Reduction of aquaculture wastewater

eutrophication by phytotreatment ponds system

I. Dissolved and particulate nitrogen and phosphorus

Salvatore Porrelloa,*, Mauro Lenzib, Emma Persiaa,Paolo Tomassettia, Maria Grazia Finoiaa

aCentral Institute for Marine Research (ICRAM), Via di Casalotti, 300-00166 Rome, ItalybLagoon Ecology and Aquaculture Laboratory (LEALab), OPL s.r.l., Orbetello, Italy

Received 22 January 2002; received in revised form 2 December 2002; accepted 4 December 2002

Abstract

The aim of the investigation was to quantify, during the daytime, the nutritional components N and

P (as dissolved and particulate) discharged into the Orbetello Lagoon (Tuscany, Italy) from a fish

farming facility equipped with a phytotreatment (lagooning) system to treat the effluent produced.

Consideration was also given to the water quality improvement capacity of the treatment system used

during the daytime phase in which Ulva rigida C. Ag develops spontaneously.

Taking into consideration the short residence time (about 8 h) of the water inside the

phytotreatment ponds, the results revealed a very active process of nitrification, an appreciable soluble

reactive phosphorus (SRP) abatement (15%), a relatively small mean annual dissolved inorganic

nitrogen (DIN) abatement (5%), sharp reduction in the organic fraction vis-a-vis the inorganic fraction

in the particulate component and an appreciable modification of the physicochemical parameters, in

particular the pH and dissolved oxygen (DO).

The DIN and SRP uptake rates were relatively low compared to literature data. The most efficient

DIN uptakes were obtained with algal densities between 700 and 1500 g/m2 wet weight (w.w.).

Discriminant statistical analysis indicates that lagooning had a strong effect on the effluent

despite the short residence time.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Water pollution treatment; Nitrogen; Phosphorus; Ulva rigida; Aquaculture wastewater

0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0044-8486(02)00656-7

* Corresponding author. Tel.: +39-6-61-57-04-16; fax: +39-6-61-56-19-06.

E-mail addresses: porrell@tin.it (S. Porrello), Lealab2@hotmail.com (M. Lenzi).

www.elsevier.com/locate/aqua-online

Aquaculture 219 (2003) 515–529

1. Introduction

Over the last 30 years, the Orbetello Lagoon area has seen the development of intensive

fish farming carried on by land-based farms which withdraw water from the lagoon itself

and from brackish water table (Fig. 1) (Lenzi, 1992).

In the Orbetello Lagoon basin (South Tuscany, Italian West Coast) (Fig. 1), there are

four intensive land-based fish farming facilities which have a wastewater inflow in the

order of 200,000 m3 day� 1, recently estimated as the equivalent of 265 kg of nitrogen

(N) day� 1 and 13 kg of phosphorus (P) day� 1 (Lenzi, personal communication). The

result is a strong environmental impact leading to an extensive cyclic growth of

opportunistic green seaweeds. When these macrochlorophyceae die during spring and

summer, they cause serious degeneration processes in the lagoon (Bombelli and Lenzi,

1996).

In 1993, the Lagoon Authority imposed the construction of lagooning ponds for the

phytotreatment of the wastewater on the four intensive fish farming facilities that

discharge into the lagoon. Although references may be found in the literature about

laboratory experiments or pilot plants designed to develop a phytotreatment technique

applicable to fish farming effluent (Ryther et al., 1977; Shpigel et al., 1993; Krom et al.,

1995; Jimenez del Rio et al., 1996), the results have not yet been transferred to a

commercial scale.

Fig. 1. Orbetello Lagoon (South Tuscany, Italy). Fish farm facilities (FFF). Fish farm studied (Nassa). Schematic

draw of rearing tanks and phytotreatment system with inflow station (E) and outflow station (U).

S. Porrello et al. / Aquaculture 219 (2003) 515–529516

The flooded areas used for phytotreatment facilitate the growth of huge quantities of

algal macrovegetation which remove N and P from the fish farm wastewater. Since

these ponds are not managed, this ‘beneficial’ effect is lost during the summer period,

when this vegetation dies, yielding up the retained N and P through mineralization

processes.

