Preliminary trials with an ultraviolet liquid sterilizer

Aquaculture, 14 (1978) 75-84 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

PRELIMINARY STERILIZER

TRIALS

WITH AN ULTRAVIOLET

75

LIQUID

EHUD SPANIER Institute of Evolution,

University of Haifa, Mount Carmel, Haifa (Israel)

(Received 26 October 1977;revised

25 January 1978)

ABSTRACT Spanier, E., 1978. Preliminary trials with an ultraviolet liquid sterilizer. Aquaculture, 14: 75- 84. The effects of a new ultraviolet liquid sterilizer were tested in fresh- and seawater aquaculture installations. Ultraviolet treatment reduced the population of aquatic microorganisms. The mortality rate of experimentally wounded fish kept in sea water treated with ultraviolet radiation was significantly lower than that of controls. A further comparison was made between ultraviolet and chemical treatment of wounded fiih. Ultraviolet radiation reduced bacterial population in a marine fiih hatchery, but the larval mortality did not decline, probably because of other environmental factors. The use of ultraviolet sterilizers in fiih culture is discussed.

INTRODUCTION

The germicidal qualities of ultraviolet radiation have been known and used for many years. However, because it can penetrate only a thin layer of liquid (Jagger, 1967), little use was made of ultraviolet light for the destruction of microorganisms in water. Recent technical improvements, which include an efficient arrangement for passing a very thin layer of liquid in close contact with the ultraviolet radiating source, now enables the use of this radiation for the sterilization of relatively large volumes of water. The potential importance of ultraviolet sterilizers in aquaculture has received little attention despite the considerable problems caused by bacterial diseases in fish culture (e.g. Papema et al., 1977). The present study attempts to test the efficiency of a small, compact ultraviolet apparatus in aquaculture installations. The first stage of the research was carried out in a closed freshwater system, and was designed to test the efficiency of the ultraviolet device in reducing the bacterial population in this medium. The second part of the study deals with the effect of ultraviolet treatment on the survival rates of experimentally wounded fish in running seawater. Finally, the possible use of ultraviolet light in rearing newly-hatched marine fish was studied.

76

MATERIALS

AND METHODS

All the experiments were done with the UVOXX UVP-150 S ultraviolet liquid sterilizer (now UVOXX model Tam&l, Yissum Company, Haifa, Israel). This compact unit (12 X 25 X 35 cm; 12 kg) utilizes four ultraviolet lamps with a maximum wavelength of 2500 to 2700 8. The maximum flow rate of this model is 60 l/min; the fluid to be sterilized enters its radiation chambers, spreads into a thin film-like flow, and thence passes between the lamp quartz jackets and the walls of the radiating chambers. These walls, made of stainless steel, are placed in close proximity to the jackets to maintain a film-like flow for a considerable distance around the periphery of the lamp. I. Freshwater investigations The experimental system (Fig. 1) consisted of two equal-sized (200 1 each) asbestos tanks (A and B). Freshwater flowed by gravitation from tank A to tank B through the UVOXX unit. The water was pumped back from tank B to A with a non-primer spiral pump (Flux, F-60 K). A flow rate of 5 l/mm was kept constant by a mercury-float-switch connected to the pump. A Gram-negative rod shaped motile bacterium was isolated from silver carp Hypophthalmichthys molitrix. (Investigations of the exact systematic definition of this bacterium are being carried out by the Laboratory for Research of Fish Diseases in Nir David in cooperation with the unit of Aquatic Pathobiology at the University of Sterling.) The appearance of haemorrhages under the skin, in the vicinity of the fins and the caudal peduncle, is a common phenomenon after handling this species. These haemorrhages develop into deep, open lesions with high concentrations of the bacterium mentioned above. This bacterium is the primary cause of death of cultured silver carp in

-_-_----_ TANK

Z‘.

B

Fig. 1. The experimental system (I, freshwater investigations) - not to scale.

