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Aquacultural Engineering 6 ( 1987) 301-322

Cyclical Fluctuations in Ammonia and Nitrite-nitrogen Resulting from the Feeding of Turbot, Scophthalmus maximus (L.), in Recirculating Systems
M. G. Poxton and S. B. Allouse*
Aquaculture Group, Department of Biological Sciences, Heriot-Watt University, Chamber~ Street, Edinburgh EH1 1HX, UK

ABSTRA CT Daily and weekly cyclical fluctuations in ammonia (NH4-N ttg litre-l) and nitrite-nitrogen (NO2-N t~g litre-l) were shown to occur in marine recirculating systems containing juvenile turbot, Scophthalmus maximus (L.). A stepwise multivariant regression analysis, computed for the prefeeding morning base levels, showed that ammonia-nitrogen concentration in the fish tanks was related to inflow ammonia-nitrogen concentration, water flow rate, water temperature, mean fish weight and fish biomass. Influent ammonia concentration and water flow rate were by far the most important variables accounting for 68"0% and 16"7% of the variation in the regression respectively. The fluctuations, which occurred at concentrations thought to be tolerated by the fish, were attributed to the amount and timing of feeding. The significance of the existence of such fluctuations was discussed together with the importance of their amplitude and frequency.

INTRODUCTION The growth of turbot, Scophthalmus maximus (L.), in recirculating systems was previously evaluated (Poxton et al., 1982) by means of a series of feeding trials. These also assessed the usefulness of readily available industrial fish such as sprats (Sprattus sprattus L.), sandeels (Ammodytes marinus Raitt) and small whiting (Merlangius merlangus L.) as food for the initial on-growing stage of cultivation (10-500 g). At the same time the design and performance of the system's biological filters
*Present address: Institute of Agricultural Technology, Department of Animal Production, At-Amyeria, Baghdad, Iraq. 301 Aquacultural Engineering 0144-8609/87/S03.50-- Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain

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M. G. Poxton, S. B. Allouse

was assessed and some preliminary results on their performance was reported by Poxton et al. (1981) whilst a beginning has been made in understanding, modelling and predicting the kinetics of nitrification by Weatherley ( 1982, 1984). Water quality criteria for marine fish such as the turbot have been reviewed by Poxton and Allouse (1982), who drew attention to the complex inter-relationships which exist between some of the most important parameters. As a result, much of the literature in this area was felt to be of little quantitative value although the levels which are completely safe or directly lethal are well identified. The water quality prevailing during the 133 days of the turbot feeding and growth experiments was summarised by Poxton et al. (1982) to demonstrate that an acceptable environment was maintained. It is very important, however, to appreciate the dynamic nature of these water quality parameters. In intensive systems daily and other cyclical variations in water quality occur depending on the stocking density, seasonal effects such as daylength, and on the amount and timing of feeding. Examples of these variations are given by Rosenthal et al. (1981) for a warm brackish-water system stocked with Tilapia, and Brett and Zala (1975) for a cold freshwater system stocked with sockeye salmon, Oncorhynchus nerka. In this paper a more detailed consideration of the variability of the water quality data for turbot in a marine system is given with the amplitude and frequency of the fluctuations being described. An attempt is then made to relate observed ammonia concentrations to operating conditions.

MATERIALS A N D METHODS

Reeirculating systems
The system design has been described by Poxton et al. (1981) but is shown again here for convenience (Fig. 1). Each system contained three polypropylene fish tanks, 0.37 m 2 in area, which were filled to a depth of 20 cm with 75 litres of seawater. A 5 cm layer of coarse (20 mm) granite chips was used to bury water collection pipes in the bottom of the biofilters. Finer gravel (6 mm) was then used to fill the beds to a depth of 30-35 cm whilst broken scallop shells were added to the top 5 cm to increase the buffering capacity. These beds had a surface area of 1 m 2 and a total substrate area available for bacteria of about 180 m 2 each. Small buckets containing marble chips were used as mechanical filters and as a further source of buffering capacity.

Ammonia and nitrite-N fluctuations in recirculating systems


head tank fish tanks

303

'mechanical
filters

primary filter
I

lm 30* isometric projection

air lifts

1 I secondary filter back w ' wash l


air lift

w~ lair lift

denitrifying algae system

Fig. 1.

Recirculatory culture unit.

The biofilters were run fully ponded, in order to provide a sufficient head of water ,for the airlifts, and as such also tended to act as reservoirs. Total volumes were 900 litres for each of the systems used (C,D,E), each of which had two biofilters connected in series. Flow rates to the fish tanks were generally between 1 and 5 litres min-1. All the water was recycled through PVC pipe but losses from sampling, cleaning and evaporation amounted to about 1% per day. Source of materials

Seawater
Seawater was obtained from the Firth of Forth at North Berwick and conveyed to Edinburgh by road tanker.

