Report of the Working Group on Marine Fish Culture (WGMAFC) Doccuments/2004/F/WGMAFC04.pdf ·...

ICES Mariculture Committee ICES CM 2004/F:03 Ref. ACME, I

Report of the Working Group on Marine Fish Culture (WGMAFC)

27–29 April 2004 Vigo, Spain

This report is not to be quoted without prior consultation with the General Secretary. The document is a report of an Expert Group under the auspices of the International Council for the Exploration of the Sea and does not necessarily represent the views of the Council.

The WGMAFC participants at the meeting in Vigo.

International Council for the Exploration of the Sea

Conseil International pour l’Exploration de la Mer

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Contents

1 Participants................................................................................................................................................................. 3 2 Terms of reference ..................................................................................................................................................... 5 3 Introduction ................................................................................................................................................................ 5 4 Activities of the WGMAFC ....................................................................................................................................... 6

4.1 Regulations regarding aquaculture.................................................................................................................. 6 4.2 Feeds in aquaculture ....................................................................................................................................... 6 4.3 Fish produced for other purposes than direct consumption............................................................................. 7 4.4 Production technology .................................................................................................................................... 8

5 Conclusions .............................................................................................................................................................. 22 6 Recommendations and meeting resolution............................................................................................................... 22 7 Annexes.................................................................................................................................................................... 24

Annex 1 List of the participants at the 2004 WGMAFC meeting in Vigo. ...................................................... 24 Annex 2 List of WGMAFC members .............................................................................................................. 25 Annex 3 Research and Technology of Single-sex Fish Production and Application to Marine Fish Culture.. 28 Annex 4 References and bibliography ............................................................................................................. 29 Annex 5 Restocking ......................................................................................................................................... 40

ICES WGMAFC Report 2004 3


1 Participants

Denmark: Josianne G. Støttrup (Rapporteur) UK: Tim Bowden Norway: Anders Mangor-Jensen (Chair) Spain: Tito Peleteiro, Oloido Cherequiri, Mercedes Olmedo, Inés Garcié de la Bondo, Fatima Lindres Cuerpo,

Victor Øiestand, Jose Iglesias. The address and e-mail of these participants is provided in Annex 1.

2 Terms of Reference

The following terms of reference were approved by the Council during the 2003 Annual Science Conference in Talinn, Estonia (2003; 2F03). The Working Group on Marine Fish Culture (WGMAFC) (Chair: Dr Anders Mangor-Jensen, Norway) met in Vigo 27–29 April 2004 to:

a) compile information on the existing regulations of individual ICES Member Countries and the EU with regard to ingredients in fish feeds;

b) compile information on the current state of the art of microdiets as a replacement for live food for larval fish;

c) review the use of live feed organisms other than rotifers and Artemia (alternative live feeds) that are used or considered for use in the culture of marine fish larvae;

d) prepare a summary of the instances where aquaculture is being used to produce fish for restocking or enhancement of wild populations in ICES Member Countries;

e) prepare a report on the status of research and technology of single-sex fish production and its application to the cultivation of marine fish, based on input from experts in the field;

f) prepare a report on existing knowledge of the effects of water quality (e.g., ozone and resulting compounds, ammonia, microbiology, and probiotics) on intensive land-based marine fish culture, included recirculation;

g) prepare a report on long- and short-term effects of gas-supersaturation in intensive marine fish cultures.

3 Introduction

Dr Anders Mangor-Jensen, Norway opened the meeting and welcomed the participants to Vigo and this meeting. Dr Alberto Gonsalez-Garces kindly welcomed us to the institute and introduced the principle activities at the Instituto Español Oceanografia at Vigo. This was ensued by an introductory round of the participants. Dr Josianne Støttrup, Denmark accepted to take on the work of rapporteur for the meeting.

The WGMFC should have met in Spain in 2003, but the meeting was cancelled due to low registration and thus an interim report was provided updating the previous years report and revising the TOR for 2004, focusing these and reducing them. Project leaders were assigned to each TOR to ensure commitment from different members. The revised TOR is listed in Section 2.

Several participants had to cancel their attendance at the last minute due to unforeseen circumstances. The reports for some of the TOR were however sent by mail to the Chair, who circulated them at the meeting for discussion and comments. For other TOR, the reports were not received and these topics were therefore not dealt with at the meeting.


4 Activities of the WGMAFC

4.1 Regulations regarding aquaculture

Tor a) The report on this topic was not completed for the meeting.

4.2 Feeds in aquaculture

Tor b) It was not possible to find a person to be responsible for this item and it was therefore not addressed at the meeting. Tor c) Live feed organisms other than rotifers and Artemia used or considered for use in the culture of marine fish larvae

The use of live feeds other than rotifers and Artemia are either widespread within a country or not used at all. The traditional live prey rotifers and Artemia are nutritionally unsuitable for marine fish larvae and lack especially essential highly unsaturated fatty acids (HUFA). Methods for feeding these organisms with emulsions containing these HUFAs have been developed and thus improved the nutritional value of these species for use in mariculture. However, hatcheries rearing marine fish larvae still experience problems with high percentages of malpigmented juveniles (McEvoy et al., 1998), and poorer survival and lower growth rates when compared to rearing on copepods (Næss and Lie 1998, Nanton and Castell, 1999). Næss and Lie (1998) demonstrated that malpigmentation problems in halibut Hippoglossus hippoglossus could be prevented by providing copepods for a short period during the larval stage.

The documented improvements have been related to higher levels of docoshexaneoic acid (DHA), eicosapentaneoic acid (EPA) and/or arachidonic acid (ARA) in the diet (Zheng et al., 1996; Sargent et al., 1997) and in particular to the level of DHA:EPA ratio (Sargent et al., 1997; Nanton and Castell, 1998). DHA levels in copepods may be up to 10 times higher than those in enriched Artemia (McEvoy et al., 1998) and the ration of DHA:EPA is generally around or greater than 2 (Nanton and Castell, 1998; Støttrup et al., 1999). These problems of dietary deficiencies seem to be exacerbated by poor husbandry, and improved experience and knowledge seem to lessen the requirements for the nutritional benefits obtained from copepods. McEvoy and Sargent (1998) suggested that the optimal EPA:ARA ratios found in copepods are responsible for helping the fish larvae to cope with stressful conditions in the hatchery. Furthermore, apart from superior fatty acid composition in copepods, they contain higher levels of polar lipids than enriched Artemia (Fraser et al., 1989) which can be double that in Artemia (McEvoy et al., 1998). Polar lipids are more easily digested by marine fish larvae enabling these to more easily assimilate DHA and other essential fatty acids when fed copepods as compared to feeding on Artemia (McEvoy et al., 1998).

The ready availability of Artemia nauplii, the short generation time of rotifers, and their relatively uncomplicated culture techniques make these organisms ideal for mariculture and it is difficult to introduce new species that may be nutritionally superior, but more difficult or complicated to culture. Comparatively little effort has been diverted to promoting or developing the culture of a copepod species or other nutritionally suitable live prey species. Efforts at developing culture systems for copepod species have been sporadic and limited to short term projects. These were unable to benefit from other projects because of the different species chosen for culture. For example the criteria for culturing many calanoids differ immensely from those for many harpacticoid species (Støttrup 1998). The same would be true if attempts were made at culturing other zooplankton species such as molluscan veliger larvae.

Alternative live prey species are, therefore, still being considered when the quality of the fish juveniles is substandard using the traditional live prey, rotifers and Artemia. Copepods are supplemented to enhance the nutritional value of the diet as for example for halibut in Norway (Næss and Lie, 1998). Additionally, production of cod in large ponds using wild zooplankton is still practised in Norway and is reported to provide better quality juveniles. Malpigmentation, incomplete eye migration, and other defects, are evident in up to 30 to 50% of the produced juveniles in situations where the larval diet has been inadequate, i.e., without zooplankton supplement. By improving enrichment of the traditional live prey, it is hoped that these problems could be eliminated. In intensive systems, many hatcheries are reluctant to introduce copepods because of the risk of introducing parasites and diseases. In Denmark and Australia, the use of alternative live feeds is either; total replacement, partial replacement such as a supplement to rotifers and Artemia, or as a main staple, but using rotifers and Artemia as a supplement.

The use of alternative live prey in marine aquaculture and the biology and production methods for copepod species are reviewed in Støttrup (2003).


Cultured copepods for feeding to marine fish In Denmark, research is conducted in developing a system for the intensive culture of a harpacticoid Tisbe holothuriae for use as live feed prey for marine fish.

In Australia, for estuary cod, coral trout and Dhufish, the calanoid Gladioferens imparipes is cultured in 500 and 1000 L conical fibreglass tanks and continuous flow (Rippingale and Payne, 2001). Several speices of microalgae are used to feed the copepods. The average daily productivity is around 450.000 /500 L tank over 409 days.

For grouper species, the calanoid Acartia sinjensis is produced in 400 L conical fibreglass tanks in batch systems (Knuckley et al. in press). Four species of microalgae are used as food and a daily production of around 600.000/tank is achieved.

Striped trumpeter is grown on a mixture of different calanoids, mainly Acartia sp. In 25–2500 L fibreglass tanks in batch or semi-continuous cultures. A mixture of 4 algal species is fed daily to the tank and daily flushing to remove settled debris and weekly transfer to clean tanks maintains hygiene. Harvest of live prey organisms for feeding marine fish In Denmark alternative live prey are used in commercial hatcheries producing juveniles for the aquaculture industry or in hatcheries producing fish for stocking purposes. Wild zooplankton bloomed in mesocosms are used for rearing turbot, Scophthalmus maximus and flounder Platichthys flesus, supplemented where necessary with Artemia. The zooplankton is allowed to bloom in the fish rearing tanks starting out from over-wintering eggs in the bottom of the mesocosm, or transferred from production ponds. The copepods are not fed directly to the fish tanks from the wild (Engell-Sørensen et al. 2003).

Harvested zooplankton is collected for feeding directly to finfish as a supplement to traditional live feeds or for stocking copepod culture tanks.

Weedy and se leafy sea dragons (Phyllopteryx taeniolatus) is fed zooplankton mainly mysids, harvested with plankton nets with or without light traps.

Striped trumpeter Latris lineate is also fed wild zooplankton > 250 µm, usually different copepod species, the most common of which are Acartia sp. Extensive systems In Denmark, turbot and flounder are reared in extensive or semi-extensive systems relying on the natural reproduction of a mixture of zooplankton in outdoor mesocosms. Copepods are supplemented from mesocosms used only for zooplankton reproduction and Artemia nauplii may also be used as a supplement.

Flame angelfish, peacock grouper, rosy snapper and golden snapper are reared in 10000–40000 L plastic or fibreglass tanks in extensive systems in Australia. The snappers are further supplemented with rotifers (from day 7–8 PH) and Artemia from day 14 or 15. Conclusions and Recommendations • Harvesting copepods directly from the wild and feeding them directly to the fish is not recommended due to the

high risk of introducing diseases and parasites. • It is, however, recommended to harvest copepods for rearing ponds and rearing at least through one generation

before using the copepods as live prey for fish larvae. • Harpacticoids, at least the species currently being cultured, are not of an adequate size for the northern species

such as cod and halibut. For these species a species with similar characteristics to most harpacticoids but with larger nauplii needs to be identified for culture.

• Collect information on different quality criteria (characteristics) for specific species reared either solely on traditional live prey or in extensive mesocosms to compare the quality of the juveniles. The characteristics need to be identified and should include biochemical parameters,

4.3 Fish produced for other purposes than direct consumption Tor d) Aquaculture production of fish for restocking or enhancement of wild populations in ICES Member Countries. Producing fish with the aim to restock or enhance wild populations is one of the more immediate applications of aquaculture. Due to the great effort that has been devoted to diversify species in the aquaculture sector, many of the experiments that have been carried out with species that at the end have been found not to be suitable for their industrial exploitation, are useful for restocking programmes. This is the case of white seabream (Diplodus vulgaris).

The implications of the introduction of cultured species in the natural environment must be carefully found out, taking into account factors such as genetic characteristics, pathologic aspects and the assessment of the impact that can cause the introduction of cultured species in the natural environment.

These aspects have been recently dealt with at the Workshop held last May 2003 in the Isla de Arosa (Pontevedra, Spain), conclusions of which are enclosed in Annex 4. In this Workshop, the following subjects were discussed:


breeding stock characteristics for restocking, size of the individuals for release, adaptation to the natural environment before the release, habitat characteristics, tagging, tracking of released individuals, legal sizes for the protection and/or regulation of fisheries and socio-economic studies of the restocking programme, etc.

Some restocking programmes carried out by different European and American countries during the last years have not produced the desired effect, due to the fact that they were not profitable from the economic point of view, or that they were carried out under scarcely monitored conditions or with unsuitable species.

To prepare this summary, information was requested to the 13 countries represented in the Working Group on Marine Fish Culture. Among all these countries, France and Canada do not currently have any ongoing restocking programme, and Denmark (Annex 1), Portugal (Annex 2) and Spain (Annex 3) do currently have restocking programmes or are preparing their implementation for the near future.

Species used in these restocking programmes are generally well-known from the point of view of their biology and behaviour, both in the natural environment and in captivity. They are even consolidated in the field of aquaculture production. There is already genetic information available about them, or it is currently being investigated. Restocking experiences are being carried out with elvers (Anguilla anguilla) in Denmark, turbot (Scophthalmus maximus) in Belgium, Denmark and Spain, sole (Solea solea) in Belgium, plaice (Platichthys flexus) in Denmark, gilthead seabream (Sparus aurata) in Spain and Portugal, and with different white seabream species in Portugal.

These restocking programmes operate in Denmark from several years, as it is the case in Belgium, Spain and Portugal. However, the economic yield does not seem to be guaranteed. Nevertheless, there are restocking programmes in other countries (i.e., Japan) in which these systems are profitable from the economic point of view when using very specific species.

These restocking programmes are generally funded with governmental funds, although in cases such as the Denmark one, part of the funds come from the granting of sport fishing licences. In Japan, financing comes both from the state and from contributions by local fishermen associations.

In Spain, the two species that have been more deeply studied from the restocking and the mass production point of view are turbot and gilthead seabream. These two species, considered to be consolidated in the aquaculture sector, have suffered important decreases in their traditional fishing grounds due to an excessive fishing effort. Nevertheless, information currently available on these two species regarding their behaviour in the natural environment after having been cultured, allows to seriously consider using them as candidate species for restocking programmes.