77

Israel. It is estimated that the destruction of about 150 000 tons of fish in Israel in 1976 was due to this bacterium (S. Sarig, personal communication, 1977). The bacteria were introduced into the system, the medium of which was enriched (dilution 1 :lOO) with Nutrient Broth (Difco Laboratories). A similar system, with no ultraviolet unit but under the same conditions (nutrients, flow, oxygen, temperature, etc.), was used as a control. Both systems were covered to prevent penetration of dust and other foreign objects into the water. Two series of experiments were conducted, operating the UVOXX unit for five and seven hours, respectively. Calorimetric readings of samples from the experimental and control systems were taken with a Klett-Summerson Photoelectric Colorimeter approximately every hour. In two further experiments, red bacteria Serratia marcescens were introduced into the experimental system; 0.2 ml samples were taken each hour, diluted to the first, second and third order and plated on agar. Bacterial counts of each plate were made and the average number of bacteria per ml was calculated. II. Seawater investigations (a) Experimentally-wounded fish. Common mullets Liza ramada (60115 mm) were wounded by removing a few scales from each side of their bodies with a scalpel. Asbestos tanks (65 X 60 X 75 cm, volume 230 1) were thoroughly cleansed of dirt and algae, and were filled with a concentrated solution of potassium permanganate in freshwater for 24 hours for the purpose of disinfection. Following this procedure, the tanks were supplied with running seawater and fish were introduced. Water flow and temperature were recorded daily. The fish were fed on an artificial diet containing 42% protein and 8% fat. The tanks were checked every day and dead fish were removed immediately with sterile nets. After the termination of each experiment, the surviving fish were transferred to plastic tanks with running water (no ultraviolet treatment) and the mortality rate was recorded for at least one additional month. In experiments Nos II 1, II 2, and II 3, wounded fish were introduced into tanks containing water treated with ultraviolet radiation. An equal number of experimentally-wounded fish served as a control: the latter were placed in water at the same temperature and flow rate, but with no ultraviolet treatment. In experiment No. II 4, an additional group of experimentally-wounded fish was dipped in 1% solution of nitrofurazone before being placed in the tank with untreated sea water (as in the control tank). Mortality rate for each of the three tanks was recorded daily. (b) Marine fish larvae. The ultraviolet sterilizer was used for treating the water in a tank containing newly hatched larvae of Sparus aurata. A very

78

high larval mortality of up to 99.9% was normally observed during the first 6-8 weeks of life. It was suspected that this mortality was due to microorganisms. Water from a cone-shaped tank containing newly-hatched larvae was pumped through a 45 pm filter to the intake pipe of the UVOXX unit and returned to the same tank after being exposed to ultraviolet radiation at a flow rate of 500 to 1000 ml/min. A similar system with no ultraviolet treatment served as a control. Water samples were taken with sterile pipettes at intervals from the test and the control tanks, and spread on Agar plates (75% seawater, 0.3% yeast extract, 0.5% peptone). It was not possible to collect bodies of dead larvae at the bottom of the tanks, since most of them had disintegrated. However, of the surviving larvae, most were concentrated just below the surface of the water. This enabled an estimate of the living larvae to be made by counting them in sections of the test and control tanks. In all experiments in seawater, Chi-square tests for statistical analysis (Maxwell, 1964) were used. RESULTS

I. Freshwater investigations The results of the calorimetric measurements are illustrated in Fig. 2. In

2

6

8

10

12 14 16 TIME Chrs)

18

20

22

24

Growth curves of bacteria in freshwater radiated with ultraviolet light, solid lines; in untreated freshwater, broken line; K.U., Klett Units. A, first series; B, second series.

Fig. 2.

79

both series a decline in Klett Units was observed after radiation. The Klett Unit (k.u.) expresses the relative abundance of microorganisms in the media, based on turbidity. Readings of the test samples speedily reached the 5 k.u. level. At the same time, there was a significantly higher reading of the control samples, which ranged above the 20 k.u. level. The later drop in k.u. level of the control was probably due to a decrease in the nutrient consumed by the growing bacterial population. The results of the bacterial counts (red bacteria) are presented in Table I. These results clearly demonstrate a lowering in number of bacteria within a given volume after relatively short exposure to ultraviolet radiation. TABLE I Numbers of red bacteria Serratia marcescens Time after the beginning of the radiation (h)

0

1 2 3 4 5 6 I 8 9 10 11

surviving ultraviolet radiation

Average count per 1 ml sample 1st series

2nd series

1265 x lo3

1490 66 34 49 83 38

83 x lo3 27 x lo*

x x x x

103 103 103 lo2

55 x lo2 130 75 10

II. Seawater investigations (a) Experimentally-wounded fish. The environmental and final mortality data of each of the four experiments (Nos II l-4) are presented in Table II. In these experiments a significant difference (P <0.05) between the control and the test was observed. While fish mortality in the test was low, under control conditions it was much higher. In all experiments the remaining fish survived at least one additional month without further ultraviolet treatment. The mortality rates of fish in the various treatments are illustrated in Fig. 3. It is obvious that in all experiments most mortalities occurred during the first three or four days after wounding. In experiment No. II 4, fish dipped in nitrofurazone had a lower mortality rate than the control, but this rate was significantly higher than that of fish treated with ultraviolet radiation.

