Aquaculture Research. 1995.26, 639-650

Nitrogenous excretion in juvenile turbot,Scophthalmus maximus {L,), under controiiedconditions

A Dosdat, R Metailler, N Tetu, F Servais, H Chartois, C Huelvan & E DesbruyeresIFREMER. Laboratoire de Physiologie des Poissons. Laboratoire de Nutrition des Poissons. Plouzane. France

Correspondence: A. Dosdat. IFREMER. Laboratoire de Physiologic des Poissons. PB 70-29280 Plouzan^. France

AbstractLaboratory experiments on juvenile turbot.Scophthalmus maximus (L.). were carried out undercontrolled temperature (12''C) and feeding regimesin a flow-through system. Monitoring of totalammonia nitrogen (TAN) and urea nitrogen (Urea-N) was performed through continuous sampling ofthe effluent sea water. The effects of ingested nitrogenlevels on TAN and Urea-N daily and hourly excretionrates were studied. Strong relationships were foundbetween ingested nitrogen, and both TAN and Urea-N excretion for both daily and hourly maximumexcretion rates. Turbot showed a low metabolicactivity, confirmed by low excretion levels. Dailypatterns for TAN and Urea-N production were dif-ferent, suggesting specific physiological phenom-enons for urea excretion mechanisms. The questionof whether turbot are partly ureotelic or ureogenic isunderlined.

Introduction

Quantification of nitrogenous excretion in fish hasbeen recognized to be of major interest by manyauthors because of the importance of water qualityin fish culture (Handy & Poxton 1993) andtransportation, and the fact that any improvementof dietary protein utilization by fish makes anevaluation of nitrogenous waste productionnecessary (Kaushik. Fauconneau & Blanc 1984).

Ammonia and urea are the two main excretoryproducts of nitrogen metabolism in teleost fish (forreviews, see Dosdat 1992; Handy & Poxton 1993).Other minor excretory products (e.g. creatinine.

creatine and uric acid) are also mentioned (Forster &Goldstein 1969). Ammonia usually represents 75-90% of the nitrogen output for aquatic teleosts livingin good-quality waters (Wood 1958; Sayer &Davenport 1987). Ammonia and urea appear to beexcreted mostly via the gills (Smith 1929; Fromm1963; Vellas. Flavin & Creach 1970; Randall &Wright 1987; Sayer & Davenport 1987).

Many factors may affect nitrogen excretion.These include: rearing conditions (Fromm &Gillette 1968; Olson & Fromm 1971; Sukumaran& Kutty 1977; Wilkie & Wood 1991); body weight(Kikuchi. Takeda. Honda & Kiyono 1990); species(Davenport. Kjorsvik&Haug 1990; Gershanovich& Pototskij 1992); intra-species families (Gallagher.Kane & Courtney 1984; Kaushik et al. 1984: Ming1985); and physiological status (Wiggs. Henderson.Saunders & Kutty 1989). However, the level ofingested nitrogen appears to be the most importantfactor because of either the quantity (Gerking1971: Savitz. Albanese. Evinger & Kolasinski 19 77:Jobling 1981: Poxton & Lloyd 1989: Kikuchi.Takeda. Honda & Kiyono 1991) or quality(Beamish & Thomas 1984: Forsberg & Summerfelt1992: Li & Lovell 1992) of the ingested ration. Infish, a post-prandial increase in ammonia excretionis systematically observed (Brett & Zaia 1975:Kaushik 1980a: Ramnarine. Piriet. Johnstone &Smith 1987). whereas no such pattern has beennoted in urea excretion (Brett & ZaIa 1975; Tatrai1981; Fivelstad.Thomassen, Smith. KjartanssonSSandol990).How-ever. Kikuchie(a/. (1991) pointout that Japanese flounder. Paralichthys oUvaceus(Temminck & Schiegel). shows fluctuations in therate of urea excretion. This feed-related excretion

639

Nitrogenous excretion in turbot A Dosdat et al. Aquaculture Research, 1995,26, 639-650

is the so-called 'exogenous excretion'. Endogenousexcretion, representing body protein and nucleicacid catabolism, is evaluated through monitoringexcretion in starved fish (Birkett 1969: Ogino, KaJdno&Chenl973;Kaushik 1980b; Salin&Williot 1991)or in fish naaintained in nitrogen balance (Jobling1981;Ra[nnarineetfll, 1987).

This study is aimed at evaluating the effect of nitrogenintake on ammonia and urea excretion levels andpatterns in juvenile turbot kept at a constanttemperature. Turbot presents a high aquaculturalinterest in Europe (Person-Le Ruyet 1993) and itsproduction is gradually increasing. Further investi-gation is necessary to evaluate the nutritionalcharacteristics of this species, which presents very highprotein utilization (Caceres-Martinez, Cadena-Roa &Metaillerl984).

Materials and methods

Rearing conditions

Turbot were obtained from the IFREMER expe-rimental hatchery in Brest. France. Six batches of 200juveniles (average weight 13.5±1.5g) wererandomly distributed in six plastic-grid baskets{height 50 cm. diameter 55 cm) with a flat bottom,placed in six identical 144-1 cylindroconical tanks(Fig. 1), In a flow-through system, sand-filtered seawater (temperature 12 ± 0.2°C. salinity 34 gl ' ) wassupplied to the tanks at a flow rate of 240 I h"'. Thedissolved oxygen concentration at the outflow wasmaintained above 80% saturation. Water inflow in eachtank was monitored twice a day, before and after everyfilter cleaning operation. Fhotoperiod was controlled on12:12 h(1900-O700h)dark: light cycle.