Experimental fish
0-group turbot were supplied by the White Fish Authority, now the Sea Fish Industry Authority, from their hatchery at Ardtoe on the west coast of Scotland. They were transported to Edinburgh in sealed plastic bags containing an oxygen atmosphere. Acclimation period After an initial 2-week acclimation period, 240 fish were weighed and placed in nine fish tanks on three (C,D,E) completely separate recirculat-

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M. G. Poxton, S. B. Allouse

ing systems, with three tanks (C/1-C/3, D / l - D / 3 , E / l - E / 3 ) per system as shown in Fig. 1. The initial stocking densities ranged from 0.3-0.4 kg m -2 (1.6 kg m-3), the individual populations ranging from 25 to 33 fish per tank.

Food and feeding


The fish were fed twice per day on weekdays and once per day on weekends, the amount and timing of the feeds differing according to the experimental design. Sprats and whiting were purchased from Firth of Forth catches and kept in a deep freeze. Prior to feeding, the food was chopped into suitably sized and shaped (elongated) pieces and allowed to thaw, the viscera and backbones being discarded. After preparation the food was dried on paper tissues to remove surface moisture and weighed. Uneaten food was recovered from the fish tanks, dried and reweighed to calculate the amount of food eaten.

Weighing the experimental fish


Every 2 weeks the fish in each tank were removed, surface dried on paper tissues, and individually weighed on a top pan balance (+ 0.1 g). The fish were kept in buckets of vigorously aerated seawater taken from the same fish tanks. After removal of the fish the tanks were drained into the filter beds/reservoirs, thoroughly cleaned and refilled from the header tanks before the fish, and water, were returned. It was not felt necessary to anaesthetise the fish, although they were not fed on the days of weighing.

Calculations offish biomass


The fish biomass was calculated daily, taking into account the weight of food eaten and using the conversion efficiencies previously determined. (Poxton et aL, 1982), in order to be able to relate it to ammonia production. Any necessary corrections were made from the 2-weekly check weighings.

Water quality control and monitoring


Salinity was maintained at 34 _+2%o by the regular addition of aerated tapwater whilst temperature control was achieved by regulating the ambient air temperature to 16+_2C. Air and water temperatures, together with salinity, were measured daily using a mercury-in-glass thermometer and a hydrometer respectively. Dissolved oxygen and free

Ammonia and nitrite-N fluctuations in recirculating systems

305

carbon dioxide were measured less frequently using an azide modification of the Winkler method and the reaction between carbon dioxide and sodium hydroxide to form sodium bicarbonate (American Public Health Association, 1965). Ammonia, nitrite and pH were determined as required by the experimental design. Ammonia was measured in triplicate using the phenol-hypochlorite method to produce indophenol blue after Koroleff (1976). The nitrite analysis was after Bendschneider and Robinson (1952) with the modification of using sulphanilamide as the diazotising agent and 1-naphthyl ethyldiamine dihydrochloride as the coupling agent giving rapid colour development with a 5% increase in sensitivity (Martin, 1972). pH was measured using a Beckman selective ion pH meter.

Sources of variability and multivariant regression analysis


Excretion rates of fish obviously depend on feeding rates and water temperatures. However, other factors such as the total biomass and average weight of the fish together with the effects of water flow rate on both the clearance of metabolites from the fish tank and on overall system performance, by affecting oxidation rates, are sources of variability and must also be considered. Because of the complexities of the situation (see Poxton and Allouse, 1982 for review) it was decided to employ multivariant regression techniques to the analysis of the data. Multiple regression is an extension of the use of the bivariate correlation coefficieflt to multivariate analysis. Whereas the linear relationship between one independent variable and a dependent variable is measured by a simple regression coefficient, multiple regression allows one to study the linear relationship between a set of independent variables and a number of dependent variables while taking into account the interrelationships among the independent variables. The basic concept of multiple regression is to produce a linear combination of independent variables which will not only correlate as highly as possible with the dependent variable but which can then be used to predict the latter. The residual is the difference between the actual value of the dependent variable and the value predicted by the linear combination of the independent variables. The regression equation is written as:

Y= blX l +

b2X2 + . . .

bsXs +(c+ r)

where Y= the dependent variable, the pre-feeding morning base level ammonia concentration in the fish tanks (ktg NH4-N litre-1), bl_ 5 = the

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M. G. Poxton, S. B. Allouse

regression coefficients, X~_5= the independent variables, X t = influent ammonia concentration (/~g NH4-N litre-~), X2 =water flow rate (litre min-~), X3ffiwater temperature (C), X4=average fish weight (g), X 5 -- total fish biomass (g), c = constant and r = the residual. Further understanding of the relation of each independent variable to the dependent variable can be gained by examining the signs of the regression coefficients. The computer automatically performs an F-test to test the overall 'goodness of fit' of the regression equation. A further discussion of the use of multiple regression for the type of application considered here is given by Alderdice (1972) while for a mathematical analysis of the method reference should be made to a statistics text which describes least-squares fitting techniques. For the present work the regressions were computed using the 'Statistical Package for the Social Sciences' multiple regression program as described by Kim and Kohout (1975). This program combines standard multiple regression and stepwise regression in a way which provides considerable control over the inclusion of independent variables.

