Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters

Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters Ricardo Jorge de Freitas José Dissertação de Mestrado em Ciências do Mar e Recursos Marinhos Especialidade em Aquacultura e Pescas 2012 Ricardo Jorge de Freitas José Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters Dissertação de Candidatura ao grau de Mestre em Ciências do Mar e Recursos Marinhos – Especialidade em. Aquacultura e Pescas submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto. Orientador – Carlos Alberto Pestana Andrade, Ph.D. Categoria – Chefe de Divisão Afiliação – Centro de Maricultura da Calheta, Direcção Regional de Pescas, Governo Regional da Madeira Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page I Resumo Nas últimas três décadas a aquacultura tem-se afirmado como uma das principais formas de produção de alimento para a população mundial. O incremento dos valores desta produção foi conseguido com a melhoria das técnicas e com o aumento do número de instalações de produção. O avanço no desenvolvimento de metodologias de cultura têm permitido a reprodução em cativeiro e o domínio dos estádios larvares de diferentes espécies de interesse comercial, fundamentais para a diversificação de espécies e para a sustentabilidade da actividade. Uma das metodologias que tem apresentado melhores resultados na produção larvar de novas espécies de peixes marinhos é o mesocosmos, de características semi-intensivas. Isto é, metodologias de produção larvar intermédias entre o intensivo e o extensivo, que utiliza tanques de cultura de grande volume entre 30 e 100 m3, e uma densidade larval de 2-8 indivíduos por litro. Os sistemas de produção larvar são sistemas dinâmicos, sujeitos a diferentes variáveis – físico-químicas e biológicas, que variam ao longo do período de cultura e influenciam o desenvolvimento larvar. Existe pouca literatura a incidir sobre o padrão dos parâmetros ambientais bióticos e abióticos durante a produção de larvas de peixes marinhos em mesocosmos e como os mesmos se distribuem no interior do tanque. Neste trabalho propomo-nos realizar a descrição espacial e temporal da evolução dos parâmetros ambientais durante a cultura de larvas de dourada (Sparus aurata) em mesocosmos de metodologias semi-intensivas. Procuramos ainda descrever o comportamento das larvas desta espécie ao longo do período de fase larvar até à fase juvenil. As larvas de dourada apresentaram uma taxa de eclosão elevada (98±0.1%), bem como uma elevada taxa de insuflação da bexiga-natatória (90%). A taxa de sobrevivência das larvas registou valores esperados para esta metodologia (60%). Um dos parâmetros utilizados para assegurar o bem-estar animal, com possibilidade de ser utilizado como parâmetro de análise de produção, é o comportamento. Na pesquisa efectuada sobre este tema, o registo existente incide sobretudo sobre a população juvenil ou adulta, com particular interesse nos comportamentos de alimentação e agressivos. Neste Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page II sentido o trabalho apresentado é pioneiro ao descrever alguns comportamentos de larva de dourada em mesocosmos. É descrita a distribuição larval na superfície e profundidade. Foram também descritos 7 comportamento, dos quais se destaca a baixa incidência de canibalismo, sendo que ocorrem comportamentos agressivos (dia 35). Foram analisados vários parâmetros e descrita a sua variação ao longo do tempo. São descritos os padrões hidrodinâmicos (velocidade e direcção da corrente), assim como de luz, pH, temperatura, oxigénio, nos momentos em que ocorram alteração do meio de cultura. Quanto aos padrões hidrodinâmicos no tanque as correntes apresentaram uma direcção paralela à parede lateral, sendo os desvios relacionados com a posição e presença de arejadores. A velocidade das correntes (entre 0.69 e 1.38 cm/s) esteve relacionada com o fluxo de entrada de água, sendo que a sua variação foi regulada de acordo com o estado de desenvolvimento larval. A temperatura é um parâmetro homogéneo no interior do tanque (20.1 ±0.1), sendo que o padrão da sua variação é igual a da água do mar. O padrão de distribuição do oxigénio (6,06±1,35 mg/L) não é homogéneo em todo o tanque de cultura. Ocorreram variações espaço - temporais deste parâmetro ambiental, com menores valores, na parte central do fundo do tanque. A salinidade (36±0.05‰) e o pH 8.6 (±0.09) foram constantes ao longo do período analisado. A luz é um dos parâmetros que apresenta maior variação (1514 ± 1444 lux), associada à fase do protocolo de cultura e ao local de amostragem. Espera-se que este trabalho permita um novo olhar sobre o mesocosmos de metodologias semi – intensivas. A compreensão da distribuição dos parâmetros ambientais, no interior do tanque de cultura, associada ao estabelecimento de padrões normais de comportamento larvar, poderão melhorar a produção em termos qualitativos e quantitativos e, no futuro servir de base para outros estudos. Palavras – Chave: Mesocosmos metodologia semi-intensiva; Sparus aurata larvas; parâmetros abióticos; parâmetros bióticos; padrões; comportamento. Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page I Abstract Aquaculture is one of the growing food supply sources in the world. Over the last years aquaculture production has grown at steady pace. This growth was achieved by improving and development of larval rearing methodologies, as well as with the increase of the number of production facilities. In the case of the rearing methodologies, the mesocosm or semiintensive methodologies has provided juveniles of higher quality of wellknown species, as well as being a methodology that is suited for the culture of “new” species, contributing for species diversification. Mesocosm of semi-intensive methodologies is a rearing methodology that is situated between intensive and extensive methodologies. Mesocosm methodologies make use of large rearing tanks, with volumes between 30 and 100 m3, and rearing densities between 2 and 8 individuals per litter. Rearing systems are dynamic due to being under the influence of several physical – chemical and biological parameters, which vary during the rearing period and with direct influence on larval development. Literature concerning the distribution of the parameters -light, dissolved oxygen, temperature, salinity, pH - in aquaculture production systems is scarce and is almost null when referring to larval stages. In this paper we propose to make the description of the spatial and temporal evolution of environmental parameters during the culture of sea bream (Sparus aurata) larvae in mesocosm of semi-intensive methodologies. We also aim to describe larvae behaviour in those culture conditions. Sparus aurata hatching rate was high (98 ± 0.1%), and the larvae presented a high rate of inflation of the swim bladder (90%). Survival rate was within the expected values for these rearing methodologies (60%). Larvae distribution patterns and behaviour in the rearing tank can be used to assess animal welfare. The distribution of larvae on the surface and depth are described. This work presents new data for Sparus aurata larvae behaviour in mesocosm. Seven behaviours are described for the entire larval phase. Despite the common aggressive behaviour of larvae from day 35 post hatching, there was low incidence of cannibalism. Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page II The different abiotic parameters showed highly variable patterns. Temperature presents a uniform pattern throughout the larval development. Dissolved oxygen presented an irregular pattern in both time and space with the existence of multiple areas of different concentrations. Light was extremely variable at surface, while in depth it varied according to the culture stage. Regarding the hydrodynamics of the larval rearing tank, the water current was parallel to the lateral wall, with alterations due to the presence of aerators. Water velocity in the rearing tank was related to the water exchange rate, according to the larvae stage and was consistently below the critical limits to the larval species. Temperature was homogenous for the entire rearing tank (20.1 ± 0.1ºC), and the variation pattern was similar to the sea water inlet. Dissolved oxygen (6.6±1.35 mg/L) presented significant differences between stations. The lowest values for this environmental parameter were registered at the central part of the bottom of the tank due to the accumulation of wastes. Both salinity (36±0.05‰) and pH (8.6 ±0.09) were constant along the rearing period. Light was the parameter that presented the highest variation (1514 ± 1444 lux), in both time and space. It is expected that this work provides a new look on mesocosm of semiintensive methodologies. A better understanding of the rearing tank environmental parameters, as well as of larvae behaviour will be useful to enhance production quality and quantity and provide a base line for further studies. Keywords: Mesocosm of semi intensive methodologies; Environmental parameters, Behaviour; Sparus aurata; Biotic and Abiotic Parameters; Patterns Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page I Agradecimentos Quero agradecer a Direcção Regional de Pescas da Madeira, por ter permitido efectuar a minha tese de dissertação no Centro de Maricultura. Ao Doutor Carlos Andrade pela ideia, orientação, confiança e conhecimentos, a sua sabedoria foi essencial para a elaboração desta tese. A todo o pessoal do Centro de Maricultura da Calheta, especialmente ao António Abreu “James”, pelo auxílio na elaboração e execução das tarefas… não me esqueci que te devo uma (s) poncha (s). Ao Emanuel Pinto, pela ajuda na construção dos materiais de apoio, ao António Branco e a Maria João pelos conhecimentos partilhados. A Lurdes Ferreira, pela companhia inesperada. Ao Bernardo Sumares, pela companhia ao longo desta jornada. A Doutora Délia Canha Departamento de Matemática e Engenharia, ao Doutor. Miguel Sequeira do Centro de Ciências da Vida, ambos da Universidade da Madeira, a Doutora Antonieta Amorim da Direcção Regional de Pescas, pelo tempo e ajuda disponibilizados para me auxiliar aspectos técnicos da tese. A minha turma de mestrado, pela amizade, brincadeira, que o contacto não se perca…. I can’t take it take it take no more\Never felt like felt like felt like this before… Ao Carlos Marques, Ana Beça, Amelia Miguel-Gilmore pelas correcções e pela amizade. Carlos vai mais um café? Para terminar aos meus pais, irmão e a Cristina sem o vosso apoio incondicional esta jornada não teria acontecido. Esta tese no fundo é-vos dedicada. Funchal, Outubro de 2012 Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page i Índex ÍNDEX...................................................................................................................................................I ACRONYMS AND ABBREVIATIONS.............................................................................................III FIGURE INDEX................................................................................................................................. IV GENERAL INTRODUCTION............................................................................................................1 1. INTRODUCTION........................................................................................................................1 1.1 GILTHEAD SEA BREAM (SPARUS AURATA)..............................................................................4 1.1.1 SPARUS AURATA LARVAL DEVELOPMENT..........................................................................4 1.1.2 SPARUS AURATA BEHAVIOUR..................................................................................................7 1.2 ENVIRONMENTAL REQUIREMENTS FOR LARVAL DEVELOPMENT............................8 1.2.1 Hydrodynamics....................................................................................................................8 1.2.2 Water Temperature.............................................................................................................9 1.2.3 Salinity....................................................................................................................................9 1.2.4 pH..........................................................................................................................................10 1.2.5 Dissolved Oxygen.............................................................................................................10 1.2.6 Light......................................................................................................................................11 1.3 REARING METHODOLOGIES.................................................................................................13 2. CENTRO DE MARICULTURA DA CALHETA (CMC)........................................................15 2.1 General layout of the hatchery .........................................................................................15 2.2 Mesocosm larval rearing tank...........................................................................................15 3. METHODOLOGY.....................................................................................................................18 3.1 LARVAE SOURCE AND CULTURE METHODOLOGY.........................................................................18 3.2 EGG AND LARVAL GROWTH PERFORMANCE..................................................................................19 3.3 BEHAVIOURAL