80

TABLE II Final mortality data of experimentally-wounded mullets Liza ramnda (Fish kept in seawater treated with ultraviolet radiation, T; fiih kept in untreated seawater, C; fish treated with 1% nitrofurazone and kept in untreated seawater, N) Experiment

No.

C

T

C

T

C

3 24

2

2

2

2

93

88

83

79

81

16

33

33

40

40

40

5

30

3

40

21

33

3

30

0

19

25 1

1

1

Average size (TL in mm)

96

89

Sample size

25

Died

24 1

16

Survived

4

5

25.5

25.5

1

2

81

85

25

16

9

14 2

11

P
p-co.05

N 5

5 25

X2

T

24

4 25

Flow rate (l/min)

C 4

4 25

Average temp. (“C)

T

24

5

Tank No.

II 4

II 3

II 2

II 1

P
P
7 P
P
(b) Marine fish larvae. The results of bacterial counts of water samples taken from the test and control tanks are illustrated in Fig. 4. A gradual decline in bacterial population was evident in the test tank, while the viable count of samples in the control was higher than that of the starting values. However, there was no significant difference between the mortality rates of larvae in the two tanks; less than 1% of the larvae survived after 52 days, regardless of treatment (Table III). When the ultraviolet device was turned off, the bacterial counts of water samples taken from the test tank reached the original high level within less than four hours (Fig. 4). DISCUSSION

The results of the first two stages of the study point to the effective action of ultraviolet treatment in reducing populations of microorganisms in fresh- and seawater. This is true both for the closed and open circulating water systems. S. Ulitzur (personal communication, 1977) has tested the killing efficiency of the same UVOXX device for certain microorganisms under controlled laboratory conditions (Escherichiu coli, Staphylococcus uureus, T4 phage, spores of Bacillus cereus T and Succharomyces cereuisiue) representing various taxonomic groups (Gram-negative bacteria, Gram-positive bacteria, viruses, spores and fungi, respectively). His findings indicated a high killing rate (over 99.9%) for all the species tested at flow rates in the

81

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32

Experiment No;-3

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8 4

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5

6

7

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Days after the beginning of the experiment

Fig. 3. Mortality rates of mullets Liza ramada plotted against the time of the experiments Fish kept in seawater radiated with ultraviolet rays, solid lines; fish kept in untreated seawater, broken lines: fiih treated with 1% nitrofurazone and kept in untreated seawater, dotted lines.

,,J , , , ,’ , 0

2

4

6

8 l0 14 16 TIME (hrs)

!.v;Of!

18

20

(

,

22

24

Fig. 4. Bacterial counts of water sampled from tanks containing larvae of Sparus aurata. Solid lines, ultraviolet treatment; broken line, no ultraviolet treatment.

82

TABLE III Number of larvae of Sparus uumta which survived in water treated with ultraviolet (test) and in untreated seawater (control) at various intervals after hatching Days after hatching

Test Temp.

Salinity

(“C)

(“I,,)

22.2

25

22.6

25.5

24.2

30

24

29

24.8

32

26

27

25

29

25.4

33

50000 1

22.4

26

22.4

26.5

23.9

29

24.4

29

24.6

31

25.8

27.5

26

29.5

26.2

34

N.S.

N.S.

N.S.

N.S.

N.S.

110

53 40

(“Ill,)

350

150 30

(“C)

600

300 20

Salinity

950

500 12

Temp.

3400

750 10

Estimated No. of surviving larvae 55000

4000 7

N.S.

73

38 52

X2

Control

Estimated No. of surviving larvae

light

N.S.

55

N.S.

order of magnitude used in the present study. This level of killing efficiency was measured for running tap water, and a recirculating system could provide an even higher killing efficiency. The above taxonomic groups contain known fish pathogens which frequently attack weak and wounded fish. Adult fishes may be sensitive to microparasite infection when wounded during handling procedures. In these. circumstances, a relatively short retention in water treated with ultraviolet light would appear to prevent infection, as can be inferred from the results of stage II a. Most fish casualties occurred during the first few days after wounding; all recovering mullets survived thereafter in water which had not been treated with ultraviolet radiation. It is recommended, therefore, that water be exposed to ultraviolet light for only a few days after wounding occurs, thus further reducing the cost of treatment. Experiment No. II 4 was carried out in order to compare the effectiveness of the ultraviolet treatment with that of conventional chemical treatment.

