Surface disinfection of Atlantic halibut and turbot eggs with glutaraldehyde: evaluation of concentrations and contact times

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1 Aquaculture International 5, Surface disinfection of Atlantic halibut and turbot eggs with glutaraldehyde: evaluation of concentrations and contact times I. Salvesen*, G. Øie and O. Vadstein SINTEF Applied Chemistry, Center of Aquaculture, N 7034 Trondheim, Norway To evaluate the effects of glutaraldehyde treatment at different disinfection temperatures, Atlantic halibut, Hippoglossus hippoglossus (L.), and turbot, Scophthalmus maximus (L.), eggs, incubated at 5 and 12 C, respectively, were disinfected with mg glutaraldehyde l 1 at three different contact times (2.5, 5 and 10 min). Egg batches of both poor and good quality were tested for halibut. Positive effects were more pronounced in poor than in good-quality batches at hatching. Egg disinfection had a highly positive effect on the viability of yolk-sac larvae in both types of batches. A level between 400 and 800 mg l 1 at a contact time of 5 10 min was optimal for halibut: at lower levels, the bactericidal effect was reduced, and at higher levels, there were indications of toxic effects. Halibut eggs disinfected with optimal doses of glutaraldehyde had furthermore a reduced hatching time and more synchronous hatching as compared with untreated eggs. Turbot was more sensitive to higher doses than halibut, and the best larval performance was obtained for mg glutaraldehyde l 1 at a contact time of 2.5 min. A further evaluation should, however, be performed before recommendations are given for species incubated at temperatures higher than 5 C. KEYWORDS: Atlantic halibut (Hippoglossus hippoglossus (L.)), Egg quality, Glutaraldehyde, Marine fish eggs, Surface disinfection, Turbot (Scophthalmus maximus (L.)). INTRODUCTION Different procedures are currently used to disinfect fish eggs to prevent transmission of diseases and to reduce problems with bacterial overgrowth in intensive egg incubation systems. In freshwater, iodophores have been recommended for surface disinfection of many species (McFadden, 1969; Schachte, 1979; Subasinghe and Sommerville, 1985). These buffered solutions of iodophores were, however, originally designed to be used in freshwater and recommended doses are not necessarily effective in seawater. Earlier studies with plaice, Pleuronectes platessa L., cod, Gadus morhua L., and Atlantic halibut, Hippoglossus hippoglossus (L.), have indicated that glutaraldehyde has a potential as a surface disinfectant for marine fish eggs (Vadstein et al., 1993; Harboe et al., 1994; Salvesen and Vadstein, 1995). Glutaraldehyde, which is called a chemosterilizer (Borick, 1968), is an attractive candidate for disinfection because of its broad spectrum of activity, its rapid *Author to whom correspondence should be addressed ( Ingrid. Salvesen@chem.sintef.no) Chapman & Hall

2 250 I. Salvesen et al. antimicrobial action and its high activity in the presence of organic matter (Block, 1977; Gorman et al., 1980). This paper reports further results on surface disinfection with the most promising doses of glutaraldehyde, using halibut and turbot incubated at different temperatures and egg batches of both poor and good quality to evaluate the effects of treatment. MATERIALS AND METHODS Disinfection procedure Disinfection solutions were prepared with filtered (0.2 m), autoclaved seawater and a buffered 25% solution of glutaraldehyde (Janssen Chimica). Concentrations refer to theoretical values and assume no consumption of the disinfectant by substances in the water. No attempt was made to evaluate this potential error. All treatment was done in darkness under a dim light source. Control groups received the same physical handling as treated groups. Both small-scale experiments with Atlantic halibut and turbot, Scophthalmus maximus (L.), and a larger-scale experiment with halibut were carried out. Before treatment the eggs were collected on a nylon mesh and washed with sterile, filtered seawater to remove organic materials. Eggs were thereafter transferred to the disinfection solution maintained in a beaker (1:10, by volume) in small-scale experiments and in a plastic bucket (1:40, by volume) in the larger-scale experiment. During disinfection, eggs were gently agitated every second minute to keep a constant concentration of the disinfectant around the eggs. After treatment, eggs in small-scale experiments were rinsed on the nylon mesh and washed 3 1 min in a beaker with sterile, filtered seawater (1:10, by volume). In the larger-scale experiment with halibut, eggs were collected on the nylon mesh before being transferred to upstream incubators (50 l) supplied with filtered seawater (0.2 m) at a dilution rate of 6 day 1. This entailed a more than 250 times dilution of the disinfectant and no washing procedure was necessary. The bactericidal effect of small-scale experiments was evaluated by an agar assay (Salvesen and Vadstein, 1995). Briefly, turbot eggs and halibut eggs were kept in sterile Petri dishes on ice after disinfection, and single eggs (turbot, n 57 60; halibut, n 40 60) were within 1 h incubated separately in M-65 seawater agar (0.5 g yeast extract (Oxoid), 0.5 g tryptone (Oxoid), 0.5 g peptone (Oxoid), 10 g agar (Difco), 800 ml aged, filtered seawater and 200 ml distilled water). The plates were incubated for 7 days at 10 C, and the eggs in the agar were characterized daily as being with or without bacterial growth. Halibut experiments Small-scale experiments with halibut were performed 4 7 days before hatching with egg batches of both poor (batch A and B) and good quality (batch C and D). Eggs were delivered from several hatcheries in Norway and batches were characterized as poor or good on arrival at the SINTEF Center of Aquaculture on the basis of characteristics such as transparency, buoyancy and mortality after transport. Eggs