In Begium a feasibility study was started in 1998 to investigate the possibility of restocking commercial important flatfish, e.g., turbot and sole. The restocking programme is build up in three different phases. The first stage was to look if cultured fish was able to adapt to the natural conditions in the northern Atlantic (North Sea and adjacent waters), with or without preconditioning. This was first carried out with turbot, since the culture biology of this species is fully understood. As tagging material the Petersen disc was used. For this, turbot was reared to a size at which tagging would be certain to result in little loss, otherwise this could mask the survival experiments. The use of Petersen disc was necessary to garantee a high reporting of tags. The restocking experiment resulted in more than 30% reporting of tags and it was clear that reared turbot in captivity can be easily adapted to natural conditions. The second phase was to see what quality of fish was needed to restock in order to obtain a high survival and reporting of tags. Next to that, a study was carried out to reveal the parental effect and the genetic loss during nursery and restocking. This phase is still going on. The third phase will be to stock large quantities of smaller juveniles to make a cost-benefit analysis and is foreseen in the near future. The problem here is that tagging with the Petersen disc is not possible, so coloration of bone structures will be used in this experiment, so other tagging methods must be investigated to obtain sufficient reporting. Belgium has recently started a similar restocking programme with sole (Solea solea), because this is the most important fish species for this country and its biological characteristics makes it a good candidate for restocking (easy hatchery and nursery conditions, slow grower in captivity, and small distribution pattern). In Norway an extended program on restocking of several species was started in early 90’ and lasted until 1997 (The PUSH- program). This program included Atlantic salmon, cod, Arctic char and European lobster. The results showed that for all species, except for the lobster, the re-catch statistics were too low to give economical or enhancement yields. In the case of lobster, most local stocks were very low due to over fishing. At the sites where experimental restocking took place the catches were more than doubled after five years. Further material provided including the conclusions from a Workshop on the subject held at Isla de Arousa-Galicia (Spain) during May 2003 is included in Annex 5.

Conclusions and recommendations The WG recommends ensuring that future stocking programmes with marine fish consider all the recommendations for responsible approach to stocking prior to starting stocking programmes. Further, it is important that other tools for stock enhancement be considered prior to embarking on fish release programmes.

4.4 Production technology Tor e) Research and technology of single-sex fish production and its application to the cultivation of marine fish


In many commercially cultured fish production of one gender is preferred due to advantages relating to growth, delayed reproduction, behavioural traits, or products such as caviar. By taking advantage of the lability of teleost sex differentiation various protocols have been developed to produce monosex populations for aquaculture (Recent reviews: Arai, 2001; Piferrer, 2001; Devlin and Nagahama, 2002; Strussmann and Nakamura, 2002; Pandian and Kirankumar, 2003). Significantly more research has been conducted on sex-reversal of freshwater fish including diadromous salmonids, however, this report will focus only on recent developments in the production of monosex marine finfish populations for aquaculture.

Production of monosex fish can be accomplished using direct and indirect hormonal induction, environmental sex reversal (ESD) or chromosome set manipulations. It is important to know a few key biological characteristics specific to the species of interest prior to developing a strategy for production of monosex populations. The timing of gonadal sex differentiation and the critically sensitive period when undifferentiated gonads are most responsive to endocrine manipulation is important for hormonal and environmental manipulation of gonad differentiation. Also knowing the species specific mechanism of sex determination is relevant since this will determine the most straightforward method of producing all-female populations which are frequently the preferred sex for many marine species such as sea bass (Dicentrarchus labrax L.), flatfish, and gadoids.

If the female is the homogametic sex (XX) then indirect feminization can be done using protocols similar to those used for commercial production of chinook salmon (Donaldson, 1986) and rainbow trout (Bye and Lincoln, 1986). Indirect feminization involves the masculinization of genotypic females (preferably from an all-female group such as those produced by gynogenesis) and sperm from these sex-reversed “neomales” are then used to fertilize eggs from normal females. The sex-reversed males that were treated with hormones using this indirect feminization technique are retained as broodstock and are not marketed for human consumption.

Gynogenesis can be used to directly produce genotypic females if females are the homogametic sex but this procedure is complicated and survival of gynogens is usually low (Felip et al., 2001). Gynogenetic fish contain only chromosomes of maternal origin. This is accomplished by destroying the genomic DNA in sperm without affecting their ability to activate egg development. Diploidy is restored by using temperature or pressure treatments to retain the second polar body (meiogyns) or to block the first mitotic cell division (mitogyns). Gynogenesis is used to demonstrate the sex determination mechanism in fish since all offspring will be female if females are the homogametic sex (Howell et al., 1995). Conversely, if females are heterogametic (WZ) then some offspring will be “superfemales” (WW), and normal females (WZ) and males (ZZ). When sex determination is also influenced by autosomal genes then sex ratios of gynogens can vary such as seen in tilapia (Beardmore et al., 2001; Ezaz et al., 2004)

Direct sex reversal using hormones, such as oestrogens or androgens, or aromatase inhibitors to alter the process of gonadal differentiation is the most common and straightforward technique. There is concern, however, that this procedure may not be acceptable to consumers since the marketable fish have been exposed to hormones although at relatively low doses during early development. Environmental sex control is also an option for some species as discussed below (Godwin et al., 2003). Sea Bass (Dicentrarchus labrax L.) Females are the preferred sex for culture of sea bass due to faster growth. Under culture conditions, however, the sex ratio is skewed towards males. More research has been done on sex control in sea bass than on any other commercially cultured marine teleost. Sex differentiation in sea bass occurs when juveniles are < 1 year old. Most studies have investigated the use of direct hormonal sex reversal (Blázquez et al., 1998). Saillant et al. (2001) produced 100% females by feeding juveniles 12.5 mg estradiol (E2)/kg feed from 90–150 dph. Blázquez et al. (2001) identified 96–126 dpf as the critical period for introduction of hormones to alter gonadogenesis. Feeding juveniles 10 mg 17α-methyltestosterone (MT) at 86 or 106 dpf for 21 d resulted in 100% masculinization. Masculinization was also successful when 17α-methyldehydrotestosterone (MDHT) was fed to sea bass at 84 dph (Chatain et al., 1999). Crossing sex-reversed males with normal females and sexing the progeny indicated that sex determination in sea bass may be influenced by autosomal or environmental factors (Blázquez et al., 1999). This was confirmed by Pavlidis et al. (2000) when exposing sea bass from embryos to 18 mm TL to low rearing temperatures (13–15°C) increased the percentage of females. Felip et al. (1998) developed techniques for the production of gynogenetic sea bass. Based on the above results direct sex-reversal using hormones, temperature or aromatase inhibitors may be the best options for all-female production of sea bass, however, these techniques have not currently been applied in commercial aquaculture industries. Small flounders Females are again the preferred sex for culture of flatfish because they grow faster and attain a larger size. Studies on Japanese flounder (Paralichthys olivaceus) (Tabata, 1991) and barfin flounder (Verasper moseri) (Goto et al., 1999) indicate that females are the homogametic sex but female heterogamety (WZ:ZZ) has been reported in Dover sole (Howell et al., 1995). Temperature has also been found to affect gametogenesis. High rearing temperatures increase the proportion of males in both the barfin flounder, Verasper moseri (Goto et al., 1999), and marbled sole, Limanda yokohamae (Goto et al., 2000). In Japanese flounder, southern flounder (Paralichthys lethostigma) and summer flounder (Paralichthys dentatus) both high and low rearing temperatures resulted in an increased proportion of males (Yamamoto, 1999; Kitano et al., 1999; Luckenbach et al., 2002; Luckenbach et al., 2003). Studies conducted by Kitano et al. (1999) showed that the effect of temperature on phenotypic sex differentiation in Japanese flounder may


result from effects on aromatase gene expression. No definitive protocols for all-female production of various flounder species has been developed for commercial use. Atlantic halibut (Hippoglossus hippoglossus) Similar to the smaller flatfish, Atlantic halibut females grow larger and mature later than males making all-female populations preferred for culture. Gonadal sex differentiation occurs prior to 38 mm FL in Atlantic halibut (Hendry et al., 2002). Larval Atlantic halibut were reared at 7, 12 and 15oC from a mean size of 20.7mm to 80mm to determine if the phenotypic sex of Atlantic halibut was affected by temperature similar to other flatfish, however, these temperatures did not significantly alter the normal 1:1 sex ratio (Hughes et al., J. Fish Biol., in review). Feeding halibut juveniles (30 mm FL) for 45 d diets sprayed with E2 (10 ppm) or MDHT (1 and 5 ppm) resulted in 70–74% females and 97–100% males, respectively (Hendry et al., 2003). Recent experiments have shown that all gynogenetic offspring of Atlantic halibut were female indicating that Atlantic halibut females are homogametic (T.J. Benfey and D.J. Martin-Robichaud, pers. comm.). Fish exposed to the MDHT treatments in experiments done by Hendry et al. (2003) have been reared to maturity (n=55) and all males were spermiating normally (D.J. Martin-Robichaud, pers. comm.). Currently, these males are being crossed with normal females and the offspring reared separately to distinguish sex reversed “neomales” from normal genotypic males based on the sex ratio of their offspring. These current results show that indirect hormonal sex-reversal of Atlantic halibut to produce all-female stocks may soon have commercial potential.

Currently there is no commercial production of monosex populations of marine finfish for aquaculture but there is international interest in the potential, and researchers are continuing to investigate various techniques for different commercially valuable species (Annex 3). Consumer acceptance and ongrowing performance of monosex populations will also need to be addressed once protocols are developed. Conclusions and recommendations

• Although the use of hormones may be perceived negatively, in effect, the WG considers that there is no risk for human consumption when hormonal treatment is administered during the early life stages. However, since these fish are not sterile, there may be environmental problems in the case of escapees and research is required to document possible consequences of adopting this procedure in aquaculture.

• Welfare and ethical issues not addressed here. Need to be addressed? Tor f) The effects of water quality (e.g., ozone and resulting compounds, ammonia, microbiology, and probiotics) on intensive land-based marine fish culture, included recirculation;

Water quality requirements The majority of marine aquaculture production is presently carried out in net cages along the coast. Net cages are open systems and are in direct contact to natural marine ecosystems. For this reason they are a potential risk and an expansion of mariculture will require improved concepts that sidestep limitations immanent in conventional mariculture installations. It is to expect that land based installations and especially closed recirculation systems will gain importance because they are allowing for a thorough after-treatment and control of effluent water. Most of the marine species can also be produced in industrial land based tank or raceway systems but the contribution to the world aquaculture production is still minor. This may change soon as more and more regulations will be imposed on aquaculture in the coastal zone.

An important step from traditional aquaculture towards an industrial production was achieved through the development of land based tank- or raceways systems. These systems allow to multiply rearing densities and at the same time to reduce land usage. An ideal would be the reduction of water consumption. It is obvious that in such systems the risk for failures is high, especially if the biological and engineering concepts do not fit to the requirements of the species. Land based systems may be operated as open systems with a continuous through flow of seawater and discharge or as nearly closed recirculation system with reduced water consumption. The first type requires a lower technical overhead than the recirculation system that is combined of several components for the treatment of the water.


Make up water flow = FM and FM ≈ FD (evaporation …) Seawater supply

Internal water flow = FI

Water treatment

(Nitrification)

∆Cammonia<0

Pump

Holding tank (Excretion)

∆Cammonia>0

Seawater discharge

Discharge water flow = FD

Figure 4.4.1. Generalised flow pattern in a recirculation system. The make up water flow (FM) << internal water flow (FI). Within the holding tank excretion of animals increases the ammonia concentration. The water treatment system must remove ammonia (nitrification) to an extent that save concentrations can be maintained in the holding tank. The water renewal in the holding tank depends on the flow rate; water exchange is a non linear function.

Water exchange The crucial point in all types of land based aquaculture systems is the water exchange. The necessary water flow into aquaculture systems depends on the species, the stocking density, and the physiological state of the animals. The water flow must be sufficient to control dissolved gas levels and to remove toxic metabolites in the holding tanks (Figure 4.4.1). While dissolved oxygen can be supplied by means of aeration or oxygenation, the control of metabolites is always a matter of dilution and removal (Figure 4.4.1).

The aimed production in an aquaculture production system can only be attained when appropriate rearing conditions are maintained throughout the rearing period. Huegenin and Colt (1989) demonstrate a simple mathematical formulation for the quantification of water flow and carrying capacity in through-flow systems. A more refined approach by means of numerical modelling requires a sound knowledge of the weight dependent metabolic processes of the target species.

The formula for calculating the water exchange rates by Kraul (1985) can be used in numerical models to simulate water quality under a given physiological state and various water flow regimes. The input variables could be weight dependent metabolic activity, ingestion rate, or excretion rate as well as stocking density, feeding rate, to list a few examples. In Figures 4.4.2 and 4.4.3 the results of two simple numerical calculations are shown that are based on a constant excretion of ammonia of the animals under culture and a continuous water exchange (Figure 4.4.1). Figure 4.4.2 and 4.4.3 show that the flow rate of water at a given biomass (stocking density) in a fish tank is determining the TAN concentration. A reduction of water flow unavoidably increases the TAN concentration even if ammonia free water is supplied. The different asymptotes reflect the non-linearity of the model. At very low exchange rates a dramatic increase of TAN concentration is to expect. Figure 4.4.3 shows the result of a numerical model assuming a constant water exchange and varying stocking densities. Again the final TAN concentration is determined by the biomass in the fish tank and overcrowding may inevitably lead to inappropriate holding conditions. Both numerical runs are based on only few assumptions and do not take any diurnal pattern, that are clearly to expect, into consideration. They simply show how the physical/chemical environment is determined by the basic engineering that very often is not based on the biological requirements.


Figure 4.4.2. Results of a model simulating animal excretion (ammonia) and the dilution through the water flow based on the formula by Kraul, 1985. The resulting equilibrium TAN concentration in a hypothetical holding tank is shown on the ordinate. Water flow rate was assumed to equal 1.0, 0.75, 0.50, and 0.25 * tank volume per hour. Stocking density was set to 50 kg * m−3 and feeding rate to 0.02 of the tank biomass. Under the assumption that the inflowing water is free of ammonia/ammonium and a constant excretion rate of 0.03 kg N * kg−1 feed the ammonia/ammonium concentration was calculated for every minute during a 48 h period (WALLER unpublished).

The result of both model runs shows the significance of a proper adjusted water flow rate through the holding tanks. Similar dependencies can be assumed for all environmental variables in holding tanks and water flow rate finally is defined by the most determining variable.

Figure 4.4.3. Results of a model simulating animal excretion (ammonia) and the dilution through the water flow based on the formula by Kraul, 1985. The resulting equilibrium TAN concentration in a hypothetical holding tank is shown on the ordinate. Water flow rate was assumed to equal one tank volume per hour. Stocking density was set to 10, 20, …, 50 kg * m−3 and feeding rate to 0.03 of the tank biomass. Under the assumption that the inflowing water is free of ammonia/ammonium and a constant excretion rate of 0.03 kg N * kg−1 feed the ammonia/ammonium concentration was calculated for every minute during a 48 h period (WALLER unpublished).

Temperature Holding temperature is determining growth and performance of poikilothermic aquatic organisms like fish. The final preferendum for temperature (Richards et al. 1977, McCauley and Casselmann 1981) is an indicator for the optimum growth temperatures (Jobling, 1981). Fish react in temperature gradients and avoid suboptimal conditions. Optimum temperature conditions are necessary to maximise growth and optimise production efficiency. In outdoor facilities with open water supply water temperature will vary with the season and may become unfavourable during seasons with extreme temperature regimes (summer, winter). In addition solar radiation may cause daily temperature fluctuations in outdoor facilities during summer depending on the flow rate or exchange rate of water respectively.


