Eel and greens production in a small-scale aquaponic system

(1)

Riassunto

Si prevede che nel 2050 la popolazione mondiale raggiungerà i 9.8 miliardi di persone, suggerendo un aumento della domanda di cibo che l’agricoltura dovrà sostenere nonostante problematiche come i cambiamenti climatici, la limitata disponibilità di fonti energetiche, di risorse idriche e di suolo. In questo contesto, l’acquacoltura rappresenta il settore zootecnico caratterizzato dalla più bassa impronta ecologica e viene indicata come l’unico settore di produzione alimentare in grado di garantire la sicurezza alimentare, intesa come disponibilità alimentare per la popolazione mondiale. Nonostante ciò, la produzione dell’acquacoltura è associata ancora ad alcuni fattori di insostenibilità confermati da studi sul Life Cycle Assessment (LCA). In uno di questi studi, viene posta l’attenzione sia sui sistemi acquaponici che sugli Integrated Multi-Trophic Aquaculture Systems (IMTA), ovvero sistemi che sono in grado di realizzare una produzione integrata di specie provenienti da livelli trofici diversi, indicandoli come opportunità per ridurre l’impatto ambientale delle produzioni ittiche.

Lo scopo del presente lavoro è stato quello di realizzare e testare un sistema acquaponico di piccola scala. Le specie prodotte sono state l’anguilla (Anguilla anguilla) per quanto riguarda i pesci, e il basilico (Ocimum basilicum) e la lattuga (Lactuca sativa), per quanto riguarda le specie vegetali. Lo studio è stato condotto in serra presso il Dipartimento di Scienze Agrarie e Agro-ambientali dell’Università di Pisa, per un periodo complessivo di 5 mesi, allevando 48 soggetti del peso medio iniziale di 133.4±11.14 g ad una densità iniziale di 6.2 kg/m3. Basilico e lattuga sono stati coltivati con il metodo floating system ad una densità di 40 piante/m2 e 30 piante/ m2 rispettivamente. Come controllo si sono impiegate le stesse specie e densità ma in coltivazione idroponica.

Sebbene la crescita ponderale delle anguille sia risultata trascurabile, giungendo a fine periodo a 181.1±16.46 g, al giorno 91 di allevamento la mortalità è risultata essere pari a solo il 14.6%, raggiungendo però il valore di 54.2% al giorno 126. Per quanto riguarda il basilico, la produzione fresca madia è stata di 26.5 g/p mentre quella della lattuga di 234.9 g/p.

In conclusione, lo studio ha messo in evidenza gravi problematiche legate al mantenimento delle caratteristiche igienico sanitarie dell’acqua di allevamento, in particolare per quanto riguarda l’accumulo di residui solidi (mangime non consumato e feci). Ciononostante, essendo questo il primo tentativo di allevamento dell’anguilla in un sistema acquaponico, lo studio ha permesso di ottenere utili indicazioni anche nell’ottica di uno specifico adeguamento del sistema.

(3)

2

Abstract

The world's population is expected to reach 9. 8 billion by 2050, suggesting an increase in the demand for food that agriculture will have to sustain despite problems such as climate change, limited availability of energy sources, water and soil resources. In this context, aquaculture represents the livestock sector with the lowest ecological footprint and is indicated as the only industry of food production capable of guaranteeing food security, understood as food availability for the world population. Nevertheless, aquaculture production is still associated with some unsustainable factors confirmed by Life Cycle Assessment (LCA) studies. In one of these studies, the focus is on both aquaponic systems and Integrated Multi-Trophic Aquaculture Systems (IMTA) that are able to achieve integrated production of species from different trophic levels, indicating them as opportunities to reduce the environmental impact of fish production.

The aim of this work was to build and test a small scale aquaponic system. The species produced were European eel (Anguilla anguilla) in the case of fish, and basil (Ocimum basilicum) and lettuce (Lactuca sativa) in the case of plant species. The study was conducted in a greenhouse at the Department of Agricultural and Agri-environmental Sciences of the University of Pisa, for a total period of 5 months, breeding 48 subjects with an average initial weight of 133.4±11.14 g at an initial density of 6. 2 kg/m3_{. Basil} and lettuce were grown using the floating system method at a density of 40 plants/m2_and 30 plants/m2_{respectively. The same species and densities were used for control but in} hydroponic cultivation.

Although eel weight growth was negligible, reaching 181.1±16.46 g at the end of the period, mortality at day 91 of rearing was only 14.6%, but reaching 54.2% at day 126. In relation to basil, the fresh production has been 26.5 g/p while that of lettuce was 234.9 g/p.

In conclusion, the study highlighted serious problems related to maintaining the hygienic and sanitary characteristics of livestock water, particularly in relation to the accumulation of solid residues (unconsumed feed and faeces). Nevertheless, since this is the first attempt to breed eels in an aquaponic system, the study has allowed to obtain useful indications also in view of a specific adaptation of the system.

(4)

3

1. Introduction

Starting from data about the growing world population within 2050, followed by an increasingly global food demand within the same time, the progress of climate change effects and the massive employment of already scarce exhaustible resources (arable and fertile lands, freshwater, oil), in 2015 we questioned what food production system we could count on in order to produce more food using fewer resources and minimizing the environmental impact (Galliano, 2015; Fronte et al., 2016). Thus, following review the available literature about sustainability and its link with food security and assign the responsibility of more sustainable food productions in agriculture, we identified in aquaculture the most suitable agricultural production system to solve the 21st-century dilemma (Galliano, 2015; Fronte et al., 2016). Indeed, aquaculture represented the major source of income for about 11 million of people (FAO, 2011) and the main source of animal protein for 1 billion of people (Tidwell and Allan, 2012); moreover, thanks to the excellent feed conversion rate (FCR), it showed the lowest carbon footprint. However, aquaculture presented some critical features in relation to environmental impact: it caused strong exploitation of fish’s wild stocks (Adler et al., 2008) due to the necessary huge inclusion of fishmeal and fish oil in the fish feed (Cataudella, 2001); moreover, aquaculture contributed to eutrophication and pollution of aquatic ecosystems (Primavera, 2006); finally, aquaculture required large quantities of water, which may vary from 3000-45000 litres/ kg of fish produced (Verdegem et al., 2006). With that in mind, we focused our attention on technological or management solutions able to lead the aquaculture sector towards more sustainable productions (Galliano, 2015; Fronte et al., 2016). Hence the interest in Integrated Aquaculture Systems (IAS) and the choice, among these, of aquaponic systems where terrestrial plants are grown in conjunction with fish (Rakocy, 2012). That system not only represented a way to obtain intensive production, contributing to improve sustainability and to reach food security goals (Fronte et al., 2016) but also an opportunity to achieve significant economic and social benefits (Somerville et al., 2014).

Since the reasons that have been led our choice towards aquaponic systems did not change so far, but we can state that they have been confirmed by recent reports and investigations; since aquaponics has caused an increasing interest in the scientific community in the last eight years and, as well as all aquaculture sector, has been undergoing new evaluations about its real sustainable production capacity, with the present work we will present the new data about global environmental, social and economic situation and the latest trends

(5)

4 of aquaculture sector. Then, we will briefly remind the basic principles of aquaponics and will deal with the new findings in this field. After that, we will introduce European eel and its farming systems.