3 Surface disinfection of eggs with glutaraldehyde 251 were treated for 2.5, 5 and 10 min with 400 and 800 mg glutaraldehyde l 1 at a disinfection temperature of 5 C. In batches B and D, 1200 mg l 1 was also included as a test concentration. After treatment, two-to-three replicates of eggs were transferred to 9 cm Petri dishes with ml of sterile, filtered seawater and incubated in darkness at 4 6 C. The hatchability was calculated as the number of hatched larvae divided by the total number of incubated eggs. This ratio integrates both the mortality of eggs and the inability of living eggs to hatch. After hatching was completed, yolk-sac larvae were transferred to new Petri dishes (n 2 3) with sterile seawater. No antibiotics were added to the water. Survival of the larvae during the yolk-sac period was recorded until day after hatching. In the larger-scale experiment, an egg batch of good quality was split in five, and two of the groups were disinfected 5 days before hatching. Eggs were treated for 10 min with 400 mg glutaraldehyde l 1 at 5 C. Egg mortality and hatchability were used as response parameters. After hatching was completed, 200 larvae were transferred to 3 l glass bowls (10 bowls for each of the five groups) with filtered seawater, and survival was recorded throughout the yolk-sac period. The temperature was 6 C during the experiment. Dead larvae were removed every week. Turbot experiment A small-scale experiment with turbot was carried out 2 days before hatching. Eggs were treated at 12 C for 2.5, 5 and 10 min with 400, 800 and 1200 mg glutaraldehyde l 1. After treatment, eggs were transferred to 250 ml Erlenmeyer flasks (n 2) with 150 ml of sterile, filtered seawater. Flasks were thereafter incubated under dim light to observe hatchability and survival until the end of the yolk-sac period. Within 24 h after hatching, temperature was gradually increased to 18 C. Dead eggs and larvae and approximately 25 ml of water were siphoned from the flasks daily until the day after hatching was completed. At the end of the yolk-sac period (day 3 after hatching), the stress tolerance of the larvae was tested by exposure to high salinity and to physical handling. At the most intense doses there were no larvae alive at this point, and a stress evaluation was therefore only performed for untreated groups and for groups disinfected with 400 and 800 mg glutaraldehyde l 1 for 2.5 and 5 min. A glass tube with an inner diameter of 6 mm was used to transfer the larvae to the test flasks. In the salinity test, larvae were exposed to concentrated seawater (50, 18 C, 100 ml), and time to 50% mortality was recorded. Observations were made every 15 min. In the physical handling test, larvae were placed in 250 ml Erlenmeyer flasks with seawater (34, C, 150 ml) on a CH-4103 Bottmingen shaking machine and shaken at 130 rotations per minute. Survival was recorded after 15 min of shaking. Statistical methods Binomial data on bactericidal effects were tested for differences in a chi-square test. Data on hatchability and survival were arcsin transformed to correct for deviations from normality before a one-way analysis of variance. In case of inhomogeneity, the Tukey test was applied for multiple comparison of means (Zar, 1984). A significance level of 5% was used in all tests.