The minimum and maximum temperature that is tolerated by a species is genetically determined. Temperature preference may be linked to basic biochemical properties (Samuelsen, et al., 1999). Over the tolerated temperature range the metabolic activity is increasing with increasing temperatures and may follow an s-shaped curve (##). Beyond the lower and upper edge of the tolerated temperature range stress is induced in several ways and mortality is likely to increase. Above the upper tolerated temperature a combination of a higher metabolic activity and a reduced dissolved oxygen concentration in the water may cause the death of animals. At the lower edge animals may not be able to maintain the vital metabolic activity. The failure of basic metabolic processes on the standard and routine level is likely to be the root cause of death. However, at the borders of the tolerated temperature range the immune-response may be weak and an outbreak of diseases may lead to considerable losses. This mechanism is well known in salmonid fishes that may suffer from bacterial infections during seasons with high water temperatures. Rapid temperature changes must also be considered as severe stress and may lead to an outbreak of latent infections.

Information on the tolerated temperature range is available for a great variety of species that are of interest for marine aquaculture. This information must be carefully taken into account during design and operation of land based systems that may be subjected to significant temperature differences compared to water based culture facilities like net cage installations. The temperature range for marine aquaculture species can be assumed between 5 and 30 °C depending on the species.

Typical cold adapted species (T < 15 °C) are cod (Gadus morhua) and halibut (Hippoglossus hippoglossus)(Le Francois et al., 2002). Temperature selection in fish is not a straightforward process. The temperature preference of cod for example is determined by various environmental factors and may be lower in juveniles during no feeding hours (Clark and Green, 1991). A reduced food availability may lower the temperature preferendum of cod in its natural distributional range because energy demand in temperature conforming fish is reduced at lower ambient temperatures (Swain and Kramer, 1995). Even if not all of these interdependencies are necessarily of relevance for aquaculture temperature preference is not a simple thermodynamic function and sub acute stress can quickly be induced in unsuitable holding conditions. Anyway, the preferred temperature is higher in juvenile cod (Waller and Boettger, 2001) compared to subadult and adult fish. The larger animals are performing better in temperatures around 10 to 14 °C and holding temperatures must be adjusted during grow out. This is an advantage of land based systems that are independent from the season.

Another cold water species is the turbot (Scophthalmus maximus) that is well investigated regarding its preferred temperature range. While larger animals have a low temperature preferendum (< 15 °C)(Hara et al., 2002), juveniles (Mallekh and Lagardere, 2002; Imsland et al., 2001; Imsland et al., 1996; Waller, 1992) and early life history stages (Gibson and Johnston, 1995) are adapted to higher temperatures (> 15 °C). In turbot the temperature preferendum may vary between populations (geographical distribution) (Imsland et al., 2000b) and genotyps (Imsland et al., 2000a) respectively. Thus the selection of brood stock is an important task and domestication (selective breeding) may improve the performance in controlled culture facilities.

Typical warm adapted marine aquaculture species (T > 20 °C) are seabass (Dicentrarchus labrax) (Alliot et al., 1983; Ayala et al., 2001; Claireaux and Lagardere, 1999; Hidalgo et al., 1987; Johnson and Katavic, 1986) and seabream (Sparus auratus). In D. labrax temperature can be a supporting feature as temperature modifications can be used to control sex ratio in seabass (Pavlidis et al., 2000; Koumoundouros et al., 2002; Blazquez et al., 1998). This is possible only in land based systems with an appropriate temperature control.

Marine prawns are tropical warm water animals with a high temperature preferendum (T > 28 °C)(Chen et al., 2001; Chen and Chen, 1999) that are more and more frequently used in aquacultures beyond their natural distributional range. This species is gaining importance in European and North American marine aquaculture. In these areas they are grown in land based systems under fully controlled conditions. Because of the preference for high environmental temperatures aquaculture under natural temperature conditions would not be possible in most of the ICES area; especially the lower growth would not allow a profitable production.

Growth in poikilothermic marine fish and invertebrates is directly under control of determining factors like temperature, salinity, or photoperiod; the temperature, however, is the most important one and directly determines growth performance. The temperature preference, therefore, must be carefully considered during system design as unsuitable temperatures will significantly reduce the capacity of a production system. Heat recovery by means of heat exchange and a low water exchange are prerequisite for successful commercial applications for warm water species in regions with low average annual temperatures. In areas with high average temperatures and/or high solar radiation it might be necessary to include cooling systems to maintain appropriate temperatures. This applies to warm water as well as to cold water species. This is often neglected because of economic considerations. Salinity Most of the marine species used in aquaculture are tolerant to a wide range of salinities. Atlantic wolfish (Anarhichas lupus) has a high tolerance to low salinity. Experiments indicated better growth performance at intermediate salinity compared to full seawater (Le Francois et al., 2003; Francois et al., 2001). American plaice (Hippoglossoides platessoides) are tolerant to reduced salinities; they can be maintained at intermediate salinities that are not lower than 14 (Munro et al., 1994). Seabass (D. labrax) have a preference for low salinities during their juvenile phase (Saillant et al., 2003) and can be grown to market size of 680 g at a salinity of 3.9 (Chervinski, 1974). Cod (G. morhua) can tolerate brackish water as well as turbot (S. maximus)(Waller, 1992).


Table 4.4.1. List of results of experiments into the response of marine fish to different levels of unionized ammonia concentrations (data of various authors). The salinity in the experiments was ranging from 33 to 41 (psu), the temperature was between 18 and 27 °C, the pH was always close to 8.

Species StageUnionized ammonia

concentrationExperimental

timeResponse

mg NH3-N * dm-3 min

Sparus auratus juvenile 0.47 480 suppressed growth, 68 % of control

Scophthalmus maximus juvenile 0.20 480 suppressed growth, 50 % of control

Solea solea juvenile 0.33 1008 suppressed growth, 50 % of control

Solea solea juvenile 0.45 1008 suppressed growth, 50 % of control

Dicentrarchus labrax juvenile 0.50 456 suppressed growth, 20 % of control

Scophthalmus maximus juvenile 0.60 672 EC50; 50 % SGR




Dicentrarchus labrax juvenile 1.69 96 LC50

Sparus auratus juvenile 2.59 96 LC50

Scophthalmus maximus juvenile 2.39 96 LC50




Sparus auratus juvenile 1.27 96 LC50


Sparus auratus larva 0.24 1440 LC50

Solea senegalensis larva 1.32 1440 LC50

Gadiropsarus capensis larva 0.46 24 LC50

Diplodus sargus larva 0.36 24 LC50

Lithognathus mormyrus larva 0.38 24 LC50

Pachymetopon blochi larva 0.42 24 LC50

The advantage of a high salinity tolerance is that under circumstances marine parasites and other germs that are adapted to full strength seawater can be inactivated by reducing the salinity without any negative effects on the target species. Recirculating systems can be operated with marine species inland without direct access to seawater; the effluents can more easily be discharged into the sewage systems.

Ammonia Ammonia is a major stressor in land-based aquaculture systems. Because stocking densities in these installations are typically high, total ammonia concentration (= [NH3] + [NH4

+]) may reach significant values during the production process. It was already mentioned above that the ammonia concentration in a land-based fish production system is determined by the water exchange rate. The most important limitation on the viability of aquaculture farms is the seawater quality, i.e., the TAC in the make up water. The equilibrium unionized ammonia concentration during operation should not exceed save limits. Inappropriate total ammonia concentrations may result in immune depression, growth retardation, reproductive problems or even death of the animals depending on the degree of the interference (unionized ammonia concentration and exposure time). Many physiological/biochemical dysfunctions are caused by increased ammonia levels in the aquatic environment. Internally, i.e., within the organism, this includes the change of pH on the cellular level and in the blood (impact on oxygen carrying system), the suppression of citric acid cycle (energy availability), damage of gill epithelium (exchange of dissolved gases and ions), and disfunction of liver, kidney and heart, to name a few examples. A complete overview is given by Tomasso (1994). The tolerance of fish to unionized ammonia may be weight depending. Larger turbot for example are more sensitive to ammonia than smaller individuals (Person-Le Ruyet et al., 1997). Fish may be able to adapt to increased ambient ammonia concentrations over time (Lemarie et al., 2004) so that under aquaculture conditions higher ammonia levels may be tolerated after a certain adaptation time.

By all means, ammonia has to be considered as a severe limiting factor with a specific threshold value in all aquaculture systems. This is obvious from one basic biological theorem: aquatic organisms are typically ammoniothelic. Values for the lethal concentration or the effective concentration are available from the literature for various species and different life history stages. A number of examples are summarized in Table 4.4.1. Figure 4.4.3


gives an overview over LC50 concentrations and the incipient limiting concentration for growth in typical aquaculture fish species based on the data in Table 4.4.1.

Exposure time and unionized ammonia level in Figure 4.4.3 do not show an obvious connection. In juvenile fish the LC50 for ammonia appears to decrease with increasing exposure times. However, if fish can adapt to ammonia levels a decreasing LC50 concentration with increasing exposure time would be unexpected. The inverse would be probable. Larvae are more sensitive (NH3-N < 1 mg * dm−3) compared to juvenile fish (NH3-N < 1 mg * dm−3) (Figure 4.4.3). Even if first physiological changes may be occur at lower ammonia concentrations, the important threshold level in aquaculture is the incipient limiting concentration for growth. This is according to the data plotted in Figure 4.4.3 in the range from 0.2 to 1.0 mg NH3-N * dm−3. To be on the safe site it is desirable to keep the unionized ammonia level as low as possible, at least below 0.1 mg NH3-N * dm−3 in aquaculture operations. In view to the S,T,pH dependence of the ratio between unionized ammonia (NH3) and the ammonium ion (NH4

+) in aqueous solutions (Bower and Bidwell, 1978; Whitfield, 1974) a threshold level of 0.1 mg NH3-N * dm-3 would compare to a total ammonia nitrogen concentration (TAN) of approximately 2 to 2.5 mg * dm−3 at S ≈ 35, T ≈18, pH ≈ 8. In view to marine recirculation systems a TAN of around 2 mg * dm−3 may allow an optimal function of the nitrifying biofilter; usually the zero order kinetic is reached at this concentration, i.e., the maximum conversion rate. Thus, lower ammonia levels may not be desirable as this would entail that the biofilter may operate at a reduced efficiency because of the ½ order kinetic that is involved at lower ammonia levels. However, Meade, 1985 assumes safe ammonia concentrations at around 0.01 NH3-N * dm−3 or approximately 0.25 NH4

+-N * dm−3

Figure 4.4.4. Unionized ammonia threshold levels for fish (Dicentrarchus labrax, Diplodus sargus, Gadiropsarus capensis, Lithognathus mormyrus, Pachymetopon blochi, Sparus auratus, Scophthalmus maximus, Solea solea, Solea senegalensis) with data listed in Table 4.4.1.

The LC50 concentrations for marine prawns are listed in Table 4.4.2 and shown in Figure 4.4.4. Higher unionized ammonia concentrations (4.7 mg NH3-N * dm−3) will probably be tolerated for short periods (24 h). If the animals were exposed for longer periods (96 h) LC50 ammonia concentration decreased to values around 0.3–1.0 mg NH3-N * dm−3. The broad range of results in Figure 4.4.4 implies that also other factors may have controlled the final toxicity of ammonia during experiments. However, in view to the highest LC50 concentrations found in the experiments prawns appears to be more tolerant as fish are (Figure 4.4.3). The lower boundary proofs that impacts are to expect already at levels around 0.3 mg NH3-N * dm−3. Thus, a safe level for prawns is possibly in the same range as for fish, i.e., below 0.1 mg NH3-N * dm−3.

The prediction of waste production in aquaculture systems, especially ammonia nitrogen, is of tremendous significance for the design of land based farming systems. Based on biological models (Lupatsch, 1998) the water flow and capacity of treatment systems can be estimated. A thorough bioengineering is prerequisite for any successful farming system.


Table 4.4.2. Results of experiments into the response of marine prawns (Penaeus sp.) to different levels of unionized ammonia concentrations (data of various authors). The average salinity in the experiments was 27 (psu), the average temperature was 27 °C, the pH was always close to 8.

Type, genus StageUnionized ammonia

concentration

Experimental time

Response

mg NH3-N * dm-3 (h)

Penaeus paulensis adult 1.1 96 LC50

Penaeus paulensis juvenile 1.1 96 LC50

Penaeus monodon postlarva 4.7 24 LC50



Penaeus paulensis postlarva 0.3 96 LC50

Penaeus indicus mysis 3.2 24 LC50

Penaeus monodon mysis 2.2 24 LC50

Penaeus paulensis mysis 0.9 96 LC50

Penaeus paulensis zoea 0.7 96 LC50

Penaeus indicus protozoea 1.0 24 LC50

Penaeus monodon protozoea 1.0 24 LC50

Penaeus indicus nauplius 0.3 24 LC50

Penaeus monodon nauplius 0.5 24 LC50

Penaeus paulensis nauplius 1.1 72 LC50

Figure 4.4.4: Unionized ammonia LC50 concentrations for marine prawns (Penaeus sp.) based on the data listed in Table 4.4.1.

Nitrite Nitrite in land based systems is usually formed during nitrification in aerobic biofilters. Thus, increased levels are particularly to expect in recirculation systems. The toxicity of nitrite in fish is linked to a failure of the oxygen carrying system through the formation of methaemoglobin, i.e., the oxidation of Fe2+ in the haemoglobin molecule. Even if nitrite impacts the oxygen transport in fish, the effective concentrations are much higher compared to ammonia. This is explainable by it that around one third of the haemoglobin in fish is usually in the form of methaemoglobin; severe effects are to expect if methaemoglobin exceeds 80 % (Noga, 2000). Because nitrite is taken up by fish via the chloride cells in the gill epithelium the nitrite uptake is coupled with the water chloride content. Nitrite is not freely passing gill membranes by diffusion processes. In freshwater where fish need a high number of chloride cells to take up the nessary Cl- ions also the internal nitrite concentration will raise quickly if NO2

- ions are available. In seawater with high


chloride availability chloride cells are less frequent and only few NO2- ions will be taken up via this pathway. For that

reason nitrite is less toxic in seawater (Atwood, et al., 2001). Only little information on nitrite toxicity in seawater fishes is available from the literature. Gilthead seabream

larvae (Sparus auratus, 12 d after hatch) tolerate 200 mg NO2-N * dm−3; 7 days old Solea sengalensis larvae resist even 2000 mg NO2-N * dm−3 for 24 hours (Parra and Yufera, 1999). Median lethal concentrations for several marine fish larvae are reported by Brownell, 1980 (Tab. 3). Larval red drum (Sciaenops ocellatus) tolerate 100 mg NO2-N * dm−3 (Holt, 1983) that is considerably lower compared to the data of Brownell.

Table 4.4.3. Nitrite toxicity to several marine fish larvae after Brownell, 1980. The salinity in the experiments was around 35 (psu), the temperature was around 15 °C, the pH was 7.8–7.9.

