Water control by filtration in closed culture systems

Aquacdture, 4(1974)369-385 0 Else-wier Scientific Publishing

WATER CONTROL

KAZUTSUGU Faculty

Amsterdam

BY FILTRATION

-

Printed

in The Netherlands

IN CLOSED CULTURE

SYSTEMS

HIRAYAMA

of Fisheries,

(Received

Company,

August

Nagasaki

12th,

University,

Nagasaki

(Japan)

19’74)

ABSTRACT Hirayama, K., 1974. 4: 369-385.

Water control

by filtration

in closed

culture

systems.

Aquaculture,

This paper describes the chemical changes in culture water in closed culture systems with sand filter. Stabilization of inorganic nitrogen after sequential changes of ammonium-N and nitrite-N in a culture system with new filters takes from 40 to 60 days. The characteristic changes in chemical composition of aged culture water, namely an accumulation of nitrate and phosphate and lowering of pH and alkalinity, are discussed. The carrying capacity in a closed culture system is determined by using oxygen consumption during filtration as an index to indicate the degree of purification by filtration.

INTRODUCTION

The key to the successful culture of aquatic animals is the control of water quality. If a plentiful supply is available, an open water system is the easiest, and often the best, to use. If, on the other hand, a satisfactory supply of water is not available all year round, purification of the water by microorganisms is the most important way of eliminating polluting substances. In Japan, for example, the water in eel or goldfish ponds is hardly changed at all. In such ponds, phytoplankton grow and release oxygen; aerobic bacteria attached to the surface of the phytoplankton assimilate and oxidize the polluting substances; and the phytoplankton in turn absorb and utilize the inorganic nitrogen and phosphate that the bacteria produce. By means of closed circulating systems dense populations of aquatic animals can be kept healthy in limited volumes of water. The water circulates between the culturing vessel and filtered bed, making it possible for the aerobic bacteria to carry out their essential role in eliminating undesirable metabolites. The passage of water through the sand bed of the filter maintains conditions that are favorable to aerobic bacterial growth. In this system, the filter bed functions to increase the surface area for bacterial attachment. The work #ofthe filter is thus similar to that of a trickling filter in a sewage treatment plant.

370

The filter provides not only the physical removal of solid wastes but also the biochemical oxidation of dissolved organic matter and ammonia. To maximize the purifying ability of the filter, the circulating water must be maintained continuously in an aerobic condition. Aerobic bacteria demand large amounts of oxygen for their oxidation activities. The oxygen demand of the bacteria in a filter is about the same as the oxygen consumed by the cultured animals (Hirayama, 1960). According to Spotte (1971), a filter bed is like a huge respiring organism. An aquarium or closed culture system requires an air supply sufficient not only to keep the animals healthy but also the filter fully active. CHEMICAL

CHANGES

IN NEW CULTURE

SYSTEMS

In a newly established aquarium or culture system with a new filter, the filter bed does not contain enough bacteria to carry out its purifying activity, and polluting substances rapidly accumulate in the water. The population of aerobic bacteria gradually builds up in the filter bed, using the metabolic products of cultured animals as nutrients, and the rate of water purification by the filter is steadily increased. Fig.1 shows the changes in chemical composition of both sea water and fresh water that took place in two experimental closed culture systems (Hirayama, 1972). These experiments were carried out under almost the same conditions in two tanks (Fig.2): surface area of filter about 300 cm2 and depth 20 cm, volume of water 50 1, water temperature 20-21°C with three fish fed 2 g of fish flesh a day. The two systems differed SEA

WATER

FRESH

.-.

PH

.-.

WATER .3

~ 0-o

NITRITE-N

.-.

__

m--I

__-

PHOSPHATE-P

.-.

~

AMMONIUM-N

NITRATE-N

~

V-----V ~0-0 ~~

o-0 ?H

CULTURE

Fig.1. Chemical

changes

PERIOD

IN

DAYS

in the water of newly established

closed

culture

systems.

371

A

Fig.2. Experimental of wa’;er.

aquarium.