Experimentaldesign
The experiments were designed to identify the existence and extent of any daily and other variations in water quality, paying particular attention to ammonia and nitrite and fluctuations resulting from feeding. Preliminary work confirmed that the water in the fish tanks was well mixed and not significantly different from that of the outlets and that no fluctuations occurred when the fish were not fed. This was felt to be necessary despite the very shallow (20 cm) depth of water as turbot spend, with the exception of their brief feeding responses, most of their time stationary on the bottom of the tanks.

Experiment 1
Seven tanks of turbot were fed at 09.30 and 14.00 h whilst one tank (C/3) received only the morning feed. Ammonia was measured hourly between 09.00 and 17.00 h to detect daily fluctuations (Fig. 2, Table 1 ).

Experiment 2
Investigated the second peak in ammonia using three tanks of turbot on system D and extended sampling until 22.00 h (Fig. 3).

Experiment 3
Investigated the hourly fluctuations in nitrite concentration in one tank of turbot on three successive days (Fig. 4).

Ammonia and nitrite-Nfluctuations in recirculatingsystems


TABLE 1

307

Temperature (C),pH, Water Flow Rate (litre min- l), Biomass (g), Mean Fish Weight (g), and Weight of Food Fed (g) to the Turbot in Each of Three Marine Recirculating Systems on 30 January 1979 (data relates to Fig. 2)

Conditions C/1 C/2 C/3

Tank number D/2 D/3 E/1 E/2 E/3

Temp. (C) 16-0 16.0 pH 7.2 7.2 Flow (litre min- ~) 2-8 2.9 Biomass(g) 210.0 463.9 Mean wt (g) 7-6 13-6 Food (g/a.m. 3"0 16"5 p.m. 5"3 10.5

16.0 15-8 15.8 15.2 15.2 15.2 7.2 7.1 7.1 7.3 7-3 7-3 2"9 2.0 2-5 2-0. 2"9 2"9 185-0 530.9 502.0 298.0 502.7 194.7 7-8 14.0 16.2 9.4 22.9 9.5 6"5 18.6 18.6 5"3 19.2 6"0 0"0 10.4 6"3 3"1 12.1 3"2

Experiment 4 Samples for both ammonia and nitrite were taken at 09.00 h, before feeding, from the infiuents and effluents of three tanks on system C from Monday to Friday to detect weekly fluctuations in base levels (Fig. 5). Experiment 5 Samples for both ammonia and nitrite were taken 4 h after feeding, from the influents and effluents of three tanks on system D from M o n d a y to Friday to detect weekly fluctuations in post-feeding levels (Fig. 6). Experiment 6. Hourly samples were taken throughout the day from system E during three periods of the study in February, April and June 1979. Temperature and water flow rates were kept constant (Table 2) so that this experiment reflects the increasing fish biomass and mean weight. Samples were taken from each of the three tanks on two successive days and the means and standard deviations were calculated (Fig. 7). Experiment 7 Flow rate was investigated by altering the flows to three tanks on system D to 1.2, 3"0 and 5.1 litres min-1, while other parameters were similar. After 48 h equilibration samples were taken throughout the third day (Fig. 8). Experiment 8 Samples were taken at 09.00 h from the influents to systems C, D and E at total flows through the biofilters of 13.0, 8-5 and 6.4 litres min -1

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M. G. Poxton, S. B. Allouse

TABLE 2 Experimental Conditions in System E During Three Periods of the Study in 1979 (data relates to Fig. 7)
Conditions February (x +.S.D.) Month April (x +_S.D.) June (x +_S.D.)

Temp. (C) pH Flow (litre min- t) Biomass (g) Food (g day-t)

17.0 + 0-3 7.2 + 0.3 2.2 +_0.8 550 + 67 27.7 __.1.1

17.2 + 0.3 6-8 + 0.2 2.2 + 0.4 977 + 76 37.4 + 6.6

17.5 + 0.5 6-5 + 0-2 2.2 + 0.5 1397 + 62 53-1 + 10.5

respectively, to investigate the effect of varying the hydraulic load (Fig. 9). Fish biomasses were 7.1-7.2, 3.7-3.9 and 2.7-2.9 kg respectively.

