Different patterns were observed at stations 4 and 5 in

May 2002 and 2003, when Ulva was present: at thisstage water chemical features were influenced by the

ng the phytotreatment ponds at station 1

NHþ4 (lM) NO�

2 (lM) NO�3 (lM) DIN (lM) PN (lM)

61.2 ± 6.5 2.4 ± 1.2 3.6 ± 1.9 67.1 ± 7.3 20.2 ± 1.857.4 ± 7.1 3.1 ± 0.9 4.7 ± 2.3 66.4 ± 8.9 22.2 ± 3.252.7 ± 7.4 2.7 ± 0.7 5.4 ± 1.8 60.7 ± 9.4 25.4 ± 5.1

erent water samples withdrawn every 3 h over a day–night cycle.= particulate nitrogen.

O2

(µM

)

0

100

200

300

0

800

1600

2400station 4station 5irradiance

DIC

(mM

)

2.4

2.5

2.6

2.7

0

800

1600

NH

4+(µ

M)

0

20

40

60

0

800

1600

6.00 18.00 6.00 18.00

NO

x-(µ

M)

0

10

20

Irradiance (µE m

-2 s-1)

0

800

1600

Fig. 2. Daily evolution of O2, DIC, NHþ4 and NO�

x concentrationsmeasured at stations 4 and 5, inlet and outlet of pond D, respectively.Samplings, carried out every 3 h, started at 10:00 on 20/05/03;continuous line represents irradiance.

1390 M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397

photosynthetic activity of the macroalgae and O2, DICand DIN evolution was strongly correlated with lightintensity. In particular, O2 and NO�

x concentrationswere increasing with light intensity whilst the oppositewas found for DIC and NHþ

4 (Fig. 2). Net daily balancesof O2, DIC, DIN and PN for ponds A and D are re-ported in Table 2. Both ponds resulted in net consumingoxygen during all surveys, with the highest O2 deficitobserved at pond A (up to �536 mol O2 d�1 measuredon 20/05/03, equivalent to an areal flux of �8 mmol

Table 2Net daily O2, DIC, DIN and PN balances calculated for ponds A and D

Net daily balance O2 (mol d�1) DIC (mol d�1) NHþ

4 (mol d�1) N

Pond A 16/05/02 �323 619 70 �26/09/02 �342 580 30 �20/05/03 �536 517 42

Pond D 16/05/02 �104 221 �217 426/09/02 �199 340 �1 220/05/03 �220 �462 �101 7

Pond A was devoid of macroalgae during the whole investigation period whilof 400 and 250 gdw m

�2 respectively.

O2 m�2 h�1). O2 consumption was coupled to DIC

regeneration; a DIC:O2 ratio > 1.6, typical of reducedenvironments with anaerobic metabolism, was measuredon 05/16/02 and 26/09/02. Despite the presence of Ulva,net O2 consumption was determined in pond D duringall campaigns; only on 05/20/03, even if O2 balancewas negative, net fixation of DIC occurred (�462mol C d�1).A production of NHþ

4 (30–70 mol N d�1) was deter-mined at pond A in all sampling dates whilst at pondD net consumption of ammonium was measured on16/05/02 and 20/05/03 (�217 and �101 mol N d�1),when Ulva was present. At pond D a significant produc-tion of NO�

x (55–203 mol N d�1) was measured duringall campaigns; in this pond NO�

x concentrations wereincreasing up to a factor of 5 (from 5 to 25 lM,measuredrespectively at stations 4 and 5 on 20/05/03, Fig. 2).Particulate nitrogen was always retained in the two

ponds even if within the same campaign higher sedimen-tation rates (ranging between �24 and �59 mol N d�1)were determined at pond D.