Feed and feeding

Fish were fed twice daily (1000 and 1600 h) crumbles(4-5 mm) of an experimental reference expanded diet(Table 1) containing 54% crude protein (N x 6.25)and 16% crude fat on a dry basis. Digestibility ofprotein (89.0%) was determined by using decantationmethod (Cho, Slinger & Bayley 1982) and chromicoxide as a marker (1% of the diet).

The six groups of fish were acclimatized for 2 weeksin the experimental tanks. During this period, the sizeof the maximum ad libilum ration was determined byfeeding the fish to satiation. Then fish were weighedand different ration levels, 20, 36. 52, 68, 84 and100% of the ad libitum ration, respectively, were

Table 1 Composition of the experimental reference diet

Ingredient

Norwegian fish meai

Soluble fish protein concentrate

Greaves mealLactic yeast

Cooked potatoes starchSoy lecithinCod liver oil

Inorganic bulk agent (zeolite)Vitamin mix

Mineral mix

Mixture (g 100 g-')

14.9 ••15.09^

12.01.0

11.95.1

1.01.9

randomly affected to the different groups. Theexperiment lasted 37 days: a 27-day feeding period,followed by a 10-<iay starvation period. The rationssupplied to fish, expressed in mg N day', were keptconstant throughout the feeding period. Fish wereweighed at the end of the feeding and the starvationperiods.

During feeding, particular attention was given tothe ingestion of the whole ration. If some remaineduneaten, these food particles were collected, countedand deducted from the distributed ration. Therefore,the distributed ration and feed intake were equal. Theactual ration levels were below the initial calculatedones because of weight increases during the feedingperiod.

Sampling and monitoring

Sampling of the outflow of the six tanks was carriedout daily during the last 10 days of the feeding periodand on each of the 10 days of the fasting period. Aseventh identical tank containing no fish was usedas a control. The sampling apparatus andmethodology have been previously described (Dosdat,Gaumet & Chartois 1994; Fig. 1). Continuoussampling for pooling and further analysis gave anaverage value of the daily concentration in the outputduring the two 10-day periods. Continuous sequentialsampling and immediate analysis gave aninstantaneous value for the outflowing concentrationfor each tank every 17 min 30 s. each analysis lasting2 min 30s.This was undertaken over 24 hat the endof the two periods. In both cases, samples werecollected through peristaltic pumps. Analyses werecarried on Technicon* Autoanalyser II. Totalammonia nitrogen (TAN) was analysed by the

640

Aquaculture Research. 1995.26, 639-650 Nitrogenous excretion in turbot A Dosdat et al.

modified indophenol blue method (Treguer & Le Corre1975) and urea nitrogen (Urea-N) by the acetyl-monoxyme method (Aminot&Kerouel 1982).

Data analysis

The continuous recording of Urea-N and TANconcentrations in effluent sea water were convertedinto Urea-N and TAN excretion rates every 17 min30 s interval using the following equation (Dosdat etal. 1994):

where Q is flow rate. C, is inlet ammoniaconcentration, C, and C, j outlet ammonia concen-tration. V is tank volume, t time between two samples.Bj the biomasse (fresh weight) and a the excretion rategiven in mg N kg FW"' h"

For the feeding period, daily excretion ratespresented in this study were the mean of the averagevalues obtained by pooling method over the last 10days. For the starvation period, they were the averageof those obtained after stabilization (the last 3 days:see 'Fasting period" below). Daily excretion ratesobtained each day by this method are not independentvariables. Therefore, no variance could be expressedon these daily values.

Results

Daily Tan and Urea-N production

Feeding period

For turbot fed the adhbitum regime, average daily TANexcretion was 158.8 mg N kg FW ' dajr' (Table 2).

representing 20% of the ingested nitrogen (i 8% of thedigestible nitrogen). Urea-N daily production for thesame regime amounted to 35.7 mgN kgFW"' day',representing 5% of the ingested nitrogen (4% of thedigestible nitrogen). Therefore. 25% of ingestednitrogen was excreted as ammonia and urea (22% ofdigestible nitrogen). For each feeding level. Urea-Nrepresented 16-22% of total TAN and Urea-Nexcretion.

Table 2 shows the daily TAN and Urea-N excretionin relation to the levets of ingested nitrogen. Thehigher the level of ingested nitrogen, the moreammonia and urea are excreted. These tworelationships are confirmed by the high adequacy oflinear regressions conducted (expressed ln mg N kgFW-' day-'):

Excreted TAN = 0.148 (ingested N) + 35.48; n = 6:r^=0.97: (1)

Excreted Urea-N = 0,030 (ingested N) + 12.04:n = 6: r = 0.95: (2)

Excreted (TAN + Urea-N) = 0.178 (ingested N) +47.52: n = 6: r^=0.98

(3).

Ninety-five per cent confidence intervals for y-axisintersections are ±17.06 and ±4.68 mg N kg FW"day-' for TAN and Urea-N excreted, respectively.

Fasting period

Figure 2 shows the changes of excretory nitrogenduring the 10-day starvation period. TAN excretion

peristalticpump

Instantaneous analysis

tank (M4 I) poolingcomputer colorimeter

Figure 1 Experimental apparatus for efnuent water monitoring from rearing tanks.