The aims of the present investigation were (a) to quantify, during the daytime, the

nutritional components N and P discharged into the lagoon (final receiving body) in

dissolved and particulate form from a fish farming facility equipped with a lagooning

system, and (b) to quantify the abatement of the same nutritional components during the

daytime phase by the treatment system in which mainly Ulva rigida C. Ag develops

spontaneously.

2. Materials and methods

2.1. Selected fish farm

The intensive fish farm investigated was located at Nassa (Fig. 1) The farm produces

about 100 tons year� 1 (ICR 1:2) of European sea bass (Dicentrarchus labrax L.) and gilt

head sea bream (Sparus aurata L.) in 450-m2 soft PVC tanks having a total volume of

9000 m3 (Fig. 1). The wastewater was converted into four phytotreatment ponds, arranged

in series. The first pond received the effluent from the rearing tanks; after undergoing

phytotreatment system, the water left the fourth pond from which it was ultimately

discharged into the nearby sea–lagoon canal.

2.2. Hydrology

In order to assess macroalgal vegetation uptake and the impact of the effluent on

the receiving environment, an assessment was made of the mean residence times

(MRT) of fish farming wastewater in the phytotreatment system. To achieve this, the

following were measured: pond size and volume, flow rate of water from the fish

farming plant using a propeller-type current meter (OTT mod. C2; Germany; data

processed using BEAVER2 software) and minimum and maximum travel times using a

coloured tracer (fluorescein) in each of the four ponds and in the rearing tanks. Lastly,

the results were processed using a mathematical model (Tchobanoglous and Schroeder,

1985).

2.3. Physicochemical parameters

The parameters, temperature (T, jC), pH, salinity (PTU) and dissolved oxygen (DO) as

percentage saturation, were measured hourly from June 1999 to May 2000. They were

measured using two multiparameter probes (HYDROLAB datasonde 3; USA) located at

the intake of the first pond (station E) for the water arriving directly from the intensive fish

farm and at the outflow of the last pond (station U) that was located after the water had

passed through all the lagooning ponds (Fig. 1).

S. Porrello et al. / Aquaculture 219 (2003) 515–529 517

2.4. Nutritional component analysis

Monthly, water samples were taken on input into phytotreatment system (first pond,

station E) and on output from phytotreatment system (fourth pond, station U) between

June 1999 and May 2000. Two field samples for each station were taken at 8:00 a.m. in

station E (E8) and at 4:00 p.m. in station U (U16). In fact, based on hydrological data

obtained, the MRT of the water mass for both the lagooning ponds and the rearing tanks

was computed throughout as 8 h each.

This experimental plan allows to verify efficiency of the phytotreatment system during

the highest macroalgal uptake rate and to analyse a sample water without variable

nutritional input. In fact, the feeding regime was considerably standardised (from 8:00

a.m. to 5:00 p.m. during winter period or to 6:00 p.m. during summer period) and the E8

and U16 water samples were deemed without wastes associated with feeding, food

wastage and metabolic excretion linked to feeding. As far as possible, in a standardized

daytime ‘‘scenario’’, we analysed samples of the same water after crossing the phytotreat-

ment system (8 h).

The four original water samples were immediately transferred to the laboratory where

they were filtered through a Whatman GF/F 0.70-Am filter paper to determine the

dissolved components and through the Millipore APFF 0.70-Am filters to determine the

particulated component. The water filtered was stored at � 20 jC before performing the

following analyses: nitrogen nitric (N–NO3), nitrogen nitrous (N–NO2), nitrogen ammo-

nium (N–NH4), total dissolved nitrogen (TDN), orthophosphates (P–PO4, soluble

reactive phosphorus (SRP)) and total dissolved phosphorus (TDP). The data reported

were the results from a set of four analytical data carried out using a continuous-flow

Autoanalyzer (BRAN+LUEBBE AA3; Germany). The dissolved inorganic nitrogen

(DIN =N–NO2 +N–NO3 +N–NH4), dissolved organic nitrogen (DON=TDN�DIN)

and dissolved organic phosphorus (DOP=TDP� SRP) were then computed. Concen-

trations were expressed in micromolars. The filters were conserved in plastic Petri capsules

at � 20 jC. After desiccation for 16 h at 70 jC and subsequent gravimetric determination,

they were used for determination of total suspended matter (mg/l, TSM), particulated

organic carbon (AM, POC), total particulated nitrogen (AM, TPN), and total particulated

phosphorus (AM, TPP). POC and TPN were determined using an elemental analyser

(CHNS Thermofinnigan model EA 1110; USA); TPP was determined according to Aspila

et al. (1976).