3.2. Benthic fluxes

3.2.1. May 2002

O2 and DIC fluxes measured in the light and darknessat pond A (respectively � �2 mmol O2 m�2 h�1 and�4 mmol DIC m�2 h�1, Fig. 3) were not significantlydifferent in the two incubation conditions (P > 0.05).Sediments were fluffy, black, smelling sulphides andcharacterised by a high respiratory quotient (�1.9, cal-culated as dark DIC/O2 fluxes). NH

þ4 fluxes, despite

being all directed from the sediment to the watercolumn, were characterised by a wide variability andaveraged 491 ± 311 and 398 ± 307 lmol N m�2 h�1

respectively in the light and dark incubations. On thecontrary NO�

2 and NO�3 fluxes were all directed from

the water column to surficial sediment with values com-prised between �6 and �32 lmol NO�

2 m�2 h�1 and

�61 and �247 lmol NO�3 m

�2 h�1.The picture was different at pond D, where in bare

sediment incubations light fluxes were close to zerowhilst in darkness O2 demand was �5.2 ± 0.7 mmolO2 m

�2 h�1 and DIC and NHþ4 effluxes were 8.4 ± 3.1

O�2 (mol d

�1) NO�3 (mol d

�1) DIN (mol d�1) PN (mol d�1)

3 3 70 �82 5 33 �134 6 52 �25

8 52 �117 �525 30 54 �246 127 102 �59st Ulva spp. was present in May 2002 and 2003 in pond D with biomass

Pond A, bare sediment

mm

olm

-2h

-1

-20

-10

0

10

20

30

LightDark

µmol

m-2

h-1

-500

-250

0

250

500

750

1000

Pond D, bare sediment

mm

olm

-2h

-1

-20

-10

0

10

20

30

Pond D, sediment +Ulva

mm

olm

-2h

-1

-200

-150

-100

-50

0

50

100

150

µmol

m-2

h-1

-1600

-800

0

800

1600

2400

µmol

m-2

h-1

-8000

-6000

-4000

-2000

0

2000

4000

6000

O2 DIC NH4+ NO2

- NO3-

O2 DIC

O2 DIC

NH4+ NO2

- NO3-

NH4+ NO2

- NO3-

Fig. 3. Benthic fluxes of O2, DIC and DIN measured in the light and in the dark at ponds A and D on 17/05/02. Measurements at pond D werecarried out on bare sediments and in sediments covered by Ulva beds. Each value is the average ± standard deviation of 4 replicates; note that graphshave different scales.

M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397 1391

and 0.75 ± 0.22 mmol m�2 h�1 respectively (Fig. 3). O2and DIC fluxes measured during light incubationsin chambers containing sediment + Ulva resulted86.2 ± 8.8 and �136.4 ± 27.8 mmol m�2 h�1 respect-ively (Fig. 3); this intense primary production wassustained by a high NHþ

4 demand (�7781 ± 602lmol N m�2 h�1). In darkness the sediment + Ulvasystem respired 12.4 ± 0.4 mmol O2 m

�2 h�1 and regen-erated 27.7 ± 6.1 mmol DIC m�2 h�1; NHþ

4 wasconsumed also in darkness (�917 ± 404 lmol m�2 h�1)but rates were considerably lower compared to thosemeasured in the light. NO�

3 fluxes measured within sed-iment + Ulva chambers were all directed to the watercolumn, with values of 903 ± 474 and 2487 ± 984lmol m�2 h�1 measured in the light and in darkness,respectively.Ulva biomass in the cores averaged 445 ± 31 gdw m

�2

and was close to in situ values; approximately 50% ofthe pond surface was covered by the macroalgae.

3.2.2. September 2002

All results from this campaign refer to dark incuba-tions due to a collapse of primary production and thedisappearance of Ulva from pond D. O2 demand andDIC efflux (respectively � �5 and 6.7 mmol m�2 h�1)were similar at the two sites, whilst NHþ

4 regenerationwas significantly higher at pond D (1015 ± 422 versus279 ± 153 lM m�2 h�1, P < 0.01) due probably to highavailability of labile organic matter from the collapseofUlva mats. All NO�

2 +NO�3 fluxes were directed from

the water to the sediment and ranged between �30and �220 lmol N m�2 h�1; rates were slightly higherat pond A but differences were not significant (P > 0.05).