4 252 I. Salvesen et al. RESULTS Small-scale experiments with halibut and turbot The fraction of eggs with bacterial growth after 7 days incubation in agar was used to evaluate the bactericidal effect of the treatments. In untreated groups, a bacterial growth zone was visible around all eggs as early as after 1 3 days of agar incubation (data not shown). The percentage of eggs with bacterial growth was significantly lower for treated groups at the end of the incubation period, with two exceptions only (0.001 p 0.05). The bactericidal effect of glutaraldehyde treatments, however, varied considerably between the four different batches of halibut (Table 1). Increasing contact time and/or concentration improved the effect of most treatments, i.e. whereas more than 80% of the eggs were characterized as surface sterile in eight out of ten treatments at a contact time of 10 min, the same bactericidal effect was only obtained in three treatments at the shortest contact time of 2.5 min. For turbot eggs, which were disinfected at a higher temperature, the bactericidal effect was good at all doses tested, and the fraction of eggs that were characterized as surface sterile varied between 95% and 100%. The amount of eggs that hatched was considerably higher for disinfected than untreated groups in poor-quality batches of halibut. In batch A, 10 min disinfection with 800 mg glutaraldehyde l 1 was the only treatment that was found to have a significant effect on the hatchability (p 0.05, Fig. 1), whereas all treatments in batch B increased the yield of larvae (p ). No larvae from the untreated groups survived throughout the yolk-sac period. The effect of surface disinfection upon survival at this stage varied between the two poor-quality batches. In batch A, live larvae were observed only in some of the disinfected groups after 4 weeks of incubation, whereas survival at higher doses in batch B was relatively good and varied between 30% and 68% at day 25 after hatching. Due to a considerable TABLE 1. Per cent eggs with bacterial growth (n 40 60) after treatment with different doses of glutaraldehyde. Temperature during disinfection was 5 C for halibut and 12 C for turbot. n.a., not available Halibut Dose Poor Good mg l 1 min A B C D Turbot Control n.a. 28 n.a n.a. 14 n.a n.a. 8 n.a. 20 0

5 Surface disinfection of eggs with glutaraldehyde 253 variation between replicates, disinfection with the most intense dose, i.e mg glutaraldehyde l 1 for 10 min, was, however, the only treatment that was found to have a significant positive effect (p 0.05). No difference in hatchability was observed between treated and untreated groups in halibut batches of good quality, with the exception of the most intense treatment in batch D, which had a negative effect on hatching (p 0.01, Fig. 2). A delayed hatching gave further indications of toxic effects at the four most intense treatments (i.e mg l 1 at all contact times and 800 mg l 1 for 10 min). Positive effects from surface disinfection of the egg surface were more pronounced during the yolk-sac period. In batch C, the best larval performance was obtained in groups treated at a contact time of 10 min. In these groups, a high survival was correlated to good reproducibility, and more than 85% of the larvae survived until day 28 after hatching. As compared with the untreated group, which had a considerable variation in survival between replicates, none of these differences were, however, found to be significant. In batch D, only 5% of the larvae in untreated groups survived throughout the yolk-sac period. Survival was significantly higher for all treated groups and averaged 67 95% at day 25 after hatching (0.001 p 0.05). For turbot, the variation in hatchability and survival between treatments was significant and clearly related to doses (Fig. 3). At all concentrations tested, a contact time of 10 min was highly toxic to turbot at this stage (p 0.001). Negative effects on hatching and survival were also observed after 5 min treatments with 800 mg glutaraldehyde l 1 and 2.5 min treatment with 1200 mg glutaraldehyde l 1 (p 0.01). The low survival was not due to an acute lethal response to disinfection, which was indicated by the fact that more than 85% of the eggs were still alive at hatching, i.e. 2 days after disinfection (data not shown). The reduction in survival was attributed to a high mortality of unhatched eggs and/or incompletely hatched larvae (only the tail or head protruding from the eggshell) after hatching was completed in the other groups. In contrast, more than 95% of the eggs hatched at lower doses (400 mg l 1 for 2.5 and 5 min and 800 mg l 1 for 2.5 min). These larvae were active with no observable morphological abnormalities and had a high survival of 87 93% throughout the yolk-sac period. The improved performance of these larvae was further confirmed in the stress tests, where larvae from groups treated for the shortest contact time (2.5 min) had the best resistance to stress (Fig. 4). Large-scale experiment with halibut A low mortality of % was observed for both groups from disinfection until hatching. Just before hatching, more than 95% of the disinfected eggs were observed in the upper part of the incubator. The untreated eggs were, in contrast, heavier and dispersed further down the water column, but at hatching, eggs and larvae from untreated groups also appeared at the surface. Both groups had a high fraction of eggs that hatched, but the period from when hatching had started (5% hatch) until it was completed (95% hatch) differed between the two groups (Fig. 5). The interval between 5% and 95% hatch was approximately five times as long in the untreated groups, i.e. 59 h as compared with 12 h in the disinfected groups. After hatching survival was high for both the disinfected and untreated groups and more

