Aquacultural Engineering 4 (1985) 59-84

Organic and Inorganic Carbon Levels in Recycled Seawater During the Culture of Tropical Prawns Penaeus sp.
J.F. Wickins
Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Experiment Station, Conwy, Gwynedd LL32 8UB, UK
ABSTRA CT Suspended particles and dissolved organic carbon in laboratory recirculation systems were broadly eategorised by size filtration and ultraviolet absorption. Their concentrations in recycled seawater increased during the culture of tropical prawns with increase in feeding rate and also with a 'load' factor calculated to take account o f the volume of water used to culture a known biomass o f prawns. In systems operated without periodic water renewal the proportion of organic matter in filterable material and the integrated ultraviolet absorbance (Z A 250-330 nm) o f filtrate increased with time. The increasing absorbanee observed, particularly in the higher wavelengths ('A 290-330nm), indicated the accumulation o f metabolically derived organic material that was resistant to microbial degradation. Mean numbers o f bacteria varied immensely from 7 to 25 000 103 m1-1 but were lower in foamed than unfoamed systems. The relation between the concentration of dissolved inorganic carbon (a measure of carbonate alkalinity) and pH in the systems varied according to the type o f media in the biological filter and the method used to control pH. Results closest to natural seawater were obtained in systems containing filters with limestone media periodically dosed with sodium hydroxide solution. Higher levels of inorganic carbon for a given pH above pH 7.9 were, however, noted when sodium hydroxide was used with plastic media filters, a feature beneficial to heavily loaded systems. INTRODUCTION During the last decade m any valuable species of Crustacea have been cultured for one or more phases o f their life-cycle in land-based culture 59 Crown Copyright, 1985.

60

J. F. Wickins

systems (Chamberlain and Lawrence, 1981 ; Conklin et al., 1981 ; Wickins, 1982). It is established that, in addition to the excretory products of the cultured species, significant amounts of dissolved and particulate material enter the water from food organisms (e.g. Arternia; Moffett and Fisher, 1978) and from prepared foods soon after immersion: around 22-30% of mass in 16 h (Forster, 1972); 31% of mass in 24 h (R. W. Sedgwick, 1979, pers. comm.); 20-30% of the protein and lipid content in 1 h (Bages and Sloane, 1981); 11% of protein in 1 h (Cuzon e t al., 1982). Large pieces of uneaten food are readily removed and such wastage may amount to as much as 32% of the daily ration (Wickins, 1985). Very few reports, however, have been published on suspended and dissolved organic wastes in marine recirculation systems containing Crustacea (Wickins, 1981) despite the problems such wastes may cause to experimenters (Bartley et al., 1980). In the first part of this paper these materials are broadly categorised by filtration and ultraviolet absorption; their concentrations are related to the daily food ration and to a 'load' factor which takes into account in its calculation the biomass of animals cultured and the total quantity of water used during the culture period. The effects of dispersed air flotation (foaming) (Wheaton et al., 1979; Lawson and Wheaton, 1980) on organic materials were observed in trials made with model recirculation systems and are also reported. Percolating biological filters are widely used in recirculation systems for the oxidation of organic wastes and ammonia (Tiews, 1981), the oxidation being accomplished by microbial slimes and bacteria coating the surfaces o f the filter media. Although carbonic acid is formed during the oxidation of organic wastes it is the continuous production of H + ions during the oxidation of ammonia which leads to a decline in inorganic carbon levels (carbonate alkalinity) in recycled seawater. In lightly stocked display aquaria the decline may take many months (Spotte, 1979; Bower et al., 1981) but under extreme conditions can occur in only a few days, particularly when senescent limestone (Siddall, 1974), plastic or otherwise inert media is used in the biological filters (Wickins and Helm, 1981 ; Wickins, 1983). In addition to more frequent seawater replacement, methods used to combat reduced pH and carbonate alkalinity in marine systems include periodic replacement of limestone or shell filter media, additions of soluble alkalis, carbonate and bicarbonates and the incorporation of de-nitrification and algal culture

Organic/inorganic C levels in recycled seawater during prawn culture

61

units. The second part of this paper reports the relationships between dissolved inorganic carbon levels and pH that were observed when different combinations of filter media and chemical additives were used during the long-term culture of tropical penaeid prawns in laboratory recirculation systems.

METHODS
Culture systems

Observations were made on 16 laboratory recirculation systems (87510 627 litres capacity) in which marine penaeid prawns were cultured for periods of 28-352 days. In addition, four model systems (190 litres capacity), also containing prawns, were used in 20 trials, each lasting 51-72 days. During some trials dispersed air flotation treatment was incorporated in two of the model systems (Fig. 1). Details of all the systems are given in Table 1.

/
Constant h e a d
chamber ~ Percolating Dispersed air 1.2 I rain-11

11.51

rain- 1 flotation column (5cm dia)

biological ~ _ filter ( 15cm dia)

// ~"

Culture tank ; r 7
i i

Air lift tube Air --

Air lift tube //

=

--

Sinterglass

/ J

air stone

l
AirJ ~ Air

Fig. 1. A model recirculation system (190 litre capacity) incorporating a percolating biological filter (4 litre capacity) and a dispersed air flotation (foam) tower (1.54 litre capacity).