RESULTS

Daily fluctuations due to feeding


Experiment 1

A m m o n i a concentrations increased in all the tanks as the direct result of feeding the fish (Fig. 2). No increase was detected in the first 30 min after the morning feed but substantial increases of up to 100% occurred within 90 min. Increases of 7 3 - 2 0 5 % (mean 143%) occurred after 3-5 h. Feeding only once (C/3) results in decreasing concentrations in the afternoon, the frequency of the ammonia cycle being greater than 7.5 h, whereas a second feed stimulates a further substantial increase. The operative conditions during this experiment are summarised in Table 1.
Experiment 2

T h e duration and extent of the second peak in ammonia concentration can be seen in Fig. 3 which indicates that it occurs 4 h after the second feed and is some 1 0 2 - 1 2 2 % (mean 112%) higher than the concentrations measured at 09.00 h on the following day.
Experiment 3

Hourly fluctuations in both the influent and effluent nitrite-nitrogen concentrations were shown to occur on three successive days (Fig. 4).

A m m o n i a and nitrite-N fluctuations in recirculating systems


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Fig. 2.

Ammonia concentrations at hourly intervals in all the fish tanks on the same day in January 1979 (see also Table 1 ).
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/L,
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Fig. 3. The amplitude and lateness of the second peak in the ammonia concentrations in three tanks of fed turbot on the same recirculating system at 18C, pH = 6.4.

310
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M. G. Poxton, S. B. Allouse
* - - * M o n (57g food) Tues(46g ) =-'"= W e d (56g )

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' 10'-00' 12"00 ' 14'.00 ' 1~00 ' 10~00 ' 12~00 ' 14!00 ' 16!00 T i m e (h)

Fig. 4. Hourlyfluctuations in the (a) influentand (b) effluentnitrite concentrationsin a tank containing 2.6 kg, 2.7 kg and 2.7 kg of turbot on three successive days at 17.5C. pH -- 6.8 and a flowrate of 4.1 litres min- t.

These are essentially similar because the nitrite does not arise from fish excretion but from the nitrification of ammonia in the biofilters. As might be expected, the increases in nitrite lag behind those of ammonia on Monday (ammonia lag is 0.5 h; nitrite lag is 1.5 h) when the system as a whole has been operating at low nutrient levels over the weekend. However, nitrite response times reduce on subsequent days, becoming very similar to those of ammonia.

Weekly fluctuations in ammonia and nitrite

Experiment 4
Influent ammonia concentrations remained low, around 15/~g NH4-N litre- 1, and more or less stable all week (Fig. 5a) apparently showing that the biofilters were oxidising the daily peaks to base levels at a total system biomass of 7.3-7"6 kg of turbot. However, it can clearly be seen (Fig. 5b-d) that in the three fish tanks concentrations steadily increased from Monday to Wednesday as the fish, which were hungry as a result of reduced weekend feeding, consumed daily rations of 156, 149 and 147 g day- 1 respectively. The base levels actually experienced by the fish prior to feeding, and before the daily increase in ammonia levels, therefore steadily increased from 35-50 /~g NH4N litre -~ on Monday to 100-150 #g NH4-N litre-1 by Wednesday. Levels in the tanks remained high on Thursday morning when the fish showed little interest in food. In fact the fish ate only 40 and 47 g of food on Thursday and Friday, 25% of that offered, despite the lower levels of ammonia present on Friday.

Ammonia and nitrite-N fluctuations in recirculating systems


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Mon Tues Wed Thur Fri Mon Tues Wed Thur Fri Time(days)

Fig. 5. Fluctuations in the ammonia ( ' . . . " ) and nitrite ( e - - - e ) concentrations measured at 09.00 h before feeding in the (a) influent and (b-d) effluents of the fish tanks on system C (17.0-18.5"C; pH = 6-8).

These results also confirm that a weekly cycle in the nitrite concentrations in the fish tanks occurred.