SpeciesNitrite

concentrationExperimental

timeResponse

mg NO2-N * dm-3 (min)

Gadiropsarus capensis 2210 24 LC50

Diplodus sargus 1360 24 LC50

Lithognathus mormyrus 1230 24 LC50

Heteromycteris capensis 2440 24 LC50

Synaptura kleini 2110 24 LC50

Gadiropsarus capensis 450 24 First feeding EC50

Diplodus sargus 330 24 First feeding EC50

Lithognathus mormyrus 360 24 First feeding EC50

Heteromycteris capensis 340 24 First feeding EC50

Synaptura kleini 350 24 First feeding EC50

The 96 h median lethal concentration in Oreochromis niloticus (4.4 g) in freshwater was 81 mg NO2-N * dm−3 (Atwood et al., 2001). In water containing 10 mg * dm−3 chloride the fish were much more tolerant. The LC50 for 96 h increased to 338 mg NO2-N * dm−3. In seabass (D. labrax) the 96 h LC50 concentration is at 23 °C 220 mg NO2-N * dm−3 (Sargolia, 1981). These values compare to the EC50 concentration for first feeding in marine fish larvae reported by Brownell (1980). Because starving fish larvae will sooner or later die the EC50 concentration resemble the LC50 concentration. One could infer from it that nitrite is toxic in seawater only at comparatively high concentrations.

In marine prawns the tolerance to nitrite appears to be lower in egg and early larval stages. For Penaeus paulensis LC50 concentrations for 24 h exposure time for eggs, nauplii, zoea, mysis, and post-larvae are reported (Ostrensky and Poersch, 1993). The LC50 nitrite concentrations amount to 0.9, 21.9, 340.6, 197.8, and 277.8 mg NO2-N * dm−3, respectively. The 96 h LC50 were 8.6, 8.2, and 10.7 NO2-N * dm−3 for zoea, mysis, and post-larvae, respectively. Chen and Chin, 1988 report 24 h LC50 concentrations for P. monodon nauplii, zoea, mysis, and postlarvae of 5.0, 13.2, 20.6, and 61.9 mg NO2-N * dm−3, respectively. During 48, 72, and 96 h exposure the LC50 for postlarvae decreased to 33.2, 20.5, and 13.5 mg NO2-N * dm−3, respectively. Thus marine prawns are more sensitive to increased nitrite concentrations as fish.

However, nitrite may mainly play a role in land-based systems in which the water quality is restored by means of biofiltration. If the two step nitrification process in the biofilter is impaired, all excreted ammonia will be converted into nitrite instead of nitrate. Thus, the malfunction of the biofilter may cause nitrite toxicity in land based seawater recirculation systems. In Figure 4.4.5 an example for a sudden failure of the biofiltration in a seabass fingerling recirculating system is shown that caused high total ammonia and nitrite concentrations. In view to the relatively low tolerance of fish to unionized ammonia and comparably high tolerance to nitrite, it is unlikely that nitrite levels can be raised to incipient limiting levels in seawater systems without harming the animals already through the elevated ammonia levels. Further, in the course of a biofilter malfunction feeding will be reduced or stopped. Thus, N-input into the system and formation of nitrogenous waste is minimized.


Figure 4.4.5. Total ammonia and nitrite concentrations in an experimental seabass (Dicentrarchus labrax) fingerling recirculation system operated with full seawater at an average temperature of 22.4 °C and 8.1 pH with unpublished data from SANDER and WALLER. At day 40, the concentration of ammonia and later the concentration of nitrite increased to values above 20 and 30 mg * dm−3 (Figure 4.4.5). This happened at a relatively low stocking density of 5 to 8 kg * m−3. During 170 days with elevated ammonia and nitrite concentrations 30 % of the initial fish population died. A rigorous exchange of seawater restored biofilter function resulting in subsequent average total ammonia concentrations of 0.8 mg N * dm−3 and nitrite concentrations of 1.7 mg N * dm−3 (Figure 4.4.4). In view to the reported tolerance limits in fish the cause of mortality was likely the unionized ammonia content in the seawater (≈ 0.8 mg NH3-N * dm−3). The nitrite concentration never approached levels in the range of 102 mg NO2-N * dm−3 that may be toxic for fish.

Nitrate The biofilter in land based recirculation systems is converting the excreted ammonia via nitrite to nitrate. Thus, considerable amounts of nitrate will be built up. Nitrate is usually mentioned to be the least toxic nitrogen compound and often no measures are taken to reduce the concentration in seawater recirculation systems. In fish nitrate is mentioned to be a stressor that may impair the immune system and lead to acute outbreaks of diseases. A failure of osmoregulation in marine fish as suspected for freshwater fish species is not likely. It is generally assumed that nitrate-N concentrations up to 1 g * dm−3 are tolerated. However, Amphiprion ocellaris survived at 100 mg NO3-N * dm−3 but growth was impaired. (Frakes, 1982).

Marine prawns are more sensitive to environmental nitrate than fish. A histological study in P. monodon revealed changes to nerve and muscle tissues at 1 mg NO3-N * dm−3. Exposure to 10 and 100 mg NO3-N * dm−3 affected further organs (Muir et al., 1991). However, the 48, 72, 96 h LC50 for nitrate in P. monodon juveniles are 2876, 1723, 1449 mg NO3-N * dm−3 in brackish water (≈ 15 (psu)) and 4970, 3525, 2316 mg NO3-N * dm−3 in full seawater (≈ 35 (psu)), respectively (Tsai and Chen, 2002). The safe level for rearing juvenile P. monodon was estimated by the same authors to be 145 and 232 mg/l nitrate-N in 15 and 35 (psu), respectively. In Australian broodstock maintenance for P. monodon maximum nitrate concentration is set to 30 mg NO3-N * dm−3 (N. Preston pers. comm.).

A typical problem in recirculating systems is that no denitrifying biofilter for the removal of nitrate is installed to save costs. It is hoped that the animals will tolerate the increasing nitrate content. Furthermore, the regular exchange of sea water in recirculation systems is very often sufficiently high to keep the nitrate content in certain limits; the bioengineering does often not allow to exchange less than 10% of the total seawater volume. Even if a denitrification filter is installed, the operation is too demanding especially when low nitrate levels are approached (< 100 mg NO3-N * dm−3). Under those circumstances a thorough understanding of the filter kinetic is necessary to maintain the required removal rate (substrate dosing). Plant filters are intensively investigated as they remove nitrate and phosphate at the same time.


Table 4.4.4. Ozone dosage and treatment time to inactivate bacteria, fungi, or virus (> 90 % inactivation)(with data of various authors).

S TOzone

concentrationExposure

timeResponse

psu °C mg * dm-3 minvarious seawater 0.12 1.0 100 % killvarious 12 27 0.75 10.0 100 % killPseudomonas, Vibrio seawater 0.90 80.0 100 % killAeromonas, Vibrio 0,15,35 11 0.20 3.0 99.99% inactivationEnterococcus 34.4 25 0.11 1.0 99 % inactivationPasteurella 34.4 25 0.06 1.0 99 % inactivationVibrio 34.4 25 0.06 1.0 99 % inactivationAeromonas, Pseudomonas, Yersinia freshwater 0.10 1.0 > 99 % killCeratomyxa freshwater 15 0.14 6.0 100 % eliminationAeromonas freshwater 25 0.15 1.0 > 99 % killBacillus (Spores) freshwater 25 1.00 10.0 > 99 % killAeromonas freshwater 20 0.01 10.0 100 % killStaphylococcus freshwater 25 0.67 1.5 90 % inactivationStaphylococcus freshwater 25 1.74 1.4 90 % inactivationStaphylococcus freshwater 25 2.35 0.5 90 % inactivationCandida freshwater 25 0.37 1.1 90 % inactivationCandida freshwater 25 0.80 1.1 90 % inactivationCandida freshwater 25 1.17 1.1 90 % inactivationCandida freshwater 25 1.56 0.5 90 % inactivationCandida freshwater 25 1.85 0.5 90 % inactivationIHNvirus freshwater 10 0.50 10.0 inactivationIPNvirus freshwater 10 0.20 10.0 inactivationStriped jack nervous necrosis virus seawater 0.10 3.0 100 % inactivationStriped jack nervous necrosis virus seawater 0.50 1.0 100 % inactivationIPN virus 0,15,35 11 0.20 1.0 99.99% inactivationNodavirus seawater 6 4.00 0.5 inactivationSJNN virus seawater 22 0.10 1.5 inactivationWSS Baculovirus seawater 25 0.50 10.0 inactivation

Bac

teri

aFu

ng

iV

iru

s

Type, genus

Ozone Ozone is a widely used disinfectant that is nowadays also applied in aquaculture. It reacts with all types of organic compounds in the water. This includes all living organisms (e.g., cell wall, mucous, epidermis) toward which the treatment may be directed. The electrophylic nature of ozone promotes reactions with a great variety of organic compounds. The typical reaction with organic compounds is the cleavage of carbon-carbon double bonds. Proteins for example are denaturised through the reaction with the disulfide bounds of the tertiary structure and amino acids are decomposed at the NH2 terminal end. It can be expected that all types of living tissues are attacked by ozone. Particularly the epithelia that are in direct contact with the ambient medium (carrying the ozone) are particularly affected. But, the effect of ozone isn't based on the ozone alone. The reaction products and particularly the free radicals formed in chain reactions in aqueous solutions leads to the break down of all kinds of compounds and can be more long-lasting than the ozone molecule itself. Radical reactions may enhance the impact on bacteria, fungi, or virus but must be coupled with an after treatment (Cho et al., 2002), especially in hatchery cultivation (Ozawa et al., 1991).

The disinfection of water is a widely used application of ozone. Table 4.4.4 lists the results of investigations into the inactivation of bacteria, fungi, and virus in aqueous solutions. This is beneficial in hatchery operation as ozone can be used to inactivate germs on the eggs (Grotmol and Totland, 2000) or to disinfect the seawater delivered into the cultures (Ozawa et al., 1991). Another important application is the improvement of larvae culture through the disinfection of live prey organisms (Theisen et al., 1998).

Figure 4.4.6 shows various combinations of treatment time and ozone concentration necessary for inactivation. In view to aquaculture operations treatment times of around 1 minute would require ozone concentrations between 0.06 and 4.00 mg O3 * dm−3. Ozone disinfection is temperature depending (Finch and Li, 1999) and lower temperatures may require longer treatment times and vice versa.


Figure 4.4.6: Ozone dosage and treatment time to inactivate bacteria, fungi, or virus (> 90 % inactivation)(with data of various authors, Table 4.4.4).

In view to the tolerance of different life history stages of fish to the exposure to ozone (Figure 4.4.7) the long term doses is at least one dimension below the doses necessary for inactivation. Granulated activated carbon (GAC) filtration is a suitable method for removing toxic ozone residuals or by-products (Chang et al., 2002; Lee and Deininger, 2000; Urfer et al., 2002 ) in the treated water. If ozone dosage is raised to levels that bromate is formed, the removal of by-products is much more difficult (Yang, 2000).

Figure 4.4.7. The effect of various combinations of treatment time and ozone dosage on different life history stages in fish (with data of various authors). Grey shaded area represents the treatment window for long term application.

Tor g) Effects of gas supersaturation in marine fish fry production Introduction Dissolved gas supersaturation (DGS) is an important issue that has received considerable attention especially in regard to transboundary rivers (see bibliography). DGS can lead to a physiological condition known as gas bubble trauma (GBT) in aquatic biota. GBT can be harmful or even fatal to aquatic organisms, as demonstrated by a number of significant fish kills in rivers used for hydropower. In aquatic environments that have an uncompensated hyperbaric total dissolved gas pressure exposed animals may suffer gas bubble disease (GBD). This is a non-infectious syndrome caused by small gas bubbles in the tissue that may interfere with both the circulatory system and nervous tissues.


Effects on fish When the fish are exposed to DGS gas from the water will diffuse into the fish down a partial pressure gradient. Inside the fish body the gas will “precipitate” and form micro bubbles in highly vascularized tissues as gills, and in eyes and skin. GBT will in extreme situations be acutely lethal, and in less severe situations only cause weaker performance among the fish. Bubble formation in the cardiovascular system may cause blockage of blood flow, respiratory gas exchange, and death GBT may cause over-inflation and possible rupture of the swim bladder in some species of juvenile (or small) fish, leading to death or problems of over buoyancy.

Further GBT may lead to extra corporeal bubble formation in gill lamella of large fish or in the buccal cavity of small fish, leading to blockage of respiratory water flow and death by asphyxiation. The micro bubbles almost always contain inert gases as nitrogen and argon.

Other signs of GBT in fish may include exophthalmia and ocular lesions (Blahm et al., 1975; Bouck, 1980; Speare, 1990), bubbles in the intestinal tract (Cornacchia and Colt, 1984), loss of swimming ability (Schiewe, 1974), altered blood chemistry (Newcomb, 1976), and reduced growth (Jensen, 1988; Krise et al., 1990), all of which may compromise the survival of fish exposed to DGS over extended periods.

DGS in intensive systems Very few papers describe the effects of long term low level DGS that typically exists in intensive aquaculture systems. Intensive systems require water treatment and pumping. In these processes the risk of making DGS is impeding. Small leaks in dry mounted pumps may act as venturies and draw gas into water under pressure. Also, in situations of uncontrolled accidental draining of header tanks high values of DGS have been recorded. Acute exposure to high values of DGS is commonly known to be dramatic in both marine and fresh water aquaculture. Under normal circumstances the problem with DGS seems to be small. A normal value for total gas pressure (TGP) in intensive open systems is typically between 100 and 102 %, i.e., far below what is in the recommended Canadian limits for river water on salmonids.

From literature information it is evident that there are differences between both species and stages of marine fish. In cold water marine aquaculture the industry has long suspected low level DGS to be one of the main causes for frequent mass mortalities and low resistance towards infectious diseases like VER caused by a noda-virus. This has typically been the case for the halibut industry. Over inflation of swim bladder among cod post larvae are commonly seen in hatcheries. This condition called “floaters” will eventually cause the death of the fish, and may on a larger scale often cause a significant loss for the hatchery.

Although the problem of “fish bends” was described already more than 60 years ago by Dannevig, the knowledge of this topic in the industry is scarce. Most of the reason for this is that good experimental data more or less is absent. Also methods for assessing gas saturation have been both expensive and inaccurate. There are also special cases of DGS in intensive systems that make them more difficult to interpret. Normally atmospheric air will dissolve in a body of water due to the partial pressures of the different gases that the atmosphere contains. DGS will therefore in “natural” situations most often also be oversaturated with Nitrogen. However, in intensive systems the use of oxygen addition in order to intensify the production units is common. Oxygen saturation levels of 120% in the inlet water is nor uncommon. Although a TPG higher than 100% include a risk of GBT, water supersaturated with oxygen but sub saturated with N2 will not necessarily represent a biohazard. The rapid growth in the intensive marine aquaculture industry therefore has created a new situation where new aspects of gas saturation must be addressed Methods for degassing Aeration of production water has been carried out in different systems for a long time. Most of these devices operate on a passive basis, e.g., equilibration of water to atmospheric air both to remove excess nitrogen, and to saturate the water with oxygen. For this purpose the surface of the water and the holding time determine the efficiency of the aerator. These systems in best cases equilibrate the water with the air, but in most cases they are not efficient to remove a supersaturation of Nitrogen gas.