641

Nitrogenous excretion in turbot A Dosdat et al. Aquaculture Research. 1995.26. 639-650

Ingested

N

(1)

766.1692.3

584.9467.8352.4

210.70.0

Excreted

TAN

(2)

158.8136.0114.8

101.3

89.369.949.3

Exogenous

TAN

(3)

109.5

86.7

65.652.0

40.020.60.0

3/1

(%)

14

1311

111110•

Excreted

urea-N

(4)

35.7

33.5

27.725.6

24.617.4

9.6

Exogenousurea-N

(5)

26.1

23.918.7

16.015.27.8

0.0

5/1

(%)

34

334

4

4/(2-Ht)

<%)

18

2019

2022

2016

Table 2 Daily excretion rates of themajor nitrogenous end products inreiatioD to nitrogen intake (mg N kgFW'' day-'). Exogenous TAN (Urea-N) represents excreted TAN (Urea-N)minus TAN (Urea-N) excreted bystarved Pish

decreases rapidly over the first day of starvation (Fig.2a) for all groups. Minimum excretion rates weresystematically observed OD the second day of thestarving period. Excretion rates stabilized after 7 daysat similar levels of 40-50 mg N kg FW^' d a y ' for alltreatments.

Urea-N excretion also decreased on the first day ofstarvation {Fig. 2b). but with a lower amplitude thanTAN excretion. On the second day of starvation. Urea-N excretion increased to an intermediate level, andthen decreased again and tended to stabilize from thefifth day at 8-11 mg N kg FW' day ' . During the firstdays of starvation. Urea-N excretion rates seemedrelated to initial feeding levels.

Endogenous excretion rates for TAN and Urea-N.calculated from the regression equations (for noingested nitrogen) were 35.5 ± 17 mg N kg FW"' day 'and 12.0± 4.7 mgNkgFW^' day ' , respectively.Thisagreed with the mean values obtained for starved fish.

of 49.3 and 9.6 mg N kg FW^' day ' , respectively. Atthe end of the starving period. Urea-N excretionrepresented 16% of the total TAN and Urea-Nproduction.

Daily TAN excretion pa t te rns

Daily patterns forTAN excretion are presented in Fig.3. At any hour, instantaneous TAN excretion rateswere higher at higher feeding rate. Thus, the excretionprofile was a function of the ingested nitrogen. Thisconfirmed the results obtained on daily productionusing the data from the pooling method, as presentedin Table 2. For the two highest feeding rates, two peaksseemed noticeable around 6 h post-feeding. A goodcorrelation occurs between the maximum hourlyexcretion rate (expressed in mg N kg FW"' h^') andthe feeding regime (expressed in mg N kg FW"' day ' ;Table 3):

160 i- (a)

2 4 6Days after starvation

2 4 6Days after starvation

Figure 2 Nitrogen excretion during the starvation period. Day 0 is the last day of feeding. Percentages refer to the feedingestion rates; (a) TAN excretion; and (b) Urea-N excretion.

AquacultureResearcii, 1995.26, 639-650 Nitrogenous excretion in lurbot A Dosdat et al.

TAN = 0.0090 (ingested N) + 1.70; n= 6: H = 0.98

(4).

Twenty-four hours after first feeding, the excretion

rates were higher for fish fed the higher ration levelthan for fish fed lower rations or starved fish.

Excretion profile for fish starved for 9 days was fairlyconstant at 1.85-2.40 mgN kg FW^'h'. representing

I i i i [ i l i ] i l i i r i t i i i i i j i l i i i X i i i l i i i l r i i i L j . , l M i J r i i l j i i l i i i J i M A i i i l i i i J r u i L r ] i l i i i J i [ [ I I I

Tinne (decimal hours)

Figure 3 Influence of ratJon size on TAN excretion patterns. The values represent the average excretion rates during successive17 min 30 s intervals. Arrows indicate time of feeding. Percentages refer to the feed ingestion rates. The dark area correspondsto the dark period. Mean endogenous TAN excretion is given as a reference.

Table 3 Parameters for TAN and Ufea-N excretion patterns (maximum and minimum values in mg N kg FW"' h"': timesin decimal hours after first meai) in relation to nitrogen intake (mg N kg FW"' day"')

IngestedN

786.1692.3584,9467.8352.4210,70,0

Maximumvalue

8,867.896.706,314,903,492.41

TAN excretion

Minimumvalue

4.212.893,082,301.931.941.85

Time-lag forfirst peakmaximum

5,85.3----

-

Time-tag torsecond peakmaximum

11,110.510.89,38,89.0

-

Urea-N excretion

Maximumvalue

4,524.433,503,193,002.131.42

Minimumvalue

0,130,110,080,070,070.090.02

Time-lagfor peakmaximum

16.016.316.316.916.917.517.2

6*3

Nitrogenous excretion in turbot A Dosdat et al. Aquaculture Research. 1995.26, 639-650

a daily excretion rate of 49 mg N kg FW"' day'.

Urea-N excretion daily patterns

The pattern for fed fish is presented in Fig. 4. For eachration level, a single peak is noticeable, the amplitudeof which is related to the ration level. A linearregression between the maximum excretion rate andingested nitrogen (Table 3) indicates the followingrelationship:

Urea-N = 0.0041 (ingested N) + 1.33: n = 6: r =0.96. (5)

The maximum excretion rates of TAN were doublethose of Urea-N at each feeding level i.e. 8.88 and4.52 mg N kg FW'' h"', respectively, for the highestfeeding regime). For Urea-N, they only occur duringa short period (1 h 30 min). The time-lag before theappearance of the peak increased with decreasingingested nitrogen from 16 to 17.5 h after the firstfeeding. Irrespective of feeding rates. 24 h after first

feeding, the excretion rates were similar.For starved fish. Fig. 5 shows a synchronized

excretion peak for each tank, irrespective of theprevious feeding rate. These peaks reach 1.2-1.5 mgN kg FW'' h~'. 8 h after the beginning of the darkperiod. Both the timing of the peak and the peakduration were similar to the fed fish. In each case, ureaseems to be excreted essentially during the darkperiod.