3.2.3. May 2003: oxic–anoxic transition

Dark O2 demand calculated over the initial 4 hof incubation was significantly higher at pond Acompared to pond D (�8.35 ± 1.05 and �4.78 ±0.24 mmol m�2 h�1 respectively, P < 0.01) (Fig. 4).

incubation time (hours)

0 10 20 30 40 50

O2,

µM

0

50

100

150

200

250

300

S2-

,µM

0

200

400

600

800

1000

O2 Pond A

O2 Pond D

S2- Pond AS2- Pond D

Fig. 4. Temporal changes of dissolved oxygen and free sulphideconcentrations in the water column of intact sediment cores sampled atponds A and D. Incubation was performed in the dark at in situtemperature, water stirring and top lids were maintained for the wholeincubation period; each value is the average ± standard deviation of 4replicates.

Pond Abare sediment

0

50

100

150

200

µmol

N m

-2 h

-1

1392 M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397

Similar results were obtained for DIC fluxes calculatedduring the oxic part of the experiment (with rates of7.55 ± 1.93 and 4.12 ± 1.61 mmol m�2 h�1 for pond Aand D respectively). After 11 h of incubation water col-umn in cores from pond A was anoxic and free sulphidesstarted diffusing from the sediment to the water column.On the contrary, in the water column of the cores frompond D, free sulphides were undetectable even after 25consecutive hours of anoxia (Fig. 4). DIC fluxes werecalculated also in the anoxic part of the incubation; a30% and 50% decrease of effluxes was determined in

Pond A Pond D

Pond A Pond D

µmol

NH

4+

m-2

h-1

0

300

600

900

1200

1500

ox ic NH4+ flux

anox ic NH4+ flux

mm

olD

ICm

-2h

-1

0

2

4

6

8

10

oxic DIC fluxanoxic DIC flux

Fig. 5. DIC and NHþ4 fluxes calculated before and after the oxic–

anoxic transition induced by prolonged dark incubation of sedimentcores; each value is the average ± standard deviation of 4 replicates.

cores from pond A and D respectively (Fig. 5). The sameapproach was used to calculate oxic and anoxic NHþ

4

fluxes; in the presence of O2, NHþ4 effluxes were

941 ± 275 (pond A) and 323 ± 77.9 (pond D)lmol N m�2 h�1, whilst during anoxia fluxes at pondA diminished (672 ± 245 lmol N m�2 h�1) and at pondD increased (686 ± 137 lmol N m�2 h�1) (Fig. 5).

3.3. Denitrification rates

3.3.1. May 2002

At pond A denitrification rates obtained from incu-bating bare sediments were low and averaged29 ± 7 lMNm�2 h�1; about 50% of the N2 flux(16 ± 6 lMNm�2 h�1) was due to nitrification-coupleddenitrification (Fig. 6) whilst less than 15 lMNm�2 h�1

was due to reduction of nitrates from the water column.At this pond, differences between light and dark Dwrates were not significant (P > 0.05) whilst dark Dn

Pond Dbare sediment

0

50

100

150

Pondsediment + Ulva

Dw L

µmol

N m

-2 h

-1µm

ol N

m-2

h-1

Dw D Dn L Dn D0

50

100

150

Fig. 6. Denitrification rates measured in May 2002 in ponds A and Din intact sediment cores. At pond D incubations were carried out withand without Ulva; macroalgal biomass was simulating in situ one.Dw = denitrification of nitrate diffusing to anoxic sediment from thewater column; Dn = denitrification of nitrate produced via nitrifica-tion; L = light incubations; D = dark incubations; each value is theaverage ± standard deviation of 4 replicates.

[15NO3-], µM

0 30 60 90 120 150 180

D15

(µm

olN

m-2

h-1)

0

200

400

600

800

1000

Pond APond D

y = 1,42x + 250,2R2 = 0,48

y = 5,3x + 14,8R2 = 0,94

y = 5.3 x + 14.8

R2 = 0.94

y = 1.4 x + 250.2R2 = 0.48

Fig. 7. Rates of 15NO�3 denitrification (D15) as function of labelled

nitrate concentration in the water column.