For this purpose the vacuum-degasser has gained increased popularity. With these setups gas is extracted to the required level. References are listed in bibliography in Annex 4. Conclusions and recommendations New equipment is now available to measure levels of super saturation of the different gases, and should be employed in fish hatcheries.

Measurement of gas saturation levels should be standard in experimental work to ensure optimal water physical conditions. This has to date been neglected and may cause bias in experimental results concerning effects on growth and survival of fish.

• There is a need to examine the effects of low level sub lethal DGS on the different marine fish species including different developmental stages

• There is a need to develop more efficient methods for removing excess nitrogen gas from production water


5 Conclusions

The group discussed the future of the WG and the work it should focus on. Fish welfare was brought foreword as a valuable topic for the group to pick up again and further develop. This topic was reviewed for the 2001 report and discussed briefly at the 2002 meeting in Faro, Portugal. The EU has started drafting conditions for fish welfare some four years ago but has not yet finalised its work. The absence of scientific assessments of welfare has led to fish welfare conditions being developed by how this is perceived rather than relying on documented data. The experience of the ICES scientists working on fish farming aspects, closely cooperating with fish farmers, also with extensive experience on what constitutes good rearing conditions for fish, would be a valuable contribution towards developing guidelines for ‘Responsible Marine Fish Farming’(ReMarF) and improve the science-based knowledge for developing guidelines for good fish welfare. It could help in identifying research needs and develop protocols for fish husbandry, fish transport, fish sorting, etc.

Topics could include improved techniques for natural spawning of marine species to provide regular production of quality eggs, measures through feeding regimes to mitigate stress and dominance behaviour and finding the balance between cost-effectiveness and fish welfare in intensive productions. This topic would focus the remit of the WG as well as utilise the expertise within the WG, i.e., fish physiology, fish behaviour and production technology expertise. It would exclude issues dealt with in other WG such as health (disease aspects), genetics and environmental effects or refer these to the relevant WG. In essence the WG has been working with different aspects of fish welfare, and has the necessary expertise to cover physiological, behavioural and technological aspects in an integrated manner and thus describe conditions which would be most favourable to fish while achieving production levels for a sustainable mariculture.

The WG suggests examining the status of Fish Welfare concerns and issues in the different ICES countries, the status of the EU draft on the conditions for fish welfare and to identify research needs for developing this field. Further the WG suggest developing guidelines for ‘Responsible Marine Fish Farming’ in different types of farming units.

6 Recommendations and meeting resolution

The Working Group on Marine Fish Culture (WGMAFC) (Chair: Dr Anders Mangor-Jensen) proposes to meet in Scotland middle may 2005 to:

a) Report on the status of fish welfare policies in the different ICES countries b) Report on the development of EU policy on fish welfare. c) Initiate work on developing guidelines for “Responsible Marine Fish Farming” aimed at publishing the

completed work as an ICES Cooperative Research Report. The guidelines will address; different species, life stages and rearing systems, and include topics such as:

• Spawning • Egg production • Feeding • Environmental parameters such as water quality • Rearing technology • Husbandry techniques • Health management and biosecurity

d) Compile information on the current state of the art of microdiets as a replacement for live food for larval fish. (This TOR is considered very important and should have been addressed at the 2004 meeting but will be addressed at the next meeting).

WGMAFC will report by 25 May 2005 for the attention of the Mariculture and the Diadromous Fish Committees, and ACME.


Supporting Information Priority: The current activities of this Group will lead ICES into issues related to the political and

biological important issue of fish welfare. The legislation within this field is under development both in EU and other ICES contries. Consequently these activities are considered to have a very high priority.

Scientific Justification :

Action Plan Nos. 2.4, 2.5, 2.6, 2.7, 3.8, 3.9, 3.10, 3.11, 3.14, and 4.7. Term of Reference a) Review and compare the present fish welfare policy within the different ICES countries. This issue is important to address in order to find how the different counties put their fish welfare legislation into practice. The use of live fish for research purposes are well regulated in most ICES countries, but guidelines for welfare in aquaculture production also need to be compared not the least for marketing purposes. Term of Reference b) The EU has started drafting conditions for fish welfare some four years ago but has not yet finalised its work. The absence of scientific assessments of welfare has led to fish welfare conditions being developed by how this is perceived rather than relying on documented data. Term of Reference c) The experience of the ICES scientists working on fish farming aspects, closely cooperating with fish farmers, also with extensive experience on what constitutes good rearing conditions for fish, would be a valuable contribution towards developing guidelines for ‘Responsible Marine Fish Farming’(ReMarF). The WGMAFC will initiate this work that is planned to continue for more than one period. This topic will utilise the expertise within the WG, i.e. fish physiology, fish behaviour and production technology expertise. It would exclude issues dealt with in other WG such as health (disease aspects), genetics and environmental effects or refer these to the relevant WG. In essence the WG has been working with different aspects of fish welfare, and has the necessary expertise to cover physiological, behavioural and technological aspects in an integrated manner and thus describe conditions which would be most favourable to fish while achieving production levels for a sustainable mariculture Term of reference d) This important question was not addressed at the 2004 meeting, and should be considered also in light of fish welfare. A formulated dry feed differs from natural food sources also in water content and texture. Small pelagic fish larvae experience an increased osmotic stress eating a dry pellet compared to natural plankton. Later stages that have fully developed extrarenal capacity will probably be able to cope. The development of formulated diets to

Resource Requirements:

The resources required to undertake the proposed activities are within the group.

Participants: The Group is normally attended by 15–20 members and guests Secretariat Facilities:

None

Financial: No financial implications Linkages To Advisory Committees:

There are no obvious direct linkages with the advisory committees


7 Annexes

Annex 1 List of the participants at the 2004 WGMAFC meeting in Vigo. Name Institute and address Contact Tito Peleteiro Instituto Español de Oceanografia, Spain [email protected]

Olvido Chereguini Instituto Español de Oceanografia, Spain [email protected] Mercedes Olmedo Instituto Español de Oceanografia de Vigo,

Spain [email protected]

Inés García de la Banda Instituto Español de Oceanografia, Spain [email protected] Fátima Linares Cuerpo Centro de Investigacións Marinas. Consellería

de Pesca. Xunta de Galicia [email protected]

Victor Øiestad Akvaplan-niva [email protected] Jose Iglesias Instituto Español de Oceanografia, Vigo,


Tim Bowden

FRS Marine Laboratory, Victoria Road, Aberdeen, AB11 9DB, Scotland, UK

[email protected]

Josianne Støttrup Danish Institute for Fisheries Research, Department of Marine Ecology and Aquaculture

[email protected]


mailto:[email protected]



Annex 2 List of WGMAFC members

List of WGMAFC members. 02-27-2004 List for WGMAFC/ Dr Craig Clarke Dr. Stephen Baynes Dept. of Fisheries & Oceans CEFAS Weymouth Laboratory Pacific Biological Station Barrack Road, The Nothe Hammond Bay Road Weymouth, Dorset DT4 8UB Nanaimo, B.C. V9R 5K6 United Kingdom Canada [email protected] [email protected] Dr David Bengtson Peter Coutteau Dept. of Fisheries, Animal and Artemia Reference Center Veterinary Science Lab. of Aquaculture, University of Ghent University of Rhode Island Rozier 44 Kingston, RI 02881 9000 Ghent USA Belgium [email protected] [email protected] Dr B. Bjornsson Daan Delbare Marine Research Institute CLO Sea Fisheries Department P.O. Box 1390 Ankerstraat 1 Skúlagata 4 B-8400 Ostende IS-l21 Reykjavík Belgium Iceland [email protected] [email protected] M. Gillespie T. Bowden Seafish Marine Farming Unit Fisheries Research Services Artoe, Acharcle, Argyll Marine Laboratory United Kingdom P.O. Box 101 [email protected] Victoria Road Aberdeen AB11 9DB Reinhold Hanel United Kingdom Institut für Meereskunde [email protected] an der Universität Kiel Düsternbrooker Weg 20 Dr Lawrence Buckley D-24105 Kiel URI/NOAA CMER Program Germany Graduate School of Oceanography [email protected] University of Rhode Island Narragansett, RI 02882 Torstein Harboe USA Inst. of Marine Research [email protected] Austevoll Aquaculture Res. Station N-5392 Storebø Ms Béatrice Chatain Norway IFREMER [email protected] Station de Palavas, Recherche Aquacole Chemin de Maguelone Dr J. Iglesias 34250 Palavas-Les-Flots Inst. Español de Oceanografía France Centro Oceanográfico de Vigo [email protected] Apdo 1552 E-36280 Vigo



Dr P. Lavens Prof. Victor Øiestad Rijksuniverstieit Ghent The Norwegian College of Rozier 44 Fishery Science 9000 Ghent University of Tromsø Belgium 9037 Tromsø [email protected] Norway [email protected] Dr Anders Mangor-Jensen Austevoll Marine Aquaculture Stat. J.B. Peleteiro 5393 Storebø Inst. Español de Oceanografía Norway Centro Oceanográfico de Vigo [email protected] Apdo 1552 E-36280 Vigo Ms Debbie Martin-Robichaud Spain Dept. of Fisheries & Oceans [email protected] Biological Station 531 Brandy Cove Road Randy Penney St Andrews, NB ESB 2L9 Research Scientist Canada Aquaculture Research Section [email protected] Ocean Programs Division Science, Oceans & Environment Branch Teje van der Meeren Dept. of Fisheries and Oceans Inst. of Marine Research P. O. Box 5667 Austevoll Aquaculture Res. Station St. John's, NL Canada A1C 5X1 N-5392 Storebø tel: 709-772-4704 Norway fax 709-772-5315 [email protected] e-mail [email protected] Dr A. Mitans Ms Jeannine Person-Le-Ruyet Latvian Fish. Res. Inst. IFREMER Daugavgrivas Street 8 Centre de Brest LV-1007 Riga BP 70 Latvia F-29280 Plouzané [email protected] France [email protected] Gerry Mouzakitis Aquaculture Development Centre Dean Perry Dept. of Zoology & Animal Ecology Milford Laboratory UCC NEFC/NMFS Lee Maltings 212 Rogers Avenue Prospect Row Milford, CT 06460 Cork USA Ireland [email protected] [email protected] P. Pousao-Ferreira Ms I. Opstad IPIMAR Inst. of Marine Research Av. 5 de Outubro Austevoll Aquaculture Res. Station 8700 Olhao N-5392 Storebø Portugal Norway [email protected] [email protected]


Dr A. Ramos Dr Uwe Waller IPIMAR Institut für Meereskunde Avenida de Brasilia an der Universität Kiel P-1449-006 Lisbon Düsternbrooker Weg 20 Portugal D-24105 Kiel [email protected] Germany [email protected] Dr P. Sorgeloos Lab of Aquaculture & Artemia Reference Center Ghent University

Antonio García Instituto Español de Oceanografía

Rozier 44 Centro Oceanográfico de Murcia B 9000 Gent Ctra. de la Azohía, s/n Belgium 30260 Mazarrón (Murcia) [email protected] SPAIN [email protected] Dr Josianne G. Støttrup Dept. of Marine Ecology and Aquaculture Mercedes Olmedo Kavalergaarden 6 Inst. Español de Oceanografía 2920 Charlottenlund Centro Oceanográfico de Vigo Denmark Cabo Estay - Canido [email protected] Apdo 1552 E-36200 Vigo Dr Ed Trippel Spain Dept. of Fisheries & Oceans [email protected] Station 531 Brandy Cove Road St Andrews, NB ESB 2L9 Canada [email protected] Simon Wadsworth Seafish Aquaculture Marine Farming Unit Ardtoe, Archacle Argyll PH36 4LD United Kingdom [email protected]


Annex 3 Research and Technology of Single-sex Fish Production and Application to Marine Fish Culture Species Common

Name Preferred Sex

Why? Commer-cial (Y/N)

Country Research Scientists/Country

Acipenser transmontanus

White sturgeon

female enhanced caviar production

no Canada direct feminization

Henry/Devlin (Canada)

Anoplopoma fimbria

Sablefish Female Faster growth, later maturation

no Canada none

Dicentrarchus labrax

Sea bass female Faster growth, later maturation

no Spain sex determination and differentiation, environmental control

Piferrer/Blázquez (Spain)

Hippoglossus hippoglossus

Atlantic halibut

female faster growth no Canada indirect feminization, gynogenesis, sex determination

Benfey/Martin-Robichaud (Canada)


Atlantic halibut

female faster growth, late maturity

no Norway temperature-induced feminization, regulation of asynchronous oocyte development

Andersen/van-Nes (Norway)


Atlantic halibut

female Faster growth in years 2–3

no UK indirect feminization, gynogenesis, sex determination

Howell/Baynes (UK)

Scophthalmus maximus

Turbot female Faster growth in years 2–3

no UK, Spain

indirect feminization, gynogenesis, sex determination, triploids

Howell/Baynes (UK) Cal R./M.Portela/Pi Ferrer (Spain)

Scophthalmus maximus

Turbot female Faster growth no Spain Indirect feminization, gynogenesis

Piferrer/Cal/Martínez (Spain)

Solea senegalensis

Senegal sole

female Faster growth no Spain Direct and indirect feminization, sex differentiation

Piferrer/Cerdà/Planas (Spain)

Solea solea Dover sole

female Faster growth in years 2–3

no UK gynogenesis, sex determination

Howell/Baynes/Thompson (UK)

Oncorhynchus tshawytscha

Chinook salmon

female removal of precocious males; use in environmental monitoring for masculinization effects of endocrine disruptors

yes Canada indirect feminization, use of Y markers for genetic sex selection

Devlin/Aquaculture producers/Environment Canada (Canada)

Oncorhynchus tshawytscha

Chinook salmon

male faster growth of males; use in environmental monitoring for feminization effects of endocrine disruptors

no Canada YY males used for production of all male populations

Devlin/Environment Canada (Canada)

Oncorhynchus kisutch

Coho salmon

female enhanced roe production

yes Canada indirect feminization, use of Y markers for genetic sex selection

Henry/Devlin (Canada)


Annex 4 References and bibliography

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estradiol-17 beta and 17 alpha-ethynylestradiol on the gonads of the gonochoristic teleost Dicentrarchus labrax. Fish Physiol. Biochem. 18:37–47.

Blázquez, M., M. Carrillo, S. Zanuy and F. Piferrer. 1999. Sex ratios in offspring of sex-reversed sea bass and the relationship between growth and phenotypic sex differentiation. J. Fish Biol. 55: 916–930.

Blázquez, M., A. Felip, S. Zanuy, M. Carrillo and F. Piferrer. 2001. Critical period of androgen-inducible sex differentiation in a teleost fish, the European sea bass. J. Fish Biol. 58: 342–358.

Bye, V.J. and R.F. Lincoln. 1986. Commercial methods for the control of sexual maturation in rainbow trout (Salmo gairdneri R.) Aquaculture 57:299–309.

Chatain, B., E. Saillant and S. Peruzzi. 1999. Production of monosex populations of European seabass, Dicentrarchus labrax L. by use of synthetic androgen 17α-mehtyldehydrotestosterone.

Devlin, R.H. and Y. Nagahama. 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208(3–4): 1–366.