The ammonium ion abatement during passage through the phytotreatment system was

calculated as (U16N–NH4�E8N–NH4

)(E8N–NH4)� 1�100.

Lastly, total nitrogen (TN=DIN +TPN) and total phosphorus (TP= SRP+TPP) com-

ponents were also computed, together with the DIN/SRP, POC/TPN/TPP and TN/TP atomic

ratios.

2.5. Macroalgal biomass determination

Monthly quantitative algal biomass samples were taken along three parallel, equidistant

transects in each pond. Three sampling stations were set up along each transect (n = 36

sampling stations in all phytotreatment ponds). U. rigida C. Ag was collected at each

S. Porrello et al. / Aquaculture 219 (2003) 515–529518

station using a 2500-cm2 enclosure system. The wet weight (w.w.) of each sample was

determined according to Bellan-Santini (1969) and statistical evaluation of biomass data

was determined according to Elliott (1971).

2.6. DIN and SRP uptake rate

In order to highlight any relation between DIN and SRP abatement between E8

and U16 and U. rigida growth in the lagooning system, DIN uptake rate and SRP

uptake rate were measured according to Jimenez del Rio et al. (1996) using the

equation:

V ¼ QðE8DIN;SRP � U16DIN;SRPÞA�1

Q =water mass crossing the system in retention time flow rate (l� 0.33 day� 1),

E8DIN,SRP=DIN or SRP inflow (mg l� 1), U16DIN,SRP=DIN or SRP outflow (mg l� 1),

A= phytotreatment surface (m2). When negative V was found, conditions of nutritional

release by the lagooning system were deemed to have prevailed (�V=R).

2.7. Statistical analysis

The physicochemical, nutritional and algal biomass data collected during the exper-

imental period were subjected to discriminant multivariate analysis (Morrison, 1976;

Mellinger, 1987) in order to detect any differences between the extreme ponds, namely, at

the first (station E8) and at the fourth (station U16).

Moreover, in order to reduce type I errors as well as the risk due to possible

interrelations among the variables contained in the discriminant model, the significance

level was suitably corrected by applying the Bonferroni correction (Hocheberg and

Tamhane, 1987). This correction was applied to the 16 simultaneous Student’s t-tests

computed for the purpose of examining the discriminant function coefficients and to show

for which variables the two compared ponds are significantly different.

3. Results

3.1. Hydrology

The lagooning area had a total area of 10,451 m2 and a volume of 7134 m3, accounting

for up to 79% of the volume of the intensive fish farm (Fig. 1).

Water intake rate into the lagooning section from the production section was 139.5 l s� 1

which gave a daily flow rate from the lagooning section of 12,052.8 m3, corresponding to

1.69 renewals day� 1 and hydraulic residence time (HRT) of 14.2 h.

Using fluorescein, the residence time of the water mass in the lagooning ponds was

computed as a minimum of 77 min and a maximum of 14 h. The mathematical model

used showed a computed MRT of 8 h for both the phytotreatment ponds and the

S. Porrello et al. / Aquaculture 219 (2003) 515–529 519

rearing tank. Thus, the water entering the fish farm plant takes about 16 h to reach the

lagoon.

3.2. Physicochemical parameters

Percentage of DO in stations E and U displayed a very wide annual range of values,

from 2 to 183 (56.6F 26.7) and from 6.4 to 199 (96.6F 41.4), respectively. There was

Fig. 2. Ammonia and dissolved organic nitrogen (DON) (average and relative standard error) during sampling

period in E8 and U16.

S. Porrello et al. / Aquaculture 219 (2003) 515–529520

also a wide annual range of pH values, from 5.08 to 8.35 (7.38F 0.64) for E, and from

5.32 to 9.58 (7.82F 0.74) for U.

The temperature and salinity plots showed no substantial variation between stations E

(18.1 jCF 5.95; 37.1 PTUF 5.06, respectively) and U (20.1F 6.7 jC; 37.6F 3.0 PTU,

respectively), because of the flow rate and the small size of the ponds. Seasonal peaks and

drops followed the tidal rates of change, which led to the periodic intake of both lagoon

water and seawater by the plant.