TABLE 1
1 1 3 953 753 4 200 85 1 000 L 26 170 160 160 156 L L L P P 182 123 213 213 190 190 52 22 44 41 59 65 80 L 216 1 515 573 663 590 637 680 1 392 2 030 845 852 438 372 400 475 54 75 L 150 P 507 2 4 2 907 1 1 2 603 4 4 1 508 1 1 1 442 3 3 1 075 1 1 1 052 1 1 875 4 20 190 186 4-5 2b 4

On t.3

The Volumes (litres) of Culture Tanks and Water Treatment Units in the L a b o r a t o r y Recirculation Systems

Number o f systems 1 1 Number of trials 3 1 Total system volume 10 627 8 565 Total volume o f water in culture tanks 10 425 6 960 Total volume a of water treatment units 202 1 605 Treatment unit volume as a percentage o f the system volume 2~ 19 Percolating filter volume (litres) 202 213 Media: L = limestone, 40-50% voids; P = plastic, 93% voids L P Hydraulic load to percolating filter (m3 m-a day -1 ) 150-200 170 Additional mechanical filtration: DE = diatomaceous earth swim pool f'dter; W -= synthetic wadding W Dispersed air flotation x/ Crustacean biomass (kg) per system 0.7-2.6 1.8 . . . 0.4 . 0.2 2.0-3.7 . . . . . upto2.0 . 1.2 DE . DE . u p t o 1.9 2-1 -

W x/ 0.04-0.8 0.02-0.05

a Includes volume occupied by percolating filter media. b Animals reared in water reservoir not in separate culture tanks.

Organic~inorganic C levels in recycled seawater during prawn culture

63

System management
Salinity and temperature ranged as follows: 26-34%o and 28 + 2C, respectively. Settled solids were removed daily by hand net or siphon and suspended solids by the mechanical filtering action of gravel, percolating filters (when used) and synthetic wool wadding which was washed daily. In some trials proprietary high-rate diatomaceous earth, swimming pool filters were used intermittently for about 2 h day -~ some 4-6 h after feeding. Such filters removed 70-80% of particles down to about 5 #m in size. Further details of the systems and their management are given in Beard and Forster (1973); Beard et al. (1977); Wickins and Beard (1978); Sedgwick ( 1979); Beard and Wickins (1980).

Feeding and calculations of gross animal load

F o o d was given once each day in the morning, and was adjusted so that some remained uneaten the next day. Generally a 5 0 : 5 0 mixture of freshly shucked mussel M y t i l u s edulis g. and frozen shrimp Crangon crangon g. was given but where pellets were fed to the prawns these were obtained from Japan (Taiyo G y o G y o Co. Ltd). Feeding rate (F) was expressed as the dry weight of food fed per day assuming an average water content of 83% for mussel, 72% for shrimp and 5% for pellets (Wickins, 1985). Dissolved and suspended wastes from pelletfed and shellfish-fed populations were not distinguished since the populations were often cultured within the same recirculation system. The gross animal load placed on a system was calculated from the weight of animals (biomass) held, divided by the total volume of water used (including the initial volume of the system) throughout the culture period. Where the biomass increased markedly due to growth of the animals, an allowance for the increased biomass was made in the calculation. For example, 577 litres of water from a 3953 litre capacity recirculation system (Table 1, column 3) were replaced with clean water twice each week during an 84-day growth experiment. The total volume of water used was therefore : 3953 + 84 = 17 801 litres

64

J. F. Wickins

During the trial biomass increased from 248 g of juvenile prawns (0.29 g mean live weight) to 3724 g of 7 g prawns. The biomass increase was therefore 3 7 2 4 - - 248 = 3476 g and the gross animal load 3476 17801 = 0-195 g litre -1

Analytical methods
The methods used for the measurement of temperature, salinity, pH, dissolved organic and inorganic carbon particulates and bacteria were the same as those described by Wickins and Helm (1981). Measurements of dissolved inorganic carbon were assumed to give a good indication of the carbonate alkalinity o f recycled seawater over the pH range 8.2-6-6, since bicarbonate and carbonate ions are the primary buffers in seawater and carbon dioxide (in equilibrium with the atmosphere) constitutes less than 2% of the total dissolved inorganic carbon present (Whitfield, 1974). For practical reasons not all of the analyses could be made on all the systems. For example, in some trials, particulates larger than 1 tam, and dissolved organic carbon, were measured once or twice weekly, while in others sub-micron particulates and ultraviolet absorption were also measured. Suspended particulate and dissolved organic matter were categorised into four components: (i) Micro-organisms and particles larger than l tam retained on Whatman GF/C filter paper. The organic content of this material was determined by ashing samples at 500C for 24 h in a muffle furnace (Strickland and Parsons, 1968). (ii) Sub-micron particulate material, which included some bacteria and colloids, retained on 0.22 tam Millipore filters. (iii) 'Dissolved' organic carbon or carbonaceous material passing through Whatman GF/C filters and measured by rapid combustion at 950C on a Beckman model 915 total carbon analyser and corrected for inorganic carbon content.

Organic~inorganic C levels in recycled seawater during prawn culture

65

(iv) 'Dissolved' organic material which passed through Whatman GF/C filters and absorbed ultraviolet light over the wavelengths 2 5 0 - 3 3 0 nm. Interference from the high nitrate levels that are expected in recycled seawater was assumed to be minimal at these wavelengths (Foster, 1974). Measurements of absorbance were made using a Unicam SP 500 spectrophotometer in 10 cm silica-faced cells against a reference cell containing photooxidised distilled water. Quantitative assessments of the integrated absorbance over selected ranges of wavelengths ( 2 5 0 - 2 9 0 and 2 9 0 - 3 3 0 nm) were made using Simpson's approximation for area under a curve: area = ~ h (Yl + 4y2 + Y3) where 2h = distance between Yl and Y3 and y = wavelength.

Statistical methods
Probability values given in the text refer to comparisons of means and rate slopes made using Student's t test. Weighted regression analysis (Draper and Smith, 1966) was applied to the inorganic carbon pH data in Figs 5(A)-(F) (Table 2). Data were grouped by pH intervals o f 0.09 and carbon concentration means and standard errors derived for each pH interval.