According to what stated by UN in its 2017 Revision of the World Population Prospects (2017), the world's population is growing by 1.10% per year and it is projected to reach 9.8 billion in 2050. Therefore, the pressure on agricultural land, water, forests, capture fisheries and biodiversity does not seem to decrease and agricultural systems will still have to improve productivity and resource-use efficiency in order to meet the rising food demand (FAO, 2017). Among the agricultural food production systems, the World Bank (2013) identified aquaculture as the only sector able to overcome the food security issue. In relation to what reported by Fronte et al. (2016), indeed, aquaculture continues to grow faster than other major food production sectors, although with a decreased average annual growth: it passed from 7.8% between 1990 and 2010 (Troell et al., 2014) to 5.8 % during the period 2000–2016 (FAO, 2018). In 2016, aquaculture contributed for the 47% at the global fish production (171 million tonnes) and inland aquaculture produced 54.1 million tonnes of farmed food fish (FAO, 2018). In 2015, fish accounted for about 17 per cent of animal protein consumed by the global population and provided about 3.2 billion people with almost 20 percent of their average per capita intake of animal protein (FAO, 2018). Since 1961, the average annual increase in global apparent food fish consumption (3.2 percent) has outpaced population growth (1.6 percent) and exceeded consumption of meat from all terrestrial animals, combined (2.8 percent) and individually (bovine, ovine, pig, other), except poultry (4.9 percent). The per capita consumption of food fish has grown from 9.0 kg in 1961 (Galliano, 2015; FAO, 2018) to 20.2 kg in 2015, at an average rate of about 1.5 percent per year (FAO, 2018). The increased production, reduce wastage and growing demand, lined with population growth, are only few factors that led the expansion in consumption (FAO, 2018). Of the 171 million tonnes of total fish production in 2016, 151 million tonnes were utilized for direct human consumption, about 36 million more than 2010 (Galliano, 2015). About 20 million tonnes was reduced to fishmeal and fish oil, in line with what registered in 2010 (Galliano, 2015).

Although from a social and economic point of view aquaculture sector seems to have a crucial role in improving food security, human nutrition and in fighting world hunger (FAO, 2018), life cycle assessment (LCA) studies give back an environmentally worrisome portrait (Bohnes et al., 2018). According to ISO (2006), LCA is an

(6)

ISO-5 standardized methodology, which quantifies the impacts on ecosystems, human health and natural resources stemming from products and systems throughout their entire life cycle, that is from the extraction of the raw materials through their production and use or operation up to their final decommissioning and disposal (ISO 2006; EC 2010). Appling that methodology to the aquaculture systems, several stages and multiple components can be recognized, each one characterized by a different contribution in relation to the environmental impact. Bohnes et al. (2018) divided those stages and components into feed production, seafood farming, e.g. hatchery and grow out, energy supply, chemical use (every medical agent or chemical product), infrastructures such as building and equipment, packaging, distribution, consumption and end-off-life (recycling or enhancement of by-products).

The production stage is identified as the main source of eutrophication impact and water dependence. According to Aubin et al. (2009), this latter specific to aquaculture category is referred to as water input relative to the fish production in the mass of biota.

As regards as feed production, the FCR influenced the cumulative energy demand, net primary production, acidification and climate change. That parameter is strictly linked with factors such as fish species and mortality at the site, feed composition, and production technology (Pelletier et al. 2009).

Since the emissions and resource uses vary according to the energy source and technology, the energy context and the composition of the electricity grid mixes (varying from a geographical situation to another) are factors that transversally influenced the environmental impact of the aquaculture systems, being them RAS or shellfish production farms (Bohnes et al., 2018).

Infrastructures and production and use of chemicals, whose Herickson et al. (2015) stated that disinfectants (chloride products) are potentially toxic for humans and local organisms, accounted for more 5% of total life cycle impacts in at least one impact category (Bohnes t al., 2018). However, all the components of a system, included the end-of-life of a product, should undergo to an LCA, due to their potential of reuse or energy recovery from disposal of food waste (Iribarren et al. 2010). In that way, the identification of the trade-offs arising from the aquaculture systems can be performed (Bohnes et al., 2018).

(7)

6 Starting by saying that the studies analysed in their work do not include intensive systems relying on state-of-the-art technologies, Bohnes et al. (2018) state that, if present, they would better optimize the resources and reduce the interactions with local ecosystems. That would lead to increase productivity and reduce impacts (Bohnes et al., 2018). Then, comparing systems with different intensities, they found that the aquatic eutrophication and water use impact of intensive systems seems to be equal or better than that of low-intensity systems (Bohnes et al., 2018) because of the water treatment (filtering) whereas, the need of more energy for moving water and the kind of source (e.g. fossil fuel) increase the climate change and acidification impacts (Bohnes et al., 2018).

From a comparison between technology types, Recirculating Aquaculture Systems (RAS) have higher impacts (cumulative energy demand and climate change) than other systems, especially in terms of eutrophication and water dependence (Bohnes et al., 2018). RAS are closed-systems developed in order to avoid the interactions between farmed and wild fish and show a better control of pathogens thanks to the sterilization treatment of incoming water (Bohnes et al., 2018), with an increasing in productivity (Klinger and Neylor, 2012).

In their review, Bohnes et al. (2018) question if there are opportunities to decrease environmental impacts of aquaculture in Integrated Multi-Trophic Aquaculture (IMTA) systems where the production of species from different trophic or nutritional levels is combined. According to Palm et al. (2018), they can be also defined as “the integration of another (secondary) species (fish, plant or algae) into a system that benefits from the main targeted species (e.g fish)”. Among the IMTA, aquaponics is a land-based aquaculture system that, counting on recirculating and treatment water technologies, utilizes the effluent water produced by rearing fish for the soilless cultivation of plants (Bohnes et al., 2018; Palm et al., 2018). Since aquaponic systems combine aquaculture for fish production and hydroponics for plant cultivation (Goddek et al., 2015), in the next chapter we will describe briefly recirculating aquaculture systems (RAS) and soilless culture. After that, we can introduce aquaponics deeper.

(8)

7

1.1 Recirculating Aquaculture Systems

According to Ebeling and Timmons (2012), closed systems where the rearing water is treated and reused, totally or partially, are referred to as Recirculating Aquaculture Systems (RAS). They are complex systems consisting of process units that correspond to a specific treatment phase (Galliano, 2015), i.e. rearing unit, mechanical filtration unit, biological filtration unit, UV treatment unit and finally aeration and oxygenation lines (Saroglia, 2001). In that way it is possible to operate at high stocking density (60-120 kg/m3_{) in a controlled environment and to reduce of 90-99% the water utilization if} compared to conventional aquaculture systems (Ebeling and Timmons, 2012). On the other hand, those systems are still producing high amount of wastes per discharged unit volume and imply high management and construction costs (Rakocy, 2012).