Discussion

Ammonia excretion

TAN excretion in turbot does not show specificdifferences from other teleost fish that have beendescribed (Mommsen & Walsh 1991; Dosdat 1992:Handy & Poxton 1993). At every feeding level,postprandial TAN excretion rate Increases rapidlybefore returning slowly to the initial level. When fedtwo meals a day. two peaks seem noticeable, as it isnoted by Kaushik (198Ga} in rainbow trout.

1

mg

Ure

a-N

8.00

8.00

7.00

6.00

5.00

4.00

3.00

2.00 -

1.00 -

0.00

1

Time (decimal hours)

Figure 4 Influence of ration size on Urea-N excretion patterns. The values represent the average excretion rates duringsuccessive 17 min 30 s Intervals. Arrows indicate time of feeding. Percentages refer to the Teed ingestion rates. The darkarea corresponds to tbe dark period. Mean endogenous Urea-N excretion is given as a reference.

AquacultureResearch. 1995.26, 639-650 Nitrogencus excretion in turbot A Dosdat et al.

Time {decimal hours)

Figure 5 Urea-N excretion pattern of fish starved for 9 days. The values represent the average excretion rates during twosuccessive 17 min 30 s intervals. Percentages refer to the previous feed Ingestion rates. The dark area corresponds to thedark period.

(Oncorhynchus mykiss (Walbaum)). and Ramnarineetfl/. (1987) In cod. Gadus morhua L.

Feeding level has a general effect on daily TANexcretion, daily pattern, maximum hourly excretionrate, prefeeding excretion level, and time-lag betweenfeeding(s) and excretion peak(s). Determinatiortcoefficients between ingested nitrogen and eithermaximum TAN values (r = 0.98) or time-lag betweensecond meal and late peak (r = 0.79) are high. Thisis in agreement with the physiological aspects ofdigestion:

(1) Gut proteolytic enzymes act more quickly on asmaller meal because of transit speed (Fange &Grove 1979). This might explain the decreasingtime lag observed when fish were fed smaller

rations.(2) Transaminating and deaminating enzymes in

the hepatocytes have the same ammonia-productive activity, given the same biologicalvalue and amino-acid balance of the diet.

The prefeeding level is also in accordance withthese aspects, indicating that, 24 h after first feeding.the whole ration had not yet been completelymetabolized, except for the three lowest regimeswhich showed values comparable to the endogenousrate (1.8-2.4mg N kg FW' h ' ) - The effect ofrelatively low water temperature (around 5 *C underthe optimum value for the species) on this aspectshould be pointed out.

Considering that endogenous excretion continues

645

Nitrogenous excretion in turbol A Dosdat et al. AquacultureResearch. 1995.26. 639-650

at the same rate during the feeding period (Jobling1981). exogenous TAN values indicate that theproportion of ingested nitrogen diverted to ammoniadecreases with decreasing feeding level, indicating animproved utilization of ingested proteins at lowfeeding levels.

The absence of any marked pattern for starved fishis also described for other species. This criteria is notmore turbot specific than TAN excretion pattern infed fish. This confirms that the postprandial patternsobserved in fed fish are caused by exogenous TANexcretion alone.

The specificity of turbot TAN excretion relies moreon the level of ammonia production than on itsmodalities. Table 4 compares levels of ammoniaproduction by different fish species raised undercontrolled conditions, particularly those concerningingested nitrogen. Turbot fed ad libitum have thelowest ratio (20%) between excreted TAN andingested nitrogen. This is comparable to the Japanese

flounder. In the same way. Davenport et al. {1990)related a daily TAN excretion of the same order ofmagnitude: 165 mg N kgFW"' day' in 450 g halibut.Hippogiossus hippogJossus L. Flatfish seem to havelower ammonia production levels than pelagicspecies.

Moreover, in turbot. the regression coefficient ofTAN excretion against ingested nitrogen (0.148 inour study) is low compared to that described in otherteleosts: 0. 160 in Japanese flounder (Kikuchi.Takeda. Honda & Kiyono 1992): 0.180 in plaice.Pkuronectes platessa L. (Jobling 1981): 0.375 inrainbow trout (Kaushik 1980b): 0.400 in largemouthbass. Micropterus salmoides Lacepede (Savitz et al.1977); and 0.492 in sea bass. DicentrarchuslabraxL.(Vitale-Lelong 1989). This is in accordance with thegood protein utilization, the low exogenous losses andthe low activity turbot demonstrate.

Excretion by fasting turbot (mean rate 49 mg N kgFW"' day') is also iow. but cannot be easily compared

Table 4 Daily nitrogen excretion rates for different fish species in relaUon to nitrogen intake (mg N kg FW"' day-')

Specln

Sea bass.Dicentrarchus labrax

Atlantic cod.Gadus morhua

Sea bream,Sparus auraia

Atlantic salmon.Salmo salar

Rainbow trout,Salmo gairdneri

3ream,Abramis brama

Japanese'louncJer.Paralichthys olivaceus

Siberian sturgeon,Acipenser baeri

Turbot.Scophthalmus maximus

Experimental

conditions

40 g

7g.

200 g,

90 g.3g.

2000 g.

IXg,

35 g.

1.8-5g.15-50g.189-575g.

270 g.

10 g.