OBSERVATIONS..........................................................................................................21 3.4 MESOCOSM ENVIRONMENTAL ANALYSIS ..........................................................................................22 3.4.1 Hydrodynamics..................................................................................................................22 3.4.2 Culture sampling stations..............................................................................................24 3.3 STATISTICAL ANALYSIS..................................................................................................................28 4. RESULTS...................................................................................................................................29 4.1 EGG AND LARVAE PERFORMANCE.................................................................................................29 4.1.2 Larvae behaviour...............................................................................................................33 4.2 ENVIRONMENTAL PARAMETERS........................................................................................................36 4.2.1 Water flow patterns..........................................................................................................36 4.2.2 Salinity..................................................................................................................................38 4.2.3 Temperature.......................................................................................................................39 4.2.4 Dissolved Oxygen.............................................................................................................41 4.2.5 pH..........................................................................................................................................43 4.2.6 Light......................................................................................................................................43 5. DISCUSSION ............................................................................................................................46 5.1 LARVAE PERFORMANCE.................................................................................................................46 5.2 ABIOTIC PARAMETERS...................................................................................................................49 5.2.1 Rearing tank hydrodynamics.........................................................................................49 Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page ii 5.2.2.pH..........................................................................................................................................50 5.2.3 Salinity..................................................................................................................................50 5.2.4 Temperature.......................................................................................................................51 5.2.5 Dissolved Oxygen.............................................................................................................51 5.2.6 Light......................................................................................................................................51 6. CONCLUSIONS........................................................................................................................53 7. REFERENCES............................................................................................................................55 Gilthead Sea Bream (Sparus aurata) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 4 1.1 GILTHEAD SEA BREAM (Sparus aurata) Sparus aurata, is a perciform teleost of the Sparidae family. It is demersal, eurythermal and euryhaline species present in a wide range of habitats, from the South of England to Mauritania. Sexually S. aurata is a protandrous hermaphrodite, achieving maturity in 2 years and, males transforming into females between 2-3 years old. During the spawning season, under good conditions, a single female can lay between 20 000 to 80 000 eggs per day (FAO, 2012a). Gilthead sea bream aquaculture began in the 1970’s, and nowadays is one of the most farmed species in European marine aquaculture (Shields, 2001). 1.1.1 Sparus aurata LARVAL DEVELOPMENT Fish larvae development can be defined as the phases a fish goes through between hatching and becoming a juvenile (Howell, Day, Ellis, & Baynes, 1998). Larval development can be divided in three main stages and two transitional stages, according to Kendal (1984) and Howell (1999): 1. Egg stage – which embraces the entire process from the fecundation until hatching. 2. Larval stage – which includes the period between, hatching and the beginning of squamation. During this stage larva undergoes transformations in terms of body shape, locomotion. There are several sub stages: a. Yolk - sac larvae – Developmental stage beginning with hatching and ending with exhausting of yolk sac; b. Preflexion larva – From the exhaustion of the yolk sac until the upward flexion of the notochord; c. Flexion larva – After the notochord has terminated its flexion until the plural bones assuming a vertical position; Gilthead Sea Bream (Sparus aurata) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 5 d. Post flexion larva - Larvae acquires the formation of the caudal fin (hypural elements vertical), as well as full external meristic complements (fin rays); 3. Juvenile stage: Completion of fin ray counts and beginning of squamation until fish enters adult population or attain sexual maturity. S. aurata larvae at hatching has 3 mm total length, the eyes become functional at the end of the stage and larvae acquire mobility (Moretti, Fernandez-Criado, Cittollin, & Guidastri, 1999). During this stage the digestive tract starts differentiation (Kendall, Ahlstrom, & Moser, 1984; Sarasquete, Polo, & Yufera, 1995) There are two moments that define the second phase of fish larval development: first feeding and the swim bladder inflation. Not being able to feed at this stage is the cause of high mortality among fish. In culture conditions S. aurata larvae are first fed rotifers of about 100 µm, which is the size of the mouth of larvae at this stage (Moretti et al., 1999). At older stages bigger size prey such as Artemia can be provided, since larvae have bigger mouth and more developed digestive tract (Fernández-Diaz & Yufera, 1995). The non-inflation of the swim bladder can be responsible for problems in buoyancy and eventually death. In S. aurata swim bladder initiates differentiation at 2 - 3 days after hatching (dah) and it is positioned dorsally between the digestive tract and the spleen (Sarasquete et al., 1995). The first