1.2 Soilless culture and hydroponic systems

Soilless culture encompasses "all those culture systems that exclude agricultural soil and in whom plant nutrition occurs through a complete in macro- and micro-nutrients, balanced nutrient solution" (Giuffrida, 2018). This technique of horticultural crop production has been introduced in protected crops to overcome some disadvantages typical of traditional techniques such as soil exhaustion, secondary salinization and soil-borne diseases (Di Lorenzo et al., 2013). After the introduction of limitations in the use of pesticides and fertilizer and of the maximum values of nitrates' content in vegetables (Malorgio et al., 2004), that technique became an effective opportunity to keep producing and to improve water and nutrients distribution as well as the control of plant growth condition (Di Lorenzo et al., 2013). In soilless cultivations, indeed, the plant's management occurs without the soil and the nutritional needs (water and minerals), are satisfied by means of a nutrient solution. Moreover, they are mainly carried out in highly productive greenhouses where water and space are employed efficiently. Finally, in the case of closed-loop systems where the nutrient solution is recycled, these systems prevent the pollution of groundwater (Resh, 2012). Despite that, the energy requirement, capital investment and the personnel's skills are remarkable (Di Lorenzo et al., 2013).

Before dealing with the classification of soilless systems, it is important to delve into the point that all kind of soilless systems shared: the nutrient solution (La Malfa, 1996).

(9)

8

The nutrient solution

The nutrient solution has the same function of the circulating soil solution (Di Lorenzo et al., 2013) and consists of irrigation water where agricultural or industrial salts and acids are added in order to satisfy nutritional requirements of growing plants (Malorgio et al., 2004). Essential aspects of the nutrient solution are its composition in terms of macro- and micro-elements (the so-called nutrient formula or nutrient recipe), pH and electrical conductivity (EC); its preparation and management, i.e the establishment of irrigation turn and volumes, as well as the destiny of surplus nutrient solution (Giuffrida, 2018). The concentration that each nutrient element should have in the nutrient solution is referred to as the nutrient formula (Malorgio et al., 2004) and, for the main horticultural crops, is obtained from trials carried out under climatic conditions typical of the temperate zone (Centre Northern Europe). Some examples of nutrient recipes are shown in Figure 1.

Fig. 1. Composition in macro- (mmol/L) e micro-nutrients (μmol/L) of the nutrient solution suitable for the main horticultural crops (Source: Giuffrida, 2018)

In the soilless culture, the quality of the irrigation water is an essential aspect and, thus, it is deeply suggested to know its composition before employing it (Malorgio et al., 2004). The evaluation should investigate parameters such as pH (most frequent values ranging from 6.5 and 8.4 in irrigation water and the optimal range for a nutrient solution is 5-6.5), total alkalinity (the concentration of bicarbonates and carbonates), electrical conductivity (EC, better if lower than 1 mS/cm) and total salts concentration as well as the concentration of all macro and micro-nutrients (essential ions) and the not-essential ions (Na, Cl, Si and heavy metals, Malorgio et al., 2004). Finally, the absence of phytopathogenic agents and the microbiological purity (for human health) are the other two aspects to assess.

The amount of each salt and acid to add to the irrigation water is obtained through a series of calculations that imply the knowledge about the composition of the irrigation water and the nutrient recipe of the target plant.

NO3- NH4+ H2PO4- SO42- K + _Ca2+ _Mg2+ _Fe3+ BO33- Cu 2+ _Zn2+ _Mn2+ MoO 2-4 11-15 1-1.5 1.5-2 3.5-4.5 5-9 3.5-5 2-2.5 20-25 30 1 5 10 0.5 13-17 1.5-2 1.5-1 1.25-2 4-6 3-3.5 2-2.5 15-20 30 1 5 10 0.5 14-17 1-1.25 1.5-2.5 1.75-2 4-7 4-5 1.5-2 20-25 30 1 7 10 0.5 16-18 1-1.25 1.25-2 1.25-2 5-8 3.5-4 1.5-2 15-20 25 1 5 10 0.5 15-18 1-1.5 1.5-2 1.75-2 5-8 3.5-4.5 2-2.5 10-15 30 1 5 10 0.5 16-19 0.5-1 1-1.75 1.25-2 5-8 4-5 1.5-2 10-15 25 1 5 10 0.5 11-13 1-1.25 1-1.75 1-1.5 4-6 3-3.5 1-1.5 20-25 15 1 7 10 0.5 Culture (mmol/L) (μmol/L) Tomato Aubergine Strawberry Melon Zucchini Cucumber Pepper

(10)

9 The macro-elements (N, P, K, Ca, Mg, S) are mainly supplied through simple salts, characterized by a high degree of purity and solubility in water. As regards as N, P and S, a rate can be provided also by means of acids, used to correct the extent of bicarbonates in the irrigation water (Malorgio et al., 2004). Metallic micro-elements (Fe, Cu, Mn, Zn) are generally supplied in form of chelates where the most utilized ligands are organic acids (DPTA, EDTA, EDDHA, HEDTA) but for Cu, Mn and Zn supply, it can be employed also simple salts (less expensive). An element can be supplied equally by two salts thus, during the choice of which salt use in the recipe, it is important to consider the solubility, the opposite ion and the price (Malorgio et al., 2004).

In commercial soilless farm, the nutrient solutions are typically prepared as concentrated stock solutions (often 100-200 times the final dilution to be applied to the plants) and diluted by use of proportional injectors (fertirrigator). Stock solutions are used to save preparation time, reduce storage space and avoiding the growth of algae as would dilute solution stored for more than a few days (Malorgio et al., 2004).

A stock nutrient solution is a 50 to 200 times concentrated solution of salts, acids and/or bases. Thus, the amount of macro- and micro-nutrients to dissolve in the stock solution's container could be calculated through the multiplication of the concentration factor. The collected stock solution is diluted with irrigation water and then provided to the plants by means of fertigators, (Malorgio et al., 2004). However, thanks to the development of innovative software, nowadays the mathematical operations are remarkably simplified and automatized. One of these software is the electronic spreadsheet (SOL-NUTRI.xtl), developed by University of Pisa ( https://www.wur.nl/en/Research-Results/Projects-and-programmes/Euphoros/Calculation-tools/Nutrient-Solution-Calculator.htm). SOL-NUTRI is enables to calculate the amount of acid and salts to added to the irrigation water to prepare the stock nutrient solutions and check the possibility of some precipitates in the stock solutions (Malorgio et al., 2004).

Soilless systems classification

A general classification of soilless systems categorizes them in relation to the kind of medium supplied as support for the plant roots (Di Lorenzo et al., 2013; Giuffrida, 2018). Therefore, there are water culture systems where the water, indeed, is the only medium whereas systems where the support is represented by substrates, inorganic or organic, is referred to as substrate culture systems (Giuffrida, 2018). The term "hydroponic" has different meanings in literature: some Authors employ it as synonymous of water culture

(11)

10 system (Malorgio et al., 2004), some others extend it to inorganic substrate culture systems (Di Lorenzo et al., 2013). In this thesis, with the term "hydroponic" we will refer to the water culture systems and, therefore, to them a specific in-depth chapter will be reserved.

Normally, the nutrient solution is dispensed to the plants in a more large amount with respect to its evapotranspiration, in order to avoiding the build-up in the substrate of some not-essential ions present in the irrigation water and to balance the different growth rate of the individual plants and the lack of uniformity of the irrigation system (Malorgio et al., 2004).