33

Nitrofurazone (5-nitro-2-furaldehyde semicarbazone) and furanace had been found to be effective chemotherapeutics for fish (Kubota and Hagita, 1963; Pearse et al., 1974). A higher mortality rate was observed in fish treated with nitrofurazone than that of fish in water treated with ultraviolet radiation (Table II). These results, however, cannot be considered as clear-cut proof that radiation offers a better treatment than nitrofurazone, since the mortality rate of fish treated with this chemical may be due partially to overdose (although the chemical was diluted ten times more than the concentration recommended in the relevant literature in view of the small size of the mullets). Nevertheless, it would seem that ultraviolet radiation can be at least as effective as nitrofurazone in preventing infection in injured fish. In the light of recent suspicions regarding the carcinogenic nature of various chemicals, ultraviolet radiation of water may be considered a preferable treatment when fish for human consumption are involved. The UVOXX sterlizer is simple to operate and requires minimum maintenance; hence it is suitable not only for large and sophisticated institutes, but also for small units and remote farms and hatcheries. The efficiency of radiation treatment can be reduced by particulate matter suspended in the water. In this case, the combined use of filtration and ultraviolet light is recommended for effective sterilization (Bullock and Stuckey, 1977). The larval stages of commercial fishes are often more vulnerable to attack by microorganisms than the adult. In the present study, ultraviolet treatment did not reduce the mortality of the larvae (Table III), though it significantly diminished the bacterial population (Fig. 4). It is possible that in this case additional factors such as physical conditions influence larval survival, although other studies (e.g. Klontz, 1973; Can-eon et al., 1976) indicate a high percentage of fish mortality in early stages due to microorganisms. The loss of fish per unit volume of water is considerable owing to the small size of the young fish concentrated in a relatively small amount of liquid. On the other hand, the limited quantities of water involved make it possible to treat the entire medium continuously with ultraviolet radiation at very low cost. ACKNOWLEDGEMENTS

Thanks are expressed to M. Lahav and I. Bejarano of the Laboratory for Research of Fish Diseases, Nir David, for their valuable assistance in running the experiments in freshwater. Appreciation is due also to H. Gordin of the Mariculture Laboratory, Israel Oceanographic and Limnological Research, Elat, and to I. Paperna of the H. Steinitz Marine Biology Laboratory, Elat, as well as other members of these two institutes, for their cooperation in conducting the seawater experiments. This study was supported by the Research Authority of the University of Haifa, and in part by a Tobias Landau Fellowship from the Department of Oceanography at the Hebrew University, Jerusalem.

84

REFERENCES Bullock, G.L. and Stuckey, H.M., 1977. Ultraviolet treatment of water for destruction of five Gram-negative bacteria pathogenic to fishes. A report of the U.S. Department of the Interior, Fish and Wildlife Service. Eastern Fish Disease Laboratory, Kearneysville, West Virginia, 27 pp. Carreon, J.A., Estocapio, F.A. and Enderez, E.M., 1976. Recommended procedures for induced spawning and fingerling production of Clarias macrocephalus Gunther. Aquaculture, 8 : 269-281. Klontz, G.W., 1973. A survey of fiih health management in Idaho. College of Forestry, Wildlife and Range Sciences, University of Idaho, Information Series No. 3, pp. l-34. Kubota, S.S. and Hagita, K., 1963. Studies on the diseases of marine cultured fishes. II. Pharmacodynamic effects of nitrofurazone for fish diseases. J. Fat. Fish. Prefect. Univ. Mie, 6: 125-144. Jagger, J., 1967. Introduction to Research in Ultraviolet Photobiology. Prentice-Hall Inc., N.J., 164 pp. Maxwell, A.E., 1964. Analysing qualitative data. John Wiley and Sons Inc., N-Y., 163 pp. Paperna, I., Colomi, A., Gordin, H. and Kissil, G.W., 1977. Diseases of Sparus aurata in marine culture at Elat. Aquaculture, 10: 195-213. Pearse, L., Pullin, R.S.V., Conroy, D.A. and McGregor, D., 1974. Observation on the use of furanace for the control of vibrio diseases in marine flatfishes. Aquaculture, 3 : 295-302.