RESULTS

Particulate and 'dissolved' organic carbon

(i) Micro-organisms and particles larger than 1 tam The concentrations (mg litre -1) of filterable material larger than 1 tam were measured one-three times each week in six culture trials which lasted 4 - 3 7 weeks. Monthly mean values from each system were plotted against the mean daily food ration, F (g dry food day-l), over the same periods and although variable, typically changed with change in feeding rate. Regression analysis (Table 3) showed that the overall mean (ln) particulate concentration was also correlated with gross animal load. The range of concentrations among individual measurements was 1 . 5 - 3 5 . 5 m g l i t r e -1 and in recirculation systems containing plastic

66

J. F. Wickins
TABLE 2

Weighted Regression Analysis and Estimate of the Proportion of the Total Variation Due to Regression: Y = Bx + C where Y= Mean Concentration of Dissolved Inorganic Carbon (rag litre -1) and x = pH

Code (see text}

B (slope)

C (intereept) Correlation coefficient, r

Probability (p <)

% Total variation due to regression

98-3 91.0 95.9 88.9 89.1 86.7 98.7

a (pH > 7-5) c e b d f (pH ~< 7.8) f(pH >1 7.8)

23.85 21.64 27.41 8-90 10.92 22.48 -24.76

- 173.28 - 151.13 - 189.97 -55-21 -66.24 - 147.43 223.66

0.991 0.954 0.979 0.943 0.944 0.931 -0.993

0.001 0.001 0.01 0.001 0.001 0.05 0.001

media filters separate, high-rate mechanical filtration was often necessary to clarify the water and reduce particulate concentration (see Discussion). The material retained on the GF/C filters was about 60% organic material with 40% o f mineral ash. Considerable variation in the organic c o n t e n t occurred between and within different recirculation systems; the overall range e n c o u n t e r e d was 10.0-92.6%. In four separate trials where the water was not changed for 56 days, the p r o p o r t i o n o f organic m a t t e r in the filterable material was found to increase linearly with time (regression eqn (1), Table 4). No proportional increase in % organic m a t t e r with time was detected in systems where regular water changes were made. The weight o f organic material varied from 0.1 to 19.1 mg litre -1 generally but two values o f 31.2 and 34.6 mg litre -1 were recorded after 44 days in one o f the four trials in which the water was not changed. The concentrations recorded overall were predom i nant l y within the limits found in particulates from the laboratory seawater supply (0. 919.4 mg litre-1). (ii) Sub-micron particulate material and bacteria The c o n c e n t r a t i o n o f filterable sub-micron material was measured three times each week in four recirculation systems o f approximately I000

Organic~inorganic C levels in recycled seawater during prawn culture

TABLE 3

67

Predictive Regression Equation Constants Describing the Increase in Particulate and 'Dissolved' Organic Matter with Increase in Feeding Rate and Gross Animal Load

Parameter

Slope

Intercept Correlation Degrees {In) coefficient of freedom

Particles > 1/.tm In mg litre -1 (monthly means) versus feeding rate 0-007 In mg litre -1 (overall mean) versus load (see Methods) 9.95 Particles < 1> 0-22/am In mg litre -1 (monthly means) versus feeding rate 0-02 In mg litre-1 (overall mean) versus load 15-27 Organic carbon in GF/C filtrate In mg litre -x (monthly means) versus feeding rate 0.005 In mg litre -1 (overall mean) versus load 3.58

3.21

0.67

23

<0-001

2.40

0.90

<0.01 > 0.001

0.77 2.48

0-54 0.95

18 4

<0.05 > 0-01 <0.01 ~ 0-001

7.07 6-53

0-64 0-81

19 9

<0.01 ~> 0.001 <0.01 > 0-001

litre capacity. Despite the i n t e r m i t t e n t use o f d i a t o m a c e o u s e a r t h filters to r e m o v e larger particles, the m o n t h l y m e a n c o n c e n t r a t i o n o f subm i c r o n particles (mg litre -1) was generally related to the m e a n feeding rate, m e a s u r e d over the same p e r i o d and, as was observed with particulate m a t t e r larger t h a n 1 ~um, the overall m e a n (ln) c o n c e n t r a t i o n o f s u b - m i c r o n particles was closely c o r r e l a t e d with gross animal load (Table 3). In m a n y r e c i r c u l a t i o n systems the t u r b i d i t y o f the seawater f l u c t u a t e d , e i t h e r gradually over a period o f days, or s u d d e n l y w h e n the water w o u l d pass f r o m c l o u d y to clear within 24 h, indicating the ' b l o o m and collapse' o f microbial p o p u l a t i o n s . Occasional m e a s u r e m e n t s o f the

68

J. F. Wickins

TABLE 4 Regression Equations Derived from Various Experimental Trials Described in the Text Textual referenee Parameter Slope Intercept Corre- Degrees lation of eoeffi- freedom cient 0.80 0.80 0.95 0.54 30 30 29 14 P

(1) (2) (3) (4) (5)

(6)

(7)

% Organic matter versus days 0 . 6 2 37.77 XA 250-330 versus days 1.07 22.56 ZA 250-290 In versus days -0.12 In 2.43 ZA 290-330 Particulates > 1 ~m (rag litre-1) versus days 0.58 8.70 ZA 250-290 versus organic carbon concentration (mg litre-1) 1.27 8.52 Rate of loss of inorganic carbon (rag litre-1 day-x) versus food nitrogen fed day-1 0-70 0-29 Rate of addition of sodium hydroxide (In g day-1) versus ln gross animal load 0-79 In 0-65

'0.001 '~0-001 ~0.001 <0.05

0.53

26

<0.01

0.91

<0.05

0.86

<0.01

numbers of bacteria present in recycled water showed wide variation from 7 to 25 000 103 m1-1 and contrasted with levels in the incoming laboratory water supply which typically contained 9 - 9 9 0 0 m l -1. Weekly estimates of bacterial numbers were made in a 3953 litre recirculation system for 16 weeks during the culture of 3.7 kg of tropical prawns (Table 1, column 3). The numbers increased erratically from 200 to 2 5 0 0 0 103 ml -I during the first 12 weeks and then decreased when feeding levels declined after animal losses. The overall mean was 7527 (standard error ( S E ) = 2 2 3 5 1 0 3 m l - l ) . In other densely stocked recirculation systems (1000-1500 litre capacity hold-