That surplus is referred to as drainage solution and if in a soilless culture system, it is collected, corrected and recycled, the system is also called as closed-loop system, while in the open-loop system, the drainage solution is never re-used on the same culture (Winsor and Schwarz, 1990).

Water culture or hydroponic systems

Since the nutrient solution is the only medium supplied to the plant roots, they can be partially (DRFS, NFT) or totally (floating) submerged in this static or recirculating liquid medium (Giuffrida, 2018). Hussain et al., (2014) classified the hydroponic systems in relation to the method employed for the nutrient solution supply to the plant roots: therefore, it is possible to recognize the deep-water culture technique, the Deep recirculating water technique, the Nutrient Film Technique (NFT), the Floating system and aeroponics (Giuffrida, 2018).

Developed by Gericke (1929), deep water culture has been the first soilless technique of commercial-scale (Malorgio et al., 2004). It consisted of tanks (15 cm height, 60 cm width and 10 m length), originally in tar paper and then in waterproofing concrete, wood or metal, filled with 10-15 cm of nutrient solution (Giuffrida, 2018). On top of them, a net with a fine mesh containing 1 cm of sand served as a growing bed (Malorgio et al., 2004; Giuffrida, 2018). Critical aspects, such as the huge static volume of the nutrient solution compared to the restricted exchange air-water surface and the low oxygen diffusion coefficient in water imposed a deep revision of that system to overcome the hypoxia problems (Malorgio et al., 2004). Thus, in the '70s there was the introduction of the deep recirculating water culture systems.

(12)

11 In deep recirculating water culture, active aeration and recirculation of the nutrient solution occur in tanks (8-10 cm height, 20-100 cm width and 3-15 cm length) made, beyond the traditional materials, of polystyrene, acrylonitrile butadiene styrene (ABS) and polypropylene (Malorgio et al., 2004; Giuffrida, 2018). Since the huge nutrient solution volume, that system enables good thermal inertia on the one end but implies more attention to appropriate aeration of radical apparatus on the other (Giuffrida et al., 2018).

The NFT has been developed by Cooper in early '70s. The system consists of tubular channels where the nutrient solution is pumped (1-3 L/min) as a thin film of a few millimetres, continuously or not (Giuffrida, 2018). In the same channels, the radical apparatus grows up and enters in contact with the nutrient solution that, finally, is collected in a tank and then reused (Giuffrida, 2018). In order to ensure proper aeration to the plant roots, it is essential that channels have 25-30 cm of width, maximum of 20 m of length and an incline between 1-2% (Giuffrida, 2018). In recent years, NFT systems are achieving success again: indeed, the development and diffusion of cultivations that employed rock wool lead to the decline of NFT systems. Nowadays, instead, they are considered suitable for the contraction of the environmental impact of protected horticulture (Giuffrida, 2018). In Mediterranean regions, however, the temperatures inside the channel and those of the nutrient solution can reach such high levels that limit a diffused implementation of them (Giuffrida, 2018).

Aeroponics is a system where, through a nebulising system, the plant roots are cultured in an environment constantly or not saturated by thin drops of nutrient solution that is then reused (Giuffrida, 2018). Vertical, incline or horizontal PVC tubes or expanded polystyrene panels are used as support for the plant growth (Giuffrida, 2018). Thanks to the lower volume of nutrient solution needed, aeroponics allows to reduce the costs for fertigation but the thermal inertia of the system and the recirculation of nutrient solution slow the application on commercial-scale (Giuffrida, 2018).

In 1976 Prof. Franco Massantini of the University of Pisa introduced a new technique in which floating expanded polystyrene trays served as support for strawberries, lettuces and cardoons (Malorgio et al., 2004). The plants had the radical apparatus totally submerged in the nutrient solution and this system was defined floating system (Malorgio et al., 2004). The floating trays have holes where the substrate employed for the sowing or the transplanting is inserted (Giuffrida, 2018). The tanks containing the nutrient solution can

(13)

12 be made of various materials and generally are 20-30 cm deep (Giuffrida, 2018). The oxygen content in nutrient solution should never climb down 5 mg/L, therefore the active aeration through oxygenation systems or recirculation of the nutrient solution by means of a pump must be implemented (Giuffrida, 2018). The management of systems like these is limited to the replenishment of consumed nutrient solution with water or other nutrient solution, in relation to the duration of cultivation cycle, to the environmental conditions and to the plant species (Giuffrida, 2018). In Italy, brief cycle crops (aromatic herbs, salads, rocket etc.) are often cultivated in floating system which, especially in basil and rocket and according to the season, permits to obtain fresh matter production higher (2-5 times) than that resulting from cultivation in soil (Incrocci et al., 2001). Moreover, in summer, the growing cycle duration (from the sowing to the harvesting time) was 2-3 weeks (Incrocci et al., 2001).

(14)

13

1.3 Aquaponics

Aquaponics is "a production system of aquatic organisms (aquaculture) and plants where the majority (<50%) of nutrients sustaining the optimal plant growth derives from waste originating from feeding the aquatic organisms" (Palm et al., 2018). As reported by Palm et al. (2018), aquaponics “in sensu stricto” is restricted to the hydroponic principle without the use of soil or substrates such as sand or gravel”. From that definition, it is also possible to state that aquaponics is an integrated aquaculture system because a secondary species (plants) benefits from the main target species, e.g fish (Palm et al., 2018). In general, aquaponic systems combine RAS technology for fish production and hydroponics for plant cultivation (Goddek et al., 2015) and essential elements are three: an aquaculture unit comprising fish tank, a filtration unit with a sludge removal device and, according to the hydroponic technique chosen, a biofiltration and a hydroponic unit that can be performed through three main techniques (DWC, NFT, ebb-and-flow tables and drip technique). However, aquaponics exists in a wide variation of systems designs and thus a revision of its terminology is necessary about it (Palm et al., 2018). There are complete recirculating units where the only fertilizer consists of fish feed, and those are referred to as closed or coupled aquaponic systems, but also the so-called decoupled aquaponic systems where aquaculture and hydroponic units are separated and the nutrient addition is significant (Palm et al., 2018). The open pond aquaponic system, due to the scares use of water pumps, water flow and aeration, is a cost-effective method that consist on an artificial pond, in the major cases of a raft system (4% of the total pond area) for plant cultivation and a facultative external filtration of the water. Fish like tilapia, catfish, herbivorous carps, tench are suitable farmed species, in mono- or polyculture, together with water spinach, eggplant, tomatoes (Palm et al., 2018). Domestic aquaponic systems include closed or coupled systems as mini and hobby installations, characterized by a single fish reservoir (e.g. aquarium) or tank, extensive stocking densities a hydroponic unit (mini-system) of maximum 2 m2 _{(Palm et al., 2018). In their simplest form, the plant} growing unit is settled in the aquarium, utilizing the internal aquarium filters, or in fish tank. Larger systems usually rely on gravity for water movement (from fish reservoir to sediment unit, plant cultivation area and finally to sump) and on a single pump that pumped back the water to the fish tank. A simple aeration, such as ait pumps or spray bars, are required to ensure fish growth and health. The space needed ranges from 2-3 m2 to 10 m2. A targeting production is not applied instead of fishkeeping that is quite common. If the purpose of fish and plant production is to supply human needs, domestic

(15)

14 aquaponic systems are referred to as backyard aquaponics (Palm et al., 2018). In that case, the space employed may varies from several ten square meters to 50 m2, still relying on a single pump for water movement (Palm et al., 2018).