Donaldson, E.M. 1986. The integrated development and application of controlled reproduction techniques in Pacific salmonid aquaculture. Fish Physiol. Biochem. 2: 9–24.

Ezaz, M.T., J.M. Myers, S.F. Powell, B.J. McAndrew and D.J. Penman. 2004. Sex ratios in the progeny of androgenetic and gynogenetic YY male Nile tilapia, Oreochromis niloticus L. Aquaculture 232: 205–214.

Felip, A., F. Piferrer, M. Carrillo and S. Zanuy. 1999. The relationship between the effects of UV light and thermal shock on gametes and the viability of early developmental stages in a marine teleost fish, the sea bass (Dicentrarchus labrax L.) Heredity 83: 387–397.

Felip, A., S. Zanuy, M. Carrillo and F. Piferrer. 2001. Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species. Genetica 111: 175–195.

Godwin, J., J.A. Luckenbach and R.J. Borski. 2003. Ecology meets endocrinology: environmental sex determination in fishes. Evol. Dev. 5(1): 40–49.

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Howell, B.R., S.M. Baynes and D. Thompson. 1995. Progress towards the identification of the sex-determining mechanism of the sole, Solea solea (L.), by the induction of diploid gynogenesis.

Luckenbach, J.A., J. Godwin, H.V. Daniels and R.J. Borski (2002) Optimization of North American flounder culture: A controlled breeding scheme. World Aquaculture 33: 40–49, 69.

Luckenbach, J.A., J. Godwin, H.V. Daniels, R.J. Borski. 2003. Gonadal differentiation and effects of temperature on sex differentiation in southern flounder (Paralichthys lethostigma). Aquaculture 216: 315–327.

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Nakamura, M. 2000. Endocrinological studies on sex differentiation and reproduction in fish. Nippon Suisan Gakkaishi 66(3); 376–379.

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Piferrer, F. 2001. Endocrine sex control strategies for the feminization of teleost fish. Aquaculture 197: 229–281. Pavlidis, M., G. Koumoundouros, A. Sterioti, S. Somarakis, P. Divanach and M. Kentouri. 2000. Evidence of

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Saillant, E., A. Fostier, B. Menu, P. Haffray and B. Chatain. 2001. Sexual growth dimorphism in sea bass Dicentrarchus labrax. Aquaculture 202: 371–387.

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Zarzosa, G., Moreno, F. 2001. Temperature effects on muscle growth in two populations (Atlantic and Mediterranean) of sea bass, Dicentrarchus labrax L. Aquaculture 202(3–4): 359–370.

Blazquez, M., Zanuy, S., Carillo, M., Piferrer, F. 1998. Effects of Rearing Temperature on Sex Differentiation in the European Sea Bass (Dicentrarchus labrax L.). J. Exp. Zool. 281(3): 207–216.

Bower, C.E., Bidwell, J.P. 1978. Ionization of ammonia in seawater: effects of temperature, pH, and salinity. J. Fish. Res. Board Can. 35: 1012–1015.

Brownell, C.L. 1980. Water quality requirements for first-feeding in marine fish larvae. 1. Ammonia, nitrite, and nitrate. J. Exp. Mar. Biol. Ecol. 44(2–3): 269–283.

Chang, E.E., Liang, C.-H., Ko, Y.-W., Chiang, P.-C. 2002. Effect of Ozone Dosage for Removal of Model Compounds by Ozone/ GAC Treatment. Ozone Science and Engineering 24: 357–367.

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Chen, H.Y., Chen, Y.L. 1999. Temperature preferendum of postlarval black tiger shrimp (Penaeus monodon). Marine and Freshwater Research 50(1): 67–70.

Chen, J.-C., Chin, T.-S. 1988. Acute toxicity of nitrite to tiger prawn, Penaeus monodon , larvae. Aquaculture 69(3–4): 253–262.

Chervinski, J. 1974. Sea bass, Dicentrarchus labrax Linne, (Pisces, Serranidae) a "police-fish" in freshwater ponds and its adaptability to various saline conditions. Bamidgeh 26(4): 110–113.

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Finch, G.R., Li, H. 1999. Inactivation of Cryptosporidium at 1 degree C Using Ozone or Chlorine Dioxide. Ozone Science & Engineering 21: 477–486.

Frakes, T., Hoff, F. H. Jr. 1982. Effect of high nitrate-N on growth and survival of juvenile and larval anemonfish, Amphiprion ocellaris. Aquaculture 29: 155–158.

Francois, N., Dutil, J.D., Blier, P., Lord, K., Chabot, D. 2001. Tolerance and growth of juvenile common wolffish (Anarhichas lupus) under low salinity and hypoxic conditions: preliminary results, pp. 57–59. Bulletin of the Aquaculture Association of Canada.

Gibson, S., Johnston, I.A. 1995. Temperature and development in larvae of the turbot Scophthalmus maximus. Mar. Biol. 124(1): 17–25.

Grotmol, S., Totland, G.K. 2000. Surface disinfection of Atlantic halibut Hippoglossus hippoglossus eggs with ozonated sea-water inactivates nodavirus and increases survival of the larvae. Dis. Aquat. Org. 39: 89–96.

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Annex 5 Restocking Stocking in Denmark. Josianne G. Støttrup In Demark releases of a number of freshwater and marine species take place yearly with the aim of enhancing wild fish stocks in local areas. Funds for the releases are provided through sports and recreational fishery licences and the Danish Institute for Marine Research is responsible for all administrative and scientific work related to stocking. An overview on Danish stocking is given by Rasmussen and Geertz-Hansen (1998). In marine waters or brackish waters, elvers Anguilla anguilla, turbot Scophthalmus maxima and flounder Platichthys flesus are released.

Turbot and flounder are reared in semi-extensive units using copepods and other zooplankton as live prey during the larval stage (Engell-Sørensen et al. in press).

Turbot has been used as a model species to examine the potential for stocking and the results showed similar growth and survival in wild and released reared fish and no replacement of the wild fish was observed (Støttrup et al., 2002). Differences in spatial and temporal dispersion after releases of similar sized fish in different areas were supposed to be related to habitat quality (Støttrup et al, 1998). Further studies to examine habitat suitability and carrying capacity have also provided data on mortality during the first few days immediately after the release (Sparrevohn et al., 2002). Further work is now focused on decreasing this post-release mortality. Work is also focused on habitat suitability and other ecological considerations, where it has been shown that the potential for stocking may differ between species depending on their population dynamics (Støttrup, 2004).

Concern for unintentional genetic impacts has resulted in cooperation with geneticists who have investigated the genetic makeup of the two marine species currently being stocked as well as looked into possible genetic consequences from stocking and recommendations for their avoidance (Hansen, 1996). Turbot was shown to belong to either the Baltic or the North Sea stock, with a mixture of these two stocks in the transitional waters of Kattegat and Skagerak and the percentage of each of the two stocks declining with distance from its origin (Nielsen et al., 2004). Work on flounder genetics is currently underway. References Engell-Sørensen, K., Støttrup, J.G., Holmstrup, M. In press. Rearing of flounder (Platichthys flesus) juveniles in semi-

extensive systems. Aquaculture. Hansen, M.M. Grundlaget for fiskeudsætninger i Danmark. 1996. DFU-rapport 28–96. pp. 59. In Danish. Nielsen, E.E., Nielsen, P.H., Meldrup, D., Hansen, M.M. 2004. Genetic population structure of turbot (Scophthalmus

maximus L.) supports the presence of multiple hybrid zones for marine fishes in the transition zone between the Baltic Sea and the North Sea.

Rasmussen, G., Geertz-Hansen, P. 1998. Stocking of fish in Denmark. In Stocking and introduction of fish, pp. 14-21. Ed. by I.G. Cowx. Blackwell Science, UK.

Sparrevohn, C.R., Nielsen, A., Støttrup, J.G. 2002. Diffusion of fish from a single release point. Canadian Journal of Fish and Aquatic Sciences, 59(5): 844–853.

Støttrup, J.G. 2004. Feats and defeats in flatfish stocking: determinants for effective stocking. In Stock Enhancement and Sea Ranching, 2nd Edition: Developments, Pitfalls and Opportunities. Ed. by K. M. Leber, S. Kitada, H. L Blankenship and T. Svåsand. Blackwell Publishing. Chapter 6.

Støttrup, J.G., K. Lehmann and H. Nicolajsen. 1998. Turbot, Scophthalmus maximus, stocking in Danish coastal waters. In Stocking and introduction of fish, Chapter 26: 301–318. Ed by I.G. Cowx.

Støttrup, J.G., Sparrevohn, C.R., Modin, J., Lehmann, K. 2002. The use of releases of reared fish to enhance natural populations. A case study on turbot Psetta maxima (Linné, 1758). Fisheries Research, 59/1–2: 161–180.

Portuguese experience on restocking using reared fish Miguel Neves dos Santos , Portugal The Portuguese experience on restocking using reared fish is recent. It only started in 1997, using a native species for which the artificial reproduction and juvenile production was a standard procedure – the gilthead seabream (Sparus aurata).

The first trials aimed the optimisation of tagging (tags type and size) and release techniques. The first restocking trial occurred in summer 1997, with the release of 600 specimens off the Ria Formosa (Algarve, southern Portugal). Later, in 2000 these experiments were extended to a new native species – the white seabream (Diplodus sargus) and to new areas. The fish started to be release at the different artificial reefs of the Algarve coast.

In 2001 a 3-year project was funded by the Fundação para a Ciência e Tecnologia – Project RESTOCK – Fish restocking associated to the Algarve artificial reefs: environmental mitigation, biodiversity and fisheries management (POCTI /1999/BSE/35608). The project objectives were related to the above mentioned practical aspects, in particular: (i) evaluate the efficiency of restocking of finfish species associated with artificial reefs, using native species where the


artificial reproduction and juvenile production are standard procedures, and (ii) develop the methodology necessary for the production in captivity of juveniles of other native with interest for restocking of artificial reefs. The evaluation of fish restocking in artificial reefs areas is done taken into account the size of the specimens, the season and the release location. Classic methodologies are in use in this project ongoing project, based on the analysis of catch (recapture) and direct observation (underwater surveys) carried out in a regular basis. This restocking initiative has been advertised to the general public, in particular fishermen and anglers, to maximise the recapture records. So far near 13,000 specimens have been released, of which 6512 correspond to the white seabream. There size ranged from 10.5–25.0cm (24–350g) and 15.6–23.2 cm (34–416g), for S. aurata and for D. sargus, respectively.

In late 2003 a new 3-year project was funded by local authorities – Project GESTPESCA – Bases científicas para a gestão de pescarias litorais (Program INTERREG III-A). This project has similar objectives and will allow to extend the restocking experiments to other species which are currently being produced at IPIMAR’s aquaculture facilities in Olhão. These include 3 other species of seabreams (D. cervinus, D. puntazzo and D. vulgaris). The trials will start in the summer of 2004. Report on the production of cultured fish to restock or improve wild stocks in Spain J.B. Peleteiro (Instituto Español de Oceanografía, Spain) In the present time, there is no general restocking programme or any programme to improve wild stocks using cultured fish in Spain. Nevertheless, a series of restocking experiments have been carried out in the northern and southern regions of the Iberian Peninsula with good results, and this makes it possible to establish a serious restocking programme.

In this sense, the Galician Autonomous Community has recently organized a Workshop to analyse the possibilities of a restocking programme based in the experience from other countries, specially Japan. The enclosed report is the result of this Workshop.

Besides, a series of experiments of tagging and releases of turbot (Scophthalmus maximus) has been carried out in the north-western region of the Iberian Peninsula (Ría de Vigo) and with gilthead seabream (Sparus aurata) in the gulf of Cadiz (south-west of the Iberian Peninsula). Turbot Works carried out with turbot in the north-western region were based in two experiments. In the first experiment, the capacity of in captivity-cultured individuals to adapt themselves to the natural environment and their recovery possibilities were studied.

With this aim, 3000 cultured individuals in age I (15 cm) were tagged using T-BAR tags, from which 9.5 % were recovered in the first year. Despite of the fact that this is a species that scarcely moves around, some individuals were found at a distance of 350 km south from the tagging area.

The second experiment was carried out to compare the mortality between wild individuals and cultured individuals that had been tagged and released in the sea, and to study the natural populations dynamics and ecology. With this aim, 438 wild individuals previously caught and 594 individuals that had been cultured using the traditional green water culturing technique were released. The individuals used in this experiment had a size of 4–6 cm. It was determined that, although tagging does not affect the individuals survival in the natural environment, the mortality rate in cultured fish was significantly higher.

It is advisable to release individuals with a size between 6 and 10 cm long that have been previously got used to the sand and predators. September is considered the best month for the release.

Gilthead seabream The Consejería de Agricultura y Pesca belonging to the Junta de Andalucía carried out a gilthead seabream restocking in the Andalusia south Atlantic coastal. With this aim, 32.414 individuals cultured in the Toruño centre (Cádiz) between 1993 and 1998 were released, and up to 6% of tagged individuals were recovered. It is advisable to use 100 g individuals for restocking.

It was proved that gilthead seabream has a high learning ability, it develops a fast antipredator ability and a clear social behaviour necessary to form shoals.

“T” tags with retention rates of 80/100 % were used.

Blackspot seabream Between 1997 and 1998, releases of blackspot seabream (Pagellus bogaraveo) were carried out exclusively using wild individuals from the natural environment. 3018 juvenile individuals 12/28 cm long were tagged, with a recapture rate of 4 %. During 2001/2002, 1602 adult blackspot seabreams 21–52 cm long (96 % < 40 cm) were tagged and the recapture rate was 10% of the total of individuals tagged.

All these recaptures were carried out in the Gibraltar strait by the commercial fleet which exploits this species. It must be highlighted that recaptures are monthly reported. This fact allows us to have an idea of the high fishing mortality of which the exploited resource is subject.

As a consequence of the excessive exploitation this stock that suffers, and due to its biological characteristics, it is a candidate species for the restocking programme.


References Iglesias, J, and Rodríguez-Ojea, G. 1994. Fitness of hatchery- reared turbot (Scophthtalmus maximus) for survival in the

sea: first results on feeding, growth and distribution. Aquaculture and Fisheries Management, 25 (1): 179–188. Iglesias, J., Ojea, G., Otero, J. J. and Fuentes, F. 2003. Comparison of mortality of wild and released reared 0-group

turbot, Scophthalmus maximus L., on an exposed beach (Ría de Vigo, NW Spain) and a study of the population dynamics and ecology of natural population. Fisheries Management and Ecology, 10:51–59.

Gil, J. and Sobrino, I. In press. La pesquería del voraz (Pagellus bogaraveo) en el estrecho de Gibraltar. Acuicultura Pesca y Marisqueo en el Golfo de Cádiz a Comienzos del Siglo XXI. Junta de Andalucía.

Sánchez de Lamadrid. 2002. Comportamiento y repoblación de la dorada (Sparus Aurata, Linnaeus 1758) en el litoral suratlantico andaluz. Universidad de Cádiz, Facultad de Ciencias del Mar. Tesis 142 pp.