Organic~inorganic C levels in recycled seawater during prawn culture

69

ing up to 1.9kg of prawns) the mean numbers of bacteria ( 103m1-1) also increased throughout the culture period:
Replicate Weeks 1-28 Weeks 29-38

1 2

1674, SE = 568 1251,SE = 448

3129, SE = 1111 4570, SE = 1645

I

In two less densely stocked model systems (190 litre capacity) numbers were low, ranging from 54-1700 103 ml -~ (replicate means 773, SE = 364 and 522, SE = 146 X 103 ml-1). An apparent relationship existed between gross animal load and bacterial numbers but was considered fortuitous in view of the wide variation of bacterial numbers from week to week within the various systems. The effect of foam treatment on bacterial numbers is described below. (iii) Carbonaceous material smaller than 1 lam and dissolved organic carbon Levels of organic carbon were measured in four trials by combustion of filtrates from GF/C filters. Regression analysis showed that this fraction too was positively correlated with both feeding rate and gross animal load and, in each of the three categories studied ((i)-(iii) above), a higher correlation coefficient was obtained when the fractions were plotted against gross animal load than feeding rate (see Table 3 and Discussion). The mean organic carbon content increased up to 16.7 mg litre -1 and was within the range normally found in the laboratory seawater supply (0-5-1 8 mg litre-1). (iv) Material absorbing in the ultraviolet wavelength 2 5 0 - 3 3 0 nm Measurements of absorbance over the wavelength range 250-330 nm were made in two trials with the model recirculation systems. In each trial 70~ of the water was renewed thrice weekly in two of the systems (group a), while in the remaining two (group b) the water was not changed for eight weeks. In group a the integrated absorbance (2; A 250330) fluctuated over the range 10.75-70.30 arbitrary units during the trial. This contrasted with group b where the absorbance increased linearly with time from around 20 to 106 units (regression eqn (2) in Table 4).

70

J. F. Wickins

When the integrated absorbance measured in the recirculation systems (group b) was divided into two portions 2; A 2 5 0 - 2 9 0 and Z A 290-330, a greater increase in the Z A 2 9 0 - 3 3 0 portion was apparent as the trial progressed, presumably as metabolically derived material accumulated (see Discussion). This is shown in Fig. 2 where the ratio 22A 2 5 0 - 2 9 0 / Z A 2 9 0 - 3 3 0 is shown to decrease with time. The best relationship for the combined data from the two trials is given by eqn (3) in Table 4. Attempts were also made with the group b data to relate the observed changes in absolute and relative absorption within the two wavelength groups to the measured particulate and 'dissolved' organic fractions in order to examine further the possible origins of the light absorbing substances. The absorbance was found to increase as the organic portion o f particulates retained on the GF/C filter papers increased, and also as the concentration o f sub-micron particulates increased. Plots of concentration against absorbance (degrees of freedom = 29 in each case) gave marginally higher correlation coefficients (r) for 22A 2 9 0 - 3 3 0 than for E A 2 5 0 - 2 9 0 (Table 5). The values of r obtained were, however, less than the value obtained when the absorbance ratio was plotted against time (r = 0.95, see eqns (2) and (3), Table 4) indicat-

2,1-

1.9._o

i
o 8

c~ .Q

1.7-

.Q <

1.5-

16

24 Days

32

40

48

Fig. 2. The temporal change in ultraviolet absorbance ratio ZA 250-290/ZA 290330 in four marine recirculation systems. The water was not renewed.

Organic~inorganic C levels in recycled seawater during prawn culture

TABLE 5

71

The Correlation Coefficient for Plots of Ultraviolet Absorbance Against (a) % Organic Content of Particulates Greater than 1 #m and (b) Concentration of SubMicron Particulates (mg litre -~)
Ordinate Abscissa r
0-64

p
< 0.001

NA 250-290 } 2;A 290-330 2 A 250-290 Y~A290-330 NA 250-290 }

2 A 290-330

% Organic content

0.70 -0.77 0.45 0.48 -0-48

<0.001 "~0.001 <0.05 > 0.01 <0.01 > 0-001 <0.01 > 0.fl01

2;A 250-290 2;A 290-330

Sub-micron particulates (mg litre-1)

ing only a general trend towards increasing absorption (1~ A 290-330) with increase in the organic content of particles greater than 1 tam and an even more variable response to concentrations of sub-micron particles. A simple relationship of absorbance to 'dissolved' organic carbon concentration could not be demonstrated because, although the integrated absorbance increased throughout the trials, carbon levels increased from 6 to 20 mg litre -1 during only the first 23 days and thereafter declined to 9 mg litre -a. The absorbance ratio tended to decline as carbon levels rose (Fig. 3) but a slight reversal at the highest carbon levels was recorded in mid-trial (see Fig. 2). Absorbance is also considered below in relation to foaming.

(v) E f f e c t o f f o a m i n g on organic material The effects of foaming on organic materials were observed in two series of experiments with the model systems. In the first series (group c) no changes of water were made for 8-10 weeks, in the second (group d) 70% of the water was renewed thrice weekly. The results are summarised in Table 6 in general terms and the differences between replicate treated and untreated systems are described below for both series in the order in which they are listed. Foaming produced a gradual increase in the concentration of particulates (mg litre -1) greater than ltam in group c with time (regression

72
2.4-

J. F. Wickins

2.2-

O00D
2.0-

o o

. 1.8o
,.Q

o o

o o

@
o o o o 0 0 0 o o o o o

o
JE~ ,<

1.6

o 00~0

1.4

1.2

1.0

,'2

,'6

2o'

2',

Organic carbon (mg I-1)

Fig. 3. The changes in the relationship between ultraviolet absorption ratio ZA 250-290/I2A 290-330 and 'dissolved' organic carbon levels in four marine recirculation systems. The water was not renewed.