Fig. 3. Nitrate and nitrite (average and relative standard error) during sampling period in E8 and U16.

S. Porrello et al. / Aquaculture 219 (2003) 515–529 521

3.3. Nutritional component analysis

Comparison between E8 and U16 allowed the daytime abatement of the nutritional load

of effluents and were evaluated after crossing through the lagooning system and through

any U. rigida bed.

The concentration of the nitrogenous compounds was characterized by the presence of a

particularly large quantity of ammonium ion in both sampling stations (Fig. 2). Conversely,

the presence of nitrites and nitrates was very low (Fig. 3). There was a strong presence of

organic nitrogen (Fig. 2) between 12% and 36% of TDN, without any significant

differences between E8 (mean 26F 6) and U16 (mean 24F 6). Ammonium ion under-

went abatement during passage through the lagooning system varying between � 1% and

� 44% (mean � 21.9F 12.4). Nitrites and nitrates increased during passage through the

ponds (Fig. 3). The nitrites, in particular, displayed a constantly high percentage increase

(mean 160F 106), with a peak of 275% in June, indicating that the nitrification process

was well under way. Overall, DIN displayed a percent abatement of � 5.5F 10.9, with

highly variable monthly values lying between a peak abatement of � 26.9% and an

increase of 10.4% (Table 1).

DON displayed abatements/increments between � 45.3% (spring) and 51% (autumn).

Phosphorus trends (SRP, DOP) are shown in Fig. 4. The percentage increase/abatement

values of this latter component varied between 100% and � 58% (� 1.42F 45.9). Table 1

shows an SRP annual mean value of � 14.7F 14.3. The maximum abatement occurred

between June and September 1999.

Figs. 5 and 6 show the trends of the various particulate components (TSM, POC, TPN,

TPP) for E8 and U16. No seasonal trends were found and abatement for all parameters

varied from 19% to 52%. The values of the POC/TPN/TPP ratio remained practically the

same between E8 and U16 (77:12:1 and 72:12:1, respectively). Instead, a change in the

Table 1

DIN and SRP monthly values in E8 and U16

DIN E8 DIN U16 % SRP E8 SRP U16 %

1999

June 65.1 58.1 � 10.8 4.2 2.8 � 33.3

July 66.5 71.5 + 7.5 5.7 4.0 � 29.8

August 64.2 70.9 + 10.5 4.4 3.4 � 22.7

September 81.4 79.6 � 2.2 7.2 4.5 � 37.5

October 91.6 92.4 + 0.9 5.2 4.9 � 5.8

November 63.7 61.6 � 3.3 4.4 3.6 � 18.2

December 51.2 47.5 � 7.2 1.9 1.4 � 26.3

2000

January 30.1 30.0 � 0.2 1.4 1.4 � 6.2

February 66.2 55.3 � 16.5 3.5 3.3 � 5.7

March 87.5 74.2 � 15.2 6.8 6.4 � 5.9

April 86.9 74.4 � 14.4 3.4 3.8 + 11.8

May 64.5 68.2 + 5.7 3.7 3.5 � 5.4

Percentage of cut (� ) or of increase (+) of DIN and SRP during the run between E8 and U16.

S. Porrello et al. / Aquaculture 219 (2003) 515–529522

nature of the particulated organic component was observed with POC annual means of

112.7F 31.5 and 65.1F 22.9 AM for E1 and U16, respectively.

In addition, as far as the dissolved inorganic fraction was concerned, the DIN/SRP ratio

did not undergo any significant change between intake and outflow from the lagooning

ponds (from 15 for E8 to 17 for U16). There was a comparatively small variation also in

the TN/TP molar ratio, which rose from 17.2 in E8 to 18.8 in U16.

Fig. 4. Soluble reactive phosphorus (SRP) and dissolved organic phosphorus (DOP) (average and relative

standard error) during sampling period in E8 and U16.

S. Porrello et al. / Aquaculture 219 (2003) 515–529 523

3.4. DIN and SRP uptake rate and macroalgal biomass determination

Table 2 shows the DIN and SRP uptake rates (V) and the DIN and SRP release rates (R)

during the period of the tests, comparing them with the macroalgal biomass values.