Fitness of hatchery-reared turbot, Scophtalmus maximus L., for survival in the sea: first year results on feeding, growth and distribution

Authors: Iglesias, J., and Rodríguez-Ojea G. Aquaculture and Fisheries Management. 1994. Vol. 25(1): 179–188. This study, carried out with 3,000 hatchery-reared I-group individuals (15 cm) released in the Ria de Vigo (NW Spain) has reached the following conclusions:

In spite of the fact of being maintained on a dry meal diet, two weeks after being released, they were actively feeding on live fishes. Fishes constitute the 96% of their diet, with a remarkable presence of Ammodytes tobianus (sand eel) in the gut contents (78%)

The juveniles released in the area of the ría where they naturally occur (exposed beaches, clean, sandy bottoms and outermost part of the ría), behave mostly in a sedentary way regarding the release area. On the contrary, if they are released in greater depths on the continental shelf (outside Cies or Ons islands), their distribution range broadens significantly, showing important migrations, up to 350 km, along Galician and Portuguese coasts.

Despite the fact of being tagged, their growth rate is not affected. Their growth at sea is similar to that obtained in captivity, increasing from 158 g to a final mean weight of 1100 g fourteen months later.

The recapture rate during the first year was 9.5%, which is acceptable and might have a positive effect on the recruitment for fisheries. Nevertheless, in order to verify this hypothesis, an large increment in the number of released fishes should be performed.

Finally, it is advisable the release of smaller-sized individuals in order to reduce the costs of a future enhancement programme. Comparison of mortality of wild and released reared 0-group turbot, Scophtalmus maximus L., on an exposed beach (Ría de Vigo, NW Spain) and a study of the population dynamics and ecology of the natural population. Authors: José Iglesias, Gonzalo Ojea, Juan José Otero, Lidia Fuentes and Tim Ellis Fisheries Management and Ecology. 2003. Vol 10: 51–59. This study continued the stock enhancement exercises with turbot in the Galician rías, consisting in the release of individuals of a smaller size than those described in the previous chapter. Therefore, 0-group individuals (4–6 cm) were released in the outermost part of the ría de Vigo. Besides, natural population dynamics was studied in the sampling area on a monthly basis.

In order to establish a comparison of mortality between wild and reared turbot, 438 individuals were captured on a beach of the Ría de Vigo, marked in the laboratory and released two days after being captured, together with 594 marked reared turbot of approximately the same age. The area was sampled at regular intervals of 40 days. According to the data obtained from the recaptures of marked wild, reared marked and unmarked wild individuals, it was possible to estimate that marking does not affect mortality in turbot juveniles, however, daily instantaneous mortality rate was found to be significantly higher (p>0.01) in reared fishes. This was probably due to a poor adaptation of reared fishes to the conditions of the natural habitat, regarding their ability to bury themselves and their greater vulnerability to predators in relation with wild individuals. A previous acclimatisation to sandy substrata and to the presence of predators would considerably reduce the mortality of reared individuals, as it has been already observed in other flatfish species, such as plaice (Pleuronectes platessa) and sole (Solea solea) when they are released in the natural environment.

Therefore, for future stock enhancement experiences it would be advisable for reared individuals to go through a period of pre-release adaptation which would include acclimatisation to sandy substrata and to the presence of predators, with the aim of reducing the high mortality observed during the first days after release.

Regarding turbot natural population, young individuals with a mean length of 3.0 mm appeared on the beach in March, staying in the area until June of the following year, when they reached between 12 and 15 cm. Once they reach this size, they abandon the shallow waters, searching for greater depths. Feeding of turbot alevins consists


fundamentally in small crustaceans (96.5% in the frequency of occurrence), polychaetes and fishes. The ingestion of fishes increases as alevins reach more than 6 cm of total length.

Taking into consideration the data belonging to mortality and natural population abundance, it is therefore recommended for future experiences to release juveniles between 6 and 10 cm of total length, previously acclimatised to sand and predators, from September to December, when the size of individuals of the natural population is similar. Stock enhancement with gilthead sea bream (Sparus aurata) on the Andalusian South Atlantic coast Author: Dr Alfonso Sánchez de Lamadrid Rey. C.I.C.E.M. El Toruño. Consejería de Agricultura y Pesca. Junta de Andalucia (Agriculture and Fisheries Counselling Bureau. Andalusian Regional Government) A greater profitability of the natural sea productivity can be achieved through stock enhancement experiences with reared fishes. This technique is common in some countries of the world, aiming at increasing fishery production within the stocked species. Nevertheless, their efficiency has not always been assessed, either because the fishes had not been tagged or the objective has not been achieved due to a lack of behavioural adaptation of the fishes to the new environment, especially regarding their ability to escape from predators and to feed on living prey.

This study has proved that reared gilthead sea bream has great learning abilities, developing in only one experience an anti-predator capacity. Furthermore, reared gilthead sea bream, principally fed on fishmeal, learn from experience whenever they have the opportunity to feed on living prey. It also proves that gilthead sea bream shows a social behaviour, needed for schooling. Behaviour in gilthead sea bream can also be modified, through conditioning by a 300 Hz sound linked to feeding and be attracted using substances such as mollusc extracts. Therefore, this species can easily adapt to the natural environment.

Several experiments were carried out to choose long-life marks for gilthead sea bream, easily detectable by fishermen. Ink marking, alevin record and operculum marking were discarded. 1.2 mm-filament “T”-tags were found to be adequate, since they present a retention rate between 80–100% after seven months. It is a plastic tag that includes a phone number to inform about the recaptures and the tag number. Besides, alizarin complexone can be used for marking in the event of restocking with gilthead sea bream eggs or larvae, which produces a fluorescent mark in the otoliths, that can be detected in the 100% of the fishes after 17-months rearing.

Once it became known that gilthead sea bream can easily adapt to the new environment and that the marking method is adequate, it was carried out a stock enhancement experience on the Andalusian South Atlantic coast, between the Guadiana river and the bay of Algeciras. Weight and release period, together with the most suitable areas to carry out restocking with gilthead sea bream, were the main aspects studied. For this reason, 32,414 marked individuals were released between 1993 and 1998, during 36 restocking exercises carried out in the main estuaries and marsh areas throughout the region. A restocking exercise is considered to be effective when high recapture rates are obtained, in spots close to the release area, during a long period and registering a high growth. The bay of Cadiz was the principal area for restocking exercises with gilthead sea bream on the Andalusian South Atlantic coast. A high proportion of fishes released in other areas than in the bay of Cadiz move towards the bay, while the 98% of those released in the bay stay in the same area. Releases performed during the summer register high growths, short movements and higher recapture rates, up to 6%. In terms of weight, fishes of 100 g show a high recapture rate and a lower cost per recaptured fish. Brackish water habitats and polluted areas are among the negative factors for carrying out gilthead sea bream restocking exercises. An important decrease in mortality among stocking fishes was also observed during the first weeks that followed their release, in spite of being under the minimum size, due to furtive and illegal fishing. Thus, in order to obtain good results from a restocking exercise, the release area must be preserved from fishing for that species. Furthermore, as a factor of attraction for gilthead sea bream, there are areas with abundant benthic infauna, especially molluscs, such as the one detected in the bay of Cadiz. It is also probable that breeding of some stocking fishes, together with wild individuals, occur in a fishing area 9 miles South of the bay of Cadiz, a possible breeding area of this species.

According to this study, it is feasible to obtain a higher yield from the natural productivity of the sea, through a stock enhancement exercise with gilthead sea bream in the Andalusian South Atlantic region.


Report on stock enhancement of commercial important flatfish species along the Belgian coast: Author: Daan Delbare (Sea Fisheries Department – CLO) The Belgian restocking programme is set up in three different phases. The first stage is to look if cultured fish was able to adapt to the natural conditions in the northern Atlantic (North Sea and adjacent waters). The second stage is to see what quality we need to stock to result in a high survival and reporting of tags. But also to analyse the genetic quality of the reared fish ready for release, e.g., parental effect. The third phase is to stock large quantities of smaller juveniles. The problem here is that tagging with the Petersen disc is not possible, so coloration of bone structures will be used in this experiment.

In 1998 the Belgian restocking program started funded by the Flemish Gouvernment and the European Commission in a 5B-project. This project was the first phase of the restocking programme, to see if reared turbot could adapt to the natural conditions. For this, 3000 juveniles were tagged after preconditioning to live feed and lower temperatures. The tagging was carried out with the Petersen disc, which garantees high reporting of taggs. Next to that, additional bonnusses were given to the fishermen when reporting. In the following years, more than 30% of the taggs were reported, revealing that these juveniles were able to adapt very well to the natural conditions. Furthermore, an analysis was made about their distribution pattern, growth rate in the wild and feeding habits. This was compared with data available from a study on the biological parameters of natural turbot stocks (Ongenae et al., 1998). In conclusion, all parameters studied (growth, condition factor, and stomach analysis) revealed that reared turbot is able to adapt itself to natural conditions, indicating a similar growth and feeding pattern as its wild counterparts. The migration pattern, however, showed that restocking of turbot can not be considered as a national activity, since the released turbot manifested a wide spread distribution area (characterised by an off-shore migration during the winter period) (Delbare and De Clerck, 2000).

In the second phase, special attention was given to the quality of the produced fish, next to crucial biological aspects in connections with the survival, growth and physiological conditions (specific quality characteristics) of the released fish. Special attention was also given to the influence of turbot culture on the environment (reduction of the use of antibiotics and effluent water). Turbot larvae were cultured in three different hatchery systems: 1) batch system and recirculation from day 8 onwards, 2) recirculation with protein skimmer with ozone and biofilter, and 3) recirculation kept in a cage. In this experiment growth, survival and pigmentation were monitored in the first 11 days post hatching (PH). High mortality in the beginning of the experiment made clear cut conclussions impossible. The flow rate in both recirculation systems caused too much stress. In a second experiment, the use of probionts was evaluated as an alternative for antibiotics. Five different bacteria werd tested: Vibrio proteolyticus, V. mediterranei, Aeromonas hydrophila, Glucanobacter sp. and a not identifiable Cluster A. From day 5 PH there was already a significant difference in the consumption of rotifers noticable between the control and the treatments with probiotics. The bacterial inoculation of Vibrio proteolyticus, Aeromonas hydrophila, and Glucanobacter sp. had a positive effect on the first colonization of the gut of fish larvae, but only became clear after 3 days PH. Bacteria added to the rotifer culture were not recovered in the rotifers. In conclusion, addition of probionts to the culture water before start feeding has a positive effect on the survival and growth of the turbot larvae. In a third experiment, the effect of vitamine C, vitamine E and fish oil additions were evaluated in turbot juveniles. Survival and growth were monitored during the experiment. Furthermore, quality was determined using a modified salinity stress test and the phagocytosis capacity. Between the different batche there was a significant size difference. Fish fed with a diet fortified with fish oil (9%), 10% Vit C and 1000 ppm Vit E were bigger than the two other groups. Fish fed with a diet fortified with fish oil and Vit C were bigger than the control group (uncoated standard granule). The survival was also in favour for the fortified diets. Results from the modified stress test showed that the fish fed with a diet high in fish oil and Vit C obtained a better stress resistance. The effect of additional Vit C could still be observed in the phagocytosis capacity. An experiment was conducted to evaluate the effect of oxidation of the oil present in the feed of Vit E in juvenile turbot. Diets were produced with different concentrations in Vit E (0 or 200 ppm) in combination with either oxidized (60 meq peroxide/kg) or unoxidezed (7 meq peroxide/kg) triglyceride oil. As contol a standard ICES weaning diet was used. Wet and dry weight of the whole body and liver, specific growth rate, hepatosomatic index and concentrations of Vit C and Vit E in the liver were measured. At the end of the experiment, the whole body weight and the specific growth of the fish fed the diet containing oxidized oil, without additional Vit E were significantly lower than the other fish. A two-way analysis of variance showed a significant effect of the oxidation, but not of the dietary Vit E level on the weigth and on the specific growth rate of the fish. The liver weight and the hepatosomatic index on the other hand were affected by the Vit E level. Diets without Vit E resulted in the highest liver weight and hepatosomatic index. 36 days after the onset of the experiment, the tocopherol level in the diets was reflected in the liver.

To guarantee a high survival rate after release the reared fish, the juveniles must have a high quality. To determine the quality of turbot larvae and juveniles three reproducible methods were used: the salinity stress test, the Cellular Energy Allocation method and the phagocytosis capacity method (D. Declerck, 2000).

The release of farmed fish into natural ecosystems has genetic implications. It is therefore necessary on the hand not to introduce genetically foreign organisms on the place of release. For this reason, one should use broodstock originating from the population present at the release site. Therefore, it is important to document the structure of natural populations of target species within its range of distribution. Genetic analysis of turbot originating from different locations throughout its distribution area revealed that turbot coming from the Irish Sea can be considered as a separate stock. There are also indications that this species can be further divided into a (sub)stock English Channel-Bay of


Biscay and a (sub)stock North Sea-Celtic Sea (Boon et al., 2000). Furthermore, it is necessary to enlarge the range of genetic diversity of the released fish, by minimizing the inbreeding, domestication and genetic drift. The parental genetic effect also determines the stress resistance and survival during rearing, consequently the genetic diversity of the juveniles and the released fish. The research on parental effect is still going on.

The third phase is to stock large quantities of smaller juveniles. The problem here is that tagging with the Petersen disc is not possible, so other tagging methods must be investigated, e.g., coloration of bone structures.

Although, the preliminary experiments demonstrated clearly the potential of restocking turbot, further research is needed to explore the possibilities of mass releases of reared turbot for stock enhancement as a long term policy in fisheries management, both on national and international level. Special attention should be paid to source of broodstock, parental effect, information on the appropriate location and season for release, size distribution of released animals, quality of the stocked fish, stocking densities, and cost-benefit analysis.

A similar restocking programme has started with sole (Solea solea). This programme is only in the first stage, where we are looking at the ability of reared sole juveniles to adapt to natural conditions. Preliminary results show that the project may have encountered some problems. First of all, sole is much more delicate concerning handling, especially during tagging. Secondly, the reporting of taggs is very poor. This is because reporting is mainly focussed on coastal fisheries, e.g., shrimp fisheries. From mouth to mouth information it is clear that coastal fishermen are not eager to report the catch of juvenile sole in their fishing gear. Approximately 3% reporting has been done in 3 years. According to the results one can conclude that also reared sole can adapt to the natural conditions. Other tagging methods must be investigated and the importance of reporting must be made clear to the Belgian coastal fishermen. References Boon, A., Delbare, D., Ongenae, E., heesen, H., de Clerck, R. and Rijnsdorp, A. 2000. By-catch species in the North

Sea flatfish fishery (data on turbot and brill) - preliminary assessment. Final Report EC-Study Contract DG XIV 97/078.

Declerck, D. 2000. Research to the phagocytosis capacity in turbot (Psetta maxima). Mededelingen van het Departement Zeevisserij – Centrum Landbouwkundig Onderzoek – Gent, Publicatienummer 255 – D/2000/0889/1.

Delbare D. and Clerck, R. de. 2000. Release of reared turbot in Belgian coastal waters as a tool for stock enhancement. 2000 ICES Annual Science Conference, 27-30 September, ICES CM 2000/O:02.