Experiment 5
The ammonia concentrations in the fish tanks 4 h after feeding (Fig. 6) showed a similar fluctuation to that seen in Fig. 5 with a peak of 240/zg NH4-N litre-l occurring on Wednesday. However, it can also be-seen that measured at this point in the daily cycle, a previously undetected weekly cycle in influent ammonia concentration also occurs. At these

312

M. G. Poxton, S. B. Allouse
(a) Influenr e-- i.....i Nit'riPe Ammonia (b) D/1
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100

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Sun Men W ~ Time (days)

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Fig. 6. Fluctuations in the ammonia (B...B) and nitrite (e---e) concentrations measured 4 h after feeding in the (a) influent and (b-d) effluents of the fish tanks on system D. peak ammonia concentrations the level of nitrite-nitrogen was relatively constant at around 50/~g NO2-N litre- 1 Sources of variability
Experiment 6

The result show (Fig. 7) that the amplitude of the daily cycles increases with increasing biomass of fish on the same recirculating system (Fig. 7). As one might expect, the base level concentrations (09.00 h) also increase with increasing loading. Although the ammonia concentration had reached an asymptote in February before the second feed, this was clearly not the case with increased biomass when the effect of the second feed became additive. The relatively higher standard deviations after 12.00 h may be due to differences in food consumption between the three fish tanks on the two days of sampling (Table 2).

Ammonia and nitrite-Nfluctuations in recirculating systems


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<

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oo

10.00

12.00 T~(h)

14.00

16.00

Fig. 7.

The means and standard deviations of the ammonia concentrations in system E at three periods of the study during 1979 (see also Table 2).

Experiment 7
The pre-feeding ammonia concentrations were greatest (120 ktg NH4-N litre- 1) in the tank with the lowest flow ( 1-2 litres min- 1) and least (55/~g NH4-N litre-~) in the tank with the highest flow rate (5"1 litres min -1) as was expected (Fig. 8). The effect of feeding the fish in D/2 and D/3 a similar amount of food was effectively the same, an approximately 100% increase in ammonia concentration occurring after 4.0 h, despite the different flow rates. A smaller (60%) increase occurred in D/1 although as a precaution against the very low flow in this tank the biomass was reduced and a second feed was not offered. Nevertheless the highest levels of ammonia (190 ~g NH4-N litre-l) were clearly associated with low flow rates. The effect of flow rate on ammonia concentration as a function of fish biomass at 16C was further studied by examining the data on the base level, pre-feeding, concentrations taken at random over a one-year period. The data was divided into three categories (< 2, 2-3 and > 3 litres min-1) according to flow rate. Data where the influent concentra-

314
200

M. G. Poxton, S. B. Allouse

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Tank No. Biomass of wr. of feed(g) Flow r a r e fish (kg) am pm (I/min) D/1 ~ DI2 e ' ' e D/3 m...4 10~00 1'2 1.4 1.5 24 28 30 0 14 15 14 O0 1.2 5'1 3.0 16 O0

12"00 Time (h)

Fig. 8. The effect of water flow rate on hourly ammonia fluctuations in three tanks on systemD at 18"C and pH = 6.6.

dons were greater than 100 #g NH4-N litre-n were excluded as inlet concentration was regarded as a separate variable in this case. The regression equations for the best fitted lines were: II1=61"04X~- 12"8
Y 2 = 2 3 . 0 6 X 2 + 13.6

(r=0"83; P < 0 . 0 0 1 ) (r=0.60; P < 0 . 0 0 1 ) ( r = 0.89; P < 0"001)

Y3= 22.90X3 + 2.7

where Y I - 3 m concentration of ammonia (gg N H : N litre -l) in the tank, X1-3---biomass (kg) up to 2 kg, 1-3 =flows of < 2, 2-3 and > 3 litre rain- 1 respectively. So, for example, at a fish biomass of 1.5 kg the base level ammonia concentration at 09.00 h would be 78, 48 or 37 gg NH4-N litre-1 at flows of less than 2, 2-3 or greater than 3 litres min- 1 respectively.
Experiment 8

It can be seen from Fig. 9 that despite having a larger biomass (7.1-7.2 kg) and receiving the most food (539 g per week) the influent ammonia

Ammonia and nitrite-N fluctuations in recirculating systems

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Fig. 9. Fluctuationsin ammonia concentration measured at 09.00 h before feedingin the influentsof systems C (e...e), D ( u - - - l ) and E (A ) at flow rates of 13.0, 8-5 and 6'4 litres min- l respectively(15.8-18.5C; pH ---6.6-6.9). concentrations in system C at 09.00 h were kept low throughout the week by the high system flow rate of 13 litres min- 1. Flows of 8"5 and 6.4 litres min-~ through the biofilters of systems D and E, which held biomasses of 3.7-3.9 kg and 2.7-2.9 kg respectively, with corresponding feed rates o f 5 1 4 and 382 g per week, allowed pre-feeding influent concentrations of 106 ~g NH4-N litre-1 to be reached on Wednesday when corresponding levels in system C were only 24 gg NHa-Nlitre- 1 The concentrations of ammonia in the fish tank effluents were higher than, but followed the same pattern as, the influent concentrations with the exception of system C where tank concentrations showed a similar weekly fluctuation to those of the other systems (Fig. 5). The maximum concentrations occurred in all three systems on Wednesday, values of 186, 175 and 156/xg NH4-N litre-1 being recorded in systems E, D and C respectively. The effect of water flow rate on tank concentrations of ammonia was standardised for fish biomass and is shown in Fig. 10. The data were analysed by calculating the common logarithms of the ammonia concentrations, the best fitted linear regression being: Log Y = l . 9 7 9 - 0 . 1 4 4 6 X (r-- - 0"87; P < 0"001)