6 254 I. Salvesen et al. FIG. 1. Per cent hatch and survival until day after hatching in two halibut batches of poor quality (filled symbols: batch A; open symbols: batch B). Concentrations of glutaraldehyde are indicated above the figure and contact times (min) below. Values for two-to-five replicates are plotted for each treatment. FIG. 2. Per cent hatch and survival until day after hatching in two halibut batches of good quality (filled symbols: batch C; open symbols: batch D). Concentrations of glutaraldehyde are indicated above the figure and contact times (min) below. Values for three-to-nine replicates are plotted for each treatment.

7 Surface disinfection of eggs with glutaraldehyde 255 FIG. 3. Per cent hatch and survival throughout the yolk-sac period (i.e. day 3 after hatching) of turbot. Concentrations of glutaraldehyde are indicated above the figure and contact times (min) below. Values for two replicates are plotted for each treatment. than 60% of the larvae survived throughout the yolk-sac period (data not shown). DISCUSSION One of the most obvious reasons why surface disinfection might be a measure to improve egg quality is that it reduces the probability of development of pathogens. Low viability of eggs and larvae is, however, not always related to the presence of FIG. 4. Evaluation of stress resistance of turbot larvae at day 3 after hatching. Eggs had earlier been treated for 2.5 min ( ) and 5 min ( ) with 400 and 800 mg glutaraldehyde l 1.

8 256 I. Salvesen et al. FIG. 5. Time to hatch in untreated (filled symbols) and treated groups (open symbols) of halibut. Treated groups were disinfected with 400 mg glutaraldehyde l 1 for 10 min. pathogens. Proliferation of excessive amounts of bacterial cells on the egg surface has been documented (Salvesen and Vadstein, 1995) and may cause problems by reducing the exchange of gases and metabolic waste between the embryo and the environment. Improved viability through surface disinfection may thus be achieved, not only by the control of pathogen transfer, but by a general reduction in bacterial colonization (Vadstein et al., 1993). The bactericidal effect of identical disinfection conditions has earlier been shown to vary between different batches and to be correlated to the initial bacterial load of the egg batch (Salvesen and Vadstein, 1995). Although no attempt was made to evaluate this in the present study, differences in initial colonization may explain the large variation in bactericidal effect between different egg batches of halibut. There was no clear relationship between the fraction of surface-sterile eggs and larval viability at a later stage. However, compared with larval performance in untreated groups, disinfection had a highly positive effect. Surface sterility is furthermore a strict criterion to use when evaluating the bactericidal effect of disinfection, which by definition is a process that is intended to kill or remove pathogenic microorganisms, and not to kill all types of microorganisms as in the process of sterilization (Gardner and Peel, 1986). For plaice, positive effects on egg survival were not seen after 10 min treatments with 1600 mg glutaraldehyde l 1, but tested batches were all of good quality with a more than 95% survival until hatching (Salvesen and Vadstein, 1995). Also for halibut, positive effects were not so pronounced at hatching in egg batches of good as of poor quality. The higher viability of treated groups during the yolk-sac period indicates, however, that surface disinfection may be a measure for quality improvement in batches of good as well as of poor quality, although disinfection may not be successful in all egg batches of poor quality. Treatment with higher doses of glutaraldehyde may improve the larval yield in some poor-quality batches, but at these higher levels there were indications of toxic effects. Further tests in the previous study with plaice indicated better larval viability for concentrations 800