inflation of the swim bladder ends around day 15, with the process being complete around 40-50 dah (Moretti et al., 1999). For gilthead Sparus aurata the development milestones of the larval stages were already established for intensive rearing at 17-18 ºC (Moretti et al., 1999) Figure 1: Gilthead Sea Bream (Sparus aurata) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 6 Day Size (mm) Characteristics 1 3 Hatching 2 3.5 Pectoral fins appear 3 3.8 Exotrophy starts 4 Eyes pigmented 60% of yolk sac absorbed 40% of oil drop absorbed 5 4 Primary swim bladder inflation 100% of the yolk sac absorbed 70% of oil drop absorbed 15 5 End of the primary swim bladder inflation 100% of the oil drop reabsorb Caudal fin 17 7 Anal fin 20 7.5 Stomach starts developing 45 11 Second dorsal fin 50 15 First dorsal and ventral fin 60-70 20 Scales 90 30 Definite morphology Figure 1 - Larval development of Sparus aurata at 17-18ºC according to Moretti, et al. (1999). Gilthead Sea Bream (Sparus aurata) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 7 1.1.2 Sparus aurata BEHAVIOUR Behaviour represents a reaction to the environment as fish perceive it and is therefore a key element of fish welfare (Martins et al., 2012). One of the most common observed behaviours of larvae fish is vertical migrations, which are a form to control light intensity and escape predators (Fiksen, Jorgensen, Kristiansen, Vikebo, & Huse, 2007). There is limited information available regarding S. aurata larvae behaviour. Moretti et al. (1999) described two types of major behaviours for S. aurata larvae in culture conditions, in 6-10 m3tanks: 1) At hatching, yolk – sac larvae have passive floating with slow and infrequent body movements without a clear posture, they sink slowly, head first and then every second swim upwards; 2) As larvae, they have a pronounced tendency to sink, with almost complete passivity accounting for a uniform dispersion in the water body. Behaviour is influenced by rearing conditions, therefore the importance of understanding the culture environment (Monk, Puvanendran, & Brown, 2008). Environmental requirements for larval development) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 8 1.2 ENVIRONMENTAL REQUIREMENTS FOR LARVAL DEVELOPMENT Water quality is of the major importance to provide a healthy environment to fish (Moschou et al., 2000). If the right parameters and environmental conditions are not met it is possible that survival of larvae is at risk or that larval development will be compromised (Andrades, Becerra, & Fernández-Llebrez, 1996; Yúfera & Darias, 2007). Environmental factors play an important role in all aspects of a fish life cycle. Parameters can be divided accordingly to their type of action: 1) if they act directly over larva development or 2) if they are limiting, if larvae require a certain range to survive (Boeuf & Payan, 2001). Temperature, salinity and light intensity were the parameters studied as part of the former category of parameters. Whereas the dissolved oxygen and pH, also analysed in the course of this thesis, need to be within a certain range for the proper development of larvae. 1.2.1 Hydrodynamics Water dynamics is responsible for water quality and for the distribution and welfare of fish (J. Oca, Masalo, & Reig, 2004; Ross, Watten, Krise, Dilauro, & Soderberg, 1995). The water exchange is responsible for assisting fish achieve a proper development, through providing oxygen and a suitable water velocity for swimming (Divanach, Papandroulakis, Anastasiadis, Koumoundouros, & Kentouri, 1997; Timmons, Summerfelt, & Vinci, 1998; Valverde, Mendiola Lopez, & de Costa Ruiz, 2005). Water current has been manipulated and used to improve feeding behaviour of sea bass (Valverde et al., 2005). The longer is the residence period of rearing water, the lower is the quality of the water (Good et al., 2009; Hougham & Moran, 2007). However, the higher the exchange rate, the higher is the velocity of the water in the rearing tank. High water velocities are responsible for fish spending more energy swimming and thus, requiring energy that otherwise could be used for growth (C. A. P. Andrade et al., 2011). Higher water velocities may also decrease the probability of larvae to feed (Canavate & Fernandez-Diaz, 2001) and is frequently the source of skeletal abnormalities (Divanach et al., 1997; J. Roo, Socorro, & Izquierdo, 2010). Environmental requirements for larval development) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 9 Aerators are commonly used in larval rearing tanks. The aerators increase water mixing improving water quality, as well as causing alterations on the water flow pattern (Shiotani, Hagiwara, Sakakura, & Chuda, 2005). According to Moretti et al. (1999) for Sparus aurata culture water velocity should be kept below 10 cm/s. 1.2.2 Water Temperature Temperature is one of the most decisive environmental variables affecting all the stages of a fish cycle, from induction of reproduction to regulating larval development. Larval stages are the most sensitive to variations of this parameter (D. Stewart Fielder, Bardsley, Allan, & Pankhurst, 2005; Green & Fisher, 2004; Koumoundouros, Divanach, & Kentouri, 2001). Inadequate temperatures can be the responsible for several abnormalities on larval development, for example: lordosis or absence of gill covers (Sveinung Fivelstad, Bergheim, Hølland, & Fjermedal, 2004) and in extreme cases cause mortality of fish (Bermudes & Ritar, 1999). Still there is a narrow range of temperatures that provide a normal development – in terms of organogenesisonly affecting growth (Bermudes & Ritar, 1999) For S. aurata larvae development water temperature should be within 14-22ºC, with optimal interval for larval development between 16-22ºC (FAO, 2012a). 1.2.3 Salinity Salinity can be defined by the amount of salts dissolved in the water (Castro & Huber, 2003). Salinity is one abiotic parameter that is exclusive of the aquatic environment (Boeuf & Payan, 2001) Osmoregulation is demanding in terms of energy. Fish species prefer conditions that allow a reduction in this process, allocating energy to other processes, like growth (Sampaio & Bianchini, 2002). Osmoregulation is carried out through ion and water regulation at different parts of the fish body. To keep homeostasis fish drink sea water and excrete the excess of salt (Tandler, Anav, & Choshniak, 1995). Environmental requirements for larval development) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 10 Salinity can affect buoyancy of eggs and larvae, as well as the ability to inflate the swim bladder. Problems with the swim bladder inflation result in high mortality reduced larval growth and skeletal deformities (D. Stewart Fielder et al., 2005; Tandler et al., 1995). Salinity is also one of the main factors influencing fish distribution (Bodinier, Sucre, Lecurieux-Belfond, Blondeau-Bidet, & Charmantier, 2010; Moustakas, Watanabe, & Copeland, 2004). S. aurata is able to tolerant a wide range of salinities, from 15 to 40‰ (Boeuf & Payan, 2001), with optimal salinity for larvae at 25‰ (Tandler et al., 1995) 1.2.4 pH pH is determinant in establishing the acid-base relations of several reactions in aquatic environment (Piedrahita & Seland, 1995) pH may have effects on fish health or be responsible for environmental problems, in either natural or controlled environments. pH is not a constant value, but is often restricted to a narrow range, despite the buffer ability of the water (Parra & Yufera, 2002; Piedrahita & Seland, 1995). S. aurata larvae have been proved to be able to develop within pH ranging from 4,88 – 9,57 (Parra & Yufera, 2002). 