Experimental data show that the best uptakes and best DIN abatements occurred between

Fig. 5. Total suspended matter (TSM) and total particulated nitrogen (TPN) (average and relative standard error)

during sampling period in E8 and U16.

S. Porrello et al. / Aquaculture 219 (2003) 515–529524

February and June and were strongly dependent on macroalgal density. Table 2 shows that

the most efficient macroalgal density for nutrient capture ranged between 700 and 1500 g/

m2. At higher densities, abatement was reduced or, as in May 2000, release prevailed since

the macroalgae began decomposing as a result of an early rise in temperature compared

with the year before. Abatement and uptake were negligible during the warmer season,

Fig. 6. Particulated organic carbon (POC) and total particulated phosphorus (TPP) (average and relative standard

error) during sampling period in E8 and U16.

S. Porrello et al. / Aquaculture 219 (2003) 515–529 525

during which nutritional release prevails, and during the autumn and first half of the

winter. The situation regarding phosphorus was different: percentage abatement was

higher between June and October, and lower during the remaining period. Only in April

did a case of release occurred. As far as this element was concerned, abatement did not

seem to be closely related to algal biomass. In fact, abatement occurred also in conditions

in which the biomass was strongly reduced. Probably, the abatement of this element was

also linked to the biogeochemical conditions of the ponds rather than to macroalgal uptake.

3.5. Statistical analysis

Discriminant analysis shows that the different parameters were significantly different

between E8 and U16 (Fig. 7). One effect of the lagooning system on the quality of the water

passing through it was reflected both in the dissolved components and in the particulate.

This analysis that was applied to the physicochemical, nutritional and biomass

parameters generated a discriminant function based on the linear combination of 16

variables considered. The function provided the best discrimination (100% of the cases

examined discriminated correctly) between the two stations. In fact, the overall test result

of the analysis, based on the Mahanobis Distance and aimed at testing for the existence of

a significant difference between the two stations for at least 1 of the 16 variables

considered, proved significant (F(16, 82) = 535, 25; p = 0.0001). Moreover, the introduc-

tion of the Bonferroni correction (a* = a/K = 0.05/16 = 0.003) on the set of simultaneous

Student’s t-tests (applied to each of the variables considered) showed that the perfect

discrimination of the two basins may be attributed to (1) a significant reduction in the

parameters determined on the particulate and a reduction in TSM; and (2) a significant

increase in nitrites, nitrates, algal biomass and pH.

Table 2

Monthly biomass estimate (g/m2 w.w.) of U. rigida that develops into phytotreatment pond system

U. rigida DIN removal efficiency SRP removal efficiency

(g/m2 w.w.)V R V R

1999

June 1800 37.3 16.5

July 57 26.6 20.1

August 10 35.8 11.8

September 20 9.6 31.3

October 21 4.3 3.5

November 20 11.2 9.4

December 50 19.7 5.9

2000

January 200 0.3 1.1

February 747 58.1 2.4

March 1100 70.9 4.7

April 1560 66.6 4.7

May 1800 19.7 2.4

Removal efficiency is reported in mg m� 2 day� 1 as DIN and SRP uptake rate (V) and as DIN and SRP release

rate (R).

S. Porrello et al. / Aquaculture 219 (2003) 515–529526

Fig. 7 shows the common intergroup correlations computed among discriminant

variables and the canonical discriminant function. These correlations were ordered

according to their absolute value. The black bars indicate the reduction in concentration

of certain parameters during the passage through the intake pond and the outflow pond,

while the grey bars indicate an increase. The vertical line separated the set of variables

found to be significant after the introduction of type I error correction from those that,

although having been included in the model, did not appear to have played any decisive

role in discrimination.

4. Discussion and conclusions

The nutrient abatement pursued through the construction of the phytotreatment system

downstream from the fish farming plant appear to have been partly attained. Indeed, the

results indicated that reduction in nutrient output is possible from an intensive land-based

aquaculture plant. Statistical analysis has indicated an appreciable differentiation of the

water masses between their inflow and their outflow from the lagooning system. However,

the sizing of the system and the water retention times were found to be insufficient to achieve

a substantial reduction in the two main eutrophizing components. The difference between

the calculated HRT of 14.2 h and the estimated MRT of 8 h meant that the water that run

through the phytotreatment system followed a preferential course and that large patch was

stagnant. Improvements of water circulation should be desirable to get closer to HRT value.