M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397 1393

(22.7 ± 5.4 lMNm�2 h�1) was significantly higher thanlight Dn (8.4 ± 2.1 l MNm�2 h�1) (P < 0.01). At pondA, denitrification rates integrated for the whole basinsurface and for day length allowed to estimate a NO�

3 re-moval of 0.94 and 1.02 mol N d�1 via Dw and Dnrespectively.At pond D, total denitrification rates were at least 5

times higher (>150 lMNm�2 h�1) and most of N2 flux(> 80%) was due to nitrification-coupled denitrification(Fig. 6). Dw values calculated on bare sediments(31 ± 7 lMNm�2 h�1) doubled Dw values calculatedfor pond A were probably due to higher nitrate watercolumn concentrations (7–9 lM). In bare sedimentincubations Dn values were significantly higher thanDw and comprised between 138 and 200 lMNm�2 h�1

(Fig. 6); dark average values for Dw and Dn wereslightly higher then the corresponding light ones butdifferences were not significant (P > 0.05). Incubationscarried out at pond D on sediments+Ulva thalli re-sulted in Dn rates (�120 lMNm�2 h�1) significantlyhigher than Dw ones (�20 lMNm�2 h�1), as shownwith sediments alone. The comparison between denitri-fication rates in sediments alone and in sediment +Ulva thalli indicated that both Dn and Dw weremarkedly diminishing when macroalgae were present;this occurred both in the light and in darkness. Inparticular, Ulva determined a decrease of Dw and Dncomprised between 26 and 34% probably due to assim-ilation of NO�

3 and NHþ4 and thus competition with

denitrifiers and nitrifiers in the closed incubationchamber (Fig. 6). At pond D denitrification rates integ-rated for the whole basin surface and for day length,assuming 50% of the surface colonised by Ulva, allowto estimate a NO�

3 removal of 1.76 mol N d�1 via Dwand an abatement of 9.11 mol N d�1 via Dn.

3.3.2. September 2002

Due to absence of macroalgae, in September 2002denitrification experiments were carried out only inthe dark on bare sediments of both ponds. Totaldenitrification rates were 54 ± 18 and 147 ± 33lmol N m�2 h�1 respectively for pond A and D. Nosignificant nitrification-coupled denitrification was mea-sured at pond A whilst at pond D Dn was detectableeven if rates (52.2 ± 25.4 lmol N m�2 h�1) were lowercompared to those measured in May when Ulva waspresent. At both ponds on the contrary Dw increasedrespectively �250 and �200% due probably to aug-mented water column nitrate concentrations (9.5 and17.0 lM). Results from the concentration series experi-ment are shown in Fig. 7; they clearly demonstrate thatdenitrification potential for the two ponds was differentwith higher nitrate reduction capacity at pond D. Atpond A denitrification seems saturated at 400lmol N m�2 h�1 whilst at pond D a saturation valuecannot be calculated being the highest D15 value

(>800 lmol N m�2 h�1) still linearly correlated withwater column [15NO�

3 ].

3.4. Ulva elemental composition and in situ growth rates

3.4.1. May 2002

Ulva discs were grown at a depth of about �30/�40 cm from the water–atmosphere interface and atvery low biomass (< 100 gdw m

�2); after 2.7 days ofincubation Ulva growth was estimated to be 4.38 ±3.11% d�1 (mean ± standard deviation, n = 4). Totalnitrogen (TN) content in Ulva thalli was 3.56 ±0.17%dw (n = 8); differences between values at thebeginning and at the end of the incubation were notsignificant (P > 0.05).