1.2.5 Dissolved Oxygen Oxygen is a fundamental variable in aquaculture production, being one of the most important factors that should be taken into account, when designing a facility. The first oxygen source is the water inlet (Merino, Piedrahita, & Conklin, 2009). In the culture environment it is not only fish who consume oxygen. The bacteria, algae and live feeds (rotifer and Artemia) also require oxygen to survive, and should be taken into account when calculating oxygen requirements (Merino et al., 2009). Oxygen requirements vary according to depth, stock density, feed and larval stage (S. Fivelstad et al., 1999; Merino, Conklin, & Piedrahita, 2011; Merino, Piedrahita, & Conklin, 2007).The effects of this variation has so far not been subject to investigation (Thetmeyer, Waller, Black, Inselmann, & Rosenthal, 1999). Environmental requirements for larval development) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 11 Low oxygen availability will constrain feeding and consequently development (Merino et al., 2011). The distribution of oxygen inside the rearing tank should be the most homogenous possible. The use of aerators besides increasing the oxygen levels, contributes to a good water mixing and prevents hypoxia situations – oxygen value below 2,0 mg/L – or anoxic situations – 0,0 mg/L (Diaz & Rosenberg, 1995; Wu, 2002). In aquaculture the parameter measured is dissolved oxygen (mg/L), which is a good indicator of water quality, due to its use in biological and chemical reactions (Mustapha, 2008). Despite differences in the adopted methodologies it is general consensus that oxygen uptake is directly proportional to water temperature and feeding ration, and inversely to fish size (Merino et al., 2009). A proper oxygen supply provides correct amounts of oxygen without compromising water velocity as it is referred to by several authors in Merino et. al. 2009. Dissolved oxygen should be within the interval of 6.48.2 mg /L for a proper development of S. aurata larvae (Navarro & Sarasquete, 1998). 1.2.6 Light Light is one of the principal parameters that help regulate fish life cycle, and is one of the most studied parameters for larval development (Boeuf & Le Bail, 1999). Light intensity can be responsible for larvae initiating feeding, since larvae require a minimum of light to feed (Boeuf & Le Bail, 1999; D. S. Fielder, Bardsley, Allan, & Pankhurst, 2002; Monk, Puvanendran, & Brown, 2006). The photoperiod, can be defined as the amount of time light is available. The more extensive is the photoperiod, the longer the time a fish may feed, allowing an increase in energy which can be used, for example for growth. The duration of day also seems to be related to swim bladder inflation (D. S. Fielder et al., 2002). Light is a highly variable parameter, varying during the day, season and medium where it interacts. In the aquatic environment light is affected by the Environmental requirements for larval development) Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 12 suspended materials that alter the depth light can reach, as well as its reflexion (Boeuf & Le Bail, 1999; Karakatsouli et al., 2010). Alterations in light can be used to alter diets, namely alterations of prey, according to their transparency (Naas, Naess, & Harboe, 1992). Light is not only beneficial for a fish, as problems related to hatching and buoyancy of eggs have been reported (Downing & Litvak, 2002). Long photoperiods can affect the larvae ability to inflate the swim bladder (D. S. Fielder et al., 2002). These effects of light are species specific (Monk et al., 2006). Light is one of the aquaculture parameters that can be manipulated, with the use of artificial sources. Light is also influenced by the colour of the tank, presence of algae in the rearing environment and is dependent of the rearing methodologies used. Sparus aurata larvae should be reared under a light intensity of at least 50-150 lux (Boeuf & Le Bail, 1999) and with optimal light intensity from 1000 to 3000 lux (Moretti et al., 1999). Rearing Methodologies Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 13 1.3 REARING METHODOLOGIES The economic viability of an aquaculture unit depends on hatcheries, which are responsible for supplying the correct quantities of juveniles at a requested moment, with reasonable prices (L. Alvarez-Lajonchère, Reina Cañez, Camacho Hernández, & Kraul, 2007). There are several types of hatcheries and these are classified according to tank volume, rearing density, prey source (Shields, 2001). One of the most common classifications is proposed by Divanach and Kentouri (2000) (Figure 2). Parameters Methodologies Extensive Mesocosm Intensive Rearing enclosures Ponds or bags Thanks or bags Tanks Localization Outdoor Indoor Indoor Rearing volume (m3) >100 30-100 <20 Rearing density (individuals per liter) 0.1-1 2-8 30-200 Food chain Endogenous Mixed Exogenous Infrastructures Light Medium Sophisticated Environment Natural Mixed Controlled Dependence on man and technique Light Medium High to very high Need for specific biological knowledge Light Medium High to very high Validity for new species Very high High Medium to low Figure 2Rearing methodologies according to Divanach et al. (2000) Every methodology presented has advantages and disadvantages. The extensive approach, provides a better higher knowledge of the biology and requirements of the species, but lacks control over the production system and has limited production capacity. The intensive approach on the other hand, allows more production with higher economical costs, due to all the technology and knowledge required. Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 20 During larval rearing daily mortality was calculated in two areas of the rearing tank, at the surface and at the bottom of the tank. Daily surface mortality was calculated by two methods. In one method a plastic square of 0.25m2was placed at the surface of the rearing tank and designated SS (Figure 7Erro! A origem da referência não foi encontrada.). A second method was tested using surface skimmers (SK) to collect all dead larvae from the water surface of the entire tank. For this method a collecting basket with a 50µm mesh net was placed at the exit of the water outlet surface (Wos). Figure 7 - Dead larvae counting devices. The X marks the positions of the squares at the surface. Daily mortality at the bottom of the tank was registered by siphoning two squares (0.25m2) painted with a white line at the bottom of the rearing tank (Figure 8). The squares (BS1 and BS2) were placed opposite each other. The squares were painted in the axel Wi – Wo, with the center at equal distance between the outlet and the center of the tank. Daily total mortality based on this method was extrapolated from the mean value of the 2 squares, as the area of each square was approximately 79 times smaller than the area of the rearing tank. The mortality rate was also calculated when siphoning of total bottom of the tank was done, as daily routine allowed. Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 21 Figure 8 - Location of the squares (BS1 BS2), at the bottom of the tank, siphoned daily to collect dead larvae. A tube with a flat end was used to reach the tank bottom squares and proceed to its siphoning. The material siphoned was collected with a 50 µm mesh net. In the case of data provided by total siphoning 1 ml counts of dead larvae was done in triplicate. The formula used to calculate mortality rate was: 3.3 Behavioural observations Behavioural observations were conducted, with the adoption of a protocol similar to the one used by Andrade et al. (2012a) which is based on the method described by Puvanendran et al. (2008). The observer placed himself on the edge of the tank and all movements and social interactions of larvae were registered for 5 minutes. Other observations were done during the daily routine around the rearing tank. Regarding larvae distribution at the water surface (Figure 9) and water column these were related to paint marks on the rearing tank (Figure 13). Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 22 Figure 9 –Tank divisions and references marks used to evaluate larvae distribution at the water surface. The criteria used for larvae distribution at surface was to register the tank quarter with the highest number of larvae. For distribution regarding depth, the criteria was to register the first time a larvae was seen near one of the side wall marks. The position of the marks are described below in Figure 14 3.4 Mesocosm environmental analysis Faced with the task of the characterization of the rearing environment a major question arised: How do we analyse a tank of this size? In order to establish the methodologies to be used, it was first necessary to decide what and when to sample. This decision requires good planning, in order to provide the less possible stress to larvae. 3.4.1 Hydrodynamics The methods used to determine flow pattern and water velocity, were the use of drogue floats and dyes. Compared to the use of sensitive flow meters, these methods require low man power, little preparation, they allow for alterations in the trials with short notice and are inexpensive (Hughes, 2002). The disadvantage of using both methods in natural conditions is the limited scope and spatial coverage, as well as the almost null probability of repeating trials (Dugan & Piotrowski, 2003). However, this is not a problem in the hatchery, as we deal with smaller masses of water and some level of control of for the environmental parameters. Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 23 The drogue float used in order to establish the flow pattern was designed and built for this purpose according to Hughes, (2002) (Figure 10). The drogue float was placed on the rearing tank, at 4 depths: -10 cm, 50 cm, 100 cm and 200 cm (Figure 11). Surface measurements were considered at 10 cm, since the floats at the surface were only 50% immersed. The drogue was always placed at the water inlet, at the planned depth. The depths were achieved by increasing the length of the rope. The drogues positions were recorded at established time intervals (5 min) by photograph using Fujifilm FinePix 5700. A ) foam; B) Rope: C) plaques; D) Weight 1 2 Figure 10 - Drogue float device 1) scheme; 2) picture of the used object. To evaluate water velocity we have used a method based on particle transport velocity (PTV), described by Oca et al (2004). A lime solution (25g/L) was placed at the water inlet (4L) and the water valve opened to reach the established water exchange rate. The entire process was video recorded using Fujifilm FinePix 5700. Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 24 Figure 11 - Scheme of the division of the water collumn, for evaluation the watter flow pattern. Atop of the surface layer (50 cm); BMiddle of the water collumn (100 cm); C - bottom of the water collumn (200cm). Lime was placed on the water inlet, on the right side of the drawing. The use of PTV derives from the fact that appropriate dyes were not available at the local market. The cost and time needed for ordering made the option of PTV more viable. 3.4.2 Culture sampling stations Sampling stations were selected to gather data about the different abiotic and biotic parameters of the entire rearing tank. Two transects were considered for the sampling stations. One transect covering the full diameter of the tank, from the inlet to the outlet of the seawater. The second transect covering the radius, from the centre of the tank to the tank border, at an equal distance from the inlet to the outlet of water. The radius transect was perpendicular to the diameter transect (Figure 12). Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 25 Figure 12 – Position of the two transect with the sampling stations used for measurements. (1 water inlet; 7 centre of the tank; 10 – water outlet; 4 border of the tank). Along the transect the water sampling and measurements were made at selected stations at the surface (10-15 cm), at 50 cm depth, middle of the water column (100 cm) and bottom of the water column (200 cm) (Figure 13). It was considered that these sampling stations provided a complete description of the environment, covering the entire rearing tank. Figure 13 – Sampling stations: the superficial layer; points at 50 cm (1,7,10,4); points at 100 cm (2,8,11, 5); points at 200 cm (3,6,9,12) points 1, 2 and 3 are next to the water inlet. Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 26 A pole was used to reach the centre sampling points of the tank. At the extremity of the pole a flat base was used to install the light meter. On the side of the pole, a hose was attached for the collecting water by siphoning 250 ml sample to a plastic cup, for salinity and pH measurements. An oxygen and temperature probe was also attached to the pole to measure central station points. Sampling points on the edge of the tank were marked with white paint in the dark background (Figure 14), before filling the rearing tank. This was done well in advance the beginning of the rearing cycle. The water was renewed for several days to wash away any harmful substances that could have any toxic effect on the larvae. Figure 14 - Marks on the wall of the rearing thank representing the depths measured. In the picture it is also possible to see, the water inlet and one of the aerators. The water collection hose and the probe to sample oxygen, temperature, pH and salinity were submerged to the appropriate depth and station. A shorter pole with a flat base was used for measuring light intensity under water with a probe, in order to obtain a more stable reading. Since the probe was not water proof, all measurements were done with the probe wrapped in a plastic sheet. The measurements were corrected from the plastic as follow, lux at any depth – lux at surface free probe – lux at surface with plastic. Preliminary tests indicated that data provided by the probe with and without the plastic, had a differences of just one or two lux. The effect of the plastic was not considered. Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 27 The daily routine measurements at surface were made near sampling point nr. 10, in area C near the water outlet. The probe is placed at a depth of 10-15 cm and the value registered assumed to be for the entire surface layer of the tank. Figure 15 - Sampling points (X) for surface light and measurements. Daily surface measurements were made around 12.00h according to Figure 15. Light measurements of the light distribution inside the rearing tank, were made at 22.00h. The reason for the late hour was to assure that there was only one light source, the fluorescent lamps. The following devices were used for the physical and chemical measurements: Water temperature and dissolved oxygen - Handy Polaris; Oxy Guard International A/S. Salinity – Refractometer Atago, S-10E pH – pH meter Hanna HI-96196 Champ pH Testes Light intensity– Testoc540 Data collection was done in a daily basis, however at the weekends there was a shortage of staff and only oxygen and temperatures were recorded. Methodology Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 28 3.3 Statistical analysis Results are presented as mean ± standard deviation (SD) Oxygen, temperature, light and pH assumption of normality adjustment and homogeneity for variance were verified using Kolmogorov-Smirnov’s and Leven’s tests, respectively with a significance level of 0.05 (Zar, 1996). If results of Kolmogorov-Smirnov were not verified, a non -parametric test was applied, (Kruskal-Wallis) to test the homogeneity along the rearing period. A mean pairwise analysis of variance was used for determining missing data. Statistical analysis was completed using IBM SPSS Statistics V.20, (Chicago Illinois, USA). Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 29 4. Results 4.1 Egg and larvae performance Egg hatching was 98%, for the floating baskets method. The hatching rate based on first siphoning of the tank (9dah) provides a slightly higher hatching rate of 99 %. Total survival rate at the end of the rearing period was 61%. The rate of normal swim bladder inflation was 90%. Larval growth performance is shown in Figure 16. Figure 16 - - Larvae growth along the rearing period. Also presented data from Andrade et al. (in Press) using similar mesocosm (40 m3) and Çoban et al (2009) in intensive production. Mortality rate was registered at surface and at the bottom of the tank ( Figure 17). Mortality decreases along time. Several peaks of mortality occurred at, 15 dah, 33 dah and 40 dah. Figure 18 presents a comparison of the values recorded and expected for the different methods to evaluate mortality. Methods SB and TB presented estimates similar to the final larvae mortality registered at the transfer to the juvenile rearing tanks. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 36 4.2 Environmental parameters 4.2.1 Water flow patterns The observed flow patterns using drogue floats are presented in Figure 24. A B C D Figure 24 - Flow pattern observed at the different depths: A -10 cm; B - 50 cm; C - 100cm; D -200 cm. Each colour represents a drogue float device. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 37 The pattern of lime dispersion in the rearing tank is presented in Figure 25. The water current calculated by the lime method, varied between 0.69 cm/s and 1.38 cm/s (Figure 26). The highest velocity was recorded by lime dispersion, with the maximum water renewal. The lowest speed was achieved by the drogue at the lowest water renewal rate. The central area of the tank presented consistently slightly higher water velocity. Figure 25 - Lime dispersion (red line) inside the rearing tank. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 38 A B Figure 26 - Water velocity calculated at different water renewal rates (% per day) A – Lime; B Drogues. 4.2.2 Salinity Salinity had a mean value of 36 ±0.05‰ for all stations and for the entire duration of the rearing period. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 39 4.2.3 Temperature Mean temperature was 20.1 ± 0.1ºC, increasing for the duration of the rearing period from 17.8ºC to 22.8ºC. The bottom of the water column was colder than the surface. Temperature had no significant variations in terms of depth or surface stations during the rearing period (Kruskal-Wallis test; ρ<0.05) (Figure 27). Figure 27 - Sea and Tank temperature along the rearing period. Temperature was registered daily. Particular emphasis was given to three moments, which corresponded to alterations of the live feed regime (Figure 28). Temperature presented a slight stratification pattern to 1.0 m depth until 21 dah. As time passed the different temperature layers became more distinct. The same pattern of distribution is observed on the lateral profiles of Figure 28 from the surface to the bottom of the tank. . Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 40 A B C D Longitudinal profile Lateral profile Figure 28 – Temperature (ºC) registered on the days that . A - Blank Tank; B - 10 dah; C - 21 dah; D -35 dah. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 41 4.2.4 Dissolved Oxygen Mean oxygen was 6.06 (±1.35) mg/l, for all stations and the entire duration of the larvae rearing period. Dissolved oxygen decreased as time passed (Figure 29). Oxygen presented significant differences (Kruskal – Wallis; ρ<0.05) in distribution, either in stations or depth (Figure 30 A and B). Figure 29 - Mean dissolved oxygen variation along the rearing period A B Figure 30 - Dissolved oxygen variation regarding depth (A) and sampling points (B) Oxygen displayed two patterns: before and after larval hatching (Figure 31). In the pre-culture moment (Figure 31A), the pattern displayed was highly variable, with two areas near the side walls with lower dissolved oxygen concentrations. After larval hatching this parameter register similar patterns with concentration decreasing as time passed (Figure 31B-D). The lateral profile evidenced that concentration decreased from the surface to the bottom of the tank. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 42 A B C D Longitudinal profile Lateral profile Figure 31 – Dissolved oxygen variation (mg/L) at different moments of the rearing period . A – Blank tank; B – 10 dah; C – 21 dah; D – 35 dah Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 43 4.2.5 pH pH determined at different stages registered a mean value of 8.6 ±0.09. pH was homogenous for the entire tank, that is no significant differences were registered between stations (Kruskal-Wallis; ρ>0.05). pH pattern increased from water inlet to water outlet (longitudinal profile (Figure 33). It also increased from the bottom to the surface (lateral profile). 4.2.6 Light Light intensity at the surface of the rearing tank was 1514 ± 1444 lux (Figure 32). Light intensity for all stations varied between 7594 lux and 99 lux. Light presented significant differences between sampling stages (KruskalWallis; ρ <0.05) Figure 34. At pre - hatching of larvae, the light intensity decreases from surface to bottom and from the center to the tank walls (Figure 34).After hatching (15, 21 and 36 dah), the pattern is similar, decreasing from the center to the tank wall. Figure 32 - Light intensity (lux) at the surface of the rearing tank, during the entire rearing period. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 44 A B C D Longitudina l profile Lateral profile Figure 33 - Variation of pH at different rearing moments . A – Blank tank; B – 10 dah; C – 21 dah; D – 35 dah. Results Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 45 A B C D Longitudinal profile Lateral profile Figure 34 - Light variation (Lux) at different rearing moments. A – Blank tank; B – 10 dah; C – 21 dah; D – 35 dah. Discussion Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 52 feed (Boeuf & Le Bail, 1999), and values inside the rearing tank were always above the light requirements. The distribution pattern of light intensity is related to the position of the artificial light and is similar to observed by Yoseda et al. (2009) and Naas et al. (1996). However, neither Yoseda et al. (2009) nor Naas et al. (1996) artificial light source was similar to the one used by at CMC. Naas et al. (1996) used light all around and parallel to the wall of the rearing tank, and Yoseda et al. (2009) tested different light sources positions in the central area of the tank. Light intensity weakens as it reaches the sides and bottom of the tank. The presence of algae is bound to affect light dispersion (Boeuf & Le Bail, 1999), and this was demonstrated by the high dispersion pattern during this phase, particularly from 323 dah. Light distribution at the surface of the tank presented a great variation, due to the influence of sun light. Conclusion Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 53 6. Conclusions When we first began this work several questions needed to be addressed in order to describe the major abiotic and biotic parameters influencing S. aurata larvae performance and distribution in mesocosm tanks. The homogeneity of abiotic and biotic water parameters in the large mesocosm tank will depend on the parameter analysed. Temperature, salinity and pH have an even distribution in the entire tank. In opposition, the light is dependent on the position of light source and dissolved oxygen depends on water renewal rate and aerators positions. Considering the group of biotic and abiotic parameters studied there were little variations inside the rearing system, therefore it can be concluded there is a single environment. The current methodologies in use for water quality control and others developed in the course of this study do provide capable answers, allowing the analysis of the selected parameters. However, methodologies can be improved for better accuracy with minor adjustments, e.g., the size of the bottom square used to measure mortality, could be complemented with more sampling stations or increase of the size of the square. It also appears necessary to improve the siphoning method. At one point no data was gathered since the high algae concentration did not allow the visualization of tank bottom. Larvae do not seem to be affected by the rearing environment, with the exception of the influence of light during the early stages of larval development. Larval behaviour is persistent during the entire rearing period, suggesting larvae were in the absence of stressful environmental factors. The size of the rearing tank is the first obstacle to overcome in a monitoring programme. It is not possible to visualize the entire tank volume from one single point. In fact, for most of the rearing period the central area is “invisible” territory due to long distance from the margin and the dark colour of the tank. The methods used in this work are easy to reproduce and allow the easy comparison with other studies. One of the limitations that can be reported to this work is the lack of duplicates. However, the tank in the present study has a big volume, with Conclusion Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 54 particular entail in several parameters. Other studies using mesocosm often provide vague data and less environmental parameters. The gathering of data of one single tank was time consuming, duplicates or triplicates, despite enriching the present work would not allow for such a complete analysis. There are few works published concerning the importance of the abiotic and biotic parameters on larval development, as well as on mesocosm of semiintensive methodologies. In addition there is a need to establish standard protocols, in order to facilitate comparison among larval rearing methodologies (Villamizar et al. 2011). It is expected that this work may contributed to increase the knowledge of the mesocosm of semi-intensive methodologies. In future works it would be interesting to deepen the relation between the abiotic and biotic parameters and larvae biology, for S. aurata and other species. References Sparus aurata larvae production in mesocosm: evaluation of abiotic and biotic parameters, Page 55 7. References Alvarez-Lajonchère, L., & Pérez-Roa, R. (2012). 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