With respect to physicochemical parameters, the maximum values recorded were found

in U in spring following the intense photosynthetic activity, and the lower values in

summer in both E and U, at the height of animal metabolic activity in the rearing tanks and

by macroalgal decomposition in the phytotreatment ponds.

Fig. 7. Common correlation within groups between discriminant variables and canonical discriminant function.

The variables are the ground of the absolute dimension of the correlation within the function.

S. Porrello et al. / Aquaculture 219 (2003) 515–529 527

Mean DIN abatement was slightly greater than 5%, rising to 7% for TDN. The situation

was improved by taking TN into consideration, the abatement of which was found to be

12.4%. This shows that the macroalgae, under the present system management conditions,

was not the most important factor. The more important abatements of TDN and TN are

probably due to bacterial activity and physical factors, such as particulate sedimentation.

The system proved more efficient for phosphorus, with an abatement of about 15% for

SRP and 21% for TP. The more abatement of P was confirmed also by the N/P atomic

ratios which increased, albeit slightly, from E8 to U16 (17.2 and 18.8, respectively). For

this element, the macroalgae factor seemed to act more selectively, even with compara-

tively small algal biomass (Table 2). The small increase in the N/P ratio in the outflow

waters did not attain the values recorded in the receiving environment, the values of which

were always >30 (Lenzi et al., 1998). This ratio is, therefore, still a disturbing source. It

expressed an availability of phosphorus compared with the limited phosphorus levels in

the receiving environment.

DON and DOP released by the phytotreatment system were probably a result of

decomposition of the abundant deposited organic matter.

The best DIN removal efficiency recorded was found to be more than 30 times smaller

than the value estimated in the pilot experiment of Jimenez del Rio et al. (1996). These

values, in perspective, could be improved and brought closer to those found by the

previous cited authors by increase of residence time and introducing a more efficient

pathway for the passage of the water masses through the series of four ponds.

Into the system studied, considering the short residence time of the water inside the

lagooning ponds, the following conclusions may be drawn.

(1) Clear-cut effects were related to the nitrification process which, during the compulsory

pathway followed by the water, was triggered to an appreciable extent (percentage

difference>200%), thus determining a reduction in ammonia load and reducing the

toxicity of the water itself. At the moment, it is impossible to quantify the influence of

denitrification process into specific environment. In fact, the rate of denitrification

depends on temperature, nitrate, organic carbon and oxygen concentrations and

denitrifying bacteria density; but, above all, in most natural aquatic systems, it can be

considered substrate-limited (Hargreaves, 1998). According to Ziemann et al. (1992)

and Tucker and van der Ploeg (1993), for aquaculture ponds, also in this case the

presence of nitrate ion (N–NO3), although with high nitrification rate, was less than to

16 AM (Fig. 2).

(2) SRP abatement was constant and appreciable.

(3) Clear effects were obtained in the particulate where the organic fraction was reduced to

a much greater extent than the inorganic fraction.

(4) The physical–chemical parameters were modified appreciably, in particular as far as

pH and DO values were concerned.

As suggested by Jimenez del Rio et al. (1996), macroalgal density should be

maintained at an optimal density level to improve DIN abatement values. At the moment,

the phytotreatment basin has never been managed, and during the warm season the

macroalgal vegetation dies, producing anoxigenic process.

S. Porrello et al. / Aquaculture 219 (2003) 515–529528

Finally, according to Shpigel et al. (1993), an improvement of environmental quality

into phytotreatment system (as absence of free ammonia and sulphide) could allow the use

into the basins of animal biomass; this will consent further nutritional parameters

abatement.

Acknowledgements

We wish to thank Mr. Fabio Savelli (ICRAM S.T.S. Chioggia) for the contribution he

made to the research regarding the CHN analysis of the sediment samples, Dr. Patrizia

Solimeno for her participation in the sampling campaign, Dr. Roberto Salvatori and Soc.

Orbetello Pesca Lagunare for making available information regarding fish farm

management.

This work was financed by a grant given by the Italian Agricultural and Forest Politics

Ministry.

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