3.4.2. May 2003

Mean weight of dry Ulva discs grown at the lowestdensity (250 gdw m

�2) augmented in three days from55 ± 12 to 62 ± 13 mg (n = 19) of �7 mg in the twouppermost cage compartments (0–40 cm depth), result-ing in a specific growth rate of about 4% d�1. Dryweight increments of discs grown in the bottom com-partments or in cages filled with higher Ulva biomasswere not significant. TN content of Ulva thalli was3.79 ± 0.16%dw (n = 6) at the beginning of the growthexperiment. In three days TN increased on average to3.89 ± 0.13 (n = 48)%dw, but no significant differenceswere observed among cages or depths. DIN concentra-tions in water samples collected within the Ulva mats in-side the cages did not vary significantly with depth oramong cages. Average concentrations of NHþ

4 , NO�2

and NO�3 were 56 ± 18, 9 ± 4 and 12 ± 6 lM, respec-

tively (n = 72 for all variables).

3.5. Sediment characterisation

At pond A sediments were black, fluffy and extremelyreduced; density, porosity and organic matter content

Table 3Concentrations of the labile organic fraction in surficial sediments from ponds A and D measured on 18/05/03 (CV = coefficient of variation)

Pond/layer (cm)

Proteins (PRT) (lg g�1) CV (%) Carbohydrates (CHO) (lg g�1) CV (%) Lipids (LIP) (lg g�1) CV (%) PRT:CHO

A 0–2 879 22 488 4 149 64 1.82–5 662 18 601 24 124 18 1.15–10 769 30 529 28 143 25 1.5

D 0–2 4640 15 17754 32 1521 50 0.32–5 1517 36 2723 44 458 7 0.65–10 754 3 1057 31 135 42 0.7

1394 M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397

along a 0–10 cm vertical profile ranged between 1.05–1.24 g cm�3, 0.81–0.93 and 14–22% respectively. Sedi-ments from pond D were muddy with a light brownupper layer; density, porosity and organic matter con-tent along a 0–10 cm vertical profile were respectivelycomprised between 1.2 and 1.5 g cm�3, 0.7% and 0.5%,8% and 13%.Concentration of carbohydrates, lipids and proteins

in surface sediments are reported in Table 3. Valuesare typical of eutrophic environments; concentrationsat pond D were on average 5 times higher than the cor-responding ones measured at pond A. Despite this, theprotein to carbohydrate ratio, was higher at pond A evi-dencing the presence of freshly produced material andhigher nutritional status of sediments at this site (Del-l�Anno et al., 2002).

4. Discussion and conclusion

Similar physico-chemical features of water outflow-ing from the fish farm during the three surveys indicatethat, despite heterogeneity of fish size and density overthe production cycle, the overall production of the plantand thus the organic matter and nutrient loads werecomparable during this investigation (Table 1). On adaily basis ponds A and D were net heterotrophyc (Ta-ble 2). At pond A, O2 was always below saturationmeaning that calculated net balances (�323 < x <�536 mol O2 d�1) were underestimates of true oxygendemand; O2 diffusing from the atmosphere to the watercolumn was in fact not taken into account. Areal fluxescomputed from such balances were comprised between�5 and �8 mmol O2 m�2 h�1, values overlapping thosemeasured by dark core incubations (Fig. 3). At pondD dissolved oxygen concentrations were below satura-tion during the night hours but far above saturation(up to 160%) during daylight, when Ulvamats were pho-tosynthesising (May 2002 and 2003); calculated daily O2balances include oxygen diffusing from the atmosphereand oxygen escaping from the water column. Due prob-ably to Ulva production (up to 86 mmol O2 m

�2 h�1), inMay 2002 and 2003 oxygen balance at pond D was lessnegative compared to that measured at pond A. In thislast pond a considerable amount of O2 was probably

consumed due to nitrification processes: in the threecampaigns net NO�

2 þNO�3 production was �100,

�55 and �204 mol N d�1 and the associated O2 demandwas �200, �110 and �408 mol d�1. This means that,excluding nitrification, oxygen balance would have beenpositive at pond D in May 2002 (96 mol d�1) and inMay 2003 (188 mol d�1) whilst it would have been lessnegative (�90 mol d�1) in September 2002. In summary,the comparison between pond A and D indicated thatthe presence of Ulva seemed to play a major role inregulating oxygen dynamics, as also evidenced by coresincubations (Fig. 3).During the period of this investigation about 780 mol