Small-scale aquaponic systems present an improvement in production areas (>50-< 100

m2) and, from a technical point of view, in functional components encompassing more than one fish tank and hydroponic subsystems, two filtration units (mechanical and biological) and at the minimum a single pump (with backup system in case of breakdown). With respect to the production in the northern hemisphere, a year around production is clearly possible as long a controlled environment (greenhouse) is provided for, but the need to minimize the energy costs imposes a seasonal production (mainly spring, summer and early autumn). According to Somerville et al. (2014), an output of 50-500 heads of lettuce should represent the production of a small-scale aquaponic system worthy of this name. Semi-commercial aquaponic systems are characterized by an intensification of the production, with a lettuce output of 500-2500 heads in less than 100 m2 _{(Somerville et al., 2014; Love et al., 2015). Aiming that plant yield, a climatic and} environmental control with CO2 air enrichment, an effective biofiltration and solid removal devices due to the high stocking density and, as a consequence, a waste and sludge management are strictly needed. Together the necessity of monitoring and pumping tools, pest management and with the high energy requirements, significant investment costs are obvious. Palm et al. (2018) proposed that operations with a production area between 100 and 500 m2 are termed a intermediate-scale commercial

aquaponic ventures while large-scale are those with a more of 500 m2 of site area. Regardless that, both typologies seek a maximum production output of fish and plants that implies the use of multiple rearing units, e.g increasing the number of plants cultured per growing area; the use of maximum stocking density for fish reared in RAS) with staggered production method for both plants (staggering or simultaneously planting with intercropping short-cycle and year-round plants) and fish reared at different ages and size ranges (Palm et al., 2018). According to Somerville et al. (2014), the production of lettuce is more than 2500 heads per annum in both intermediate- and large- scale commercial ventures. Such an optimization of production, marketing and sales can be performed considering an adoption of mechanization (such as automatic climate and environmental control), a reuse of water with cold traps, biogas and photovoltaic systems are all arrangements suitable for highly engineered operations of temperate climatic zones (Kloas et al., 2012; Palm et al., 2018).

(16)

15

1.3.1 Theoretical and technical basis of aquaponic systems: from fish to

vegetables

In aquaculture, about 5% of feed is not consumed by farmed fish, while the remaining 95% is ingested and digested (Khakyzadeh et al., 2014); of this share, 30-40% is retained and converted into new biomass, while the 60-70% is released in form of faeces, urine and ammonia (Somerville et al., 2014). Studies refer that 1 kg of feed (30% crude protein) globally release about 27.6 g of N as well as 1 kg of fish releases about 577 g of BOD, 90.4 g of N and 10.5 g of P (Tyson et al., 2011). In aquaponic the waste products of the fish metabolism and uneaten feed are used as fertilizers for crop production, transforming a waste into a valuable resource (Figure 2).

Fig. 2. The main actors playing in aquaponics and nitrogen cycle (Source:

https://www.google.com/search?q=nitrogen+cycle+aquaponics&tbm=isch&source=iu&ict x=1&fir=H7shIAfvufCBhM%253A%252C5bBVyXXlfOi4dM%252C_&vet=1&usg=AI4_

-kTzPuGrOWUTueNI7RJe4-ROol86fw&sa=X&ved=2ahUKEwjTtezZq-7hAhVS_aQKHdq1DZIQ9QEwBHoECAgQCg#imgrc=H7shIAfvufCBhM:&vet=1 ) In this transformation, the role of bacteria is crucial. The output water from the fish tank is conveyed to a mechanical filter for the separation and removal of the most large solid particles; afterwards, the water reaches a biofilter, primary site of bacteria nitrification; this process consist in the oxidation of ammonia (NH3) and the ammonium ion (NH4+) to nitrates (NO3-), more accessible form of nitrogen for plants (Somerville et al., 2014; Rakocy, 2012). The conversion takes place through two successive reactions and involves two distinct groups of nitrifying bacteria: Nitrosomonas, by which the ammonium ion is converted into nitrite (NO2-); Nitrobacter, which finally transform the nitrite to nitrate (Saroglia, 2001). Nitrification and a vital bacterial colony are therefore essential conditions for an aquaponic system to work properly. Another important group of aerobic

(17)

16 bacteria is heterotrophic bacteria that are involved in the mineralization of solid waste (Somerville et al., 2014). Hence, the nitrates and other nutrients enriched water leaves the biological filter and circulates towards the hydroponic section where the phytoremediation process takes place (Somerville et al., 2014) and nitrates in the water may be reduced by more than 97% (Lennard, 2006); a final UV sterilization allows the water to return in the fish tanks. Studies have shown that on average, for every 60-100 grams of feed supplied, a square meter hydroponics culture is required for a just poor water purification (Rakocy et al., 2006); also, a square meter of hydroponic culture can remove 0.83 g of N and 0.17 g total P (Tyson et al., 2011). In relation to the technical basis of aquaponic systems, there is a wide variety of aquaponic systems even though they all share some essential components: a fish tank, a mechanical filter, a biofilter, a hydroponic container for plant growth and a sump (Somerville et al, 2014). In the fish tanks, the stocking density may vary from 20 kg/m3 (Somerville et al, 2014) up to 70-80 kg/m3; just in some specific cases it seems to be possible to reach stocking density of about 140-200 kg/m3 but the water retention time could not be longer than 1.2 hours in order to avoid the accumulation of ammonia after feed administration (Pantanella, 2012). Among mechanical filters, the most used is “clarifier” (FAO, 2014) that removes about 59% of total solid waste, with a water retention time of 20 minutes (Pantanella, 2012) and a volume of 10-30% to the rearing tank (Somerville et al., 2014). According to Somerville et al. (2014), the minimum volume of the container should be 1/6 of the fish tanks and the most frequently used substrates are Bioballs® (500-700 m2/m3) and volcanic gravel (300 m2/m3). The various types of aquaponic systems are named after the hydroponic technique used for the cultivation of plants. MBT is the most common method adopted in small-scale aquaponic production (Rakocy, 2012), and the porous substrate provides support to the plants and it works not only as mechanical but also as “bio” filter (Somerville et al., 2014). Conversely, the NFT method and DWC are suitable for commercial aquaponic systems but, unlike the method with the growth beds, they require a mechanical filter and a biofilter. The water output from hydroponic containers by gravity falls into the sump, a water collection tank (Rakocy, 2012), where is located the submersible pump. The Feed Rate Ratio (FRR) is the ratio between the amount of feed administered on a daily base, and the area used for hydroponic cultures. The calculation of the FRR strictly affects the rate and extent of accumulation and removal of nutrients from the fish tanks as well as the integration of macro- and micro-nutrients required in aquaponics to maximize the yield of the crop production (Lam et al., 2015). According to Rakocy (2012), the optimal ratio is 57 g of feed per day per meter of hydroponics

(18)

17 surface area; it is also recommended to maintain a ratio between rearing tank and hydroponic containers of 1:7.3. As regards the water quality, according to Somerville et al. (2014) a good compromise is achieved when the system ensures temperatures between 18-30 °C, a pH of 6-7, ammonia and nitrates less than 1 mg/L and DO exceeding 5, and through the administration of 60-100 g of feed per m2 of hydroponic surface area (Rakocy et al., 2006).