14=023''C

24°C1302

24°C24''C

12°C

1S-1S

IngestetN

7782000

325

9982899

641

C1143

Tan excretion

rateStarved

216

156801

—

—

64

15''C 400 60(Zooplankton)

2O''C20-02O''C

17°C

12''C

2280990723

1125

786

1835448

32

49

Fed

5481160

244

€2

3531032

144

517

220

517205267

315

158

TAN/Ingest. N

(%)

7058

75

3536

22

45

55

23

2137

28

20

Urea-N excretion

rateStarved

-50

—

—

—

20

10

211710

—

10

Fed

180

-

00

22

83

25

663830

—

36

Urea-N/

Ingest. N

(%)

9

-

00

3

7

6

344

—

5

References

Spyridakis (1989)Vitale-Lelong (1989)

RamnarinBetal.(1987)

Porter ef a/. (1987)

Fivelstadflfa/. (1990)

Kaushik (1980b)

Tatrai(1981)

Kikuchi eraf. (1990)

Kikuchi ef a/. (1991)Kikuchi ef a/. (1992)

Salin&Willlot(1991)

Present study

646

AquacultureResearch. 1995.26, 639-650 Nitrogenous excretion in turbot A Dosdat et al.

to other species, as metabolic rates rise with decreasingindividual weight and increasing temperature (Jobling1981:Tatrai 1981: Vitale-Lelong 1989; Kikuchietfli.1990: Davenport etfli. 1990).

The maintenance level of nitrogen that can bededuced from Eqn 3 when excreted (TAN + Urea-N)is equal to ingested N (Jobling 1981) is 58 mg N kgFW-'

Urea excretion

In turbot fed ad libitum, excreted Urea-N is equivalentto that found in other species, except for juvenilesea-bass (Table 4). Porter. Krom. Robbins. Briskell &Davidson (1987) did not notice any urea excretionfor sea bream. Sparus aurata L. On the other hand.Guerin-Ancey (1976a) noted a very high ureaexcretion in sea bass (confirmed by Vitale-Lelong1989). as did James. Probyn & Seiderer (1989) inCape anchovy,'Engraulis caperisis Gilchrist.

Contrary to the data published for salmonids (Brett& Zaia 1975: Kaushik 1980b; Beamish & Thomas1984). bream. Abramis brama L. (Tatrai 1981). orsturgeon. Acipenser ruthenus L. (Gershanovich &Pototskij 1992). in our study, turbot urea excretionis meal size dependant as shown by the gooddetermination coefficient of the regression given inEqn 2: r = 0.9 5. This also seems to be true in Japaneseflounder (Kikuchi et al. 1992). although a lowdetermination coefficient (H = 0.44) was obtained intheir study. Although it is impossible to qualifybecause of the absence of nitrogen intakequantification in the study of Guerin-Ancey (1976a).this also seems to be the case in sea bass. For thisspecies, a relationship between urea excretion andfeeding is reported by Vitale-Leiong (1989).

Urea production in fed turbot represents 4-8 ofingested nitrogen (which is higher than in otherspecies. Table 4). and 18-22 of total TAN -t- Urea-Nproduction. The latter ratio is relatively highcompared to other flatfish species, i.e. 19% inJapanese flounder (Kikuchi et al. 1991) and 12% instarry flounder. Platichthys stellatus (Pallas), (Wood1958). salmonids. i.e. 14% in rainbow trout (Kaushik1980b) and 13% in Atlantic salmon. Salmo salar L.(Fivelstad et al. 1990), and sea bass. i.e. 16-21% injuveniles (Guerin-Ancey 1976a).

For starved fish, compared to other species,endogenous urea excretion in turbot is low. as shownin Table 4. Nevertheless, urea contribution to totalTAN + Urea-N production is comparable in turbot

(17%) to other species, i.e. 10-15% in sea bass(Guerin-Ancey 1976b), 24% in rainbow trout(Kaushik 1980b). 10-24% in Japanese flounder(Kikuchi et al. 1990) and 20% in carp, Cyprinus carpioL. (Vellas et al. 1970). This relatively high valueencountered in turbot is a consequence of a com-paratively lower endogenous ammonia production.

The strong specificity of turbot urea excretion relieson the exceptional and unexpectedly daily patternobserved. Turbot. whether fed or not. show a singlehigh excretion peak approximately 17 h after firstfeeding and 8 h after the beginning of the dark phase.This peak also occurs 10 h after the first ammoniamaximum excretion rate. The time lag for itsapparition was dependant on nitrogen intake. Thedelay between the first meal and the urea excretionpeak is inversely related to the feeding level. Thisseems to indicate that urea excretion by turbot doesnot correspond to the biochemical phenomenadescribed by most of the authors (Forster & Goldstein1969: for review, see Mommsen & Walsh 1991).Indeed, urea production by teleosts includes twometabolic pathways:

(1) uricolysis through degradation of purines (e.g.adenine and guanine) into uric acid andallantoVn: and

(2) synthesis through the ornithine-urea cycle andarginase activity.

These two metabolic routes occur in the hepatocytes.It is generally assumed that urea released byammoniotelic freshwater teleosts is produced mainlythrough uricolysis and corresponds to an endogenousrhythm. It is reported to be linked to the constant bodyturnover (Brett&Zala 1975: Mommsen& Walsh 1992)and dependant on metabolic activity. Few examples of afunctional ornithine-urea cycle are reported amongteleosts (Read 1971). In extreme conditions, as fortilapia. Oreochromisalcalicusgrahami (Boulenger). in veryalktUine waters (Randall. Wood, Perry, Bergman. Maloiy,Mommsen & Wright 1989: Wood, Perry. Wright.Bergman & Randal! 1989) or air-breathing fish(Mommsen & Walsh 1989; Sayer & Davenport 1987).key enzymes of the ornithine-urea cycle may havesignificant activities.