A: culture

tank;

F: filter;

E: air lift. Arrows

indicate

flow

in the density of the water, the species of fish (goby and goldfish), and the total weight of the animals (67 g and 90 g, respectively). The filters were composed of the same sand collected from a river bank with some shells and coral, all of them washed so thoroughly with sodium hypochloride as to have no initial capacity for purification. In both the marine and fresh-water aquaria, ammonium-N accumulated very rapidly until the 8th-10th day, making the aquarium water somewhat alkalne. The filter bed then acquired the ability to oxidize ammonia to nitrite, and the ammonia almost disappeared from the water. At the same time, nitrite began to build up, gradually making the aquarium water acidic. This accumulation reached its highest value on the 35th day in both aquaria, and almost disappeared by the 50th day in the sea-water aquarium and by the 44th day in the fresh-water one. As a result of the activity of nitrifying bacteria in the filter bed, the ammonia and nitrite disappeared from the aquarium water quite suddenly within a few days. Well-conditioned aquarium water thus contains little ammonia and nitrite. The stabilization of the nitrogen sequence in a culture system with a new filter takes from 40 to 60 days. The pattern of change in chemical composition of both the seawater and fresh water was almost the same. Other investigators (Saeki, 1963; Kawai et al., 1964; Liao and Mayo, 1974) have obtained similar patterns under various experimental conditions, even though the actual levels of accumulation of ammonia and nitrite differed in each experiment. The sea water system of the Suma Aquarium, Kobe City, Japan, was filtered through an experimental filter with clean sand that had no capacity for purification and the oxygen consumption during filtration was measured each day (Hirayama, 1960). The results are shown in Fig.3. Until the 6th or 7th day after the start of filtration, there was no oxygen consumption at all, but then the filter began to consume oxygen. Oxygen consumption was greatest on the 15th day, and this may be closely connected with the fact that by about the 14th day, the filter bed had acquired enough oxidizing capacity to have removed almost all of the ammonia from the water. Kawai

372

;A$

I 4

IL

DAIS

L1~L 6

,‘,

20

FROM

_ ZR

THF

Fig.3. Oxygen consumption system by newly established

ST!ihi

52

3F

,b

‘+u

4.1

411

32

F~LT%ATiCh

during filtration filter.

of the culture

water in a large sea-water

et al. (1964) have shown that the bacterial population in the filter bed changes greatly during the early stages of cultivation and reaches an equilibrium in about 2 months. These facts also seem to be in accord with the observation that nitrite is hardly detectable in culture water after 40-60 days. CHEMICAL

CHANGES

IN AGED

CULTURE

In closed-system aquaria, the culture istic changes in chemical composition.

WATER

water gradually

undergoes

character-

Lowering of pH and alkalinity As long as the filter retains its full activity, the aquarium water tends to become gradually more acid due to the accumulation of nitrate and the decrease of alkali since part of the magnesium precipitates with the excess in phosphate (Saeki, 1963). To verify this acidification phenomenon, black porgies (Mylio macrocephalus) were cultured and fed at different rates in a well-conditioned aquarium with a filter from which the calcium (shells and coral) had been completely eliminated by strong acid, and the daily changes in pH and alkalinity were examined (Hirayama, 1970). From these experimental data, a linear relation was obtained between the rate of acidification of the aquarium water V (equivalents per day) and the rate of feeding F (g/day): v = 0.92F

x 1o-3

(1)

373

This relation is shown in Fig.4. According to Kawai et al. (1965), the nitrifying activty of a filter is greatest at a pH of about 9.0 and decreases with any further increase in hydrogen ion concentration. Nevertheless, when black porgies were fed 2 g of shrimp per day, no accumulation of ammonia and no decrease in the rate of acidification were observed, even at a pH of 7.0 and an al:kalinity approaching 0.2 meq/l. This suggests that a filter bed can retain enough nitrifying capacity to keep the ammonia at a low level, even when the pH and alkalinity reach very low values. The acidification of culture water can be prevented to some extent by the addition of alkali (Ca(OH),, CaO, or NaHC03) to the water itself or shells or coral (CaCO,) to the filter medium.

ilrElCHT!;:'?F

Fig.4

Relation

AcczLmulation

of rate of acidification

F:.,?l PER

of aquarium

D;i

IF)

water to feeding rate.

of nitrate

As the final oxide of ammonia derived from metabolic products or uneaten food, nitrate reaches remarkably high concentrations in culture water. The accumulation of nitrate-N usually exceeds 100 ppm in aged aquarium water. There is a limit to the amount of nitrate that accumulates, however, because of the considerable number of denitrifying bacteria in the filter bed (Kawai et al., 1964), and the undoubted presence of anaerobic microspheres there despite an adequate amount of aeration. Harmful effects of accumulated nitrate have rarely been reported, but the accumulation of nitrate, together with the fall of pH and alkalinity, can adversely affect octopus respiration (Hirayama, 1966c). This may be one of the reasons why it is difficult to culture octopus in aged aquarium water. In