1.3.2 Fish and plant species

A standardized aquaponic system can produce 7 kg of edible plants per kg of farmed fish (Graber and Junge, 2009). Fish species that have been proven to be suitable for aquaponic production are Nile tilapia, trout, barramundi, murray cod, African catfish and Koi carp (Kloas et al., 2015; Palm et al., 2014; Roosta, 2014; Endut et al., 2011; Nelson, 2007; Lennard and Leonard, 2006; Rakocy et al., 2006; Savidov, 2005; Adler et al., 2000). Among the cultivated plant species, the most widespread are lettuce, tomato, basil, eggplant, pepper and water spinach (Khater et al., 2015; Palm et al., 2014; Endut et al., 2011; Nelson, 2007; Rakocy et al., 2006; Savidov, 2005; Adler et al., 2000). For further information, refer to Galliano (2015).

1.3.3 New acquisitions about aquaponic system

A great step forward towards improving sustainability and profitability of aquaponics has been carried out in 2015 through the introduction of a completely new concept, sustained by unique system design. Since that time, the general system design was a single recirculating aquaponic system (SRAPS) where aquaculture and hydroponic units were directly connected in order to achieve bioremediation of water and production of fish and plants. Thus, Kloas et al. (2015) developed and tested a system prototype consisting of a double recirculation aquaponic system (DRAPS): in such a system, a RAS and a recirculating hydroponic unit are connected by two ways. The first directs the water flow from the aquaculture to the plant growing unit by a one-way valve. An air conditioning system, after condensation of the evapo-transpired water, conserves and feed it back to the fish tank. The system relies on renewable energy (photovoltaics) that powers the irrigation, heating and air conditioning systems (Kloas et al., 2015). In that way, Kloas et al. (2015) not only obtained the highest productivity rate (75.9 kg/m3_{in 7.91 m}3_{of rearing} volume) for tilapia reared in aquaponic systems and lowest freshwater use (220.6 L per kg fish produced) in comparison to the water needed by conventional RAS without a denitrification unit (30-300 L per kg fish biomass), according to Martins et al. (2010). As

(19)

18 regards plant production, tomato yield (8.89 kg/plant) obtained by the hydroponic unit of ASTAF-PRO revealed the potential to reach the high performance (15.88-20.41 kg/plant) obtained by specialized greenhouse using NFT, if an optimization of the illumination conditions, and of the hydroponic fertilizers are established.

Finally, vertical systems, other new forms of aquaponics, represent an opportunity to maximize the production output and the design possibilities (Fernández-Cañero et al., 2015) by using DWC, NFT, ebb and flood or growing towers with aeroponics (Pattillo, 2017) but taking in advantage of the vertical space (Palm et al., 2018). They are mainly located in urban areas but Love et al. (2015) stated that 29% of commercial aquaponic producers used vertical towers.

(20)

19

1.4 European eel

1.4.1 Anguilla anguilla: taxonomy, morphology and quick reference of

physiology (Jacoby et Gollock, 2014)

Kingdom Animalia Phylum Chordata Class Actynopterygii Order Anguilliformes Family Anguillidae

Genus Anguilla Fig. 3. European eel specimens (Source: Species Anguilla anguilla (Linnaeus, 1758)

European eel (Figure 3) is a warmwater, euryhaline catadromous fish species belonging to the temperate zones of the world. As member of the Order of Anguilliformes, European eel body appears elongated, cylindrical anteriorly and compressed posteriorly, covered by a thick and secretory integument. The skin's thickness varies widely according to the body region (thinnest in the caudal area and around the pectoral fins), the age (yellow eels show a thinner skin than silvery eels) and to the sex (the female's skin is thicker than the male's skin). Over the whole skin, many substances with a protective function are secreted by both mucus and club cells. Embedded in the skin, there are rudimentary scales, whose develop somewhat late in ontogeny (after the sixth year of life according to Cognetti and De Angelis,1980), in an irregular manner and, in some region, with a parquet flooring distribution. From the development of the first and the last scales, it occurs 2 or 3 years. Analysing the part of the body that showed scales with the highest number of annual rings, it has been possible to recognize with the oral region the first of scales' development. The lips, the throat and the pectoral fins bases are scale less. Although classified as cycloid, the eels' scale appears elliptical with concentric lines (circuli). With respect to the number of vertebrae, a diagnostic means at the species level and connected with myosepta e segmental musculature, European eel has 110-119 vertebrae (Cognetti

https://www.google.com/url?sa=i&source=images &cd=&cad=rja&uact=8&ved=2ahUKEwi_wsq2ru 7hAhVSDOwKHdZSDjMQjRx6BAgBEAU&url= https%3A%2F%2Fwww.dw.com%2Fen%)2Feels -released- ))as-europe-tries-to-replenish-decimated- fishery%2Fa-47880482&psig=AOvVaw2slcM-Ff_JqtS8nZ4YXx_S&ust=1556388813964324

(21)

20 and De Angelis, 1980). The pigmentation is widely different from an ontogenetic stage to another and, thus, used as recognising means. In the freshwater environment, adult specimens not yet sexually mature show a greenish-brown livery colouration and a

conspicuous lateral line

(http://www.fao.org/fishery/culturedspecies/Anguilla_anguilla/en). As regards the fins, the ventral is absent, just like the pelvic girdle. Despite the pectoral girdle is present, it lacks connection with the skull and these features, together with a reduced post-temporal, is unique of eels. The unpaired fins, the dorsal and anal fin, are confluent with caudal fin whereas the pectoral fins are rounded and small and change in shape shortly before the gonads’ maturation (or: "during the later stage before becoming adults"). Dorsal fin origins from the middle of the back whereas the anal fin behind the anus. Conforming to the body shape, the head is elongated (useful for hiding in sand, mud and narrow holes) but, especially in Anguilla anguilla, can vary in shape. Indeed, we can recognize broad- and narrow-headed European eel (Tesch, 2003), although intermediate shapes are present (Cognetti and De Angelis, 1980). The eyes are round and the size seems to depend on an individual (e.g age, sex) and/or abiotic (light intensity in the water column) factors. The two nostrils appear unusually far apart from one to another and lie from the anterior margin of the nasal cavity (anterior nostril) to the anterior border of the eye (posterior nostril). The mouth is composed by a lower jaw longer than the upper (http://www.fao.org/fishery/culturedspecies/Anguilla_anguilla/en), houses setiform teeth set in bands in both jaws and in a patch on vomer. They change in form during the different developmental stages but they are less useful for classification at species level before the eels reach 12-15 cm of body length. The very long gill apparatus, accommodated to the elongated form, is displaced behind the skull (Tesch, 2003) with small and vertical gill openings restricted to the side. A peculiarity of the eels is the double row of the gill lamellae on the filament.