DIN and 270 mol PN were discharged every day to thephytotreatment ponds from the fish farm. At stations 1and 2, inlet and outlet of pond A, most of DIN was rep-resented by NHþ

4 whilst a slight but significant decreaseof the NHþ

4 /DIN ratio was evidenced in all campaignsat station 4 and 5 due to nitrification processes occurringat ponds B, C and D (Fig. 2).At pond A sediments were sulphidic resulting in

ammonification of organic nitrogen uncoupled to signif-icant nitrification rates and positive net daily balance ofDIN (Table 2); 70, 33 and 52 mol DIN d�1 were regener-ated respectively in May and September 2002 and May2003, mostly due to sediment–water fluxes of NHþ

4 asconfirmed by core incubations (Table 2, Fig. 3). At pondA mean daily NHþ

4 production largely exceeded PNabatement (Table 2) indicating that part of the ammo-nium came probably from dissolved organic nitrogen.The oxic–anoxic transition had a dramatically differ-

ent effect on the sediments of the two ponds: at station Ait determined the rapid release of toxic sulphides to thewater column but had little effect on nutrient dynamics.It is likely that in this dystrophic pond most oxygen con-sumption was due to reoxidation of reduced compoundsat the sediment–water interface; once stopped such com-pounds can diffuse upwards (i.e. sulphides). At the moreoxidised station D the set up of anoxia had no effect onsulphides but a marked effect on the efficiency of C min-eralisation (Figs. 4 and 5); higher NHþ

4 regeneration wasprobably a consequence of stopped nitrification. Theseresults are probably linked to different quality of organicmatter within ponds (Table 3) and the lack of an activeiron buffer at pond A.

days

0 10 20 30 40 50 60 70 80 90

Ulva biom

ass (gdw

m-2)

0

50

100

150

200

250

300

mol

Nd-1

pon

d-1

0

20

40

60

80

100

120

Ulva biomassN uptake

Fig. 8. Theoretical N uptake by Ulva. The dashed line shows theevolution of Ulva biomass according to a logistic growth curve(r = 0.2 d�1, K = 300 gdw m

�2); the continuous line represents theassociated nitrogen demand, calculated assuming an Ulva N content of4%dw. Nitrogen demand sustaining Ulva growth equals nitrogenremoved per day per pond. Maximum removal occurs when Ulva

biomass reaches 150 gdw m�2.

M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397 1395

At pond A, both Dw and Dn rates were low(< 60 lmol N m�2 h�1) and in the period of this investi-gation N abatement in the pond was comprised between2 and 3 mol N d�1 equivalent to 0.3–0.4% of the DINdaily load. Such values are explained by extremelylimited sediment oxygen penetration in the sulphidicsediments (<0.5 mm, data not shown) and low watercolumn nitrate concentrations (�3–4 lM). Furthermore,due probably to toxic porewaters, the potential for deni-trification at pond A was low as demonstrated byincreasing water column nitrate concentrations (Fig. 7).At pond D, oxygen evolution was mostly regulated

by Ulva when present, and nitrification was probablyassociated with the macroalgae, acting as a physical sup-port for bacteria. Ammonium concentration in thewater column was high enough (>50 lM) to supportboth macroalgal uptake and nitrification activity.Despite this quantitatively important oxidation NO�

3

concentration was higher than that measured at pondA but still below 25 lM and Dw was probably limitedby the low concentration of the substrate. Dn was signif-icantly higher than Dw in May 2002, when Ulva waspresent, whilst the opposite was determined in Sept-ember 2002, after the collapse of the macroalgalproduction.N abatement at pond D was �12 mol N d�1 equiva-

lent to �1.5% of the incoming DIN load but the poten-tial for denitrification at this pond was much highercompared to that found at pond A and linearly corre-lated with the substrate concentration (Fig. 7). Overall,assuming similar rates for Ulva colonised ponds B, Cand D, the N abatement through denitrification in thephytotreatment plant has been estimated in �38mol N d�1 equivalent to �4.9% of the incoming DINload. This low percentage could be enhanced by improv-ing nitrification activity (i.e. by biofilters in between thefishfarm and the phytotreatment plant or by night aera-tion of the ponds water column).Ammonium uptake by Ulva has been estimated in