374

eel ponds, however, nitrate is scarcely ever detected since the phytoplankton remove it by assimilation. Attempts have been made to use seaweeds to purify the culture water in marine aquaria (Shelbourne et al., 1963; Goldman et al., 1974; Siddall, 1974). Accumulation

of phosphate

Phosphate is accumulated in aquarium water as one of the final products of the destruction of organic substances containing phosphorus. It scarcely reaches concentrations of more than 6 ppm because of the precipitation with magnesium or calcium (Saeki, 1963). Additional

changes

Generally, it is difficult to culture most invertebrates in aged aquarium water. The accumulation of nitrate accompanied by acidification seems to be one reason for this. The accumulation of organic substances should be also recognized as one of the restrictions on the culture of certain invertebrates. It is well known that aged aquarium water becomes yellowish. Although there is a stable yellow substance that occurs in natural sea water (Kalle, 1966), it is probably significant that organic substances like this yellow substance accumulate in an aquarium. In fact, the author has measured concentrations of organic carbon of more than 50 ppm in an experimental aquarium. DETERMINATION

Purification

OF CARRYING

CAPACITY

index

One of the important aspects of biological filtration is its effect on carrying capacity. Carrying capacity is defined as the animal load that a system can support. Although Saeki (1958) proposed a relationship between carrying capacity and weight of the filter sand by using the ammonia content as an index, critical decisions as to the carrying capacity of any given culture system have had to depend almost entirely on the culturist’s experience. In fresh-water culture systems, BOD is known to be an excellent index to evaluate the degree of pollution. Oxygen consumption during filtration (OCF), which results from the biochemical oxidation carried out by the bacteria in the filter bed, might be used as an index to indicate the degree of purification for the same reasons that BOD has been used. Special experimental filters were designed to make possible the sampling of water from four different depths of the filter bed (Fig.5). The water from a closed system in a public aquarium was filtered through this filter, and water was sampled at each depth. The water was then analyzed to investigate the relation of oxygen consumption to decrease in BOD during filtration (Fig.6) and to the oxygen consumed in nitrification (Fig.7). OCF has such close

375

WATER SUPPLY 1

VATER SUPPLY

Fig.5. Experimental filters. Type A: water can be taken from 4 different depths of sand. Type 13: four filters with different depths of sand (12, 24, 36 and 48 cm). A: screw valve for controlling the flow of water. B: screw valve for sampling water.

0

0. c

\

05-p

t

0

t?1

.

0

F

-m

a’ 403

-

0 0

l

I

OCF

(mg/l)

Fig.6. Relation of OCF to decrease in BOD during filtration of culture water. Correlation coefficient between OCF and decrease in BOD = 0.63, which is statistically significant

(p < 0.01).

376

Fig.7. OCF and oxygen consumption in nitrification during filtration through various depths of sand. Solid line, Experiment 1; broken line, Experiment 2; solid circles, observed OCF; empty circles, calculated oxygen consumption in nitrification.

relationships with these indicators (Hirayama, 1965a). Amount

of purification

that it can be used as an index itself

by filtration

The relation of the amount of water purification to the depth of sand was examined. Two examples of 13 experiments for OCF obtained at four different depths of sand of filter bed (Fig.5A) are shown in Fig.& In general, the BOD data fit the equation of a first-order chemical reaction. Since OCF, like BOD, results from biochemical oxidation, it is possible that the OCF data fit one of the equations of chemical reaction. As shown in Fig.8, the relation of OCF to depth of sand layer fitted a second-order reaction formula very well compared with a first-order reaction formula (Hirayama, 1965b). In order to determine this relation in greater detail, the culture water was filtered through four filters which differed only in the depth of sand of the filter bed (Fig.5B). The time taken for the water passage through sand of the four filters was made equal by adjusting the filtering velocity of each filter. As shown in Fig.9, all of the OCF values obtained were almost the same, even though the depth of sand varied. This fact indicates that the time taken for the water to pass through the sand is correlated directly with the OCF and that the depth of the filter bed is only indirectly connected with the OCF.

TIME

TAKEN

PASSAGE

FOR

WATER

THROUGH

SAND

TIME PASSAGE

TAKEN

FOR THROUGH

WATER SAND

Fig.8. IRelation between OCF and the time taken for water passage through sand. The time taken for water passage through 12 cm of the sand used as 1 unit of time (T=l). Solid circles, observed OCF. Broken line, fitted curve of a first-order reaction; solid line, fitted curve of a second-order reaction.