As previously announced, European eel is an euryhaline fish, therefore it shows a remarkable ability to shift its osmoregulatory system (Maciver et al. 2009) according to the environmental osmolarity, at whatever developmental stage (van de Thillart et al., 2009) and without incurring any osmotic shock (Cognetti and De Angelis, 1980). Indeed, in a hyperosmotic environment (salt water) euryhaline fishes 1) eliminate the monovalent ions in excess through the gills to fence the raising ions concentration in blood 2) to minimise the loss of water through passive diffusion, drink large volume of seawater and produce small quantities of urine (Roberts and Ellis, 2012); whereas in freshwater they

(22)

21 display the opposite mechanism (Cao et al., 2018) and maintain the intern osmotic pressure almost constant. As European eel migrates from these two widely different environments, the gill, kidney and intestine maintain of ions and water homeostasis (Kültz 2015; Peh et al. 2009; Comrie et al. 2001) under endocrine system control (Roberts and Ellis, 2012). The gills are not only involved in the respiratory function: indeed, their epithelium presents specialized, mitochondrion-rich cells that primarily pump salt inwards in fresh-water and outwards in seawater from the body fluids. These, discovered for the first time exactly in European eel by Keys and Willmer in 1932, referred to as "ionocyte" (Hiroia and McCormik, 2012). As regards the kidney, it plays a role in osmoregulation of body fluids by means numerous nephrons whom structure varies considerably between freshwater, euryhaline and marine fishes. As a euryhaline fish, European eel has a small nephron whose structure is a combination of both types: indeed, it consists of a glomerulus, a neck segment, two or three proximal, intermediate segments, as well as marine teleost but in addiction presents a distal segment (Roberts and Ellis, 2012). The homeostasis is ensured by a low/high plasma filtration rate and a small/large production of concentrated/dilute urine, in saltwater and freshwater respectively (Cao et al., 2018). The gut walls host numerous sodium, potassium and chloride co-transporters for adsorption of these three ions in parallel with the adsorption of water from intestinal lumen (Schettino and Lionetto 2003; Yuge and Takei 2007), precisely the rectum. With respect to the resistance to survive in air, Tesch (2003) stated that it is linked to the ability of adult eels to employ both branchial and cutaneous mode of respiratory gas exchange. Although the benthic habits, that exposes the eels to hypoxic zones of water at low temperature, can suggest a specific resistance of this species to poorly oxygenated water, it seems its resistance is like that shown by other fishes (Tesch, 2003) but, it is strictly linked to the temperature. Indeed, study conduct by Hill (1969) revealed that eels were able to withstand at 2,5 mg/L O2 in 21°C water, confirmed by Querellou (1974) who defined 3 mg/L O2 as the lower level below whom suffering signs are evident on eels at a temperature between 16-27°C. According to Cognetti and De Angelis (1980), when that value further decreases specimens undergo easily fall into sickness although survive. As regards cutaneous mode, can survive 12 hours in a moist environment and out of the water (Cognetti and De Angelis,1980).

The life cycle of European eel is complex for the multiple and different developmental stages that follow one another, for the unique pathway that it chases and covered by the mystery, mainly about the spawning ground and reproduction, almost until the early 20th

(23)

22 century (van Ginneken and Maes, 2005). An explaining picture is given below (Figure 4). Since the majority of the European eel life history's events occur in the ocean, its completion depends strongly on ocean condition: when certain lunar phases and atmospheric conditions occur, yellow eels move to the ocean, following the Canary and North-equatorial currents. According to van Ginneken and Maes (2005), male European silver eels of approximately 40 cm depart earlier (August) than female silver eels of >50 cm (September-October) from the European coast to the Sargasso Sea.

Fig. 4. Biological life cycle of the European eel (Source:

https://www.google.com/url?sa=i&source=images&cd=&cad=rja&uact=8&ved=2ahUKEwjZ6Im9r O7hAhUEyKQKHfrrAAYQjRx6BAgBEAU&url=https%3A%2F%2Fwww.researchgate.net%2Ffi

gure%2FBiological-cycle-of-the-European-eel-Anguilla-anguilla_fig1_43002497&psig=AOvVaw2G5HAU7zTr0c1_B-5BDS4G&ust=1556388261346996 ) The reasons of that difference in migration time, corresponding to approximately 1 month, between males and females can be explained by the different pre-migration site and the different cruising speed. Tesch (1977) stated that female eels use to occupy up-rivers instead of male eels that are predominantly found in lower coastal areas and lagoons. Another reason is the different body length of males and females that influences the cruising speed. Thus, female eels would perform the 6000-km journey in 139 days and males in 174 days. Indeed, assuming a swimming speed of 0.5 and 1.0 body length per second, this journey takes 6-7 months, after whom the males and females will meet each other in the Sargasso Sea (Schmidt, 1923). During that catadromous migration, European eel gonads’ maturation occurs, triggered by endocrinological factors whose

(24)

23 mechanisms are still covered of mystery (van Ginneken and Maes, 2005). Dufour (1994) described that the sexual immaturity in silver eels is caused by a low content of gonadotropin-releasing hormone (GnRH) and a inhibitory action of dopamine. That induces a dual blockage in the hypothalamous-pituitary axis of the brain. However, observations of animals kept in captivity demonstrated that external environmental stimuli trigger the maturation and development of the gonads (van Ginneken and Maes, 2005). Together with the most common environmental factors influencing the onset of maturation in fish species (temperature, light, salinity, pressure), the physical exercise has been suggested and studied by the Leiden group in large Blazka swim-tunnels and, from unpublished results, they concluded that a period of prolonged swimming might trigger the maturation of the gonads (van Ginneken and Maes, 2005). About the releasing gametes behaviour, three types (female-female; male-female; male-male interactions) and forms (approaching the head region of female; touching the operculum; approaching the urogenital area by males) of spawning behaviour were documented by van Ginneken et al. (2005). European eel is referred to as a monocyclic species because, after breeding, the specimens die (van Ginneken and Maes, 2005). The non-sticky pelagic eggs, with a speed rising of 2.24±0.33 m/h (van Ginneken and Maes, 2005), did not received a parental care as well as in the non-guarding behaviour (van Ginneken et al., 2005d). From the hatching of the pelagic eggs (March-June), the willow leafed larvae, named leptocephali, are passively transported for other 8-9 months by the Gulf Stream and North-Atlantic Drift, until arriving at the eastern Atlantic shelves. There took place the first metamorphosis to an adult-like specimen, the glass eel. At this no pigmentation ontogeny stage, the glass eels return continental waters where remain for 6-10 years. Complete this time, the not yet sexually mature eels are ready for the migration to the spawning site. The spawning period is from March to June, with a peak in spawning in April (McCleave et al. 1987) and, based on the hatched leptocephali founded, the spawning area seems to be the so-called subtropical convergence zone (STCZ) due to its features such as chemical-physical factors that may act as signals to spawn. The spawning depth is few hundred meters. As previously said, larvae of < 5 mm (7 days old specimens) cannot display an active transport mechanism (swimming) because of they a primitive morphology. However, an active transport can be assumed for > 5 mm larvae. From studies conducted by Lecomte-Finiger (1994), European eel leptocephali seem to spend less than a year for transoceanic migration. Age at metamorphosis is around 18 months, with a mean age of glass eel of 190-280 days. They grow of 0.26-0.30 mm per day.