Given such a specific urea production by turbotheld under controlled conditions, the question of theorigin of this excreted urea must be asked. A numberof hypotheses may be put forward;(1) Urea excretion could be the consequence of an

active urea cycle that would explain the linkagebetween urea production and nitrogen intake. In

647

Nitrogenous excretion in turbot A Dosdat et al. Aquaculture Research. 1995.26. 639-650

teleosts. the first key enzyme of the cycle, themitochondrial carbomoyl phosphate synthetase(CPS HI), is glutamine dependant (glutaminecomes from accretion of ammonia on glutamicacid), although CPS Is ammonia dependant invertebrates. This specificity of fish, added to thedelay caused by the time that the urea cycle takesto produce urea, could explain the time lagbetween ammonia and urea excretion peaks. The

I question if urea cycle enzymes are active and

I turbot are partly ureotelic remains unanswered.(2) Urea excretion could be the consequence of a

high affinity of mitochondrial arginase in theI turbot liver for dietary arginine. Arginase is the

last enzyme of the ornithine-urea cycie thathydrolyses arginine into ornithine and urea. Asarginine is an essential amino-acid. this routemight not be the most important one.

(3) Intermittent urea excretion might be caused bya pulsatile release of urea stored in the urinarybladder, as in the ureogenic toadfish, Opsanusbeta Goode & Bean. {Walsh. Danulat & Mommsen1990). Discontinuous urination is also reportedin the plaice, a flatfish (Fletcher 1990). The

I emptying during the night of such a bladder inturbot is still to be confirmed. This hypothesissuppose that urea excretion by turbot isperformed by the kidney instead of gills for tworeasons: (a) The bladder is linked directly to thekidney throught the urinary duct. Moreover, theexcretory function of the bladder is low{Cleveland. Hickman & Trump 1969). (b) Theexcretion rates observed beyond the excretionpeak are very low. So other routes, for ureaexcretion, particularly the gills, would be weakin case of urinary involvement.

(4) Urea could also be voided through the digestivetrack along with faeces, which seemed to beeliminated mostly during the same period thanurea. Urea has been found in faeces from turbotculture {G. Arzul. personal communication).

I This could explain both the association with, nitrogen intake and the rhythm of release.

Excretion by the skin should also be considered:however, this does not easily fit the observedphenomena.

The uniqueness of turbot concerning theirnitrogenous excretion indicates that further studiesshould be conducted in order to understand themetabolic pathways of ammonia and ureaproduction. This metabolic specificity has con-sequences in intensive fish farming, particularly in

recirculating systems in which Urea-N and TANshould be considered in the treatment process.Behavioural studies are necessary in order toinvestigate the role of nychthemeral rhythm on theobserved urea excretion pattern. Low nitrogenousexcretion rates in juvenile turbot were observed in thisstudy. This confirms the aquacultural interest of thisspecies, which utilizes dietary proteins very well.

References

Aminot A. & Kerouel R. (1982) Dosage automatique del'uree dans I'eau de mer: une methode trbs sensible a ladiacetylmonoxime. Canadian journal of Fisheries andAquatic Science Z9, 174-183.

Beamish F.W. & Thomas E. (1984) EITects of dietary proteinand lipid on nitrogen losses in rainbow trout, Saimogairdneri. Aquacukure'il, 359-371.

Birkett L. (1969) The nitrogen balance in plaice, sole andperch. Journal of Experimental Biology 50, 375-386.

Brett I.R. & Zala C.A. {1975) Daily pattern of nitrogenexcretion and oxygen consumption of sokeye salmon(Oncorhynchus nerka) under controlled conditions. Journalof the Fisheries Research Boardof Canada Z2,2479-2486.

Caceres-MartinezM.. Cadena-Roa M. & Metailler R. (1984)Nutritional requirements of turbot {Scophthalmusmaximus}. I. A. preliminary study of protein and lipidutilization, journal of tbe World Mariculture Society 15,191-202.

Cho C.Y.. SUnger S.J. & Bayley H.S. (1982) Bioenergetlcs ofsaimonid fishes: energy intake, expenditure andproductivity. Comparative Biochemistry and Physiology73B. 25 -41 .

Cleveland P.. Hickman I.R. & Trump B.F. {1969) The kidney.In: Fish Physiology. (Vol. 1 ed. by W.S. Hoar & D.J. Randail).pp. 91-239. Academic Press. New York.

Davenport I.. Kjorsvik E. & Haug T. {1990) Appetite, guttransit, oxygen uptake and nitrogen excretion in captiveAtlantic halibut. Hippoglossus hippoglossus L.. and lemonsole, Microstomus kitt {Walbaum). Aquaculture 90, 2 6 7 -277.

Dosdat A. {1992) Lexcretion chez les poissons teleosteens I.L'azote. La Pisciculture Frarii;aise 108 . 2 5—10.

Dosdat A.. Gaumet F. & Chartois H. (1994) Marine aquacultureeffluent monitoring; methodological approach to theevaluation of nitrogen and phosphorus excretion by fish.Aquacultural F,ngineering 14, 59 -84 .

FangeR&GroveD.(1979)Digestion.[n:Fis/iPhysio/ogy.(Vol. 8ed. by W.S. Hoar. D.J. Randall & I.R. Brett), pp. 162-241.Academic Press, New York.

