Internship in Safiestela S.A.: Implementation of Fish Welfare Assurance System (FWAS) And Effect of Light Spectrum in The Larvae Development of The Solea senegalensis

João Filipe Macedo Pereira Internship in Safiestela S.A.: Implementation of Fish Welfare Assurance System (FWAS) and Effect of light spectrum in the larvae development of the Solea senegalensis Dissertação de Candidatura ao grau de Mestre em Ciências do Mar – Recursos Marinhos, especialização em Aquacultura e Pescas, submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto. Orientador – Prof. Doutor José Fernando Magalhães Gonçalves Categoria – Professor auxiliar Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Co-orientadora – Drª. Renata Maria Neto Serradeiro Categoria – Administradora da AQUACRIA PISCÍCOLAS, S.A., Diretora Geral & de ID da AQUACRIA PISCÍCOLAS, S.A. e da SAFIESTELA, S.A. Afiliação – Sea8. Dedico esta tese aos meus pais, à minha irmã, à minha namorada e a todos os meus amigos. Agradecimentos: A toda a equipa da Sea8, em especial ao pessoal da Safiestela S.A. por me terem recebido tão bem, são pessoas fantásticas e que me integraram melhor do que poderia imaginar. Um especial agradecimento ao Prof. Doutor José Fernando e à Drª. Renata Serradeiro por terem aceite este desafio e por me terem realmente orientado durante este tempo (espero não ter sido muito difícil). Esta etapa foi uma das que nunca me arrependerei de ter ingressado, pois, com esta experiência, consegui crescer a nível académico e pessoal. Ao Dr. JW (Hans) van de Vis que foi de grande ajuda em momentos mais difíceis deste projeto. Aos meus colegas, e acima de tudo, amigos, que me acompanharam durante este ano: Filipe Furtado, Luis Baião e Tiago Sá. Sem o vosso poderia ter feito o mesmo, mas não era a mesma coisa. À minha namorada, Rafaela Santos, que me acompanhou e aturou, e muito, durante esta etapa. Ajudasteme a encontrar o rumo deste-me a estabilidade emocional necessária para realizar este trabalho. Por último, mas não menos importante, agradeço aos meus pais e à minha irmã que me apoiaram para que pudesse alcançar este patamar e que me ajudaram a pensar de outra forma para ultrapassar problemas encontrados nesta etapa, espero ter-vos deixado orgulhosos. Resumo Atualmente, e cada vez mais, a sociedade preocupa-se com as condições de bem-estar animal em produção, com o aumento da aquacultura esta preocupação é cada vez mais direcionada para este sector. Devido a isso, existem agora autoridades especializadas em bem-estar animal em produção, que criam certificações que levam a uma vantagem competitiva. Tais vantagens levaram a que a direção da Safiestela, S.A. estivesse interessada na implementação de um plano Fish Welfare Assurance System (FWAS), com o intuito de melhorar as condições de produção para salvaguardar o bemestar animal, este plano irá ajudar a instalação na obtenção de uma futura certificação para o bem-estar e na competição em mercados internacionais. A aquacultura de Solea senegalensis é algo relativamente recente. Vários estudos foram realizados, e outros ainda estão em progresso, para ultrapassar alguns desafios na produção desta espécie. Neste contexto, o desenvolvimento larvar desta espécie é um destes desafios. Devido a tal parte deste trabalho foi focado no experimento de novas condições de produção, para tentar melhorar o desenvolvimento larvar do linguado-do-Senegal. Assim, neste trabalho foram realizados 2 experimentos, para tentar avaliar os efeitos de 2 espectros diferentes (um usado conforme os protocolos de desenvolvimento larvar da Sea8 e outro com um comprimento de onda de 435-500 nm, que corresponde à luz azul) no desenvolvimento larvar. Resultados deste primeiro experimento não foram conclusivos, no entanto alguns parâmetros diferiram significativamente (p<0,05) entre grupos: despigmentação, peso seco, estádio da metamorfose e intensidade luminosa. Em relação ás taxas de despigmentação, o grupo de tratamento apresentou valores mais altos (41%), comparativamente com o grupo de controlo (11%). Outro parâmetro que diferiu entre grupos foi o peso seco; que foi maior nos grupos de tratamento (0,241 mg e 0,747 mg no 7º e 12º dia após eclosão, respectivamente) comparativamente com o grupo de controlo (0,205 mg e 0,671 mg no 7º e 12º dia após eclosão, respectivamente). Foi também avaliado o estádio de metamorfose; as larvas do grupo de tratamento apresentaram um desenvolvimento mais rápido (o estádio 1 foi observável no segundo dia da experiência, 8% das larvas, enquanto que no grupo de controlo este estádio foi apenas observável no sexto dia da experiência, 10% das larvas). Em relação à intensidade luminosa, o grupo de tratamento apresentou menores valores (621 lux, em média), comparando com o grupo de controlo (média de 1027 lux, em média). Os resultados do segundo experimento mostraram que alguns parâmetros ainda apresentavam diferenças significativas (p<0,05) entre grupos (intensidade luminosa e despigmentação). A intensidade luminosa continuou a ser menor no grupo de tratamento (760 lux, em média), comparativamente com o grupo de controlo (1167 lux, em média), apesar dos melhoramentos do protocolo. Em relação à despigmentação, este parâmetro apresentou maiores percentagens no grupo de tratamento (0%, 80% e 50% no 2º, 5º e 12º dia após eclosão, respectivamente), enquanto que o grupo de controlo as percentagens foram significativamente menores (2,5%, 15% e 7,5% no 2º, 5º e 12º dia após eclosão, respectivamente). Estes experimentos revelaram que a luz azul pode melhorar as condições em cativeiro, no entanto não há evidências de que, sob condições de produção, o desenvolvimento larvar seja melhorado. Ao longo destes trabalhos, havia também o objetivo de realizar um estágio nesta instalação, com a finalidade de perceber como é o trabalho rotineiro numa aquacultura. Abstract Currently, the society is concerned about the animal welfare conditions in production. This problematic is increasingly more focused on aquaculture. Due to that, there are now authorities specialized in animal welfare in production, these create certifications that potentially lead to a competitive advantage. Such advantages led the direction of Safiestela, S.A. to be interested in the implementation of a Fish Welfare Assurance System (FWAS) plan, in order to improve the production conditions, to safeguard the animal welfare. This plan will help the facility to acquire a future welfare certification and compete in international markets. The aquaculture of Solea senegalensis is relatively recent. Various works were done, and others are still in progress, in order to surpass some challenges in the production of this species. In this context, one of the production challenges is the larvae development. Consequently, part of this work was focused in experiment new holding conditions. In order to attempt to improve the larvae development; 2 experimental works were conducted, in order to evaluate the effects of 2 different light spectrums (one used according to the Sea8 protocols and another with a wavelength of 435-500 nm that correspond to a blue light) in the larvae development. Results of this preliminary experiment were not conclusive, although some parameters significantly differ (p<0.05) between groups: depigmentation, dry weight, metamorphosis stage and light intensity. Regarding the depigmentation rates, the treatment group presented higher values (41%) comparatively with the control group (11%). Another parameter that differed between groups was the dry weight, that was greater in the treatment groups (0.241 mg and 0.747 mg in the 7 and 12 dph, respectively), comparatively with the control group (0.205 mg and 0.671 mg on the 7 and 12 dph, respectively). Was also assessed the metamorphosis stage: the larvae from the treatment group presented faster development (the stage 1 was observable on the second day of experiment, in 8% of the larvae, while this stage in the control group only was observable on the sixth day of experiment, in 10% of the larvae). Regarding the light intensity, the treatment group presented lower values (average of 621 lux), comparing to the control group (average of 1027 lux). The results from the second experiment showed that some parameters presented significant differences (p<0.05) between groups (light intensity and depigmentation). The light intensity continued to be lower in the treatment groups (760 lux), comparatively with the control group (mean of 1167 lux), despite the protocols improvements. Regarding the depigmentation, this parameter presented higher percentages in the treatment group (0%, 80%, and 50% in the 2, 7 and 12 dph, respectively), while in the control group these values were significantly lower (2.5%, 15% and 7.5% in the 2, 7 and 12 dph, respectively). These experiments revealed that the blue light can improve the captivity conditions, however, there is no evidence that, under production conditions, the larval development is ameliorated. Along with these works, there was also the objective to realize an internship in this facility, in order to integrate the routine work in an aquaculture. Index Resumo _______________________________________________________ 4 Abstract _______________________________________________________ 5 Chapter 1: Introduction __________________________________________ 1 1.1 State of world aquaculture __________________________________ 1 1.2 Aquaculture in Portugal _____________________________________ 3 1.2.1 Establishments and productions systems _____________________ 4 1.3 Animal welfare in aquaculture ________________________________ 5 1.3.1 Definition of animal welfare ________________________________ 6 1.4 Welfare and husbandry _____________________________________ 8 1.4.1 Rearing densities ________________________________________ 9 1.4.2 Grading ______________________________________________ 10 1.4.3 Feeding ______________________________________________ 11 1.4.4 Transportation of live fish _________________________________ 13 1.4.5 Breeding ______________________________________________ 16 1.4.6 Slaughter _____________________________________________ 18 1.5 Good aquaculture practices ________________________________ 19 1.5.1 Site selection __________________________________________ 20 1.5.2 Water quality __________________________________________ 20 1.5.3 Source of animals ______________________________________ 21 1.5.4 Husbandry operations ___________________________________ 21 1.6 Biology of Solea senegalensis ______________________________ 21 1.6.1 Aquaculture ___________________________________________ 22 1.7 Thesis objectives _________________________________________ 23 Chapter 3: Implementation of a Fish Welfare Assurance System (FWAS) in Safiestela S.A. ______________________________________________________ 24 3.1 Introduction ______________________________________________ 24 3.2 Conduct a hazard analysis and risk assessment _______________ 30 3.3 Determine critical control points (CCP) _______________________ 31 3.4 Establish critical and target limits ___________________________ 31 ___________________________________________________________ 32 3.5 Establish monitoring procedures ____________________________ 32 3.6 Establish corrective actions ________________________________ 33 3.7 Establish verification procedures ____________________________ 34 3.8 Establish a record keeping system __________________________ 35 3.9 Verification on site ________________________________________ 37 3.9.1 Receiving of breeders ___________________________________ 37 3.9.2 Broodstock in RAS and in flow through system ________________ 38 3.9.3 Transference of larvae to the weaning area __________________ 39 3.9.4 Grading in weaning area _________________________________ 40 3.9.5 Fish transference from the weaning to the pre-ongrowing area ___ 41 3.9.6 Grading in pre-ongrowing area ____________________________ 43 _________________________________________________________ 43 3.9.7 Water quality in weaning and pre-ongrowing areas _____________ 43 3.9.8 Conclusion ____________________________________________ 44 Chapter 4: Effect of light spectrum in the larvae development of the Solea senegalensis _______________________________________________________ 46 4.1 Introduction ______________________________________________ 46 4.2 Materials and methods of the preliminary experiment ___________ 47 4.3 Results __________________________________________________ 48 4.3.1 Total length ___________________________________________ 48 4.3.2 Dry weight ____________________________________________ 49 4.3.3 Depigmentation ________________________________________ 50 4.3.4 Metamorphose stage ____________________________________ 51 4.3.5 Stomach content _______________________________________ 53 4.3.6 Light intensity __________________________________________ 53 4.4 Discussion ______________________________________________ 54 4.5 Materials and methods of the second experiment ______________ 55 4.6 Results __________________________________________________ 56 4.6.1 Total length ___________________________________________ 56 4.6.2 Dry weight ____________________________________________ 57 4.6.3 Depigmentation ________________________________________ 58 4.6.4 Metamorphose stage ____________________________________ 59 4.6.5 Stomach content _______________________________________ 60 4.6.6 Light intensity __________________________________________ 60 4.6.7 Relation between dry weight and total length _________________ 61 4.7 Discussion and conclusions ________________________________ 61 Chapter 5: Internship report in the Safiestela S.A. ___________________ 63 5.1 Introduction ______________________________________________ 63 5.2 Broodstock area __________________________________________ 63 5.3 Incubation room __________________________________________ 65 5.4 Larvae room _____________________________________________ 66 5.5 Live feed room ___________________________________________ 68 5.5.1 Rotifers room __________________________________________ 68 5.5.2 Artemia room __________________________________________ 69 5.5 Weaning area ____________________________________________ 70 5.6 Pre-ongrowing area _______________________________________ 71 5.7 Daily routines in Safiestela S.A. _____________________________ 73 5.7.1 Weaning area __________________________________________ 73 5.7.2 Live feed rooms ________________________________________ 74 5.7.3 Broodstock area ________________________________________ 75 5.7.4 Pre-ongrowing area _____________________________________ 75 5.8 Recirculating aquaculture systems (RAS) _____________________ 76 5.8.1 Maintenance of RAS ____________________________________ 77 5.8.2 RAS in the Safiestela S.A. ________________________________ 78 References ___________________________________________________ 79 Annexes _____________________________________________________ 87 Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 3 pathogen outbreaks or the closure of establishments; nonetheless, these causes are momentary, and the production numbers can recover in the future. The global growth rate of aquaculture, between 2000 and 2012, was 6.2% per year, which is lower than the 10.8% in the period of 1980 to 1990. This trend is expected, since aquaculture production have a tendency to stabilize (Bostock et al., 2010, Volpe et al., 2013). 1.2 Aquaculture in Portugal Globally aquaculture is conditioned by a group of factors and their implications. These are: (1) existent technology; countries or regions with scarce resources are unable to produce at the same tecnological leve, when compared with more resourceful countries; (2) institutional system; since aquaculture is a novelty in some countries and, this system may present lacunas; (3) environmental conditions; the production will be less expensive and simplest if the species are comfortable with the range of environment parameters existing in the region; (4) human resources; in some areas the specialized manpower is scarce; (5) the market demand; which is different according to the region, since each region have its traditions and preferences regarding to the consumption of aquatic products. In Europe, the aquaculture has been challenged in the last years, and that affected the production, some countries showed a decrease in their productivity (such as Spain and France). This summarize the incapability of Europe to compete with another markets (figure 2) (Bostock et al., 2010, Volpe et al., 2013, FAO, 2014, Troell et al., 2014). Figure 2 -European aquaculture production between 1990 and 2012 (FAO 2014). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 4 The socio-economic crisis that Europe is going through affected many activities, including aquaculture. In the Portugal case, this crisis has been particularly severe, and as consequence, the aquaculture production decreased. According to INE (Instituto Nacional de Estatística) in 2013 the aquaculture sector decreased 9.0%, comparing with the year before (figure 3), this decline was due to the reduction in Scophthalmus maximus production (-46.6%). This reduction produced an opportunity in the market for other species, such as sole. Nonetheless, the value of the aquaculture products presented an increase of 3.1% (figure 3). Figure 3 - Portugal aquaculture in 2012 and 2013 (INE, 2014). The Portuguese aquaculture has a significant focus on marine and brackish species. The main fish species produced in 2012 were turbot (Scophthalmus maximus) and gilthead sea bream (Sparus aurata), representing 85% of the entire national production in marine and brackish waters. Half of aquaculture production in Portugal is due to molluscs, where clam (Ruditapes decussatus) and mussel (Mytilus edulis) are the most produced species. Regarding the fresh water production, in 2013 and 2012 Portugal only produced one species, rainbow trout (Oncorhynchus mykiss) (INE, 2014). 1.2.1 Establishments and productions systems Comparing 2013 with the previous year, aquaculture facilities in Portugal increased to a total of 1,522. This increase led to a rise of area used for this sector of 62.6%, this significant increase was also due to the authorization for offshore production. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 5 The majority of the facilities (88.2%) are intended for the production of molluscs. Regarding the fish production, only 9.2% of the establishments were intended for that purpose. Concerning to the type of production system used in Portugal, in marine and brackish waters 54.7% of the facilities produce in extensive systems while 34.7% of the facilities operate in the intensive system. The entire fresh water production comes from intensive production systems (figure 4) (INE, 2014). Figure 4 - Aquaculture production according to the production system and type of water. (INE, 2014). 1.3 Animal welfare in aquaculture The increase of production in aquaculture sector is higher than any other animal production (FAO, 2014). In recent years,The rise in production was followed by the rise of some concerns, namely, the conditions of culture and, consequently, the animal welfare. (Frewer et al., 2005, Huntingford et al., 2006, Ingenbleek et al., 2012, van de Vis et al., 2012, Bovenkerk and Meijboom, 2013, Huntingford and Kadri, 2014, Mellor, 2014, Mustapha, 2014). There are now authorities that inspect these aspects in different kinds of production, including aquaculture. One example, in the United Kingdom, the Royal Society for the Prevention of Cruelty to Animals (RSPCA) (King, 2009, Bovenkerk and Meijboom, 2013). This type of associations promote actions amongst producers, with the objective of improving the animal welfare in production. Additionally, the general public is also creating, increasingly, more pressure in this sector, due to the increase about this ethical concern. Nowadays, in some cases, people prefer to pay more for a product that is certified in welfare. This pressure from the public made appear the certification processes in aquaculture, namely in countries in the North of Europe (Diana et al., 2013, Harvey and Hubbard, 2013, Ingenbleek et al., 2013, Rossi and Garner, 2014). Brackish and marine water Fresh water Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 6 The European population is one of the most aware about the animal welfare, in particular, the people from United Kingdom and Nordic countries. Thus, it is most likely that this kind of certification processes will also appear in other countries as well, such as Portugal. Though, the certification process requires certain points. Firstly it is necessary to define what welfare is, how to measure it, how to improve the culture conditions and also how to improve practices for each species production (van de Vis et al., 2012, Ingenbleek et al., 2013, Rossi and Garner, 2014, Colson et al., 2015, Ellingsen et al., 2015). 1.3.1 Definition of animal welfare In fish farming the welfare is complicated to define; this concept creates complex questions between normative and ethical actions. Fish welfare can be defined in different ways, depending on the perspectives (function-based, feeling-based and nature-based) (van de Vis et al., 2012, Bovenkerk and Meijboom, 2013, Huntingford and Kadri, 2014). The definition of welfare has changed through the time: firstly, the welfare was safeguarded if the physiological equilibrium of the animal was reached. However recently this concept has been defined in numerous ways. One of the most used references to measure animal welfare in production is through the five freedoms (freedom from hunger, freedom from pain, disease or injury, freedom from discomfort, freedom from fear and stress and freedom to express their natural behavior) of the Farm Animal Welfare Council (Bovenkerk and Meijboom, 2013). The majority of the authorities use this definition; however, there are authors who claim that this definition is too static and cannot measure the welfare effectively in all cases (McEwen and Wingfield, 2003, Bovenkerk and Meijboom, 2013). The five freedoms concept assume that welfare can only be assessed if the production guarantees all the freedoms. However in some authors perspective the freedoms can be antagonistic. In the example used in Ohl and van der Staay (2012), it is explained that a natural behavior, such as exploring the environment can implicate stress in the animal, and not all of the five freedoms statements can be assured at the same time. Such critics has conducted to the appearance of a new concept, allostasis (McEwen and Wingfield, 2003, Schreck, 2010, Ohl and van der Staay, 2012, Turnbull and Huntingford, 2012). In this concept, the main modification in the definition of welfare was, the capacity to change and adapt to new conditions. Accordingly to Fraser (2003), there are mainly 3 ways to define animal welfare, these are: (1) function-based (the main question of this view is if the fish can adapt to the farming conditions); (2) feeling-based (in this perspective the central bottleneck is if the fish have feelings, assuming that they have, the welfare can only be achieved if the feelings of the fish are take into account, in order to avoid any distress or pain to the Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 7 animal); (3) nature-based (in this view, welfare of fish only can exist if they can express their natural behavior). Other complex questions are implicated in the definition of welfare in fish, such as: What is the natural behavior? This issue is highly difficult to answer, since in animal production, usually, there is sustainability. In other words, the animals in production come from selected broodstock; this selection of specific traits can indirectly or directly change the animal behavior through generations (Teletchea and Fontaine, 2014). According to Bovenkerk and Meijboom (2013), in 2009 it was conducted an inquiry, by Wageningen UR Livestock Research, in order to know the opinion of part of the population related to cow production. The conclusion of this investigation was that, for the general opinion the best way to produce cow was in open field, however welfare specialists defend that indoor production can promote better welfare. At least in some matters, for example in this type of production, the pathogenic spread is highly controlled, in relation to outdoors production. Another point defended by the specialists, is that the cows are domesticated animals and the definition of what is their natural behavior can be different from the general assumption. These inconsistencies in the definition of animal welfare are complicated in terrestrial animal productions but in fish production are even more complex. The knowledge regarding to this group of animals presents many gaps, for example in Solea senegalensis the optimal parameters in culture are not all defined. Despite of that, the culture of this species is already undergoing, and many researchers efforts have being done in the recent years (Villamizar et al., 2011, van de Vis et al., 2012). In conclusion, there are several definitions of what welfare is, and there are mainly three ways to analyze it: function-base, emotion-base, and nature-base. The other major problem is how welfare should be quantified objectively. Generally in aquaculture is performed the register some parameters that could affect the fish welfare, such as: water quality parameters and animals numbers with tissue damages (van de Vis et al., 2012). However, the fish farmers only observe the effects of poor welfare conditions and not the cause, so in this matter it is important to act before the appearance of side effects. To achieve this, it is important to implement a system that safeguard the fish welfare and promote preventive actions (van de Vis et al., 2012). In this work, it was tried to take into account all these perspectives to implement a Fish Welfare Assurance System (FWAS), to improve animal welfare in Safiestela S.A. This system will be explained in detail further in this document. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 8 1.4 Welfare and husbandry Nowadays, the concept of animal welfare is increasingly associated with the production of animals for consumption, but in many cases it is targeted to mammalians, such as pig farming and cow farming. Nonetheless, in the past decade studies concerning about this thematic in fish farming, appeared. This fact is caused by the increase of this sector in meat production and because of the popularization of aquaculture in general (Frewer et al., 2005, Mesquita, 2011). On the other hand, pressure of the public to apply husbandry systems that improve animal welfare has been caused by the increase of ethical and moral concerns; either due to the higher educational qualifications, either by the increasing in the access to information (Ingenbleek et al., 2013, Van Loo et al., 2014). This pressure is reflected at economic level, thus, a new market demand appeared (particularly in North of Europe), which is, products that are certified in animal welfare (Frewer et al., 2005, Ingenbleek et al., 2012, Ingenbleek et al., 2013, Mellor, 2014) . In fish farming, there are routines that can compromise the animal welfare (transport, slaughter, grading). To mitigate this, it is important to know the species requirements and natural behaviors. In extensive productions, usually, a welfare friendly husbandry system exists (Håstein et al., 2005, Håstein, 2007, Roberts et al., 2008). Husbandry systems that promote welfare are being studied, planned and implemented in intensive aquaculture. One example of that is the relatively new system to improve animal welfare in aquaculture, the FWAS plan. This is an ongoing plan that can be applied in any aquaculture, to reduce and eliminate various problems that affect the welfare of fish, such as in the slaughter process, the environmental conditions and stockmanship (van de Vis et al., 2012). Improved husbandry systems in intensive aquaculture can be a challenge. The production methods have a strict dynamic and any minor alteration require a detailed study of the consequences in the production chain and profitability (Huntingford and Kadri, 2014). In the literature, there are authors that defend that the concern about animal welfare shall come in the planning of the facility and with the choice of the species to produce. Since, different species have different requirements and behaviours, for example, there are species that are more resilient to handling than others, and it is more likely to produce these more resilient species (Jarman et al., 1976, Håstein, 2007, Roberts et al., 2008, Mesquita, 2011, Huntingford and Kadri, 2014). Huntingford et al. (2006) describes a series of husbandry operations that can compromise the animal welfare in aquaculture facilities, these are: (1) Stocking densities; Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 9 (2) water quality; (3) photoperiod; (4) stockmanship; (5) transport; (6) disease and subsequent treatment; (7) slaughter; (8) food administration; and (9) crowding. 1.4.1 Rearing densities In aquaculture, some operations can affect the production and the animal welfare, as described above. Thus, stocking density requires a well-established management plan, to maximize the profitability and to maintain the welfare conditions in acceptable levels. This thematic has been studied in a number of species (salmonids, turbot, and Senegalese sole, and Atlantic cod) (Iguchi et al., 2003, van de Nieuwegiessen et al., 2009, Wunderink et al., 2011, Sánchez et al., 2013, Villanueva et al., 2013, Creţu et al., 2014, Andrade et al., 2015, Menezes et al., 2015). The effects of rearing densities are different in accordance with the species, the phase of the life cycle, season, type of exploration and type of water circulating system. In extensive and semi-intensive aquacultures the stocking densities, usually, do not affect negatively neither growth rate, neither welfare. That’s because these type of aquaculture systems depends on environmental conditions, favors the natural behavior of animals, and the rearing densities are quite low when compared with intensive systems. On the other hand, intensive aquaculture, by definition, is a type of production that has high stock densities and tend to control the production parameters. For instance, in the recirculating aquaculture system (RAS), it is possible to manipulate several water parameters, such as temperature and oxygen, required for the production. On the contrary, in flow through less parameters can be manipulated. These different systems l influence the rearing densities (Zhang et al., 2011, Kolarevic et al., 2014, Colson et al., 2015). Regarding stock density in fish farming, no legislation determine the minimum area that each animal requires, which could generate unhealthy husbandry conditions. Nevertheless, the profitability of an aquaculture, namely in those that use the intensive exploration, is strictly connected to the proper husbandry conditions. Stocking densities correlate with many of these conditions, so it is imperative to define the proper rearing density. Thus, is in the best interest of the producer to improve the holding conditions (Iguchi et al., 2003, Saillant et al., 2003, Richard and Flemming, 2007, Andrade et al., 2015). According to the species and to the life stage, there must be a careful plan, based on investigation studies, to implement the rearing density. In literature, it is described species that are not affected by high stocking densities and other species that prefer lower stocking densities (Daniels et al., 1996, Iguchi et al., 2003, Saillant et al., 2003, Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 10 Turnbull et al., 2005, Salas-Leiton et al., 2011, Luo et al., 2013, Andrade et al., 2015, Menezes et al., 2015). Commonly high rearing densities are connected to detrimental welfare. However some studies prove that, depending on the species and life stage, high rearing densities improve the animal welfare. For example, in juveniles of African catfish high stocking densities improve welfare conditions, which is reflected by the increase in growth rate and decrease in aggression between specimens (van de Nieuwegiessen et al., 2009). Another species that do not appear to be damaged with high stocking densities is the Senegalese sole (Sánchez et al., 2010, Wunderink et al., 2011, Sánchez et al., 2013, Andrade et al., 2015). In table 2 there are some examples of species reared at different densities and their effects. Table 2 -Examples of stocking densities studies of different species. Species Initial Densities Measure Conclusion Senegalese sole (Solea senegalensis) 7, 17, 24 kg/m2 Growth performance and stress hormones High stocking densities did not affect growth performance or levels of stress hormones (Andrade et al., 2015). Jade perch (Scortum barcoo) 120, 180, 270 fish/m3 Physiological Best performance at the density of 180 fish m3 (Luo et al., 2013). Ayu sweetfish (Plecoglossus altivelis) 100, 400, 1250 fish/m3 Stress hormones Moderate stocking densities are better for the production of this species (Iguchi et al., 2003). Silver catfish (Rhamdia quelen) 8, 16, 32 kg/m3 Physiological The moderate and the higher density are more suitable for aquaculture of this species (Menezes et al., 2015). 