316

M. G..Poxton, S. B. Allouse

2OO

O--O ~

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2"0

~
.=
O~

160

1.5

t2o

O .d

6~

oo

1"O

<C

0.5
O

=,
, .~ , ~. , ~ , "'-~

W a t e r flow rate C l / m i n / k g o f f i s h )

Fig. 10.

A m m o n i a concentrations in fed fish tanks at different water flow rates.

Log Y= log10 tank ammonia concentration (pg NH4-N litre- ~) X-- water flow rate (litre min- ~kg- ] of turbot) The equation was converted into the original form after Snedecor and Cochran (1973), the resultant exponential being: Y-- 95.2796e- 334x
M u l t i v a r i a n t r e g r e s s i o n analysis

A multivariant regression analysis was computed for the pre-feeding morning base levels of ammonia concentrations in the fish tanks (Y) using the data in Table 3. The fitted equation was: Y= 0.967(X1 ) - 13-621(X2)+ 12.924(X3)+ 0"296(X4)+ 0"004(S 5)
- 158-255

The tank biomass correlation (X5) is the smallest, but this is perhaps not surprising as, given an adequate flow rate through the individual fish tanks, the carrying capacity of the recirculating system is mainly

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318

M. G. Poxton, So B. Allouse

ammonia concentration (X~) and water flow rate (X2) were clearly the most significant factors accounting for 68.1% and 16.7% of the variation in the regression respectively. The estimated F-value of 58.578 with these five variables, and 26 degrees of freedom, was highly significant at the 1% level. General water quality considerations Unionised ammonia concentrations depend on the temperature and pH (Hampson, 1976) and ranged from 0"07 to 0.31% of the total ammonia concentrations during these experiments. For instance, at the highest concentration of total ammonia (508 ~g NH4-N litre-l), which occurred at 20.00 h at a water temperature of 18C and pH 6-7, the unionised concentration was only 0.9/ag NHa-N litre- ~. This level only lasted for a few hours and was unlikely to be the cause of any stress (Alderson, 1979). The highest nitrite concentration was 94 ~g NO2-N litre-i at which time the total and unionised ammonia concentrations were 127 /~g NHg-N litre- 1 and 0-203/~g NH3-N litre- ~respectively. Dissolved oxygen (DO) concentrations remained close to 100% saturation throughout the study, ranging from 6"6 to 7.6 mg O 2 litre- ~at 160C for flow rates through the fish tanks of 1"5-5.0 litres min- 1 kg- ~of fish. Little or no changes in DO occurred within the fish tanks or in relation to feeding. The buffering capacity of the materials used in the filter beds was insufficient to maintain the normal pH (8"0-8.4) of seawater and the pH gradually decreased, by 0.2 units per month, from 7.4 in January to 6.4 in June 1979 after which it remained fairly constant. Free carbon dioxide concentrations in the fish tanks ranged from 6"0 to 8"5 mg litreTemperature and salinity ranged from 15.0 to 18-0C and 34 to 39%o respectively. Food conversion factors were of the order of 2.5:1 on a wet:live weight basis. The fish remained in good condition, with only one mortality occurring throughout the study, achieving good growth rates similar to those already described (Poxton et al., 1982).

DISCUSSION These results show that ammonia concentrations do constantly fluctuate in a marine recirculating system stocked with carnivorous fish fed on high protein diets. While the timing of these fluctuations are clearly related to feeding, the actual concentration of ammonia experienced by the fish was shown to be related to inflow concentration, flow rate,