Fivelstad S.. Thomassen J.M.. Smith M.I.. Kjartansson H. &Sando A.B. (1990) Metabolite production rates fromatlantic salmon [Salmosalar L) and artic char {Salvelinusalpinus L.) reared in single pass land-based brackish waterand seawater systems. Aquacultural Engineering 9. 1-21.

648

Aquaculture Research, 1995,26, 639-650 Nitrogenous excretion in turbot A Dosdat et al.

Fletcher C.R. (1990) Urine production and urination in thepiaice Pleuronectes platessa. Comparative Biochemistry andPhysiology 96A. 123-129.

Forsberg J.A. & Sumraerfelt R.C. (1992) Aminonia excretionby fingerlings walleyes fed two formulated diets.Progressive Fish-Culturist 54. 45-48.

Forster R.P, & Goldstein L. (1969) Formation of excretoryproducts. In: Fish Physiology, (Vol. 1 ed. by W.S. Hoar &D.J. Randall), pp. 313-350.

Fromm P.O. (1963) Studies on renal and extra-renalexcretion in a freshwater teleost. Salmo gairdneri.Comparative Biochemistry and Physiology 10. 121-128.

Fromm PO. & Gillette J.R. (1968) Effect of ambient ammoniaon blood ammonia and nitrogen excretion of rainbowtrout (Salmo gairdneri). Comparative Biochemistry andPhysiology 26. 887-8S6.

GallagherM.L.,KaneE.&CourtneyI.(1984)Differencesinoxygen consumption and ammonia production amongamericanelvers [Anguilla rostrata). Aquaculture40, 183-187.

GerkingS.D. (1971) Influence of rate of feeding and bodyweight on protein metabolism of bluegill sunfish.Physiology and Zoology 44. 9-19.

GershanovitchA.D. A PototskijI.V.(1992)Thepeculiaritiesof nitrogen excretion in sturgeons (Adpenser ruthenus){Pisces. Adpenseridae) - I. The influence of ration size.Comparative Biochemistry and Physiology 103A, 609-612.

Guerin-Ancey 0. (1976a) Etude experimentale deI'excretion azotee du bar (Dicentrarchus labrax) en coursde croissance I. Effets de la temperature sur I'excretiond'ammoniaque et d'uree. Aquaculture 9, 71-80.

Guerin-Ancey 0. (1976b) Etude experimentale del'excretLon azotee du bar (Dkentrarchus labrax) en coursde croissance. II. Effetts du jeune sur I'excretiond'ammoniaque et d'uree. Aquaculture 9. 187-194.

Handy R.D. & Poxton M.G. (1993) Nitrogen pollution inmariculture: toxicity and excretion of nitrogenouscompounds by marine fish. Reviews in Fish Biology andFisheries i. 205-24:1.

James A. G.. Probyn T. & Seiderer L.J. (1989) Nitrogenexcretion and absorption efTiciencies of the Cape anchovyEngraulis capensis Giichrist fed upon a variety of planctondiets. Journal of Experimental Marine Biology and EcologyU M O l - 1 2 4 .

Jobling M. (19 81) Some effects of temperature, feeding andbody weight on nitrogenous excretion in young plaicePleuronectes platessa L. Journal of Fish Biology 18.8 7-96.

Kaushik S.J. (1980a) Influence of a rise in temperature onthe nitrogen excretion of rainbow trout {Salmo gairdneriR.). In: Symposium on Nfw Developments in the Utilizationof Heated Effluents and Recirculalion Systems for IntensiveAquaculture. Stavanger. Norway. 28-30 May 1980. EIFAC;E:6, 32. 23 pp.

Kaushik S.J. (1980b) Influence of nutritional status on thedaily patterns of nitrogen excretion in the carp [Cyprinuscarpio L.) and the rainbow trout [Samo gairdneri R.).

Reproduction. Nutrition, Diveloppement 20,1751-1765.Kaushik S., Fauconneau B. & Blanc J.M. (1984) A study of

nitrogen excretion and oxygen consumption in five half-sib families of rainbow trout. Reproduction. Nutrition,Deveioppement 24. 431-438.

KikuchiK..TakedaS.. Honda H.&KiyonoM.{1990)Oxygenconsumption and nitrogenous excretion of starvedJapanese flounder Paralichthys olivaceus. Nippon SuisanGakkaishi 56.1S91.

Kikuchi K.. Takeda S.. Honda H. & Kiyono M. (1991) Effectof feeding on nitrogen excretion of juvenile and youngJapanese flounder. Nippon Suisan Gakkaishi 57. 2059-2064.

Kikuchi K.. Takeda S.. Honda H. & Kiyono M. (1992)Nitrogenous excretion of juvenile and young Japaneseflounder. Nippon Suisan Gakkaishi 58. 2329-2333.

Li M. & Lovell R.T. (1992) Effect of dietary proteinconcentration on nitrogenous waste in intensively fedcatVishponds. journal of the World Aquaculture Society 2i.122-127.

Ming F.W. (1985) Ammonia excretion rate as an index forcomparing efficiency of dietary protein utilization amongrainbow trout {Salmo gairdneri) of different strains.Aquaculture46. 27-35.

Mommsen T.P. & Walsh P.J. (1989) Evolution of ureasynyhesis in vertebrates: the piscine connection. Science245, 72-75.

Mommsen T.P & Walsh PJ. (1991) Urea synthesis in fishes:evolutionary and biochemical perspectives, (ed, byHochachka & T.P. Mommsen). In: Biochemistry andMolecular Biology of Fishes. Wo]. 1 pp. 137-163.