1.4.2 Grading Grading is also an operation that can compromise the productivity and the animal welfare. Nevertheless, this operation is fundamental in intensive aquaculture, since in high stocking densities, commonly found in these systems, there are an increasing competition for food between animals, spread of diseases, accidental (damage due to the contact with other individuals or with equipment), aggressiveness and space competition (Saillant et al., 2003, Qu et al., 2009, Overton et al., 2010, González et al., 2011, Petrović et al., 2011, Slavík et al., 2011, Daly et al., 2012, Barron et al., 2013). All of these problems can be ameliorated when a frequently grading processes is implemented, which regulate the size of the specimens within cages, tanks or ponds. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 11 These consequently affect the profitability of the aquaculture (Daly et al., 2012, Barron et al., 2013). Another productivity benefit in grading is the size control in each compartment, this regulation allows to improve other husbandry operations, like feeding, disease treatment and transport. Grading also help the management of the facility to estimate more accurately the biomass in the production, since along with the grading process it is possible to recalculate the biomass that is going through that process (Saillant et al., 2003, Petrović et al., 2011, Barron et al., 2013, Lekang, 2013). In this operation, disadvantages are also incremented, for instance, grading requires handling, this can cause damages along with stress issues, and this can lead to disease spread and consequently decrease in productivity (Qu et al., 2009, Slavík et al., 2011, Mustapha, 2014). On the other hand, according to Slavík et al. (2011), energy consumption of graded European catfish were higher when they were exposed to unfamiliar individuals, than when they were exposed to familiar individuals. This can compromise not only the welfare but also the growth rate since the energy of the animal is used to other physiological processes than growth. This study concludes that to improve welfare in aquacultures, it is necessary to take into account the path and social connections of each species. In another study to improve animal welfare through grading, Barron et al. (2013) showed that in larvae of Lota lota maculosa, the survival in this life stage was increased with regular grading. The results of this study showed a survival of 74.3% when grading was performed and 59.3% when not graded. Salas-Leiton et al. (2010) conducted another experiment regarding how grading benefits depend on which species is analyzed. In this case the species studied was Solea senegalensis and it was concluded that the growth was higher when there was heterogeneity in sizes. This is explained due to social relationships and hierarchy in this species, this is, the size distribution is caused by the hierarchy established. This might suggest that it is necessary to change the way this species is produced, to improve profitability and the welfare. In conclusion, these studies remark the importance and the dangers of husbandry operations. It is important to know the species requirements, to improve the animal welfare and also productivity. 1.4.3 Feeding One of the significant bottlenecks in aquaculture of any species is the feeding. This operation not only require the knowledge of what are the feed requirements, but also to know the physiologic processes of each species (Mesquita, 2011, Boglino et al., 2012, López-Olmeda et al., 2012, Turnbull and Huntingford, 2012, Luo et al., 2013, Marinho et al., 2014, Trejchel et al., 2014). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 12 This husbandry operation is highly related to welfare, since in farming conditions one of the first signs that farmers usually are aware, when the welfare is compromised, is the lack of feed intake by the animals or the increased amount of wasted feed. Thus, this operation, like other husbandry operations, is related and it is crucial to know how these relations work for each species production, in order to improve productivity and welfare conditions (Mesquita, 2011, Turnbull and Huntingford, 2012, Liu et al., 2014, Mustapha, 2014). Feed intake is regulated by the hypothalamus; this portion of the brain is responsible for receiving and interpreting inner and outer stimulus and then transmit the appropriate response. These responses, include the regulation of hormone secretion, hormones that regulate hunger, temperature and others physiological processes (Kulczykowska and Sánchez-Vázquez, 2010). Therefore, the endocrine system not only is controlled by internal stimulus but external ones also regulate it. Roughly, all species, including the ones in the aquaculture, are controlled by their circadian cycle. Thus, this cycle controls a series of behaviors, such as feeding rhythms. In practically all species, this particular behavior occurs only at day or at night, depending on the species. However in many species, the circadian cycle is flexible and can be modified in farming conditions through time, when environment conditions are manipulated (Kulczykowska and Sánchez-Vázquez, 2010, López-Olmeda et al., 2012, Ganzon-Naret, 2013, Liu et al., 2014). The food anticipatory activity (FAA) is a behavior that affect several species under farming conditions, this behavior require that the animal stays in aquaculture long enough to synchronize their cycle with the implemented feed regime (Kulczykowska and Sánchez-Vázquez, 2010). In aquaculture, namely in intensive systems, the feed intake is conditioned by some husbandry operations, including the artificial feed. These operations can potentially interfere in the animal welfare, and consequently inhibit the feed intake and ultimately the productivity. On the other hand, the feed residues left in a tank, can potentially alter the water quality to a point which will be adverse to the animals health and welfare (Kulczykowska and Sánchez-Vázquez, 2010, Nilsson and Torgersen, 2010, Mesquita, 2011, López-Olmeda et al., 2012, Turnbull and Huntingford, 2012, Ganzon- Naret, 2013, Liu et al., 2014, Marinho et al., 2014, Bonaldo et al., 2015, Summerfelt et al., 2015). In the majority of the aquaculture facilities, the manual feeding distribution is used because is cheaper, since it is required a minor investment than in automatic systems, although it requires more labour. This feeding system can be less effective, once it depends on the regularity of the distributions, and the amount of feed and the knowledge of the operator (Mesquita, 2011). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 19 method is prohibited from the European Union), or a stunning method, to avoid unecessary disconfort or pain (King, 2009, Lines and Spence, 2012, Lines and Spence, 2014, Mustapha, 2014, Bermejo-Poza et al., 2015). The processes that are commonly used are, the death by hypothermia (using the live chilling process) or by bleeding (Håstein et al., 2005). Nonetheless, according by several studies none of these process is considered to be human, that is, this methods does not protect the animal welfare, avoiding unnecessary pain and stress (Håstein et al., 2005, Bovenkerk and Meijboom, 2013, Lines and Spence, 2014, Mustapha, 2014). In fact, in this sector little was done to improve this process regarding the welfare. According to the literature the only produced species that altered significant the husbandry alterations was the Atlantic salmon aquaculture (Lines and Spence, 2014). The various works regarding this thematic link the slaughter methods to the quality of the final product (Lines and Spence, 2012, Lines and Spence, 2014, Mustapha, 2014). Since the work presented was integrated entirely in an aquaculture nursery, the slaughter for consumption is not a process implemented in the husbandry operations of this facility. Nonetheless, another less studied type of slaughter present in this facility is the elimination of selected animals (Håstein et al., 2005). The selected fish in aquaculture is a process where the animals with illness, lower growth rates, or any damage that compromise the commercial value are discarded, this will improve the productivity by various ways like: disease control, less expense in food, and increasing the average economic value of the biomass (Håstein et al., 2005). This theme is much less studied than the previously mentioned because the animals are not for consumption and to cut expenses, usually, the slaughter is done in a most inexpensive way. This careless for the slaughter of selected animals will reflect in the welfare, since this methods, most probably, do not avoid distress and pain. Concluding, the best method of slaughter should inflict the lesser stress and pain possible, for that it is considerate by some authors that sedation or stunning before slaughter, when properly applied, are the best possibilities to guarantee a friendlywelfare operation (Håstein et al., 2005, Bovenkerk and Meijboom, 2013, Mustapha, 2014). 1.5 Good aquaculture practices As already referred, the aquaculture is an activity in a fast expansion across the globe. Thus, the implementation of good aquaculture practices is, more and more, important, to respond to the society demands regarding food quality, animal welfare, and ecological concerns. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 20 The aquaculture practices will influence: (1) the site selection of the facility; (2) the water quality, since this factor is depending on the local that the facility is constructed; (3) the source of animals, the source of animals will depend on the management plan implemented; (4) and the husbandry operations (Khuraibet and Al Attar, 2002, Boyd, 2003, Sapkota et al., 2008, Broughton and Walker, 2010, Bosma and Verdegem, 2011, Moss et al., 2012, Diana et al., 2013, Mondal et al., 2013, Boison and Turnipseed, 2015). 1.5.1 Site selection The location of an aquaculture is dependent of parameters, such as: (1) what species is going to produce; (2) what production system is going to be used (for example offshore aquaculture will need specific environment conditions, although, inland aquacultures have other requisites); (3) the access to natural resources (water); (4) access to the aquaculture facility (for example: roads); (5) surrounding activities that can compromise the production (for example: agriculture leakages). For these reasons, the site selection of an aquaculture facility must be careful deliberated to assure its success (Soto et al., 2013). 1.5.2 Water quality Usually, the aquaculture products are for human consumption. Thus, the safety protocols of these are more rigorous. In this activity, one of the most control factors is the water quality control, in which the animal develops. This factor is even more relevant when it comes to intensive aquaculture, namely, in facilities that use RAS, in this case, the water treatment system must guarantee the control of the water quality within the favorable levels for the species produced. Although, this system complexity can create management problems, nowadays there are more and more facilities that implement these systems, due to the advantages (more efficient, more ecological, safeguard the animal welfare and increase productivity). Despite the system used, the water must be treated within the appropriate levels for the produced species. This control is fundamental to prevent any diseases and outbreaks in the facility and subsequently safeguard the productivity and animal welfare. The water source shall be free from any contamination, either by other production activities, either by urban effluents (Yang et al., 2001, Bossù et al., 2013, Ababouch, 2014, Colson et al., 2015). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 21 1.5.3 Source of animals In the aquaculture the import/transfer of animals from others facilities, or the wild is widely common. However, this is one step that could endanger the production, since any introduction of live biomass can translate to an outbreak of pathogens in the facility, thus, any introduction of animals should pass through the quarantine facilities (Broughton and Walker, 2010, Ababouch, 2014). 1.5.4 Husbandry operations Regarding the good practices in the husbandry operations, these were already covered in the sections above. In summary, there are a number of practices that must be implemented, in order to safeguard the productivity and the animals: (1) good hygiene practices, this should cover the disinfection and sanitary practices in the holding facilities, equipment, and staff; (2) the production design should be adequate for the species in production; (3) minimize the stress in the animals, the stress can be caused by the handling in the routine operations or by other factor such as water quality or transportation; (4) use exclusively feeds that are certified by an authority, this can prevent diseases dissemination and contaminations in the facility; (5) protect the fish in production from any predators or other animals that can harm the fish in production, this can also be a source of pathogens; (6) control holding conditions, in order to increase the animal welfare; and (7) do not exceed the carrying capacity of the aquaculture (Khuraibet and Al Attar, 2002, Frewer et al., 2005, Sapkota et al., 2008, Broughton and Walker, 2010, López-Olmeda et al., 2012, Vázquez-Rowe et al., 2012, Ababouch, 2014, Huntingford and Kadri, 2014, Boison and Turnipseed, 2015). All of these practices will depend on the species that is produce, the live stage and the type of production system used. 1.6 Biology of Solea senegalensis Regarding the distribution of this species, it is commonly found from north to south in the Atlantic east, has a high market value and, for that reason, it is considered a species of great interest to produce. This rise promoted an increase in the number of studies with this species inthe last decades, with the objective to know therequirements for aquaculture (Bedoui, 1995, Dinis et al., 1999, Imsland et al., 2003). Solea senegalensis is a benthonic fish, regarding the morphology it is a flatfish with the eyes on the right side. It presents an asymmetric oval shape and has the anal and dorsal fins connected with the caudal one. It reaches maturity approximately at 30 Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 22 cm of length, which corresponds to 4-5 years of age. The reproduction season initiate in May-June when the waters warm up to 18-20º C (Dinis et al., 1999). This animal normally inhabits littoral ecosystems with sandy or muddy bottoms in depths inferiors to 100 m. In nature, this species feeds upon polychaetes, small crustaceans, and other benthonic invertebrates. 