In accordance with the observations described above, the relation of OCF y (mg/l) to the rate of filtration (filtering velocity) V (cm/min) and to the depth of sand D (cm) is shown by the following formula: 1 -=_ Y

1 co

+

V COZKZI

where Co and K are the constants representing the condition of the culture water. The relationship between OCF and grain size of the filter sand was also examined (Hirayama, 1966a). Purification in a filter is accomplished mainly by bacteria attached to the surface of the particles. It is assumed that the purifying capacity of a filter is closely connected with the area to which these bacteria can attach. The total surface area of all the grains in a filter containing a definite volume of sand is proportional to the reciprocal of the average grain diameter. One index is proposed as coefficient (G), which is expressed by the formula:

G = -+ X1 + 1

(Xl

+ x2

$

X2 2

+ . . ..X.=lOO)

+.. . . j& Xn n

378

?irn

23in

3icm

4&m+

i!‘hlD

DEPTM

Fig.9. Relation between OCF and the depth of sand layer, when the time taken for the water passage through the sand beds are made equal in four experimental filters. Solid circles, observed n days after the beginning of filtration where n = number alongside; empty circles, mean.

where mean grain size and weight percentage of each part of sand are shown by R and X, respectively. Thus, the grain-size coefficient (G) is 100 times the reciprocal number of the diameter (mm) of grain. On the assumption that K (constant) in the above equation can be expressed as CYG,eq.2 may be rewritten as. 1 -=co+ Y

1

V Co’aGD

(4)

To determine whether or not this assumption is correct, the following experiments were performed. By means of three experimental filters of the type shown in Fig.5A, with the sand differing only in grain size, the water of a closed system at a public aquarium was filtered with the same rate of speed and the OCF was measured at four different depths of sand in each filter. Fig.10 illustrates the results of two of these experiments, which involve the relation of the velocity constant (K) of the equation with which observed OCF values are fitted to the grain-size coefficient of the filter sand. As shown in Fig.10, the results support the assumption. In the case of a closed system with W (m’) of filter surface, the purifying

379

t

HO

AT 42nd DAY AFTER BEGINNING OF FILTRATION

20

40

IG)

60

80

100

HO

(G)

Fig.10. Relation between velocity constant (K) of the OCF reaction and grain-size coefficient (G) of filter sand. Broken line, relation between K and G; solid line, line estimated on the assumption that it passes through the 0 point.

capacity formula:

per minute,

shown by OCF Y (mg/min),

is expressed

by the following

10 WV

y =-

(5) -_1. CO

v Co’crGD

The average values of Co and (Y for the culture water in an aquarium with the 1ar:ge population of animals (the Suma Aquarium) were estimated to be 1.42 X 10m3 and 0.52 X 10d3, respectively. If these values represent the culture condition of a safe limit to keep animals healthy, maximum OCF (the possible purifying capacity) obtained by filtration of the culture water may be expressed as the following equation: 10 w

y =0.70 _V

+ 0.95 x lo3 GD

(6)

The depth of sand at which the degree of purification of culture water reaches half of the total attainable value in a sand filter 100 cm deep at various filtering velocities is calculated in Table I. In filters with smaller sand grains and slc’wer rates of filtration, purification occurs nearer the surface, most of the purification taking place in the upper layers of the filter bed.

380

TABLE

I

Depth of sand (in cm) at which the degree of purification of culture of the total attainable value, in a sand filter of 100 cm in depth Grain-size coefficient

10 20 40 80 160

Rate of filtration

(cm/min)

0.5

1.0

2.0

4.0

8.0

29 20 13 7 4

36 29 20 13 7

-~42 36 29 20 13

46 42 36 29 20

48 46 42 36 29

Degree of pollution

water attains

one-half

due to fish rearing

If aquatic animals are to be reared properly in a culturing system for prolonged periods, a balance must be established between the purification of the culture water by means of filtration and the polluting of the water as a result of the feeding and excretion of the animals. In such a system, the oxygen consumption during filtration (OCF) must correspond to the rate of pollution of the water. To demonstrate this, red sea breams (Chrysophrys major) were reared in an experimental aquarium (Hirayama, 196613). The OCF per min was measured as the rate of pollution, by culturing fish of different weights without feeding.

Fig.11. Relation between oxygen consumption during filtration (OCF), and the body weight of a fish kept in the experimental aquarium without feeding. Rate of filtration was maintained at 3.5 cm/min.