eqn (4), Table 4). No such relationship was seen in group d and no differences in the levels of sub-micron particulates due to the treatment were found in either group (see Discussion). Levels of 'dissolved' organic carbon (in samples of GF/C filtrate) increased less rapidly in foamed than in unfoamed systems in group c; however, in contrast, regular, partial water changes masked the effect in group d. The calculated rate slopes and 9 5 ~ confidence limits for plots of concentration against time were:
Foamed Not foamed

Water not renewed (data from eight experiments) Degrees of freedom Water regularly renewed (data from four experiments) Degrees of freedom

0.14 (0-11-0.18) 87 0.13 (0.04-0.26) 29

0.28 (0.24-0.32) 117 0.15 (0.09-0.21) 28

Organic/inorganic C levels in recycled seawater during prawn culture TABLE 6 Some Effects of Dispersed Air Flotation Treatment on Organic Materials in Recycled Seawater Foam ed Group c (water not renewed) Correlation between particulates > 1/am and time Particulates < 1 tzm Correlation between 'dissolved' organic carbon and time Correlation between ultraviolet absorption and time Numbers of bacteria Group d (water periodically renewed) No t Jbarn ed

73

+ve ND Slow increase ND Low ND ND ND Low High +ve Low

No correlation ND Fast increase ND High ND ND ND High Low No correlation High

Particulates > 1/~m Particulates < 1/~m 'Dissolved' organic carbon Ultraviolet absorption, 2;A 250-290 and 2;A 290-330 (means) NA 250-290 Ultraviolet absorption, ' NA 290-330 (ratxo) Correlation between ZA 250-290 and 'dissolved' organic carbon Numbers of bacteria ND, no differences observed.

Thus, in systems where the water was not changed foaming tended to slightly increase the levels of filterable particulates and reduce the rate of accumulation o f 'dissolved' organic carbon. Ultraviolet light absorption increased equally with time in both foamed and unfoamed systems in group c, but in group d absorbance fluctuated and mean values tended to be lower in foamed systems (p > 0.05 < 0.1). The ratio of the integrated absorbances in group d was higher in foamed than in unfoamed systems and was also less variable (Table 7). Possibly foaming removed some of the light absorbing material and also proportionately more of the fraction (of metabolic origin) that absorbed strongly at the higher wavelengths. In this context

74

J. F. Wickins

TABLE 7 The Effect of Dispersed Air Flotation Treatment on Absorbance of Ultraviolet Light by Recycled Seawater
Foamed Not foamed

Number of experiments (group d, water regularly renewed) Mean absorbance, ZA 250-290 95% Confidence limits of mean Degrees of freedom Mean absorbance, ZA 290-330 95% Confidence limits of mean Degrees of freedom Z A 250-290 Ratio, XA 290-330 95% Confidence limits of mean Degrees of freedom Coefficient of variation (%)

2 14.58 12.44-16.72 31 7.56 6.00-9.12 31 2.04 1.96-2.12 30 11.4

2 19.13 16.21-22.05 31 11.80 9.11-14.49 29 1.81 1.69-1.93 30 18.2

it was noticed that the integrated absorbance at the lower wavelengths ( Z A 250-290 but not Z A 290-330) generally varied proportionately with 'dissolved' organic carbon levels in the foamed but not in the unfoamed systems (see Discussion). The regression eqn (5) describing the relationship is given in Table 4. Despite the wide variation in bacterial numbers, a series of counts made at intervals of at least one week indicated that foaming either expelled or inhibited the development of bacteria in both groups c and d:
Foamed Not foamed

Number of bacteria ml -a (mean of two replicates) 56 t statistic Degrees of freedom = 26, p < 0-001

595 3.75

Carbonate alkalinity and pH

(i) R e d u c t i o n in carbonate alkalinity Changes in levels o f pH and inorganic carbon that occurred in the model recirculation systems during the culture of 26-57 g of tropical

Organic~inorganic C levels in recycled seawater during prawn culture

75

prawns (1-5 g live weight) are shown in Fig. 4. In two trials a thrice weekly replacement of 70% o f the seawater was sufficient to prevent major changes in pH and inorganic carbon levels over a 56 day period (Fig. 4, lines (a) and (c)). In four other trials where the water was not

301
251

0
o,, a

I ~k---._.
~O

o ~."

b
0 4 ; 1'2 1; 2; 214 2'8 312 3v6 4; 4'4 4'8 ;2 .56
Days

80!
7.8 7.67.4,o

1 a.2 J~.lk..

8.4

O0 0 ~ ~ 0 ~ 0 0

~0"~ 0 ~
o

0
o

6.6
6.66.4-

d
0 ~'----~0
0 00

0
0

'6.2 6.0

' ' 1 '8 2' 0 4

' 2 4 26 . .32. 36 . .40. 44 48 5~ 1 16 20 2 56

Days

Fig. 4. The changes in levels of (A) inorganic carbon and (B) pH during nitrification in recirculation systems with regular seawater renewal (lines (a) and (c)) and with no renewal (lines (b) and (d)).