1.6.1 Aquaculture The production of this species has encountered various challenges, such as, high susceptibility to pathologies, technological limitation and captivity reproduction (Dinis et al., 1999). The time that is required to this species achieve the commercial size lasts, usually, two years (125 g). Concrete or fiberglass can compose the production tanks, depending on the production process that is used, this will also influence the density used (Dinis et al., 1999, Dias et al., 2004, Salas-Leiton et al., 2008, Andrade et al., 2015). For the production, the environmental parameters vary according to the management plan, however regarding the temperature this can range between the 22- 27º C (Dinis et al., 1999). The feed administered can be natural (fresh or frozen polychaetes and other invertebrates) or artificial (commercial pellets) (Bedoui, 1995, Dinis et al., 1999, Imsland et al., 2003, Dias et al., 2004, Salas-Leiton et al., 2008, Marinho et al., 2014). In farming, the reproduction is controlled by temperature and photoperiod, to produce eggs according to the management plan, each female can produce 140,000- 200,000 eggs/kg (Imsland et al., 2003), and the incubation time is normally of 800 degrees-hours at 19º C (Dinis et al., 1999). Depending on the characteristics of the gametes and the environmental conditions, the fertilization rate can range between 50- 100% (Bedoui, 1995) and the hatching between the 30-80% (Dinis et al., 1999). The larvae are pelagic, and begin metamorphosis at 2,2-2,9 mm (Dinis et al., 1999, Blanco-Vives et al., 2010, Blanco-Vives et al., 2011). Normally, the provided feed in this stage is live feed from 3 dph until the 35 dph (Dinis et al., 1999, Pedro Cañavate et al., 2006). Posteriorly the larvae stage, the animals pass through a weaning phase to acclimatize to inert feed, this process can produce mortality between the 40-80% (Dinis et al., 1999, Imsland et al., 2003). The market demand for this species is due, namely, to his organoleptic properties and due to their nutritional benefits (Dinis et al., 1999, Imsland et al., 2003). Another bottleneck in the aquaculture of the Senegalese sole is, the susceptibility to pathologies, normally these can be treated and the fish can survive, but the physical damages caused by the pathologies result in an economical losses, for example the Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 23 Photobacterium damselae ssp piscicida causes an erosion in the caudal fin and even if the fish are treated the economic value will decrease, because the retailers and consumers have certain esthetic standards (Dinis et al., 1999, Imsland et al., 2003). Other blockages include the formulation of adequate feeds, which usually translate in a poor FCR and consequent poor growth, and the need to capture wild breeders to assure the production cycle, because the animals that are produced do not have the best qualities to became breeders (for example: low production of sperm) (Dinis et al., 1999, Brown, 2002, Imsland et al., 2003). The increase of investigation works and the holding conditions improvement can reduce and solve these problems. 1.7 Thesis objectives The goals of this work were: 1. Integration in the routine practices in Safiestela S.A., to apply theoretical knowledge in a realistic context; 2. Implementation of a FWAS plan in the facility; 3. Realization of an experiment in the larval stage, to compare holding conditions at a commercial level. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 24 Chapter 3: Implementation of a Fish Welfare Assurance System (FWAS) in Safiestela S.A. 3.1 Introduction Following other animal productions, aquaculture has a need to implement a plan that safeguard the animal welfare and the production itself. Since 2006, all food producers that compete in the European market are obliged to implement HACCP (hazards analysis and critical control points) into their production safety plan, this regulation created an increase in the concern about the products quality from animal production (Ingenbleek et al., 2012, Ingenbleek et al., 2013). Another increasing concern among the generally population is, the holding conditions that the animals are subject under production, in other words, the animal welfare (van de Vis et al., 2012). These concerns together generated pressure on the market for aquaculture products, creating a demand for products that are animal welfare certified. This demand were first applied in terrestrial animal productions, nonetheless, with the increase of aquaculture, these concerns are, nowadays, more focused on this sector. To supply the demand generated, it was created a welfare assurance system for fish farming, FWAS. This system follows the same seven principles of HACCP. Nonetheless, each step was altered to be applied to animal welfare in aquaculture. In order to create a strong and simple FWAS plan, it is necessary to have already established a code of good aquaculture practices (GAP) and a biosecurity plan in the aquaculture, this underlies that exist good hygiene, good maintenance and control of other operation practices. If these pre-requirements are established, it is possible that minor adjustments are enough to reach the main goal. If not, greater changes are needed, consequently the production could be affected. Because new practices need to be implemented, and the workers need time to adjust to these, which could influence the productivity of the company. For the reasons mentioned above it has been implemented a FWAS plan in Safiestela S.A. In this chapter it is presented part of the work done in this facility, the complete flow diagram and FWAS plan are present in the annex one and two, respectively. The United Kingdom was one of the first countries to implement this certification and to create associations, such as, RSPCA, to acknowledge and come to agreement of which rules need to be applied to the fish farmers (Bovenkerk and Meijboom, 2013). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 25 The FWAS diverge from the common system, HACCP. This last system allows the production strengthen, not only by improving the product quality, but also by optimization of the production (Fernández-Segovia et al., 2014, Psomas and Kafetzopoulos, 2015). The HACCP is based on seven principles, with 13 steps within them, and according to van de Vis et al. (2012) it is possible to adapt these to fish welfare in aquaculture, these steps are: (1) Assemble the FWAS team; (2) Describe the product; (3) Identify intended use; (4) Construct a flow diagram; (5) Assess on site; (6) Identify hazards and preventive measures; (7) Identify critical control points; (8) Determine target and critical levels for each critical control point; (9) Establish monitoring system; (10) Establish corrective measures; (11) Establish verification procedures; (12) Establish a record keeping system; (13) Review the FWAS plan. These steps or stages can differ depending on the type of production and on it is implemented. For example, in United Kingdom there are 14 stages instead of the 13 presented above, the difference in the UK system is the addition of a first step (define the terms of the reference, in other words, define what the plan is going to focus). This first step is often implemented to prevent an exaggerated plan that probably is not going to be fully implemented, therefore, will be ineffective (Dillon et al., 1996). In this case, the first step is to assemble a team. Depending on the size of the company, several members can compose this group, normally never exceeding six members, or only by one person (Dillon et al., 1996). The team selection can be done by one external identity specialist in HACCP plans, or by an internal one. Nevertheless, the knowledge of the team regarding the quality control, routine works, HACCP plan and identify hazards is always essential (Dillon et al., 1996, Jahncke et al., 2002, Miget, 2004). The next stage, describes the product, is essential that the team is provided with the information about the production, to evaluate in the best way possible all the hazards and consequently improve the quality. The third stage referred is, the identification of the intended use, in other words, identify the target population of the product and if there are any danger concerning the product. In the elaboration of a flow diagram (step four), is crucial understand all the production process and to analyze possible hazards along the process, allowing an overview of the production cycle. There is a group of rules to create a flow diagram, each symbol represents one feature, component or piece of the production cycle (figure 5). In figure 6 is represented an example of a part of the flow diagram from the Safiestela S.A., that was result from the implementation of the FWAS plan. (Annex1). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 26 The next step will be confirming on site the information provided by the flow diagram. Thus, it is needed that every member of the team assesses the events described by the flow diagram, at different times and with different workers in the same event. This will provide a more realistic evaluation of the production routines and consequently a more accurate and better plan (van de Vis et al., 2012). In the point 3.9 it is specified the work done regarding this stage in, Safiestela S.A. The sixth stage is, to identify any hazard along the production and implement preventive measures, this step in the plan is crucial, and it needs to be thorough. At this step, an inexperienced person will have difficulties to identify the hazards. Therefore, a person who has experience in this plans is required on the team, to guide the other members. The identification of hazards normally is done by categories, for example, in a FWAS plan it is common to use four categories to identify hazards: biotic, abiotic, managerial and environmental (van de Vis et al., 2012). Posteriorly the identification of all hazards present in the production cycle it is required to assess which are critical control points (CCP). A CCP is a point where the control is essential, to guarantee the objective of the plan, either if the aim is food safety or animal welfare (Dillon et al., 1996, van de Vis et al., 2012). To identify these CCP it is used a scheme that guide through a series of questions, to separate these critical control points from others problems (figure 7). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 27 Figure 5 - Example of a flow diagram, regarding the incubation and larvae production, developed for Safiestela S.A. Annex 1. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 28 Figure 6 - Decision tree to identify critical control points (CCP) (van de Vis et al, 2012). The next stage is to establish target and limit levels for each CCP. Although, this process can be relatively straightforward in some productions, regarding the food safety and quality, in a FWAS plan this can be challenging. Due to the subjectivity of the concept of animal welfare, nonetheless, in either case, this step must be implemented. In the case of a FWAS plan, the target or limit levels can be defined in some ways, depending on the point that is being analyzed. For example, in aquaculture, acceptable levels , as well as limit levels, are determined for various parameters, such as the concentration of ammonia levels in the tanks of production, to protect the animal welfare. Nonetheless, these boundaries will differ from the species produced, life stage, and the production system. In this stage is fundamental analyze a significant period, to collect enough data to recognize any pattern of the variation of these levels, because any pattern can be an evidence of a problem in the production process. The monitoring system will be a tool to prevent the origin of a CCP and will help in the control of any CCP in production. The monitoring system needs to specify what is monitoring, by whom, the frequency of the monitoring and how the monitoring can be done (table 3) (Dillon et al., 1996, van de Vis et al., 2012). Establishing corrective Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 35 Table 5 - Example of verification procedures developed for Safiestela S.A. Annex 2. 3.8 Establish a record keeping system A record keeping system is essential to guarantee that the FWAS plan is working properly. Another advantage of a record keeping system is to create dynamism in the FWAS plan, to reduce the CCP over time and find others problems that could only be identified through a significant period. According to van de Vis et al. (2012), there are a list of points that help to understand if the record keeping system is effective or not, these are: (1) Point the person responsible; (2) Due diligence requirements; (3) Pre-requisite programs controlled and monitored; (4) Documentation of the procedures; (5) Records of sanitary procedures in a manner to control CCP; (6) Corrective actions documented; (7) Document to determine when, by who and how the FWAS plan is reviewed. In the next image is part of the FWAS plan from Safiestela S.A., while in annex 2 there is the whole plan. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 36 Table 6 - Example of fish welfare assurance system (FWAS) plan for hatching eggs of Senegalese sole, developed for Safiestela S.A. Annex 2. m M What How Frequency Who Hatching eggs Biotic Disease Environment and bad management of holding conditions Monitor egg/larvae quality; verify if the UV treatment is working properly Use of biosecurity plan Here m=M/ Make sure the biosecurity plan is followed Assess that instructions are followed Inspect work done Every hatching of eggs Hatchery manager Train the workers A sampling plan is used to monitor the fish keep records of instructions, analysis and corrective actions Abiotic Fish exposed to deteriored water quality Bad management of holding conditions Instruct the workers Instructions for operation of system are followed Operating limits for operation in instructions Critical limits for water quality in instructions Monitor water quality as specified in the instructions Inspect work done Every hatching of eggs Hatchery manager Train the workers A sampling plan is used for water analysis keep records of analysis, instructions and corrective actions Managerial Eggs/larvae handled roughly during hatching Bad management of hatching conditions Instruct the workers Use of instructions for hatching of the Here m=M / avoid rough handling of Assess that instructions are followed Inspect work done Every hatching of eggs Hatchery manager Train the workers Verify the ecclosion rate keep records of the ecclosion rate Environmental Intake of polluted water to holding tanks Bad management of water intake Instruct the workers Make sure instructions are followed Here m=M/ Avoid intake water if polluted Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for analysis of intake water Keep records of analysis, instructions and corrective actions Monitoring Corrective actions Verification Records Step in process Hazard Source Preventive mesures CCP Criteria Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 37 3.9 Verification on site The critical control points (CCP) identification was done primarily by assessing the production cycle and then on the site. Nonetheless, this first verification in the local was done simultaneously with the integration in the routines of the production, and further attention was needed to confirm the CCP. To complete this task, it was elaborated several verification lists (one per each CCP) with a series of parameters that were discussed by the FWAS team. These lists are checklists that needed to be measured,and posteriorly registered (e.g. time that the fish were exposed to air or the time of the cleaning of the tank). In order to create an average for each point, this average take into account the operator that was in the particular task. The results are posteriorly compared to the limits, which were pre-established by the FWAS team, to indicate if the operations are respecting the limits or not. These verifications create a record to, not only, percept eventual problems in the production but, as well to assure that the previous CCP were, in fact, well identified. Since, some of them can be eliminated or subject to alteration after this thorough analysis on site. Next it will be presented some examples of this verification procedures taken in Safiestela S.A. Not all of the processes were verified due to the time limitations. Nonetheless, the verification templates were done. 3.9.1 Receiving of breeders In the process of bringing new broodstock to the aquaculture, it is usually captured new individuals from the wild or extensive and semi-intensives aquacultures. During this process the animals can suffer damage, due to the potential rough handling, other problems may include parasitic contamination, so analysis must be done to ensure the health state. Regarding the transportation this is done with the animal out of the water, this method do not compromise the animal welfare (Ignacio Martín and Rasines, 2014). The next tables are templates that were created for Safiestela S.A., each one as a checklist necessary in this CCP. Firstly it is recorded the date, if there are any visible damages, the origin of the animals, this allow to the manager to follow the animals and prevent receiving animals from facilities where there are historic of diseases. Finally the operator responsible for the record must sign, this is important