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temperature, mean weight and biomass of the fish, efficiency of and hydraulic loading on the biofilters and day of the week. The particular experiments reported here were designed to explore the amplitude and frequency of the 'nitrogen cycles' with a view to improving the practical management of recirculating systems. In these particular systems the biofilters reduced the ammonia concentration to a very low level as a result of low weekend feeding so that prefeeding levels on Monday morning could be as low as 10 ~tg NH4-N litre-~. For the same reason the fish were generally very hungry and consumed their full ration of food. The daily ammonia cycle showed an initial lag followed by a steady rise in concentration reaching a peak 73-205% greater than the pre-feeding concentration, 3-5-4.0 h later. If fed only once, ammonia levels decline in the afternoon but if given a second feed then they rise to a second higher peak after a further 3.5-4.0 h period. This pattern of feeding is typical of both industrial and academic situations and it is therefore important to note from these results that the highest daily ammonia concentration does not occur until late at night when everyone has gone home. Since concentrations have usually reached a base level in the morning, when they are often checked by research personnel, the actual concentrations experienced by the fish can easily be seriously underestimated. Later in the week the fish appear less hungry while nitrogen levels in the system gradually increase. This weekly cycle, which is not apparent in the influent if only measured at 09.00 h, but which can be detected 4 h after feeding~ has the daily cycle superimposed on it. Maximum concentrations occur 4 h after the second feed on a Wednesday when the levels may be 20-30 times those of Monday morning. Nitrite cycles arise due to the conversion of the ammonia by nitrifying bacteria in the biofilters of recycle systems. On a Monday there is a distinct lag between the nitrite and ammonia cycle but as the nitrifiers increase their populations in response to the increasing nutrients this time lag reduces. The significance of these fluctuations is that they indicate that water quality monitoring carried out in a random manner is almost meaningless. Similarly, monitoring carried out at one particular time of day and then used to indicate the prevailing system conditions (Otte and Rosenthal. 1979; Poxton et al., 1982) can be very misleading if not adequately related to the daily and other cycles. The amplitudes of the fluctuations depend on fish biomass (Expt 6) although it is clear from the results that they can be mediated to some degree by altering the flow rates both to the fish tanks themselves (Expt 7) and through the biofilters (Expt 8). The implication of this is that

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systems must be designed to allow the flow through the biofilters to be controlled independently of the flow through the fish tanks. Rosenthal et al. (1984) also concluded that daily nitrogen cycles in recycle systems could be counteracted by adjusting the hydraulic load. They showed for a rotating bionet biological filter that ammonia and nitrite oxidation rates could be increased at times of peak loading by doubling retention times. It should be a main ahn of future research to try to stabilise the environment as this would not only benefit the fish but would also improve the efficiency of the biofilters. Clearly, though, studies which assume that biofilters will be operated under steady state conditions are unreali.~tic in practice at the present time. Fluctuations of small amplitude and constant frequency may convey some resistance to the fish but equally even minor fluctuations may have drastic consequences if tolerance levels are exceeded even in the short term (Rosenthal, 1983). Burrows (1964) demonstrated that prolonged but intermittent exposure to unionised ammonia resulted in reduced growth rate and physical stamina in chinook salmon (Oncorhynchus tshawytscha). He suspected that gill hyperplasia, resulting from the exposure to unionised ammonia, was a precursor of bacterial gill disease. Future toxicity work would therefore be most useful if it examined the chronic effects of fluctuating concentrations of mixtures of ammonia and nitrite within the range tolerated by the fish. Rychly and Marina (1977) found that there were fluctuations in the ammonia excretion of both fed and starved rainbow trout (Salmo gairdneri Richardson) kept in small containers with static water during a 24 h period. Their results indicated the possible existence of a circadian rhythm in both ammonia excretion and nitrogen metabolism. Aspects of the ammonia production from the cultured turbot during these experiments will be examined in a further paper; however, it is interesting to note that Brett and Zala (1975) also showed that ammonia excretion rates peaked 4-0-4.5 h after feeding sockeye salmon on Oregon moist pellets and, as in these experiments, fell to a baseline between 02.00 and 08.00 h. They also showed that metabolic rate rose prior to feeding, the rate of ascent being almost parallel to that of ammonia excretion, and that it began to fall prior to the ammonia excretion peak being reached in harmony with the gastric evacuation rate. However, they concluded that the large pulse in ammonia excretion was entirely the result, of food intake and not the product of an increased metabolic rate. The influence of feeding on the metabolic rate of fish has been reviewed by Jobling (1981). Although concentrating on the fact that oxygen consumption increases following feeding, rather than nitrogen excretion, the review is still useful in pointing out the duration and

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magnitude of the effect and the way in which it is related to meal size and quality and temperature. Following feeding oxygen consumption quickly doubles, within 4 h according to Flowerdew and Grove (1980), and since similar increases take place in the biofllter one must expect that DO cycles will also be present in recycle systems. These may be significant in that the toxicity of the ammonia and nitrite mixture is likely to be greater at lower DO concentrations. In experiments involving the multiple feeding of tilapia fingerlings in a recycle system, Rosenthal et al. (1981 ) also reported fluctuating nitrogen, pH and DO levels. Using trickling filters they noted that the biofilter quickly responded to increased ammonia levels but that nitrite concentrations increased to levels that were regarded as being close to critical. Despite the fluctuating conditions reported in the present study the maximum levels of ammonia and nitrite and the minimum levels of pH and DO recorded were always well within the criteria identified by Poxton and Allouse (1982).