76

J. F. Wickins

changed (lines (b) and (d)), the pH fell from 8.2 to 6.6 and carbon from 25 to 2 mg litre -1 in 40 days, a loss of about 0.6 mg carbon litre -1 day -1. The mean rate o f carbon loss C (rag litre -I day -~) was linearly related to the mean weight o f food fed calculated in terms of its nitrogen content, F N (g day-l; Wickins, 1985) and is described by eqn (6) in Table 4. (ii) Inorganic carbon/pH relationships When all recirculation systems were categorised according to the type of filter medium and chemicals used to control pH and alkalinity it was noticed that the relation between the concentration of dissolved inorganic carbon and pH varied (Fig. 5). In most o f the systems a proportion o f the water was changed each week. Six conditions were identified by the following codes:
Filter medium None Chemical additions Sodium carbonate/ bicarbonate Sodium hydroxide

Plastic Limestone gravel

A (water not renewed) B

C D

E F

The large number of data points and the frequent association of values of the independent variable (pH) with more than one value of the dependent variable (inorganic carbon concentration) called for grouping and the use o f weighted regression analysis in order to present a clear picture for each condition (see Methods). Considering firstly systems in which the filter media did not contribute to the alkaline reserve, i.e. plastic media, as pH decreased carbon levels fell markedly (Figs 5(A), (C) and (E)) and there were no significant differences between the slopes above pH 7-4regardless of chemical additions. However, the combined data could not be represented by a c o m m o n line since the three intercepts were significantly different (t test, p < 0 - 0 5 ) ; for example, at pH 8.0 dissolved inorganic carbon levels were highest (30 mg litre -1) when sodium hydroxide was used (Fig. 5(E)); less (22 mg litre -1) with sodium carbonate and bicarbonate (Fig. 5(C)); and lowest (17.5 mg litre -1) when 11o chemicals were added (Fig. 5(A)).

Organic~inorganic C levels in recycled seawater during prawn culture B

30

77

20

10-

0 c3 c o c 30.

10

O 6.6

J
i J

/
i i l i i

f
, v

7.0

7.4

7.8

8.2

7.0

7.4

7.8 pH

8.2

7.0

7,4

7.8 pH

8.2

pH

Fig. 5. The relationship between pH and inorganic carbon concentration in seawater recycled from (A) plastic media filters; (B) limestone gravel filters; (C) plastic media filters dosed with sodium carbonate/bicarbonate solution; (D) limestone gravel filters dosed with sodium carbonate/bicarbonate solution; (E) plastic media filters dosed with sodium hydroxide solution; and (F) limestone gravel filters dosed with sodium hydroxide solution.

In filters containing a limestone medium, carbon levels fell less markedly than in filters with a plastic medium following a reduction in pH. This was probably due to contributions made by the limestone to seawater alkalinity. There were no differences between slopes among systems not dosed and systems receiving sodium carbonate and bicarbonate, and although carbon levels tended to be higher in dosed systems they were not significantly so. Plastic and limestone containing systems dosed with sodium hydroxide showed a similar relationship between pH and carbon over the pH range 7.3-7.8. Above about pH 7.85 in plastic filters (Fig. 5(E)) the relationship with carbon concentration continued linearly. In limestone filters, however, a pronounced reversal occurred (Fig. 5(F)) and levels of dissolved inorganic carbon declined with increasing pH (see Discussion).

78

J. F. Wickins

In systems containing filters of plastic media where chemicals were not added and pH was allowed to fall below pH 7.4 (Fig. 5(A)), an inflexion in the pH inorganic carbon relationship occurred at around pH 7.45 (see Discussion). No such inflexion occurred in limestone filters, again, probably due to the contribution of the limestone to carbonate alkalinity. (iii) Control o f pH and carbonate alkalinity Using data from 11 culture trials, made with plastic media filters, the quantities of sodium hydroxide added to maintain pH (and, indirectly, carbonate alkalinity) were plotted against gross animal load. The In weight (g) added per day per 100 litres of water used throughout the culture period increased in proportion to In load according to the regression eqn (7) in Table 4. (iv) Effect o f foaming on carbon dioxide and pH The vigorous aeration employed in dispersed air flotation columns typically assisted the loss of excess carbon dioxide from the water which tended to normalise pH levels. This occurred in systems where dissolved inorganic carbon levels were maintained (for example, by regular partial water changes) at 16-21 mg litre -~ during normal nitrification (Table 8).

DISCUSSION

Particulate and 'dissolved' organic material

Organic substances arise not only from the cultured animals and their uneaten food but also from the metabolic activity of microorganisms attached to suspended solids and filter surfaces. Most of the microorganisms are likely to be heterotrophic and will increase in numbers in proportion to the available organic (nutrient) substrate concentration (SSrner, 1981). In a number of systems, levels of particulates increased with time as the daily feeding rate increased. When animals were removed from systems for experiments, food input was reduced accordingly. For this reason levels of particulates, dissolved organic carbon and perhaps

Organic~inorganic C levels in recycled seawater during prawn culture TABLE 8

79

The Effect of Foaming on Carbon Dioxide and pH Levels

Replicate a Foamed b Not foamed c d

Carbon dioxide (mean mg CO2-C litre-1) 95% Confidence limits of mean Mean pH calculated from [H] 95% Confidence limits of mean

0.08 0.07 0.07-0.09 0.06-0.07 8.18 8.11 8-1%8-19 8.08-8-15

0-14 0-12 0.12-0.16 0-10-0.14 8.00 7.97 7.98-8-03 7.94-8-01

Degrees of freedom = 24 in each case.

bacteria were more closely related to feeding rate than to time. The low (though significant) correlation coefficients obtained (0.54-0.67) from plots of particle concentration against feeding rate were probably due to differing water renewal rates and to the intermittent use o f diatomaceous earth filters in some of the systems. Only a general trend towards higher concentrations with increased feeding rate was therefore shown. Higher correlation coefficients (0.81, 0.84) were obtained when the overall mean concentrations were compared with gross animal loads in the systems, a figure which accounted for different rates o f water renewal. At high gross animal loads mechanical filters that operated independently of biological filters were convenient to use because filtered material could be removed without disturbing biologically active slimes. Trials made w i t h o u t periodic water renewal Under comparable conditions of animal load, whether the water was changed or not seemed to have little effect on concentration of particulates and dissolved organic carbon. When the water was not changed, however, the proportion of organic matter in material retained on GF/C filters and the integrated ultraviolet absorbance (particularly I A 290330) increased with time. Changes in the composition or origin of dissolved organic material in seawater have been indicated from seasonal changes which occur in