May 2002 by cores incubation in 7.8 mmol N m�2 h�1

which is an impressive value equivalent to a theoreticalabatement of 280 mol N pond�1 d�1 (�36% of theincoming DIN load, Fig. 3). This result is quite far fromDIN balances calculated at pond D (Table 2); it isapparent that such uptake is overestimated due proba-bly to optimal light and water circulation conditions in-side incubation cores. Still, Ulva has a great potential forN removal but its activity is strongly dependent on thesurrounding physical environment and on its physiolog-ical status; excess biomass for example limits light pene-tration and alters water microcirculation resulting in selflimitation. Here as in other environments, the lack of ac-tive Ulva management as, for example, a periodic har-vesting of the surplus biomass, results in a collapse ofthe production, determines a sudden input of fresh or-ganic matter to the sediments, and dramatic release of

accumulated nutrients to the water column, setting tozero the phytotreatment action.In May of both years Ulva was still growing but at

slow rates. Overall, therefore, nitrogen removal by Ulvabeds was low, and the treatment system abated only asmall fraction of the inorganic nitrogen load from thefish farm (Table 2). Ulva growth was most likely limitedby light penetration in the algal mats, due to the highdensity, rather than by nitrogen availability, since DINconcentration was on average higher than 70 lM duringthe 3-day growth experiments. Total N content of Ulvathalli, around 4%dw, and C:N ratios of about 9 alsoindicate that the alga was not nitrogen limited, with re-spect to carbon (Atkinson and Smith, 1983).Clearly, Ulva density in the ponds was too high to al-

low an efficient N removal. Higher N abatement ratescould be obtained if Ulva were regularly harvested andmaintained at an optimal density. We tried to estimatethe optimal value of Ulva biomass for the maximum re-moval of inorganic nitrogen from the water. The tempo-ral evolution of macroalgal biomass and N removalfrom water have been simulated with a logistic modelassuming an instantaneous growth rate of 0.2 d�1 anda carrying capacity of the environment of 300 gdw m

�2.We also assumed for healthy Ulva growing in a not lim-iting environment a constant nitrogen content of 4%dw.The amount of nitrogen removed per pond per day wasthen calculated as a function of the in situ Ulva biomass(Fig. 8). With the chosen logistic model and parameterswe calculated that the maximum inorganic nitrogen re-moval (about 110 mol pond�1 d�1) was attained whenUlva biomass reaches 150 gdw m

�2 and growth rate is0.1 d�1. Considering the whole system, Ulva could abate400–450 mol N d�1, which corresponds to about 50% of

1396 M. Bartoli et al. / Marine Pollution Bulletin 50 (2005) 1386–1397

the DIN loading from the fish farm. The assumptionsfor the growth model are realistic: Ulva N content mea-sured in May 2002 and 2003 ranged from 3.4 to 4.1%dw;moreover, with the chosen instantaneous growth rateand carrying capacity values, Ulva should grow about3.4% d�1 at 250 gdw m

�2, which is close to the rate mea-sured in 2003 in the whole cage with the lowest biomass(2.6% d�1 at 250 gdw m

�2). What should be carefullyconsidered by the fishermen is that Ulva biomass needsto be managed to avoid collapse of the production andto sustain its phytodepuration capacity; once it reaches150 gdw m

�2 in fact the equivalent of �43 kgdw shouldbe removed from each pond every day to keep N abate-ment at its maximum level. For the whole lagooning sys-tem this is equivalent to about 1700 kg of fresh Ulva perday which means further costs for the fish farm.

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