Ongenae, E., Delbare, D. and Clerck, R. de. 1998. Collection and modelling of biological data for turbot stock assessment in support of the Common Fisheries Policy. Final Report EC-Study Contract DG XIV 95/039.

Workshop on Stock Enhancement in the Galician Rías IGAFA, Illa de Arousa - Galicia, Spain – 6–7 May 2003 Organised by: Xunta de Galicia (Galicia Regional Government), and Instituto Español de Oceanografía. Centro Oceanográfico de Vigo 1 GENERAL CONDITIONS FOR A STOCK ENHANCEMENT PROGRAMME The following aspects must be taken into consideration when implementing a stock enhancement programme: 1.1 Broodstock characteristics • Broodstock must be autochthonous, preferably captured in the area where they will be released thereafter. It is

important that the quality of the individuals is certified by a centre that would also perform the necessary sanitary and genetic controls.

• Broodstock must be composed of a sufficient number of individuals so as to ensure that genetic diversity is maintained.

1.2 Release size

• A minimum size for each species and the capability to incorporate them into fisheries must be established in order

to obtain significative recaptures. • It is advisable to take into account the following: recapture rates obtained with that specific size, growing

potential, production costs and patterns of migratory behaviour among the designated species. 1.3 Adaptation to environment prior to release

• This is an important aspect, since it is during the first weeks immediately after release that the highest mortality

rates are recorded among the released individuals. Therefore, it is advisable to carry out adaptation experiments on


these species, either in laboratories or in the natural environment, since they register diverse behaviours (e.g., flatfishes require sandy bottoms and must become familiarised with the presence of predators; lobsters need habitats offering them a variety of shelters,…)

1.4 Characteristics of the habitat • A complete study of the potential release areas must be carried out, taking into account, among others, the

following factors: predator density, competitors, food availability, as well as the reaction faced by the ecosystem with the release of new individuals.

1.5 Tagging • For an effective monitoring of the recaptures, a tagging exercise prior to release must be performed on all the

individuals. • When using external tags, it is of great concern the high rate of tags lost, becoming entangled with seaweed, or

swallowed by other individuals. • Magnetic tagging has proved to be both useful and effective in detecting marks. In order to implement this

method, fish markets should be equipped accordingly. 1.6 Monitoring released individuals • It is fundamental the presence of a monitoring team in the area of release carrying out studies on growth,

mortality, migrations, depth distribution, sanitary controls and recapture rates. In order to perform this, research and/or commercial surveys must be carried out so as to establish a relationship between recaptured individuals both tagged and wild.

1.7 Legal measures for fisheries protection and/or regulation • It is necessary to point out that a stock enhancement programme will lose effectiveness unless strict accompanying

measures are adopted, related to fisheries legal protection and regulation. Commitment on the side of the extractive sector in this kind of activities is also essential.

1.8 Socio-economic study of the stocking programme • Socio-economic studies must be carried out, following the example of other countries that are already

implementing stocking programmes, in order to assess the cost-benefit analysis involved in this type of projects. The indicators traditionally used relate the economic benefic provided to fisheries to production and release costs.

2 ASSESSMENT OF SPECIES TYPE IN GALICIA Once identified the general characteristics that a stocking programme must fulfil, an assessment of three target species in Galicia (lobster, turbot and sea bream) follows, on the basis of previous culture and enhancement experiences. 2.1 Crustaceans 2.1.1 Target species: • From the point of view of the feasibility of the released individuals, spiny spider crab (Maja squinado) is a species

that should be taken into consideration, since it presents high growth rates, a not too demanding behaviour regarding the required substratum or shelters and both lower number of competitors and intensity of competitiveness than European lobster (Homarus gammarus). Nevertheless, production techniques of hatchery-reared larval individuals are not ready yet. Besides, it should be considered whether, according to the present status of the natural stocks, it is advisable the implementation of a stocking programme or the management of the catches is preferred instead.

• As for lobster, weight data belonging to individuals sold in Galician fish markets for the last 10 years show a steep decrease and very low annual selling figures. Although data of catch per unit of effort would be more conclusive, according to the present situation of the commercial fishery, the need for an enhancement of the natural populations through stocking with hatchery-reared juveniles should be considered. Besides, hatchery production techniques are sufficiently developed.


2.1.2 Areas of release: As an initial measure, areas with the following characteristics should be preferred:

• Those where good rates of catches are, or used to be, obtained • Areas with hydrodynamic retention in order to avoid the dispersion of released individuals • Those whose substratas offer shelter and food • Selected spots should be studied as for their number of predators, competitors and food availability for a minimum

period of one year. 2.1.3 Broodstock • Local breeding females are required, keeping their health under control and rejecting ill or injured individuals • The minimum effective number of breeders must be determined in order to ensure the conservation of genetic

variability • Females preferably with a carapace length between 28 and 35 cm should be selected so as to ensure spawning

quality • Female maintenance conditions in the facilities must be cared for, in order to reduce losses of valuable individuals

extracted from the natural environment • It is desirable to carry out a study on natural genetic variability so as to determine the feasibility of employing

captured breeders in spots away from the potential release areas, without interfering with natural genetic diversity.

2.1.4 Larval rearing Intensive production techniques of juveniles are well developed, however survival results are still be variable and unpredictable. Therefore:

• It is advisable to carry on with larval rearing studies in order to achieve better results, more predictable, and less

variable, which could mean important savings in production costs.

2.1.5 Quality of the juveniles produced

In order to maximise the results, the aim must be set to produce competitive juveniles, adapted to the environment in which they will be released, with a normal morphology in which respects pigmentation and claws dimension. Therefore: • It would be appropriate to stimulate natural escape behaviour in the presence of predators, search and use of

shelters, interaction with other individuals,… prior to their release.

2.1.6 Release methods

• A total length of 6–7 cm is considered to be the minimum release size to assure positive results • The best season for spawning will fundamentally depend on the predator activity that has been detected in the

studies mentioned in point 2. It must also be taken into account the development level of annual recruitment in the environment.

• Individuals must be released in shallow waters (short time in the water column subject to predation) having been previously acclimated to the phisicochemical conditions of the environment.

• Predator activity must be controlled in the first two hours after the first releases are made, in order to determine the losses.

• It is advisable not to carry out too many releases, nor to release a great number of individuals in the same spot so as to avoid that natural recruitment is negatively affected by stocking, or that the carrying capacity of the environment is exceeded.

2.1.7 Control of recaptures • It is necessary to perform a tagging exercise on all released individuals • At the moment, the best tagging method for lobster is the use of coded-wire microtags containing a binary code,

which implies the setting out of detectors, both in those spots designed for monitoring the catches and where they are first sold.


• In order to achieve this, it is necessary to obtain collaboration from the extractive sector so as to ensure the control of the catches.

2.1.8 All these measures should be accompanied by strict regulations for the management and protection of

resources if pre-existing stocks are to be recovered 2.2 Flatfishes 2.2.1 Target species Turbot (Psetta maxima) is chosen as a species type, given its commercial interest and the low annual catch rates observed (around 100 tons). Besides, another positive element presented by this species is the fact that culture techniques, biology and is bathymetric distribution in the rias are well-known.

2.2.2. Broodstock Broodstock must be autoctonous and captured in the wild. In order to guarantee their genetic variability, the availability of a minimum effective number of breeders must be assured. It is important the existence of a centre that certifies the quality of the breeders, carrying out the necessary sanitary and genetic controls on the stock.

The artificial fertilisation process must be carried out on an individual basis (one male and one female per fertilisation) with tagged individuals providing a well-identified history. They should be included in genetic selection programmes so as to enhance broodstock. 2.2.3. Larval rearing Semi-intensive and intensive larval production techniques are already developed, nevertheless, further research must be conducted on larval culture in this species, with the aim of enhancing the survival rates that are currently obtained. 2.2.4. Larval quality In order to avoid genotypical alterations, it is important not to release individuals presenting malformations or morphologic anomalies (lack of the operculum, albinism, etc.)

Pathologic and genetic studies on the wild population must be conducted prior to release, aimed at assuring the non-interference of the released individuals in the essential characteristics of the wild population. 2.2.5. Release areas and seasons In reference to the ecological study of the release area, it is advisable to release 0-group individuals, between 6 and 10 cm, on beaches with a sandy substratum and great wave exposure, located on the outer areas of the rias. Once these areas are spotted, a study on the carrying capacity and the presence of predators and competitors must be conducted.

The most appropriate season for release is from September to December, when individuals from the same wild population have reached the same size. 2.2.6. Release methods With regard to the adequate size for release, taking into account previous experiences, related to growing capacity, movement, recapture rates and cost, the most adequate size to perform stock enhancement exercises is between 6 and 10 cm, but it is advisable to carry out previous experiments with these sizes before conducting any massive releases, since there are no records in Galicia with such sizes.

As for the conditioning prior to release, one of the main problems observed in previous experiences of turbot stocking is the high mortality rate of this species immediately after being released, so it is considered as a priority to reduce such mortality subjecting individuals to a period of previous adaptation, taking into account the following: 1) Identification and defense mechanisms in the presence of predators 2) Development of colour adaptation and burying capacities Thus, it is suggested to carry out experiments with individuals in both wild and reared environments, in closed areas, with the aim at improving their capacity of adaptation to the natural environment.

Despite being the tagging method most widely used, “T”-bar anchor tags present high rates of loss, which are necessary to reduce in order to increase recapture rates. Thus, it is recommended to essay other tags, of magnetic type, aiming at improving the final effectiveness of the stock enhancement programme.

Regarding transport and release techniques of individuals, it is essential to carry out previous studies to determine which is the most adequate and less stressing method for this species. 2.2.7. Control of recaptures Every stock enhancement programme calls for a continuous release process, for five years at least, so it can have any effects on the fishery. After this period, its possible positive effect on fishery recruitment can be assessed.


As an informative reference, it should be pointed out that in previous experiences with plaice carried out in Japan, between 100,000 and 400,000 individuals were released on an annual basis, for a five-year period, subsequently observing a possitive effect on the commercial catches.

It is essential the existence of a monitoring team in the natural environment conducting studies on growth, mortality, migrations, depth distribution, sanitary controls and recapture rates. Thus, it is necessary to carry out research and/or commercial surveys so as to establish a relationship between the characteristics of tagged recaptured individuals and those belonging to autochthonous wild stocks.

Finally, with regard to the impact on fisheries, it is fundamental to involve of the extractive sector in the stocking programme, to carry out socio-economic studies of the programme and to analyse its contribution to the fishery’s total catches. A final analysis of the stocking programme, regarding its costs in relation with the benefits provided to the fishery, will finally allow for the assessment on the effectiveness of the selected stock enhancement method. 2.3. Roundfishes 2.3.1. Target species. The following species were considered, red seabream (Pagellus bogaraveo), pollack (Pollachius pollachius), white seabream (Diplodus sargus) and European seabass (Dicentrarchus labrax), as potential candidates for stocking, according to their commercial importance, the state of development of their culture and the state of natural stocks in Galicia.

Among the main requirements to carry out a stocking programme, have been analysed those shown in the tables below, where an evaluation of these species for future stocking programmes is made.

According to the above-mentioned criteria, Red seabream (Pagellus bogaraveo) was selected as a target species for the first phase of a stocking programme in Galicia. 2.3.2. Broodstock Broodstock must be autochthonous, captured in the wild environment. A minimum effective number of broodstock must be assured in order to guarantee genetic variability.

At present, red seabream wild broodstock are available with information on their genetic variability and sanitary controlled. Spontaneous spawning is obtained between January and May.

It is essential the existence of a centre certifying broodstock quality and conducting sanitary and genetic controls. 2.3.3. Larval rearing Techniques for the intensive production of juveniles are already developed, however, it is advisable to carry out further studies on larval rearing enhancement to obtain an improvement on survival and a greater predictability of the results. 2.3.4. Quality of the juveniles produced It is important that none of the released individuals present morphologic or pathologic anomalies (lack of operculum, lordosis,…)

To optimise the results it is important to release juveniles adapted to the environment and competitive within the natural stock. 2.3.5.Release areas and seasons With regard to the release area, even though no previous records of red seabream larvae releases can be found, we have received information from fishermen about certain nursery areas, where it would be adequate to carry out the first releases.

The best stocking season observed for other sparids was Spring–summer, thus this seems the most advisable season to us, although we do not hold sufficient information on red seabream. It would be desirable to perform adaptation tests to the natural environment prior to release. 2.3.6. Release methods According to previous stocking experiences with other sparids in the Gulf of Cadiz, an initial release size could be determined between 30 and 100 g. In any case, it is regarded as essential to conduct experiences aimed at determining the most suitable release size.

Juveniles transport and release techniques are well-known, since they have been carried out successfully for other purposes. 2.3.7. Control of recaptures In order to perform these stocking programmes, it is necessary to complete tagging systems for this species. “T”-tags are habitual in stocking programmes, successfully employed on wild stocks of this same species.

Once the stocking programme has been accomplished, growth in the natural environment, recapture rates, movements, length of time in which recaptures appear, relationship between wild and released recaptures in experimental fishings, and cost/benefit analysis must be subject to control by a monitoring team.


With regard to the other three proposed species, pollack, white seabream and European seabass, the following evaluation tables are enclosed for reference in future stocking programmes.

Broodstock Culture Stocking

Species Existing in Galicia

Availability

Genetic and pathologic studies

Breeding control

Spontaneous spawning

Larval rearing Growth Release

season Tagging techniques

Transport techniques

Release techniques

Red seabream YES YES DONE NO YES YES YES Spring–

summer Microchip “T”-tags YES YES

(cages)

Pollack NO YES Non-existing NO YES YES YES Unknown NO YES NO

White seabream NO YES Non-

existing NO YES YES YES Unknown “T”-tags YES YES

European seabass NO YES Non-

existing YES YES YES YES Unknown NO YES NO

Biology and Ecology

Species Migrations Knowledge of hatchery habitat Ichthyoplankton surveys

Red seabream Migratory YES ¿? Pollack Migratory NO ¿? White seabream Non-migratory YES ¿? European seabass Migratory YES ¿?

3 Workshop conclusions 1) Stocking programmes must be addressed within a long-term project, and they need to have a purpose of annual

continuity. 2) Few species fulfil the necessary requirements to be included in a stocking programme. The most important are:

• culture techniques well-known • commercial catches showing a significant decline in evolution • being of high commercial interest • released juvenile must remain within the release area

3) The following aspects must be taken into account in a stocking programme:

• Breedstock characteristics • Release size • Knowledge of the potential habitats for release • Adaptation to environment prior to release • Carrying capacity • Tagging systems • Monitoring of released individuals • Area and resource protection measures after the release • Socio-economic study on the programme. Cost-benefit analysis

4) Stocking possibilites concerning three species have been assessed: two fishes (turbot and red seabream) and one

crustacean (lobster), taking into account previous release experiences and known culture techniques. 5) Multidisciplinary studies in collaboration with other teams and institutions in order to develop stocking

programmes adequately. 6) Once the species suitable for stocking are identified, contacts with other world stocking centres should be

established and meetings with experts working on these species could be held. 7) It is essential the involvement of the concerned fishing sector in the stocking design and its actions.

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