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the ultraviolet absorption characteristics of natural seawater (Foster and Morris, 1971 ; Foster and Foster, 1977). In systems where the water was not changed there were also significant but more variable positive correlations between 2; A 290-330 and the percentage of organic matter in material retained on the GF/C filters and between ~ A 290-330 and levels of sub-micron particulates. Although little can be said concerning the characterisation of the substances absorbing the ultraviolet light, the results seem to confirm the findings of Foster and Morris (1971) who suggested that the greater absorbance which occurred at longer wavelengths in coastal seawater during autumn (~; A 275-300) was due to organic matter derived from in s i t u biological activity rather than from land drainage material which absorbed strongly at lower wavelengths. The accumulation of dissolved organic material, 'yellow substance', derived from fish foods which absorbed strongly at 315 nm and also of coloured, filterable particulate matter in very densely stocked recirculation systems was described by Rosenthal e t al. (1978), who used the dissolved 'yellow substance' as an indicator of the levels of non-biodegradable compounds which accumulated in the water. Although the water did not appear yellow in the present marine systems run with water renewal, the integrated absorbance Y.A 290-330 included a component due to the accumulation of metabolically-derived material which was resistant to microbial degradation both by slimes in the biological filters and by suspended microorganisms. Ozone was used successfully by Rosenthal e t al. (1978) to degrade such refractory organic substances into components which were then oxidised in percolating biological filters. The reversal in the relationship between absorbance ratio and 'dissolved' organic carbon shown in Fig. 3 was probably an artifact caused by the temporary increase in the ratio during days 19-31 (see Fig. 2). The increase indicated that absorbance by material of metabolic origin might have become temporarily of less importance in the presence of material from other (unidentified) sources. Examples of possible sources might include contaminated distilled water added to counteract evaporative losses or organic aggregations released from filters.

Carbonate alkalinity and pH

Loss of alkalinity during water treatment in marine aquaria was quantified by Hirayama (1974) who showed that the rate of loss V(equivalents

Organic~inorganic C levels in recycled seawater during prawn culture

81

day-1) was related to the weight of food fed F (g day-l): V -- (2.52 + 0.92F) x 10-3. A similar slope value (0.79) was found for the relationship between weight of sodium hydroxide used to maintain pH and animal load in the laboratory systems. The ratio of inorganic carbon loss to nitrogen input rates in the present trials was 0.99, close to that predicted stoichiometrically (0-948) and within the range 0.76-1.24 found in model marine filters dosed with ammonium chloride solutions (Wickins, 1983). It is important to consider the relationship between pH and inorganic carbon in recycled seawater with reference to the method of pH control and nature of the filter media. In the laboratory filters neither medium employed prevented acidification: but it was found that results closest to natural seawater, which at pH 8.0 contains about 26 mg inorganic carbon litre -1, were obtained with limestone filters dosed with sodium hydroxide solution (25 mg carbon litre-1). Greater reserves of carbonate alkalinity (30 mg carbon litre -1) were obtained at the same pH when sodium hydroxide was used with plastic media filters, presumably as conditions favoured the entry of respiratory and atmospheric carbon dioxide into the bicarbonate-carbonate system (Wickins, 1983). Two features of Figs 5(A) and (F) are worthy of comment. The inflexion in the pH/inorganic carbon relationships shown in Fig. 5(A) is an artifact of the regression analysis. The relationship is perhaps better illustrated by a curve over the pH range 7.4-7.7 within which seawater buffering is minimal (Whitfield, 1974). In Fig. 5(F) the decline in dissolved inorganic carbon levels with increasing pH above pH 7.85 was probably due to carbonates crystallising out of solution onto the limestone surfaces of the filter media (Weyl, 1967; Berner, 1975). Because of this, and the tendency of limestone filters to become blocked, the use of sodium hydroxide with plastic media filters proved more reliable and predictable in laboratory systems where gross animal loads were high.

Dispersed air flotation

Perhaps the most useful effects of dispersed air flotation were the marked reduction in bacterial numbers and the tendency to stabilise pH. In single pass or through flow dispersed air flotation units (Wheaton et al., 1979), foaming generally results in a decrease in the concentration of particulate material retained on GF/C filters and an increase in that retained on the finer GF/F grade (Wickins and Helm, 1981). Under

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J. F. Wickins

recirculation conditions the fraction retained on GF/C filters increased but that retained on 0.22/am Millipore filters did not. In the system of Rosenthal et al. (1978), the 0.22/am fraction was reduced after dispersed air/ozone flotation treatment. It was possible that in recycled seawater the fine particlek formed coarser aggregations or that the conditions of foaming actually gave rise to larger particles (Johnson and Cooke, 1980). The effect of foam treatment on ultraviolet absorption is not clear although the results suggest that foaming might have removed some dissolved organic material which absorbed in the range ~ A 2 9 0 - 3 3 0 nm. The variable but significant (p < 0.01) correlation between integrated absorption Z A 2 5 0 - 2 9 0 and dissolved organic carbon levels in foamed systems leads to speculation that foaming produced material that absorbed strongly at the shorter wavelengths concurrently increasing the absorbance ratio Z A 2 5 0 - 2 9 0 / Z A 2 9 0 - 3 3 0 in treated systems. Despite the present uncertainties concerning the action of foaming in seawater the incorporation o f dispersed air flotation into marine recirculation systems seems to provide benefits w o r t h y of further study.

REFERENCES Bages, M. & Sloane, L. (1981). Effects of dietary protein and starch levels on growth and survival of Penaeus monodon (Fabricius) post-larvae. Aquaculture, 25, 117-28. Bartley, D. M., Carlberg, J. M., Van Olst, J. C. & Ford, R. F. (1980). Growth and conversion efficiency of juvenile American lobsters (Homarus americanus) in relation to temperature and feeding level. Proc. 11th Ann. Meeting Wld. Maricult. Soc., New Orleans, Louisiana, USA, 5-8 March, Louisiana State University, pp. 355-68. Beard, T. W. & Forster, J. R. M. (1973). A growth experiment withPenaeus monodon Fab. in a closed system. ICES, Shellfish and Benthos Committee, CM 1973 K: 39, 6 pp. (mimeo). Beard, T. W. & Wickins J. F. (1980). The breeding ofPenaeus rnonodon Fabricius in laboratory recirculation systems. Aquaculture, 20, 79-89. Beard, T. W., Wickins, J. F. & Arnstein, D. R. (1977). The breeding and growth of Penaeus merguiensis de Man in laboratory recirculation systems. Aquaculture, 10, 275-89. Berner, R. W. (1975). The role of magnesium in the crystal growth of calcite and aragonite from seawater. Geochim. cosmochim. Acta, 39,489-504.