9 Surface disinfection of eggs with glutaraldehyde 257 mg glutaraldehyde l 1 at a contact time of 10 min. A concentration between 400 and 800 mg glutaraldehyde l 1 at a contact time of 5 10 min seems to apply well for halibut also. Lower or higher levels are not recommended, because of a reduction in the bactericidal effect at shorter contact times and indications of toxic effect at higher concentrations. Glutaraldehyde solutions interact with proteins by forming intra- and intermolecular bridges (Block, 1977; Gorman et al., 1980), and treatments with higher doses of glutaraldehyde (1600 mg l 1 for 10 min) have earlier been shown to increase the hardness of the eggshell (Salvesen and Vadstein, 1995). This increase in egg hardness might be a reason for the delayed hatching of halibut at the most intense treatments in the present study. In the larger-scale experiment with halibut, a more synchronous and reduced hatching period was obtained after treatment with a lower glutaraldehyde dose (400 mg l 1 for 10 min). If the larvae are to be transferred to yolk-sac incubators after hatching, a reduced time interval between 5% and 95% hatch is an advantage, from a practical point of view, because it facilitates the transfer of larvae. Furthermore, the high amounts of organic matter that are released during hatching give a significant increase in bacterial growth, and a shorter hatching period will reduce the time that the larvae spend in this poor-quality water. In untreated groups, massive overgrowth of obligate aerobic bacteria on the egg surface could, by consuming much of the available oxygen, create hypoxic conditions for the embryo, which for halibut may delay hatching. Further positive effects of disinfection were not revealed throughout the yolk-sac period in this large-scale experiment. An earlier study with Atlantic halibut has, however, shown that such effects might first be manifested at the first-feeding stage (Harboe et al., 1994). The sensitivity to the more intense doses of glutaraldehyde was higher for turbot than for halibut, but this effect might rather be explained by the difference in temperature during disinfection than by a difference in sensitivity between species. The activity of aldehydes is highly affected by temperature, i.e. a 10 C increase in temperature effects a fourfold reduction in the time to kill (Gardner and Peel, 1986). For turbot, the best larval performance was obtained for concentrations of mg l 1 at 2.5 min contact time. The results showed furthermore the importance of an evaluation of larval stress resistance before any doses are recommended. Such tests might reveal differences in larval quality that are not expressed as mortality during early larval stages. RECOMMENDATIONS 1. Surface disinfection of Atlantic halibut eggs with doses of glutaraldehyde between 400 and 800 mg l 1 for 5 10 min may improve hatchability and larval survival in egg batches of both poor and good quality. 2. A further evaluation of contact time and concentrations should, however, be performed before recommendations are given for species incubated at temperatures higher than 4 6 C.

10 258 I. Salvesen et al. ACKNOWLEDGEMENTS This study was supported by the Norwegian Research Council (formerly the Royal Norwegian Council for Scientific and Industrial Research, NTNF), FINA exploration Norway and Hydro Seafood. REFERENCES Block, S.S. (1977) Disinfection, Sterilization and Preservation (2nd edition). Leax Febiger: Philadelphia, 1049 pp. Borick, P.M. (1968) Chemical sterilizers (chemosterilizers). Advances in Applied Microbiology 10, Gardner, J.F. and Peel, M.M. (1986) Introduction to Sterilization and Disinfection. Churchill Livingstone: Melbourne, 183 pp. Gorman, S.P., Scott, E.M. and Russel, A.D. (1980) Antimicrobial activity, uses and mechanism of action of glutaraldehyde. Journal of Applied Bacteriology 48, Harboe, T., Huse, I. and Øie, G. (1994) Effects of egg disinfection on yolk sac and first feeding stages of halibut (Hippoglossus hippoglossus L.) larvae. Aquaculture 119, McFadden, T.W. (1969) Effective disinfection of trout eggs to prevent egg transmission of Aeromonas liquefaciens. Journal of the Fisheries Research Board of Canada 26, Salvesen, I. and Vadstein, O. (1995) Surface disinfection of eggs from marine fish: evaluation of four chemicals. Aquaculture International 3, Schachte, J.H. (1979) Iodophor disinfection of muskellunge eggs under intensive culture in hatcheries. The Progressive Fish-Culturist 41, Subasinghe, R.P. and Sommerville, C. (1985) Disinfection of Oreochromis mossambicus (Peters) eggs against commonly occurring potentially pathogenic bacteria and fungi under artificial hatchery conditions. Aquaculture and Fisheries Management 16, Vadstein, O., Øie, G., Olsen, Y., Salvesen, I. and Skjermo, J. (1993) A strategy to obtain microbial control during larval development of marine fish. In: Fish Farming Technology Proceedings of the First International Conference on Fish Farming Technology (eds H. Reinertsen, L.A. Dahle, L. Jørgensen and K. Tvinnereim) Balkema: Rotterdam, pp Zar, H.J. (1984) Biostatistical Analysis (2nd edition). Prentice Hall International Editions: Englewood Cliffs, New Jersey, 718 pp.