Mommsen T.P & Walsh P.J. (1992) Biochemical andenvironmental perspectives on nitrogen metabolism infishes. Experientia4S. 583-593.

Ogino C., Kakino J. & Chen M.S. (1973) Protein nutrition infish - II. Determination of metabolic fecal nitrogen andendogenous nitrogen excretion in carp. Bulletin of theJapenese Society of Scientific Fisheries i9, 519-523,

Olson K.R. & Fromm P.O. (1971) Excretion of urea by twoteieosts exposed to different concentrations of ambientammonia. Comparative Biochemistry and Physiology 40A,997-1007.

Person-Le Ruyet J. (1993) L'elevage du turbot en Europe.La Pisciculture Frangaise 112. 5-22.

Porter C.B.. Krom M.D. Robbins M.G.. Brickell L. & DavidsonA. (1987) Ammonia excretion and total N budget forgilthead seabream {Sparus aurata) and its effect on waterquality Aquaculture 66. 287-297.

Poxton M.G. & Lloyd N.J. (1989) Fluctuations in ammoniaproduction by eels {Anguilla anguilla L.) as a result offeeding strategy. In: Aquaculture - A Biotechnology inProgress. Vol. 16 (ed. by N. De Pauw. E. Jaspers, H. Actaifors& N. Wilkins), European Aquaculture Society, Bredene.pp. 1125-1135.

Ramnarine I.W., Piriet ].M.. Johnstone A.D.F. & Smith G.W.(1987)The influence of ration size and feeding frequency

649

Nitrogenous excretion in turbot A Dosdat et al. AquacultureResearch. 1995.26. 639-650

on ammonia excretion by juvenile Atlantic cod. Gadus

morhua L. Journal of Fish Biology 31, 545-559.Randall D.J.. Wood CM., Perry S.F.. Bergman H.. Maloiy

G.M.O.. Mommsen T.R & Wright P.A. (1989) Ureaexcretion as a strategy for survival in a lish living in a

very alkaline environment. Nature 337.165-166.Randall D.J. & Wright P.A. (1987) Ammonia distribution

and excretion in fish. Fish Physiology and Biochemistry 3.107-120.

Read L.J. (1971) The presence of high ornithine-urea cycle

enzyme activity in the teleost Opsanus tau. Comparative

Biochemistry and Physiology i9'R.A0')-m.

Salin D. & Williot P. (1991) Endogenous excretion of Siberiansturgeon. Acipenser baeri Brandt. Aquatic Living Resources

4.149-153.Savitz J., Albanese E.. Evinger M.]. & Kolasinski P. (1977)

Effect of ration level on nitrogen excretion, nitrogenretention and efficiency of nitrogen utilization for growth

in largemouth bass (Micropterus salmoides). Journal of Fish

Biology 11,185-192.

Sayer M.D. & Davenport J. (1987) The relative Importance

of the gills to ammonia and urea excretion in five seawater

and one freshwater teleost species. Journal of Fish Biology

31, 561-570.

Smith H.W. (1929) The excretion of ammonia and urea bythe gills of the fish. Journal of Biological Chemistry, 81 .

727-742.

Spyridakis P. (19 89) Utilisation desproteines alimentaires chez

le bar (Dicentrarchus labrax). Ph.D. Thesis. University de

Bretagne Occidental Brest. France.

Sukumaran N. & Kutty M.N. (1977) Oxygen consumption

and ammonia excretion in the catfish Mystus armatus.

with special reference to swimming speed and ambient

oxygen. Proceedings of the Indian Academy of Science 86B.

195-206.

Tatrail. (1981) Diurnal pattern of the ammonia and urea

excretion of feeding and starved bream. Abramis brama L.

Comparative Biochemistry and Physiology 70A. 211-215.

Treguer P. & Le Corre P (1975) Manuel d'Analyse de Sels

Nutritifs dans I'Eau de Mer(Utilisation de I'AutoanalyseurTechnicon*), 2nd ed. Laboratoire d'Oceanographie

Chimique. Univea ite de Bretagne Occidentale Brest. France.

Vellas E, Flavin M. & Creach Y. (1970) L'ur^echezqueiques

teleosteens d'eau douce. Archives Scientifiques de

Physiologie 24. iO7-m.

Vitale-Lelong D. (1989) Bilan azote du loup {Dicentrarchus

labrax L) en coursdepregrossissement. Vie Marine 11, 111.

Walsh PI.. Danulat E. & Mommsen T.R (1990) Variation inurea excretion in the Gulf toadfish Opsanus beta. Marine

Biology 106. 323-328.Wi^s A.J.. Henderson E.B.. Saunders R.L. & Kutty M.N. (1989)

Activity, respiration, and excretion of ammonia by Atlantic

salmon (Salmo salar) smolt and postsmolt. Canadian Journal

of Fisheries and Aquatic Science '^e, 79O-79S.

Wilkie M.P. & Wood CM. (1991) Nitrogenous waste

excretion, acid-base regulation, and ionoregulation in

rainbow trout {Oncorhynhus mykiss) exposed to extremely

alkaiine water, Physiological Zoology 64, 1069-1086.

Wood CM.. Perry S.F.. Wright P.A.. Bergman H. & Randall

D.[. (1989) Ammonia and urea dynamics in the Laice

Magadi tilapia. a ureotelic teleost fish adapted to an

extremely alkaiine environment. Respiration Physiology

77. 1-20.

Wood J.D. (1958) Nitrogen excretion in some marine,

teleosts. Canadian Jouranl of Biochemistry and Physiology

36. 1237-1242.

650