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Bower, C. E., Turner, D. T. & Spotte, S. (1981). pH maintenance in closed sea water culture systems: limitations of calcareous filtrants. Aquaculture, 23, 211-17. Chamberlain, G. W. & Lawrence, A. L. (1981). Maturation, reproduction and growth of Penaeus vannamei and P. stylirostris fed natural diets. J. Wld. Marlcult. Soc., 12 (1), 209-24. Conklin, D. E., Bordner, C. E., Garrett, R. E. & Coffer R. J. (19811). Improved facilities for experimental culture of lobsters. J. Wld. Maricult. Soc., 12 (1), 59-63. Cuzon, G., Hew Meng, Cognie, D. & Soletchnik, P. (1982). Time lag effect of feeding on growth of juvenile shrimp Penaeus ]aponicus Bate. Aquaculture, 29, 33-4. Draper, N. R. & Smith, H. (1966). Applied Regression Analysis, John Wiley & Sons, New York, pp. 77-81. Forster, J. R. M. (1972). Some methods of binding prawn diets and their effect on growth and assimilation. J. Cons. perm. int. Explor. Mer., 34 (2), 200-16. Foster, P. (1974). Ultra-violet absorption characteristics of natural waters. War. Res., 8, 137-42. Foster, P. & Foster, G. M. (1977). Ultra-violet absorption characteristics of waters in an industrialized estuary. Wat. Res., 11, 351-4. Foster, P. & Morris, A. W. (1971). The use of ultra-violet absorption measurements for the estimation of organic pollution in inshore sea waters. Wat. Res., 5, 19-27. Hirayama, K. (1974). Water control by filtration in closed culture systems. Aquaculture, 4,369-85. Johnson, B. D. & Cooke, R. C. (1980). Organic particle and aggregate formation resulting from the dissolution of bubbles in sea water. Limnol. Oceanogr., 25 (4), 653-61. Lawson, T. B. & Wheaton, F. W. (1980). Removal of organics from fish culture water by foam fractionation. Proc. l l th Ann. Meeting Wld. Maricult. Soc., New Orleans. Louisiana, USA, 5-8 March, Lousiana State University, pp. 128-34. Moffett, W. g. & Fisher, W. S. (1978). Ammonia production rates ofArtemia salina under various culture conditions. J. bi'sh. Res. Bd Can., 35, 1643-8. Rosenthal, H., Krfiner, G. & Otte, G. (1978). Effects of ozone treatment on recirculating water in a closed fish culture system. ICES, Mariculture Committee, CM 1978 F: 9, 16 pp. (mimeo). S~irner, E. (1981). Removal of dissolved and particulate organic matter in high-rate trickling filters. Wat. Res., 15,671-8. Sedgwick, R. W. (1979). Effect of ration size and feeding frequency on the growth and food conversion of juvenile Penaeus merguiensis de Man. Aquaculture, 16, 279-98. Siddall, S. E. (1974). Studies of closed marine culture systems. Prog. Fish. Cult., 36 (I), 8-15.

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Spotte, S. (1979). Seawater Aquariums, the Captive Environment, Wiley Interscience, New York, 413 pp. Strickland, J. D. H. & Parsons, T. R. (1968). A practical handbook of seawater analysis. Bull. Fish. Res. Bd Can., 167, 1-311. Tiews, K. (Ed.) (1981). Aquaculture in Heated Effluents and Recirculation Systems, Vols 1 and 2, Heenemann Verlagsgesellschaft, Berlin. Weyl, P. K. (1967). The solution behaviour of carbonate materials in seawater. Stud. Trop. Oceanogr., 5, 178-228. Wheaton, F. W., Lawson, T. B. & Lomax, K. M. (1979). Foam fractionation applied to aquacultural systems. Proc. 10th Ann. Meeting Wld. Maricult. Soc., Honolulu, Hawaii, USA, 22-26 January, Louisiana State University, pp. 795-808. Whitfield, M. (1974). The ion-association model and the buffer capacity of the carbon dioxide system in seawater at 25C and 1 atmosphere total pressure. Limnol. Oeeanogr., 19,235-48. Wickins, J. F. (1981). Water quality requirements for intensive aquaculture: a review. In: Aquaculture in Heated Effluents and Recireulation Systems, Vol. 1, ed. K. Tiews, Heenemann Verlagsgesellschaft, Berlin, pp. 17-37. Wickins, J. F. (1982) Opportunities for farming Crustaceans in western temperate regions. In: Recent Advanees in Aquaculture, eds J. F. Muir and R. J. Roberts, Croom Helm, London, pp. 87-177. Wickins, J. F. (1983). Studies on marine biological filters: model filters. War. Res., 17, 1769-80. Wickins, J. F. (1985). Ammonia production and oxidation during the culture of marine prawns and lobsters in laboratory recirculation systems. Aquacultural Engineering, 4 (in press). Wickins, J. F. & Beard, T. W. (1978). Ministry of Agriculture, Fisheries and Food, Prawn Culture Research, Lab. Leafl., MAFF Direct. Fish. Res., Lowestoft (42), 41 pp. Wickins, J. F. & Helm, M. M. (1981). Seawater treatment. In: Aquarium Systems, ed. A. D. Hawkins, Academic Press, London, pp. 63-128.