ACKNOWLEDGEMENTS We acknowledge the help of Andy Pooley in the methodology of water quality analysis. Whilst the work was supported by Heriot-Watt University one of us, S.B. Allouse, was self-f'manced.

REFERENCES Alderdice, D. E (1972). Factor combinations. In: Marine Ecology, Vol. 1, ed. O. Kinne, Wiley, London, pp. 1659-722. Alderson, R. (1979). The effect of ammonia on the growth of juvenile Dover sole (Solea solea) and turbot (Scophthalmus maximus). Aquaculture, 17, 291-309. American Public Health Association (1965). Standard Methods for the Determination of Water and Wastewater, 12th Edn, American Public Health Association, New York. 769 pp. Bendschneider, K. & Robinson, R. J. (1952). A new spectrophotometric method for the determination of nitrite in sea water. J. Mar. Res., 11, 87-96. Brett, J. R. & Zala, C. A. (1975). Daily pattern of nitrogen excretion and oxygen consumption of sockeye salmon, Oncorhynchus nerka, under controlled conditions. J. Fish. Res. Bd Can., 32, 2479-86. Burrows, R. E. (1964). Effects of accumulated excretory products on hatchery reared salmonids. US. Dept. of Interior, Bureau of Sport, Fisheries and Wildlife. Research report. 66, 12 pp. Flowerdew, M. W. & Grove, D. J. (1980). An energy budget for juvenile thicklipped mullet, Crenimugil labrosus (Risso ). J. Fish Biol., 17,395-410.

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Hampson, B. L. (1976). Ammonia concentration in relation to ammonia toxicity during a rainbow trout rearing experiment in a closed freshwater-seawater system. Aquaculture, 9, 61-70. Jobling, M. (1981). The influences of feeding on the metabolic rate of fishes: a short review. J. Fish Biol., 18, 385-400. Kim, J. O. & Kohout, E J. (1975). Multiple regression analysis: subprogram regression. In: Statistical Package for the Social Sciences, 2nd Edn, eds. N. H. Nie, C. H. Hull, J. G. Jenkins, K. Steinbrenner & D. H. Brent, McGraw-Hill, New York, pp. 320-67. Koroleff, F. (1976). Determination of ammonia. In: Methods of Sea Water Analysis, ed. K. Grasshoff, Chemie. Weinbeim, New York, pp. 126-33. Martin, D. F. (1972). Marine Chemistry, Vol. 1, Marcel Dekker, New York. Otte, G. & Rosenthal, H. (1979). Management of a closed brackish water system for high-density fish culture by biological and chemical water treatment. Aquaculture, 18, 169-81. Poxton, M. G. & Allouse, S. B, (1982). Water quality criteria for marine fisheries. Aquacultural Engng, 1, 153-91. Poxton, M. G., Murray, K. R., Linfoot, B. T. & Pooley, A. B. W. (1981 ). The design and performance of biological filters in an experimental mariculture facility. In: Proc. World Syrup. on Aquaculture in Heated Effluents and Recirculation Systems, Vol. 1, ed. K. Tiews, Heenemann Verlagsgesellschaft mbH, Berlin, pp. 370-82. Poxton, M. G., Murray, K. R. & Linfoot, B. T. (1982). The growth of turbot (Scophthalmus maximus) in recirculating systems. Aquacultural Engng, 1, 23-34. Rosenthal, H. (1983). Diumal fluctuations in water quality levels in relation to different pond conditions and their significance in intensive fish culture. Unpublished discussion paper, Biologische Anstalt Helgoland, Hamburg, 46 PP. Rosenthal, H., Andjus, R. & Kruner, G. (1981). Daily variations of water quality parameters under intensive culture conditions in a recycling system. In: Proc. World Symp. on Aquaculture in Heated Effluents and Recirculation Systems, Vol. 1, ed. K. Tiews, Heenemann Verlagsgesellschaft mbH, Berlin, pp. 113-20. Rosenthal, H., Chiba, K. & Kruner, G. (1984). Daily fluctuation of water quality in a recirculating system and its influence on the performance of a rotating bionet biological filter. I.C.E.S. Statutory meeting, C.M., 1984, F:20, 1-11. Rychly, J. & Marina, B. A. (1977). The ammonia excretion of trout during a 24hour period. Aquaculture, 11,173-8. Snedecor, G. W. & Cochran, W. G. (1973). Statistical Methods, Iowa State University Press, Ames. Weatherley, L. R. (1982). Application of simple dynamic response analysis to a recirculating aquaculture s y s t e m - A preview. Aquacultural Engng, 1, 93-113. Weatherley, L. R. (1984). Rate models for a marine biological filter. Aquacultural Engng, 3, 15-29.