because people perception of visible damages may differ. Because the analysis are only performed after acclimation, it was needed to create two separate tables at this point. In table 8, it is Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 38 recorded the date, the animal (in this step the breeder is identifiable by introducing an electronic tag), and the operator responsible for the record. At this stage the animals are in good conditions or not, there is not a medium scenario. Table 7 - Template required in the integration of new income breeders developed for Safiestela S.A. Table 8 - Template required in the integration of new income breeders developed for Safiestela S.A. 3.9.2 Broodstock in RAS and in flow through system The maintenance of broodstock in aquaculture must be regulated thoroughly. Since, in this part the animals are induced to different thermoperiods (according to which room: Spring, summer, autumn and winter) to control the spawning according with the facility needs. Due to that, a template was created to verify if the parameters in production are the best ones to the animals, according to the FWAS plan. To guarantee the animal welfare, limits regarding oxygen concentration and temperature were created, to assess if there were anomalies that could compromise the welfare. The next table represents part of the verification record in the broodstock area, in this table it is only presented the room (spring), however, for the other rooms the verification is similar. In this record must be specified the date, the limits that were established by the FWAS team, and finally the actual values of temperature and oxygen concentration in each tank. Date Visible damages OK/Not OK Origin Operator Date Animal Analysis OK/Not OK Operator Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 39 *Limits according to the Sea8 protocol 3.9.3 Transference of larvae to the weaning area In order to confirm if this step was actually a CCP it was necessary to assess on local some parameters, like the time that the larvae were subject to handling, the temperatures of the origin and destiny tanks and which worker was responsible for the operation (table 10). In this case, it was followed ten operations to assess if the limits, established by the team, were respected or if this was, in fact, a critical control point. The results are demonstrated in the next graph (figure 8). Because normally the transference is done in two days (the first day is transferred half of the tank and the next day the other half) in the results there are two separate moments. It is noticeable that in the second transference the average time that the fish is handled is higher (1361 seconds), than in the first moment (719 seconds). This difference was caused by the husbandry operation itself, since in the second half of each tanks, the operator must be more thorough to guarantee that all the larvae are transferred. Nevertheless, it seems that the larvae were not affected by the time of transference, since there was no mortality in the next two days, and the feeding behavior was normal. Table 10 -Template for transference of larvae to the weaning area developed for Safiestela S.A. *Limits according to the Sea8 protocol Temperature Temperature Temperatura >Temperature>* >Oxygen>* Oxygen Oxygen Oxygen Spring 1 2 3 Date Operator <Temperature> | <Oxigénio> | Comportamento OK/Not OK Limits Date Operator Origin tank Destiny tank Time of handling (s) Temperature DAH Limit max * Origin Destiny Table 9 - Template for broodstock developed for Safiestela S.A. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 40 Figure 8 -Time for each moment of the larvae transference to the weaning area. 3.9.4 Grading in weaning area The grading is a common operation in the aquaculture sector and helps to improve the productivity as mentioned in the 1.4.2 chapter. However, this induces stress in animals due to the handling and, can be a critical point that can potentially compromise the productivity. It is important to define limits, such as time of handling, to avoid any decrease in productivity. In the weaning area it was followed this operation for six times and established the average duration that the fish were handled (time that the fish was packed, plus the time that the fish were out of the water). Other records were made: (1) tank that were graded, (2) destiny tanks, (3) tanks that were potentially disturbed by the operation, (4) operators involved, (5) date of the operation, and (6) mortalities in the day of operation and the next day (table 11). The averages times are presented in figure 9, and it is clearl that the animals are out of the water less time (20.4 seconds, in average), than when they are packed (200.4 seconds, in average). Because the grading process in this area is performed with equipment with a screen and the animals are easily separated by size. The animals exposed to air did not present any signs of distress. Thus, grading in this area is normally rapid and seems not to influence the fish negatively. Nonetheless, further studies are needed to define a limit time for the operation. 700 820 782 701 620 690 840 772 677 606 1456 1542 1658 1700 1683 1438 1628 1669 1619 1765 0 200 400 600 800 1000 1200 1400 1600 1800 2000 1 2 3 4 5 6 7 8 9 10 Time (s) Events Time of handling in the transference of the larvae to the weaning area First half Second half Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 41 Figure 9 - Time of handling during grading in weaning area. 3.9.5 Fish transference from the weaning to the pre-ongrowing area When the fish reach the appropriated size (around 1 g) these are transferred to the pre-ongrowing area, where there are different husbandry conditions. This operation needs to be done rapidly to avoid any decrease in productivity. In this step it was measured: (1) handling time (packed time plus time out of the water), (2) origin tank, (3) destiny tank, (4) operators involved and (5) mortalities in the day of operation and the next day (table 12). These measurements were taken in five moments. The averages time of handling showed that the time out of the water was lower than the time packed (figure 10). Regarding the effects of this operation, it seems that the fish are negatively affected. This was concluded when analysed the mortalities (figure 11), these were higher in the day after the operation (average of 45 fish), than in the day of the operation (average of 15 fish).Thus, a limit must be defined to established, and further studies are needed to assess if the animal welfare is compromised. Table 11- Template for grading in weaning area developed for Safiestela S.A. 170,0 211,0 190,0 221,8 210,8 199,0 11,3 12,8 21,4 24,2 24,2 28,4 0,0 50,0 100,0 150,0 200,0 250,0 1 2 3 4 5 6 Time (s) Events Time of handling during grading Packed time Out of the water Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 42 Figure 10 - Average time of handling during transference. Figure 11 - Average of mortalities in the day of transference and in the day after.. 15,2 45 0 5 10 15 20 25 30 35 40 45 50 Number of individuals Average of mortalities Average of mortalities in the day of operation Average of mortalities in the day after Table 12 - Template for the transference from weaning to pre-ongrowing area developed for Safiestela S.A. 131,5 183,5 152 138,25 168,7 16,2 19,25 19,7 20 19 0 50 100 150 200 250 1 2 3 4 5 Time (s) Events Handling time during the transference of fish from the weaning to the pre-ongrowing area Packed Out of the water Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 43 3.9.6 Grading in pre-ongrowing area As mentioned before, grading can potentially decrease productivity. In this case, it was followed 3 events, and it were registered the next parameters: (1) date, (2) operators involved, (3) handling time, (4) origin tank, (5) destiny tanks, (6) tanks that were potentially disturbed by the operation and (7) mortalities on the day of operation and the next day. In this case, it was not differentiated the time out of the water with the packed time. Because this operation is done using an automatic calibrator, which complicated the following of each fish when it is out of the water. Nonetheless, the time of handling is this case was, in average, 110 seconds. The average mortalities were slightly higher on the following day (52 fish on the day of operation and 60 fish in the next day). Thus, in this case, it is not clear if the grading is affecting the fish negatively or not. Nonetheless, a extended study must be done to reach conclusions. 3.9.7 Water quality in weaning and pre-ongrowing areas In aquaculture, water parameters need to be in optimal values, to increase productivity and to safeguard the animal welfare. In this case, it was performed a compilation of data from six months (from November of 2014 to April of 2015) that had values outside the limits established. It was concluded that in Safiestela S.A., there was only one parameter that exceed these limits, this was the ammonia concentration, for this parameter it was defined an operating limit of 0.45 mg/L. Other parameters were analyzed, and limits were established, such as: temperature, oxygen concentration, nitrites and bromine (table14). In figure 12 is represented the percentage of days, in each month analyzed, that the ammonia concentration was higher than the limits established by the FWAS team. It is clear that the optimal levels were not reached between this months. However, this does not seem to affect the productivity negatively. One explanation is that the operating limit established, according with the literature, did not corresponded to the best ranges for this species. Nevertheless little work with this species was done, to discover which Table 13 – Template of grading in pre-ongrowing are developed for Safiestela S.A. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 44 ammonia concentration levels are beneficial and which are harmful. Additionally, the ammonia values were tested for correlation with the temperature levels. This was an attempt to find a pattern that could lead to the cause of the non-optimal levels. The results of this correlation showed that the temperature fluctuations does not were the cause for the higher levels of ammonia ( the correlation value was 0.116 for the weaning area and 0.41 for the pre-ongrowing area, and p>0.05). 3.9.8 Conclusion With this verification process, it was possible to eliminate some of the initial established CCP. Since, it was confirmed that some of these points did not compromise Table 14 – Template, in which was compiled the water quality data, developed for Safiestela S.A. Figure 11 - Percentage of days, per month, that ammonia levels were higher than 0.45 mg/L. 86,7 71,0 77,4 60,7 67,7 36,7 46,7 38,7 100,0 100,0 100,0 90,0 0 10 20 30 40 50 60 70 80 90 100 November December January February March April Percentage [NH3] Not OK in weaning and pre-ongrowing areas weaning pre-ongrowing Limits OK/ Not OK Limits OK/ Not OK Limits OK/ Not OK Limits OK/ Not OK Date Temperature Temperature Temperature Temperature 18º C < Temp <22º C 0.45 mg/L 1.5 mg/L 0.08 mg/L Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 51 Figure 15 – Percentage of larvae on the stage 0 of metamorphosis. Figure 14 - Percentage of depigmentation from each group. 4.3.4 Metamorphose stage The stage of metamorphosis between the two groups was significantly different (p<0.05) when performed the ANOVA test. Tanks with the treatment light had a faster development, comparing with the tanks in the control group (figure 16, 17, 18, 19 and 20). This is more marked in figure 17 related with the stage 1, in this case, was observable larvae from the treatment group at the second day of the experiment. In the control group the same stage of development appeared on the sixth day of the experiment. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 1 2 3 4 5 6 7 8 9 10 11 12 13 Depigmentation control treatment 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 12345678910 11 12 13 percentage days Stage 0 control treatment Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 52 Figure 16 - Percentage of larvae on the stage 1 of metamorphosis. Figure 18 - Percentage of larvae on the stage 3 of metamorphosis. 0% 20% 40% 60% 80% 100% 1 2 3 4 5 6 7 8 9 10 11 12 13 percentage days Stage 1 control treatment 0% 20% 40% 60% 80% 100% 1 2 3 4 5 6 7 8 9 10 11 12 13 percentage days Stage 2 control treatment 0% 20% 40% 60% 80% 100% 1 2 3 4 5 6 7 8 9 10 11 12 13 percentage days Stage 3 control treatment Figure 17 - Percentage of larvae on the stage 2 of metamorphosis. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 53 Figure 19 - Percentage of larvae on the stage 4 of metamorphosis. 4.3.5 Stomach content In all tanks it seems that the presence of stomach content fluctuated with the same pattern. Although, the treatment group showed a delayed (figure 21). The statistical analysis did not reveal any significant differences between the groups, according by the ANOVA test (p>0.05). Figure 20 - Percentage of larvae with stomach content, comparing the two groups. 4.3.6 Light intensity There were significant differences between the two groups when performed ANOVA test, regarding the light intensity (p<0.05). The tanks from the control group had a light intensity higher (average of 1027 lux), than the tanks from the group of treatment (average of 621 lux). 0% 20% 40% 60% 80% 100% 1 2 3 4 5 6 7 8 9 10 11 12 13 Stomach content control treatment 0% 20% 40% 60% 80% 100% 1 2 3 4 5 6 7 8 9 10 11 12 13 percentage days Stage 4 control treatment Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 54 4.4 Discussion This experiment was an attempt to replicate some results described by Villamizar et al. (2011), and serve as well to amend the experimental design. In the experimented presented in this work the results were not conclusive, due to the number of variables that the experiment had. For instance the light intensity between the control group and the treatment group differ, and the results were probably, influenced by this. The treatment group had low light intensity because it was used only one lamp per tank; additionaly, the type of lamps used was designed for tanks with small diameters (one lamp per 0.2-0.3 m2), and the production tanks had a diameter larger than the recommended, this affected the distribution of light and consequently the distribution of the live feed. Since, the Artemia spp,. that was used as feeding during part of the experiment, has a positive phototaxis and were concentrated in the center of the tank where the light intensity was higher. This may induced an additional energy expenditure for the larvae, in order to find the feed. Another fact that may have affected the development of the larvae in the tanks under the blue spectrum, was the light interference, due to the ack of isolation in the tanks under blue spectrum, since these tanks were in the same room with other tanks of the production. Hence, other light spectrums may have interfered in the tanks under treatment. It was not possible to amend this problem during the experiment, because, under production, it is quite challenging manipulate any holding conditions without affecting the productivity of the facility. 0 200 400 600 800 1000 1200 1400 1 2 3 4 5 6 7 8 9 10 lux Days Light intensity treatment control Figure 21 - Light intensity in each tank during the experiment. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 55 Since, larvae from the two groups were not from the same breeders and spawning, this was another factor that influenced the results. Because different breeders, and even different spawning from the same breeders, can influence the quality of the eggs and larvae (Dinis et al., 1999, Guzmán et al., 2009). It was not possible to amend this problem during the experiment, because, under production it is very challenging manipulate any holding condition without affecting the productivity of the aquaculture facility. Despite these facts described above, in general, seems that the development of the larvae was positively affected by the blue spectrum, namely, when analyzed the dry weight and the metamorphosis stages. Regarding the depigmentation, this parameter presented the highest percentages in the tanks under the blue spectrum. And as in other species like in Pagrus pagrus, this can be related to better holding conditions and better welfare conditions (Szisch et al., 2002, López-Patiño et al., 2013). Although, this fact is not confirmed for the Solea senegalensis, the larvae under blue spectrum presented a faster development in the metamorphosis stages, possibly corroborating the better holding conditions in these tanks. In both groups the larvae transited from pelagic to benthonic stage at the 11 dph, and were then transferred to other tanks with different light regime, so the measure of the light intensity ended on that day. To confirm the results from this experiment, it was important to revise the protocol, in order to amend some flaws from this preliminary work. Nonetheless, in this experiment seems like the blues spectrum beneficiate the development of the larvae, due to the faster development (figure 16, 17, 18, 19 and 20) and the higher dry weights (figure 14). These results can be due to better holding conditions that promoted higher synthesis rate of hormones (Daniels et al., 1996, Szisch et al., 2002, Blanco-Vives et al., 2011, Villamizar et al., 2011, Wunderink et al., 2011, López-Patiño et al., 2013), and/or by higher rates of feeding. Since, the live feed administered could be more visible in tanks under blue light, however, no significant differences were found when analyzed the stomachs content (figure 21). 4.5 Materials and methods of the second experiment In this second experiment, it were used three tanks with a volume of 2.7 m3 each, the same type of tanks as the preliminary experiment. However, in this case, two of the three tanks represent the control group, and the third one was the group of treatment. This last tank was properly isolated to avoid any light interference from other tanks in the production and, this time it was used two lamps with blue spectrum, (435-500 nm) from Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 56 the same brand and model (AQUARAY® AquaBeam 1000), these were installed at 80 cm from the water surface. In the tanks under control, the spectrum used was according to the protocol from the Sea8. The density in each tank differed from the first experiment. There were 36,000 larvae per tank, and the larvae in all tanks were from the same breeders. The rest of the holding conditions were similar to the first experiment (according to the Sea8 protocol). In this experiment the larvae were monitored since the day two post hatching until the 12 dph, 3 samplings were done during this time: On the first day (2 dph); on the fifth day (7 dph); and on the tenth day (12 dph). In each sampling 60 larvae per tank were collected and crio-preserved in eppendorfs for posterior lyophilization. However, in this sampling it was measured the total length and assessed anomalies in pigmentation, thestomach content and the metamorphosis stage from 20 larvae from each thank. The statistical analysis was realized with the software IBM SPSS Statistics 22. 4.6 Results 4.6.1 Total length The total lengths evaluation, as for the other parameters, was done in 3 distinct phases: 2 dph, 7 dph and 12 dph. Thus, it was assessed the evolution in each tank through a linear regression analysis (figure 23). The next graph shows the total lengths of all larvae from each tank in every sampling point. No significant differences were found (p>0.05), between the control group (2.84 mm at 2dph, 5.12 mm at 7 dph and 7.26 mm at 12 dph) and the group of treatment (2.81 mm at 2dph, 4.97 mm at 7 dph and 7.35 mm at 12 dph). The R square values are 0.952 (treatment) and 0.954 (control), they are very similar and explain the evolution of total lengths through time, revealing that the development was highly correlated with the passage of time. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 57 Figure 22 - Linear regression of total lengths (TL) from each group. 4.6.2 Dry weight For statistics analysis of the dry weight, it was also done a linear regression analysis (figure 24). There were no significant differences between the dry weights of the larvae from the two groups (means of the control group were: 0.035 mg at 2 dph, 0.428 mg at 7 dph and 0.738 mg at 12 dph; means of the treatment group were: 0.035 mg at dph, 0.427 mg at 7 dph and 0.747 mg) (p>0.05). The value of R square is higher in the treatment group (0.885) while this value in the control group was 0.835. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 58 4.6.3 Depigmentation Regarding depigmentation, there were significant differences between the control group and the treatment group (p<0.05). In this case, it was done an ANOVA test to compare both groups and tanks. In figure 25 it is noticeable that the larvae that were under treatment presented higher rates of depigmentation: 0% at 2 dph, 80% at 7 dph and 50% at 12 dph. While, the control group had lower percentages: 2.5% at 2 dph, 15% at 7dph and 7.5% at 12 dph. Figure 24 - Percentages of depigmentation of each tank. Figure 23 - Linear regression of the dry weights from each group. 0% 80% 50% 2,5% 15,0% 7,5% 0% 20% 40% 60% 80% 100% 2 7 12 Percentage DAH Percentage of depigmentation Treatment control Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 59 Figure 25 – Percentage of larvae from each group in each stage of metamorphose, from each group at 2 dph. 4.6.4 Metamorphose stage In this experiment, there were no significant differences between the larvae from the two groups (p>0.05), when performed the ANOVA test. It appears that the blue light had not effect, in this parameter, when comparing to the control group (figures 26, 27 and 28). Figure 26 - Percentage of larvae from each group in each stage of metamorphose, from each group at 7 dph. 100% 0% 0% 0% 0% 100% 0% 0% 0% 0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 1 2 3 4 Percentages Stages Stages on 2 dph treatment control 0% 80% 20% 0% 0%0% 85% 13% 3% 0% 0% 20% 40% 60% 80% 100% 0 1 2 3 4 Percentages Stages Stages on 7 dph treatment control Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 60 4.6.5 Stomach content Regarding the stomach content, no significant differences were present between the control and treatment groups (p>0.05). On the first day of experiment (2 dph) the larvae were not feed, so there were any stomach content (figure 29). On the other days of sampling the stomach content was present in the majority of the larvae from the both groups (the percentages at 7 dph were, 100% in the treatment group and 98% in the control group; in the 12 dph they were 100% in both groups). 4.6.6 Light intensity During the experiment, the light intensity was measured in the same days as the samplings, and there were significant differences between the two groups (p>0.05), Figure 28 - Percentage of larvae with stomach content in each group at the time of the samplings. 0% 0% 5% 80% 15% 0% 0% 8% 80% 13% 0% 20% 40% 60% 80% 100% 0 1 2 3 4 Percentages Stages Stages on 12 dph treatment control Figure 27 - Percentage of larvae from each group in each stage of metamorphose, from each group at 12 dph. 0% 100% 100% 0% 98% 100% 0% 20% 40% 60% 80% 100% 2 7 12 Percentage DAH Stomach content treatment control Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 67 About feeding concerns, the larvae present diurnal behaviors so they are fed when the light conditions are favorable. Firstly, they are fed with rotifers, and after a few days they are fed with brine shrimps (according with the Sea8 protocols). At this stage, it is crucial to administrate feed that will fulfill the larvae requirements. Since, the larvae rely on visual perception to predate the live feed, it is fundamental to provide the proper environment conditions, this can be achieve by produce larvae in green waters (this enhance the contrast of the preys with the surround environment) (Villamizar et al., 2011). Another requirement rely on the size of the preys, these need to have the proper size for the mouth size of the larvae (Villamizar et al., 2011). The quality of feeding is essential to provide proper development and to increase ultimately the productivity. Since, it is at this point that the future development will be more influenced, in other words, if the larvae are in good conditions, this will promote an increase in productivity, if not, the productivity will face more challenges, and their future development will be slower and more challenging. Due to that, the larvae development must be followed, every day three to six larvae are sampled per tank, in order to assess their total length, stomach content and anomalies. These samplings provide valuable information to the production management, such as: if the larvae are feeding properly (since adaptation to the feed can be problematic), if there is any tank that has pathogens or any other problems in the holding conditions. Another routine that is essential in this stage is the water parameters control, namely, the temperature and oxygen. Regarding other parameters, these include ammonia, nitrites, salinity and pH. To ensure the proper levels in the various parameters it is periodically done the filters maintenance (backwash of the sand filter and verification of the UV filters). The tanks cleaning is performed in a non-intrusive way, since, the tanks are integrated into a flow through system the metabolites and any dead animal are eliminated with the water flow (the caudal used is stipulated by the company protocols, and it has to be higher enough to properly renovate the water and lower enough to not disturb the larvae). When the larvae starts to perform metamorphosis (10-13 dph) they will sink in the tank and became benthonic, posteriorly it will be transferred to the weaning area. This process is very similar to the transfer between the incubation room and the larvae room. Thus, in this case, the larvae will be siphoned from the bottom of the tank, to a bucket like equipment with a filter in the center. When all the larvae are restrained, they are distributed between, usually, two weaning tanks. To guarantee that the distribution is equivalent between weaning tanks, half of the volume of the bucket like equipment shall Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 68 be distributed in each tank (since the larvae are homogenously distributed in the bucket, due to the turbulence provided by an air diffuser). 5.5 Live feed room Regarding the live feed production, this aquaculture is equipped with the necessary means. Since, the larvae are fed with live feed it is required that this production is synchronized with the larvae cycle, to fulfill the larvae feeding requirements (such as the size of the feed). The feeding of larvae is done manually and it is required to prepare enriched live feed in the morning for the rest of the day. This production area is sub-divided in two parts/rooms, being one focused in the rotifers production and the other in the artemia production. 5.5.1 Rotifers room Rotifers are produced in cylindrical tanks with a conic bottom to facilitate husbandry operations. Concerning about the parameters that need to be followed, the rotifers are maintained at a temperature of 28º C with a salinity level similar to the fish production conditions and an oxygen saturation of 80%. The oxygen is provided by air diffusers that, as in the larvae and incubation room, need to have a particular flow (according to the Sea8 protocol), to maintain the homogenous distribution of animals and feed. The feed of rotifers is composed of algae and yeast and it is administrated several times per day (Lavens and Sorgeloos, 1996). However, when the rotifers achieve the proper size for feeding the larvae, it is required to enrich them. This is done by a commercial formula (according to the Sea8 protocol), which has high levels of fatty acids (Lavens and Sorgeloos, 1996). Routines tasks that need to be done to maintain the cycle of production. It is required: (1) the control of oxygen levels in intervals of four hours; (2) cleaning, equipment and room disinfection and cleaning is required to prevent the development of pathogens that can contaminate the rotifer and fish productions (rotifers are, mainly, affected by bacteria and parasites) (Mondal et al., 2013, Volpe et al., 2013, Boison and Turnipseed, 2015); (3) every day is necessary to assess the biomass, this is done by collecting a sampling and make a count through a magnifier, and estimating the population for the volume of the tank. In this process it can be assessed if the rotifers have any parasite that can compromise the population (Lavens and Sorgeloos, 1996); (4) when the rotifers reach the 3 dph it is necessary to harvest them by filtering, this process is similar to what is done in the incubation and larvae rooms, and (5) conserve Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 69 the recent enriched rotifers in a cold place (an adapted milkmaid equipment), this lower the metabolism and avoid the loss of properties through metabolization. 5.5.2 Artemia room The Artemia spp. has unique properties that allowed it to become the most used type of feed for larvae in many species. The production of brine shrimp is done under similar circumstances comparing with the rotifers production. They are produced in cylindrical with a conic bottom tanks, oxygen is provided by the same air system as what happens in the rotifers room, with a saturation of 80%. There are other parameters that are in the same values, when comparing with the rotifers production, these are: the temperature and the salinity . However, there are some differences between the two productions, for instance, in order to maintain the pH at the adequate level (pH = 8), it is administrated sodium hydroxide (NaOH), which prevent the pH level to decrease to unfavorable levels (Lavens and Sorgeloos, 1996). Normally the hatching of artemia happens in 24h (Lavens and Sorgeloos, 1996) and, to separate the cysts from the brine shrimp it is directed the water through a filtering system that will restrain the cysts, due the action of a magnet field. Since, the cysts obtained were passed through a technique that make possible to restrain them with this method (Naessens-Foucquaert et al., 2009). Once the filtering is complete, the brine shrimp are relocated in other tank, in these they will develop until they reach the proper size, to feed the larvae. In the preparation to feed the larvae, it is required enrich the brine shrimp with a commercial formula (according the Sea8 protocol) rich in fatty acids (Lavens and Sorgeloos, 1996). This enrichment is crucial to increase the nutritional value of the brine shrimp and, therefore, satisfy the larve nutritional requirements. Regarding the routines for the production of brine shrimp, these are quite similar to what happens in the production of rotifers: It is required (1) the control of oxygen levels every four hours; (2) cleaning, disinfection and cleaning of the room is required to prevent the development of pathogens that can contaminate the artemia and fish productions; (3) Separate the cysts from the recent incubated artemia, through the technique mentioned previously; and (4) conserve the recent enriched artemia in a cold place (an adapted milkmaid equipment), this lower the metabolism and avoid the loss of properties through metabolization. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 70 5.5 Weaning area The weaning area is the part with the higher area, and it is composed of 49 square tanks (each tank has a small water column) and another small room (laboratory), where all the water parameter tests from the facility are performed. The area where the tanks are implanted is subdivided into two minor areas (although there is none physical barrier) weaning one and weaning two. The main difference between this two areas is the type of system in which each one is integrated, the weaning one area is in flow through and the weaning two is in a recirculating system. The transference from the larvae room is usually done to the weaning one area, this allows the production to make sure that recently transferred fishes will not transmit pathogens to the rest of the production. It is called weaning area, due to the acclimation to the artificial feed, this is done between the 25-30 dph, and it is a crucial phase in the production cycle. Since, if the larvae, for some reason, do not habituate to this new feed all the production is compromised, or if there is a problem during this transition, the condition of the fish could be affected for the rest of the cycle, and therefore, compromise the productivity. In this area there are a numbers of routines that need to be done every day: (1) water parameters analysis, these analysis are regarding the temperature, oxygen, redox, ammonia, salinity, and nitrites. The temperature is measured at the water entry of a tank with a thermometer and the oxygen with a oximeter (this is measured in every tank, near to the water exit, where the value is lower and where it could be more problems related to lower and high levels of oxygen). While, the rest of the parameters is measured by collecting water samples (normally these samples are from a water entry from one tank), for each parameter there are a protocol to follow, which normally involve adding a reagent and posteriorly pass the sample in a spectrophotometer. The limits for each level are established according to the Sea8 protocols; (2) cleaning, the cleaning and disinfection of equipment and this part of the facility is essential, these are practices that are integrated in the GAP (Mondal et al., 2013, Volpe et al., 2013, Boison and Turnipseed, 2015), every day it is done the cleaning of the tanks (while is doing that, it is simultaneously assessed visible damages in the fish), with a brush for each tank to avoid contamination. The disinfection of the floor and footbaths is also done every day; (3) feeding, this operation is made in an automated way, since, in this area, there is a feeder in each tank, these are connected to a silo like machine that is programmed to deliver an pre-determine amount of feed regarding the biomass of the tank. Nonetheless, in tanks were it is required live feed this operation is done manually; (4) and assess the Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 71 general condition of the fish in the culture tanks, this is done through observation when the tanks are cleaned or during grading. Any damage or ill fish is removed from the tank and register in the production register system. Tanks with excessive rates of illness/damage fish are treated according to Sea8 protocol. Additionally, there are other operations that need to be done with other periodicity, these are: (1) grading, this operation must be done regularly to separate fishes by size and to evaluate the health state, since this species present a great disparity in size of fish with the same age (Blonk et al., 2010, Overton et al., 2010). Normally, this husbandry operation, at this life stage, is made manually with the help of a screen that have a specific mesh, the fishes that pass this mesh are the ones that did not grow as expected and are discarded, the rest of the fishes are distributed according to the management plan; (2) estimate the biomass of a tank, this process consist in weight a determined amount of fishes and estimate the mean weight, this provide to the management an approximate biomass and allow the increase of productivity, since the quantity of feed is determined by the estimation of biomass in each tank, on the other hand, it helps in the traceability process ; (3) and another operation that is done in the weaning area, is the transfer to the pre-ongrowing area, this operation require an excellent management, since the fishes need to be in perfect conditions (to not contaminate the animals in the pre-ongrowing area). When the transference is schedule the daily routines need to be done in the fastest way possible, without compromise the welfare of the animals. The transference, usually, is made when the animals reach the proper size (according with the Sea8 protocol). This company promotes the GAP to increase the productivity. 5.6 Pre-ongrowing area This area is composed by 56 raceway tanks, there are four levels, and each is divided into two parts (north and south) with seven tanks each (figure 31). After the transference from the weaning area to this part of the aquaculture, it is expected that the fish will grow until 40 g and therefore, transferred to the ongowing unit of the Sea8, Aquacria Píscicolas S.A., located in Estarreja. In this area similar processes as what happen in the weaning area are done. For instance the daily routines are the same (water parameter analysis, cleaning, feeding and assess the fish condition) with the difference, that is, the larger proportions that are implied in this area. For example, it is needed to provide higher amounts of feed, due to the superior dimensions of the fish and due to the higher biomass presented in each tank. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 72 The holding conditions of the animals are also similar to the weaning area, with the temperature between the 18-20º C, the salinity between 30-35 ‰ and oxygen above the 80% of saturation. There are other operations that are required, in order to maintain the productivity, these are also similar to what happens in the weaning area (grading and transport). However, as mention before, the larger dimensions of the animals and the higher biomass present in each tank in this area, results in higher duration of these operations. Regarding the grading process, this is done with the aid of a calibrator that split the fishes by three sizes (small, medium and larger). Normally, the smaller fishes are discarded. The transportation management plan required that the fishes pass through a starvation phase (24-48h). This time is in accordance with what is found in the literature (Lekang, 2013), and this will prevent the deterioration of the water during transportation. This process, usually is done once per month by land (truck) and, depending on the biomass that is needed to transport, can be performed on two consecutive days. In the day when the transportation occurs, the personal of the pre-ongrowing area have to expedite the daily routines, without compromise the production and the welfare. For that, it is required an excellent management plan to alleviate, for that day, these routines. This operation must be performed in the fastest way possible, without compromising the animal welfare. The fish distribution in the vehice shall be equivalent between the several compartments. Thus, the nursery manager calculate the biomass during the loading. Figure 30 - Example of a raceway tank (Imsland et al., 2003). Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 73 5.7 Daily routines in Safiestela S.A. Since this facility is composed of six parts (broodstock area, incubation room, larvae room, live feed room, weaning area, and pre-ongrowing area), it is natural that group of workers are established to work in different areas. Nonetheless, there are parts of this facility that are not used continuously (larvae room and incubation room), since in these areas the biomass is transferred quite rapidly (in some days). The description of one day of routine work in this facility englobes the areas in which is needed workers permanently. 5.7.1 Weaning area As mentioned already, this part is separated into three sub-parts (weaning one, weaning two and laboratory). The weaning one receive animals from the larvae room and, for that, it is required to administrate live feed in some of the tanks, until the animals reach the proper age to acclimatize to the inert feed. In the tanks that live feed is administered, the tanks cleaning routines and the animal health state assessment are not performed, since the fishes are too small and could be damaged during this procedure, additionally, during feeding the remain brine shrimps are wash with the water flow. Otherwise, in the rest of the tanks it is required the cleaning and health state assessment of the animals. Thus, on a normal day the chronological routines are: (1) first it is assessed if the recirculating system is working properly (if the filters working properly, it is assessed the skimmer level, and verified the water pumps, the heat exanchers and the ozone generator) ; (2) If there are larvae in the correspondent room it is necessary to feed and take samples of these from each tank, to posterior evaluation for the parameters described in the point 5.4; (3) clean the tanks and assess the health state of the fishes (during this operation it is necessary to remove any ill or dead fish from each tank and report the amount in the register board, for posterior record in the database); (4) the next task that is done is the water analysis, there are parameters that are analyzed in all tanks (oxygen saturation and temperature) and other parameters that are assessed at only one point (redox, ammonia and nitrites), the methods to evaluate these parameters have been already described in the point 5.5; (5) there are a number operations that can be performed after, like grading and consequent distribution, assessing the biomass by calculate the mean weight in each thank, the transference of animals to the preongrowing area, apply treatment in tanks were disease was confirmed during the assessment of the health state of the fishes, and the cleaning and disinfection of the weaning area. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 74 Due to the fish age dispersion, grading is a common practice, while the biomass estimation and the transference to the pre-ongrowing area are performed with another periodicity (in accordance with the Sea8 protocols). The cleaning and disinfection are performed every day, nonetheless, these can be performed more or less thoroughly depending on the time spent on other operations. However, there are minimal tasks in this step that need to be performed to guarantee that the biosecurity protocol is respected; (6) verification of the recirculating system and their components (as already described in the point one); and (7) register the data in the facility digital database. 5.7.2 Live feed rooms In the production of live feed there are two separated rooms, one is for artemia production, and the other is for rotifers production. Although, the production of larvae is not a continuous process, the production of live feed needs to be continuous. Since, even after the metamorphosis the animals require live feed, until they reach the proper age to pass through the weaning process (according to the Sea8 protocols). The daily routines described include the production of both live feeds, and can happen simultaneously. Regarding the artemia production these are the chronologic tasks: (1) in order to provide the larvae first feed of the day, it is required that the previously enriched artemia are filtered, and then put them in the cold (milkmaid); then (2) is necessary to estimate the amount of brine shrimp cysts that is going to be needed for the next 48 hours; (3) in the artemia that need to be enriched it is added sodium hydroxide ever six hours. Every time that this is added it is required to register this in the correspondent sheet; the next task (4) is the calculation of the biomass in each tank, this is done by taking a sampling from the production tank. Posteriorly, it is counted the number of individuals from 1 mL from the diluted sample, knowing this, it is estimated the number of individuals for the volume where the sample was taken. These estimations of biomass allow the personal of this area to calculate the volume needed for each feeding (according with the Sea8 protocols). Thus, any other worker can feed the larvae with the proper instructions from the live feed personal, this is more relevant for the night turns workers; and (5) clean and disinfection of the area, this is performed everyday, in accordance with the Sea8 protocol. Otherwise, the tasks to produce rotifers are these: (1) place the previoulsy enriched rotifers in a filtering system, to admnisterate the first feed of the larvae; (2) initiate a new batch in a clean tank, according with the Sea8 protocols; (3) it is required to calculate the biomass in each tank. To perform this, it is collected a sample from each Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 75 tank, then to calculate the number of rotifers present in this volume it is collected 100 µL from the sample, and it is counted the number of individuals. Like what happen in the artemia counting, it is estimated the biomass from this count; and (5) clean and disinfect the area, this is performed everyday, in accordance with the Sea8 protocol. 5.7.3 Broodstock area Regarding the broodstock daily routines, these are quite simple and normally they are performed in a rapid way, in order to not disturb the animals. The person in charge of the breeders have to: (1) firstly it is assessed if the RAS system and the components are working properly; then (2) it is assessed the state of the animals and verify if there is any eggs in the collectors; (3) clean the collectors and the tanks (this is done according with the Sea8 protocols). While the tanks are cleaned it is assessed the health state of the fishes, as well as, if there are any indications that the fishes are producing gametes; (4) then it is verify the water parameters (referred in the point 5.2); (5) next it is admnistrate feed to each tank. The feeding is usually composed of inert feed, and to administrate it is done a calculation (this is made by the broodstock manager in accordance with the Sea8 protocol), that have to take into account the biomass in each tank, then it is distributed the feed manually; (6) it is performed the cleaning and the disinfection of the area. This step can be done more thoroughly or not depending on the time that the worker have. Nonetheless, there are cleanning procedures that need to be performed to respect the biosecurity protocol; (7) it is registered in the database the water parameters and any other valuable information (like mortalities or presence of gametes, on the animals or collectors); at the end (8) it is verify if the recirculation system is working properly, as described in the point 5.2. The management of the environments parameters allow to manipulate the breeders spawning timing, normally, this operation involves the adjustment of the water temperature (according with the Sea8 protocol). 5.7.4 Pre-ongrowing area As mentioned already in the point 5.6, the pre-ongrowing area has four levels. The daily routines in this area, generally, take more time than in any other part in the production. Thus, at the beginning of the day the workers have to: (1) proceed to inspection of the recirculating system, similarly to what happens in the weaning area; then (2) the workers need to clean all of the tanks, this operation is performed in the lower and upper levels at the same time. While the cleaning is performed it is required to remove any ill Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 76 or dead fish, and register on the board the amount of fishes that were removed. After the tanks cleaning it is remove particles or any fish from the floor; (3) transport of animals to the ongrowing facility when it is needed; (4) grading if necessary, (5) water analysis, (6) cleaning and disinfection, (7) treatment in tanks where it was verified disease outbreaks, (8) verification of the recirculating systems and (9) it is realized the preparations to receive animals from the weaning area, when necessary. Due to the large proportions and the fish age dispersion present in this part, the grading operation is performed regularly. However, this operation lasts a longer time than in the weaning area. During this operation, some fishes are selected, due to the presence of illness, damage or because they are slow growers. Regarding the water analysis, there are parameters that are measured in every tank (oxygen saturation and temperature) and parameters that are measured at one point (redox, ammonia and nitrites). The disinfection and cleaning of this area are performed similarly as what take place in the weaning area, which is conditioning by the available time. Nonetheless, this step needs to guarantee the sanitary conditions defined by the biosecurity protocol. At the end of the day, it is verified the recirculating system as already described in the point 5.7.1. There are operations that are performed sporadically, these are: the internal transport of animals within the facility (from the weaning to the pre-ongrowing area) and the external transport (that is, animals that are transported to the ongrowing installation, Aquacria Piscicolas, S.A.). Both of this transports are described previously, in the points 5.5 and 5.6, respectively. 