381

The relationship between OCF and the weight of a fish, kept without feeding, is shown in Fig.11 and the results can be summarized in the following formula: log L = 0.544 1ogB - 1.739

(7)

where .C and B represent OCF (mg/min) and body weight of fish (g), respectively. Further experiments in which the fish was fed showed that the load (OCF) on the filter, brought about by the metabolism of the fish, was reduced to about 55% of the load in the former case in which the fish was maintained without feeding. The load derived from eq.7 can be expressed as:

LX 0.55 = B".s44 X

10-2

(8)

In order to examine the rate of pollution that is associated with uneaten food, minced, Japanese mackerel flesh was introduced into an aquarium equipped with a well-conditioned filter, both with and without live fish being present. The changes in OCF were measured over the next 24 h. The mean OCF per min a (mgjmin) obtained with various weights of food F (g/day) is shown i.n Fig.12. The relationship between mean OCF and weight of food, no live fish. being present, can be represented by the following formula: a = 0.0908 F - 0.082

(9)

r

IO-

0

i 0

_L_L..__/__l~

_I

3

6 WEIGHT

OF

FOOD

_L_1+ 9

(Fg/doy)

Fig.12. Relation between amount of food per day and mean OCF value per minute. Solid circles, mean OCF value (a) when food remains in the water without being eaten by fish. Empty circles, mean OCF value (0’) when fish eat food. Mean value of d/a is 0.56.

382

As shown in Fig.12, the load (OCF), when the food was eaten by the fish, was 56% of the load when the mackerel flesh remained uneaten. Thus, the load by the food which was fed to fish can be expressed as: a X 0.56

= 0.051F

- 0.046

(IO)

When red sea breams are cultured at about 20°C including feeding with the flesh of Japanese mackerel, the load on the filter, expressed in OCF per minute X (mg/min), is described by the following formula: 4

x

=

c

(Bjo-4

x 10-z)

+ 0.051F

(11)

j=l

where Q is the number of fish. This indicates that the amount of pollution the culture water depends mainly on the amount of food given per day. Carrying capacity

of

of culture systems

Unless the pollution process exceeds the purification one, culture can succeed. The following equation for carrying capacity in an aquarium is derived from the rates of pollution and possible purification in a closed culture system or aquarium, obtained as mentioned above: 10 Wi 2 i=l

0.70 vi+

0.95 x lo3 Gi Di

5 j-i

(Bjo.544 x 10-z)

+ 0.051F

(12)

in which W, V, D, G and p represent the area of the filter (m2), rate of filtration (cm/min), depth of sand (cm), grain-size coefficient and number of filters respectively. If the above equation holds for a culture system, fish can be maintained in it in good condition for an indefinite period of time. DISCUSSION

The calculation of the carrying capacity of various aquaria by using the proposed equations is somewhat complicated, but Fig.13 and 14, which are derived from these equations, may be easily used to estimate the carrying capacity. For instance, in the case of culturing five red seabreams, three weighing 50 g each and two 100 g each, feeding at a rate of 3% of their total body weight per day, the rate of pollution (load on the filter) can be estimated from Fig.13 as 0.16 X 3 + 0.28 X 2 = 1.04 mg/min (in OCF). If the fish are maintained in an aquarium in which the filter sand consists of grains 2.5 mm in diameter and has an average depth of 80 cm and the rate of filtration is 2.0 cm/min, the capacity of purification of a square meter of filter area can be estimated from Fig.14 as 15 mg/min (in OCF). It may be supposed that a

383 -12% //_9%

20 0

FOOD

WEIGHT

BODY

10.0

/ DAY

WEIGHT

Fig.13. Rate of pollution brought about fed various amounts of food per day.

by the cultured

fish of different

VELOCITY

FILTERING

VELOCITY

icm/min)

50

40 30 20 10

50 DEPT-1

Fig.14.