5.8 Recirculating aquaculture systems (RAS) In animal production, the environmental contamination it is one of the greatest threats, and aquaculture is no exception. In this sector, there are different systems such as flow through and RAS that offer different levels of protection against pathogen outbreaks. In the last decades aquaculture experienced the greatest rise in production, this brought a series of concerns and problems, for example: access to fresh water; effluent discharges; and land resources (Zhang et al., 2011, Badiola et al., 2012, Dalsgaard et al., 2013, Powell et al., 2015, Schroeder et al., 2015, Summerfelt et al., 2015). The RAS is considered the most ecological and the most efficient system for this activity (Zhang et al., 2011). This system helps to control the water parameters that could potentially improve the efficiency and could improve the welfare of the animals within the facility. In a study comparing flow through and RAS, relatively to welfare in rainbow trout Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 83 López-Patiño, M. A., Conde-Sieira, M., Gesto, M., Librán-Pérez, M., Soengas, J. L. & Míguez, J. M. 2013. Melatonin partially minimizes the adverse stress effects in Senegalese sole (Solea senegalensis). 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Chronic and acute stress responses in Senegalese sole (Solea senegalensis): The involvement of cortisol, CRH and CRH-BP. General and Comparative Endocrinology, 171, 203-210. Yang, L., Chou, L.-S. & Shieh, W. K. 2001. Biofilter treatment of aquaculture water for reuse applications. Water Research, 35, 3097-3108. Yuqing, L., Yanxiang, W. & Liling, C. 2014. The Design of Automatic Control System for Industrialized Recirculating Aquaculture. Advanced Materials Research, 246. Zhang, S.-Y., Li, G., Wu, H.-B., Liu, X.-G., Yao, Y.-H., Tao, L. & Liu, H. 2011. An integrated recirculating aquaculture system (RAS) for land-based fish farming: The effects on water quality and fish production. Aquacultural Engineering, 45, 93-102. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 87 Annexes Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 88 Annex 1: Complete flow chart of the production cycle, supporting figures 5 and 6. Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 89 Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 90 Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 91 Annex 2: Entire FWAS plan supporting tables 3, 4, 5 and 6 m M What How Frequency Who Biotic Diseases Natural environment Monitor animals at source Selection of fishing site Here m=M / Health condition (fish without illness/damage or fish with illness/damage) Assess that the fish is healthy Health condition analysis, swmiming, outer appearance response to feed given At each purchase Broodstock manager Purchase from another site/season Report of labaratory analisys keep records of analysis from each supplier Fish may be contaminated with pollutants in previous holding tanks Natural environment Monitor animals at source Analysis result Here m=M / Health condition (fish without illness/damage or fish with illness/damage) Assess that the fish is healthy Perform water analysis At each purchase Broodstock manager Purchase from another site/season Report of labaratory analisys keep records of analysis from each supplier Abiotic Transport conditions Badly managed on transportation Monitor transport parameters Use of Good manufacturing practices to use transport equipment Here m=M / Make sure Good manufacturing practices are being used Assess that Good manufactuing practices are being followed Inspect work done Every transport Broodstock manager Train workers Assess knowledge and verify if transport plan is followed monotoring equipment during transportation Managerial Rough handling during catch Badly managed catching Instruct the workers Intructions for handling during catching are followed Here m=M / Fish condition (fish without damage or fish with damage) Assess that instructions are followed Inspect work done At each capture or purchase Broodstock manager Train workers Check external damage and long term survival (2 weeks) keep records of sample analysed, corrective actions Environmental not applicable Corrective actions Verification Records Receive breeders from the wild Monitoring Criteria Step in process Hazard Source Preventive mesures CCP Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 92 m M What How Frequency Who Biotic Disease Environment and bad management of holding conditions Monitor fish health in case it has not been done at catching site Use of biosecurity plan Here m=M/ Make sure the biosecurity plan is followed Assess that the fish is healthy Check if the workers are following the biosecurity plan properly Daily Broodstock manager Train the workers A sampling plan is used to monitor the fish keep records of analysis, corrective actions Abiotic Fish exposed to deteriorated water quality Bad management of holding conditions Instruct the workers Instructions for operation of quarantine holding tanks are followed O2 < 60% O2 >160% Monitor water quality as specified in the instructions Inspect work done Daily Broodstock manager Train the workers A sampling plan is used for water analysis keep records of analysis, instructions and corrective actions Managerial Adapt fish to artificial feed Badly managed transition to artificial feed Instruct the workers; feed of special composition (atractants) Use of instructions given to adapt fish to inert feed Here m=M/ Make sure the instruction are followed Asses that instruction for inert feed adaptation are followed Inspect work done Daily Broodstock manager Train the workers; change feed type/supplier Assess knowledge and use of instructions; verify feed used keep records of analysis, instructions and corrective actions Disturbance Caused by visitors and/or workers Sound management; Instruct workers/visito rs how to aproach the area Use of Instructions given for handling of fish and quarantine area Here m=M/ Fish behaviour (fish is swimming constantly or fish is settle at bottom) Assess that instructions are followed Inspect work done When visitors/worke rs approach the fish Broodstock manager Train the workers/Plan proper instructions to visitors Verify the disturbance when workers/visito rs approach the fish Keep records of instructions and assessments Environmental Intake of polluted water to holding tanks Bad management of water intake Instruct the workers Make sure instructions are followed Here m=M/ Avoid intake water if polluted Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for analysis of intake water Keep records of analysis, instructions and corrective actions Corrective actions Verification Records Quarantine and acclimation to inert food pellets Monitoring Criteria Step in process Hazard Source Preventive mesures CCP Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 99 m M What How Frequency Who Environmental Intake of polluted water to live feed production tanks Bad management of water intake Instruct the workers Make sure instructions are followed Here m=M/ Avoid intake water if polluted Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for analysis of intake water Keep records of analysis, instructions and corrective actions Weaning Biotic Disease Environment and bad management of holding conditions Monitor fish health; verify if the UV treatment is working properly Use of biosecurity plan Here m=M/ Make sure the biosecurity plan is followed Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used to monitor the larvae keep records of instructions, analysis and corrective actions Abiotic Inadequate inert feed quality Irregular feed quality supplied Selection of supplier based on quality standards Acquisition of tested weaning diets Here m=M/ only tested diets to be used Assess feed quality Check fish respons e to feed; check fish growth Daily Hatchery manager Change supplier Verify feed quality, fish response and growth Keep records of feed quality and corrective actions Fish exposed to deteriored water quality Bad management of holding conditions Instruct the workers Instructions for operation of weaning system are followed O2 < 60%; ammonia 0,75mg/l; nitrite 1,5 mg/l O2 >160% ; ammonia 1mg/l; nitrite 1,8 mg/l Monitor water quality as specified in the instructions Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for water analysis keep records of analysis, instructions and corrective actions Monitoring Corrective actions Verification Records Step in process Hazard Source Preventive mesures CCP Criteria Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 100 m M What How Frequency Who Managerial Innapropriate feed regime Bad management of feed regime Instruct the workers Use of feeding instructions Here m=M/ Make sure the instructions are followed Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers Assess knowledge and use of instructions; Keep records of instructions and assessments Elimination of selected fish Selection of bad growers and defective fish Instruct the workers Use of instructions to kill selected fish Here m=M/ Make sure the instructions are Asses the instructions are followed Inspect work done Every time fish is eliminated Hatchery manager Train the workers Assess knowledge and use of instructions; Keep records of instructions and assessments Disturbance Caused by visitors and/or workers; light regime Sound management; light regime; Instruct workers/visitors how to aproach the area Use of Instructions Here m=M/ larvae behaviour and condition Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers/Pla n proper instructions to visitors Verify the disturbance when workers/visit ors approach the fish Keep records of instructions and assessments Environmental Intake of polluted water to weaning system Bad management of water intake Instruct the workers Make sure instructions are followed Here m=M/ Avoid intake water if polluted Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for analysis of intake water Keep records of analysis, instructions and corrective actions Live feed grown in polluted water Bad management of water intake Instruct the workers Make sure instructions are followed Here m=M/ Avoid intake water if polluted Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for analysis of intake water Keep records of analysis, instructions and corrective actions CCP Preventive mesures Source Hazard Step in process Records Verification Corrective actions Monitoring Criteria Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 101 m M What How Frequency Who Biotic not applicable Abiotic not applicable Managerial Elimination of selected fish Selection of bad growers and defective fish Instruct the workers Use of instructions to kill selected fish Here m=M/ Make sure the instructions are Asses the instructions are followed Inspect work done Every time fish is eliminated Hatchery manager Train the workers Assess knowledge and use of instructions; Keep records of instructions and assessments Rough handling of the fish or exposed to air for too long badly managed Instruct the workers Use of instructions Here m=M/ Make sure the instructions are followed Asses the instructions are followed Inspect work done Every time fish is handled Hatchery manager Train the workers Assess knowledge and use of instructions; Keep records of instructions and assessments Environmental not applicable Pre ongrowing Biotic Disease Environment and bad management of holding conditions Monitor fish health; verify if the UV treatment is working properly Use of biosecurity plan Here m=M/ Make sure the biosecurity plan is followed Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used to monitor the larvae keep records of instructions, analysis and corrective actions CCP Preventive mesures Source Hazard Step in process Records Verification Corrective actions Monitoring Criteria Grading, selection and transfer Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 102 m M What How Frequency Who Abiotic Inadequate feed quality Irregular feed quality supplied Selection of supplier based on quality standards Acquisition of tested diets Here m=M/ only tested diets to be used Assess feed quality Check fish response to feed; check fish growth Daily Hatchery manager Change supplier Verify feed quality, fish response and growth Keep records of feed quality and corrective actions Fish exposed to deteriored water quality Bad management of holding conditions Instruct the workers Instructions for operation of preongrowing are followed O2 < 60%; ammonia 0,75mg/l; nitrite 1,5 mg/l O2 >160% ; ammonia 1mg/l; nitrite 1,8 mg/l Monitor water quality as specified in the instructions Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for water analysis keep records of analysis, instructions and corrective actions Managerial Innapropriate feed regime Bad management of feed regime Instruct the workers Use of feeding instructions Here m=M/ Make sure the instructions are Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers Assess knowledge and use of instructions; Keep records of instructions and assessments Elimination of selected fish Selection of bad growers and defective fish Instruct the workers Use of instructions to kill selected fish Here m=M/ Make sure the instructions are Asses the instructions are followed Inspect work done Every time fish is eliminated Hatchery manager Train the workers Assess knowledge and use of instructions; Keep records of instructions and assessments Disturbance Caused by visitors and/or workers; light regime Sound management; light regime; Instruct workers/visitors how to aproach the area Use of Instructions Here m=M/ larvae behaviour and condition Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers/Pla n proper instructions to visitors Verify the disturbance when workers/visitor s approach the fish Keep records of instructions and assessments CCP Preventive mesures Source Hazard Step in process Records Verification Corrective actions Monitoring Criteria Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 103 m M What How Frequency Who Environmental Intake of polluted water to weaning system Bad management of water intake Instruct the workers Make sure instructions are followed Here m=M/ Avoid intake water if polluted Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for analysis of intake water Keep records of analysis, instructions and corrective actions Starvation and transfer to ongrowing facilities Biotic Disease Environment and bad management of holding conditions Monitor fish health; verify if the UV treatment is working properly Use of biosecurity plan Here m=M/ Make sure the biosecurity plan is followed Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used to monitor the larvae keep records of instructions, analysis and corrective actions Abiotic Fish exposed to deteriored water quality Bad management of holding conditions Instruct the workers Instructions for operation of preongrowing are followed O2 < 60%; ammonia 0,75mg/l; nitrite 1,5 mg/l O2 >160% ; ammonia 1mg/l; nitrite 1,8 mg/l Monitor water quality as specified in the instructions Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for water analysis keep records of analysis, instructions and corrective actions CCP Preventive mesures Source Hazard Step in process Records Verification Corrective actions Monitoring Criteria Mestrado em Ciências do Mar - Recursos Marinhos 2014/2015 João Filipe Macedo Pereira 104 m M What How Frequency Who Managerial Rough handling of the fish badly managed Instruct the workers Make sure instructions are followed Here m=M/ Fish shoud not reveal excessive physical traumas Assess that the handling is gentle Inspect the fish in ongrowing facility and inspect handling at Every transfer Hatchery manager Train the workers verify the condition of the fishes in ongrowing facility keep record the condition of the fishes, instructions and assessments Environmental Intake of polluted water to weaning system Bad management of water intake Instruct the workers Make sure instructions are followed Here m=M/ Avoid intake water if polluted Assess that instructions are followed Inspect work done Daily Hatchery manager Train the workers A sampling plan is used for analysis of intake water Keep records of analysis, instructions and corrective actions CCP Preventive mesures Source Hazard Step in process Records Verification Corrective actions Monitoring Criteria