OF

SAND

Purification

(err

)

capacity

DEPTH

OF

of a filter

ID0

SAND

(cm

under

)

various

conditions.

body

weights

384

filter area of 30 X 30 = 900 cm’ can support these fish easily, since the capacity of purification in this filter has been estimated as 1.38 mg/min (in OCF). If, however, the filter has an area of only 20 X 20 = 400 cm2, its purification capacity (0.62 mg/min, OCF) is too small to support them. If the filter sand is replaced with finer grains of 1.25 mm diameter and the filtering velocity is adjusted to 4 cm/min, such a filter can maintain the fish in good condition, because its purification capacity now reaches 1.23 mg/min (in OCF). This method of estimating carrying capacity suffers from some restrictions. For example, the purification has been assumed to occur only on the surface of the sand grains. Moreover, the experiments were performed with one species of aquatic animal fed with one kind of food and under a definite culturing condition (i.e., at about 20°C). Nevertheless, it may be said that at least as far as this equation holds true for a culture system, fish may be reared safely in it for long periods of time, unless they belong to species extremely difficult to keep in captivity. ACKNOWLEDGEMENTS

The author wishes to express his sincere thanks to Dr James W. Atz of the American Museum of Natural History and Dr Won Tack Yang of School of Marine and Atmospheric Sciences, University of Miami, for their kind reading of the manuscript and valuable criticism. REFERENCES Goldman, J.C., Tenore, K.R., Ryther, J.H. and Corwin, N., 1974. Inorganic nitrogen removal in a combined tertiary treatment-marine aquaculture system - I. Removal efficiencies. Water Res., 8: 45-54 Hirayama, K., 1960. On operation of filter of marine closed-system aquarium. Suisan Zoshoku, 8: 123-132 Hirayama, K., 1965a. Studies on water control by filtration through sand bed in a marine aquarium with closed circulating system - I. Oxygen consumption during filtration as an index in evaluating the degree of purification of breeding water. Bull. Jap. Sot. Sci. Fish., 31: 977-982 Hirayama, K., 1965b. Studies on water control by filtration through sand bed in a marine aquarium with closed circulating system - II. Relation of filtering velocity and depth of sand layer to purification of breeding water. Bull. Jap. Sot. Sci. Fish., 31: 983-990 Hirayama, K., 1966a. Studies on water control by filtration through sand bed in a marine aquarium with closed circulating system - III. Relation of grain size of filter sand to purification of breeding water. Bull. Jap. Sot. Sci. Fish., 32: 11-19 Hirayama, K., 196613. Studies on water control by filtration through sand bed in a marine aquarium with closed circulating system - IV. Rate of pollution of water by fish, and possible number and weight of fish kept in an aquarium. Bull. Jap. Sot. Sci. Fish., 32: 20-27 Hirayama, K., 1966c. Influences of nitrate accumulated in culturing water on Octopus uulgaris. Bull. Jap. Sot. Sci. Fish., 32: 105-111 Hirayama, K., 1970. Studies on water control by filtration through sand bed in a marine aquarium with closed circulating system - VI. Acidification of aquarium water. Bull. Jap. Sot. Sci. Fish., 36: 26-34

385

Hirayama, K., 1972. Circulatory system for rearing aquatic animals and related water quality control. Baioteku (Technol. Biol.), 3: 605-609 Kalle, K., 1966. The problem of the gelbstoff in the sea water. In: H. Barnes (Editor), Oceanogr. Marine Biol. Ann. Rev. Vol. 4. Allen and Unwin, London, pp.91-104 Kawai, A., Yoshida, Y. and Kimata, M., 1964. Biochemical studies on the bacteria in aquarium with circulating system - I. Change of the qualities of breeding water and bacterial population of the aquarium during fish cultivation. Bull. Jap. Sot. Sci. Fish., 30: 55-62 Kawai, A., Yoshida, Y. and Kimata, M., 1965. Biochemical studies on the bacteria in aquarium with circulating system - II. Nitrifying activity of the filter-sand. Bull. Jap. Sot. Sci. Fish., 31: 65-71 Liao, P.B. and Mayo, P.D., 1974. Intensified fish culture combining water reconditioning with pollution abatement. Aquaculture, 3: 61-85 Saeki, A., 1958. Studies on fish culture in the aquarium of closed-circulating system. Its fundamental theory and standard plan. Bull. Jap. Sot. Sci. Fish., 23: 684-695 Saeki, A., 1963. The composition and some chemical control of the sea water of the closed circulation aquarium. Bull. Mar. Biol. Stn Asamushi, Tohoku Univ., 11: 99-104 Shelbourne, J.E., Riley, J.D. and Thacker, G.T., 1963. Marine fish culture in Britain. 1. Plaice rearing in closed circulation at Lowestoft. J. Conseil, 28: 50-69 Siddal’, SE., 1974. Studies of closed marine culture systems. Pro. Fish-Culturist, 36: 8-14 Spotte,, S.H., 1971. Fish and Invertebrate Culture. Wiley-Interscience, New York, N.Y., P.8