(25)

24

1.4.2 European eel farming technologies in Europe and Italy

Before the 19th century, the eel farming in Europe was of minor importance if compared with the fast development that occurred in Japan and only in France and Italy some form of eel farming was present in the lagoons of Arcachon and Comacchio (Tesch, 2003). In this latter area, the extensive farming system was referred to as "vallicultura" and consisted in stocking glass eels or elvers in polyculture with other brackish species such as mullet and sea bass (Tesch, 2003). In the 1960s, parasitic diseases such as argulosis and white spot diseases considerably affected the industry (Gousset, 1990; Tesch, 2003; Parisi et al., 2014). At the same time, relying on the Japanese experiences, a thrust towards the intensification of the production was valued and in the '70s the outdoor pond culture with earthen or concrete tanks, aeration, artificial feed and heated effluents was employed (Tesch, 2003). That technique was suitable in temperate zones, such as Mediterranean countries, where the optimum temperature for European eel growth (23°-28°C) was ensured (Gousset, 1990; Tesch, 2003). That was not economically viable in Nordic countries where the limiting climatic conditions imposed the development of a system that combined the heating of water and energy conservation. Thus in early 1980, Jaspersen and Hodal (1980) proposed an advanced recirculation system relying on the waste heat from power station to warm the water (Kamstra and Davidse, 1991; Tesch, 2003). From that time, the intensive farming of European eel in Europe was performed mainly through two different methodologies: the recirculation technology, widely developed in Northern Europe particularly in The Netherlands and in Denmark, who still has a forefront position within commercial RAS eel farming among the Nordic countries (Tesch, 2003; Dalsgaards et al., 2013); the open loop system, which in Italy is the prevailing and consolidated type.

A brief touch upon RAS technology basic principles has been made in the introduction of aquaponic systems, therefore now the employment of this technology in the eel farming will be dealt with. According to Dalsgaards et al. (2013), European eel is a species suitable for intensive production systems such as RAS due to its relatively wide endurance to acceptable water quality parameters (pH, NH4-N and CO2) and to high rearing density (200 kg/m2), provided that optimal oxygen levels are ensured (Tesch, 2003). The stocks are reared in both raceways, circular and rectangular tanks and juveline and on-growing eels RAS systems are nearly similar, but great attention must be placed in rearing density: indeed, 0.2-0.3 kg/m2 are suitable densities for the quarantine of wild-caught glass eels (Huertas and Cerda, 2006) and McCarthy et al. (1996) stated that the

(26)

25 stocking density of elvers should not exceed 25 Kg/m3_{, with a survival from glass eel to} pre-adult stage of 85-90% (Buchman et al., 2011); instead, with respect to on-growing stage, stocking density can exceed 120 kg/m3 (Dalsgaards et al., 2013). The requirements of European eel, in terms of water quality parameters, are shown in Table.

Regular grading is important in every developmental stage and must be performed with increasing frequency at the increasing of individual differences in growth rate. Feeding systems and type of chosen feed varies according to the rearing phase: indeed, glass eels are primarily fed with cod roe and secondary weaned on a granular feed of 0.3-0.8 mm, administered by means of belt feeders; subsequently, they are reared at the market size (200-400 g) on pellets of 1-2,5 mm through demand or electrical feeders (Delsagaards et al., 2013). The feeding is continuous for 8-10 h/day and is characterised by small meals followed by resting periods and stomach excretion. According to Mortensen (1996) and Seymour (1989), that technique seems to improve feed utilization. The feed loading is about 10-20 kg feed/m3 make up water that is around 4-8% per day (Delsagaards et al., 2013). Tesch (2003) stated a water consumption of 500 L/kg feed. In order to reach the market size, it takes on average 1.5-2 years (Delsagaards et al., 2013). With respect to the design features, the facilities generally consist of a well-insulated building and rely on advanced water treatment that enables to be independent of waste heat. Both raceways, circular and rectangular tanks are employed (Delsagaards et al., 2013) and vary in size from 4 m2_{for glass eels to 50 m}2_{for on-growing (Tesch, 2003).}

1.5 Aim of the work

The aim of the present study was to build up and run a small-scale aquaponic system for rearing eel and producing greens (lettuce and basil) for both eel wild population restocking (eels) and human consumption (eels and greens). Through these tasks, data related to growth performances of eel and greens have been obtained for the very first time.

(27)

26

2. Materials and Methods

The experiment was carried out in Pisa, Tuscany (Italy) with the scientific partnership of the Department of Veterinary Science and the Department of Agricultural, Food and Agro-Environmental Sciences, where the experiment took place. In a 200 m2 greenhouse (Figure 5), two experimental systems were set up: a small-scale aquaponic and a hydroponic system, with the latter used as control system. With respect to the plant growing component, the hydroponic technique chosen was the floating technique in both systems.

Fig. 5.View of the greenhouse hosting the aquaponic system (Source: Google maps) In order to perform a proper sizing of each unit and to obtain a balanced aquaponic system, the component calculations and ratios reported by Somerville et al. (2014) have been followed. The resulting configuration of the system is shown in the following schematic Drawing 1. The system cycling took about three months, from November to January when the first fish weighing was carried out.

(28)

27 Dwg. 1. Top view of the small-scale aquaponic system: (A) fish tank; (B) swirl clarifier;

(C) biological filter; (D) centrifugal pump; (E) hydroponic unit (floating sheets)

2.1. Small-scale aquaponic system design

The total water volume in the aquaponics prototype (fish tank, mechanical clarifier, biological filter, and plant growing unit) was about 1400-1600 L under a flow rate of 1500 L/h, giving a water retention time of 60 minutes in fish tank and 30 minutes in floating troughs. The system consisted of several components, below described.

Fish tank and plumbing. An Intermediate Bulk Container (IBC, high-density

polyethylene) tank of 1 m3 _{was insulated with polystyrene sheets and covered by an} agricultural shading net, in order to further avoiding light stress to the fish and reducing algae proliferation. Moreover, a plastic netting along the edges of the tank was set up to prevent the wall climbing and consequent escape of fish (Figure 6). Along the bottom surface, a sludge removal system was made of three 5 cm PVC horizontal pipes with several small slits, combined each other with a vertical pipe towards the exit hole.

(29)

28

Fig. 6.Details of the insulation and shading tools

Thus, the solid wastes were collected in the pipes laying on the bottom and drained out the fish tank by the vertical outlet pipe. On top of the fish tank, a 2.5 cm polyethylene pipe provided the inlet water.

Swirl clarifier. The water flowed by gravity from the fish tank to the swirl clarifier

through another 5 cm pipe entering near the lower-middle of a 250 L PVC tank. Due to the tangentially position in relation to the tank, the inlet pipe enabled a circulation motion of the water that caused the collection of the solid wastes due to the ogival shape at the bottom of the tank. A drainage valve at the bottom of the tank allowed the flushing out. Finally, water exited the swirl clarifier at the top through two 5 cm pipes and reached a secondary mechanical filtration unit.

Biological filter. Inside the biofilter tank, two cylindrical plastic buckets full of non-toxic

synthetic white fibres were placed in order to trap the residual solid wastes. Then, the water fell into the 250 L bio-filter tank, filled for 2/3 with filter medium foam sponge cubes of specific surface area, SSA, of 600 m2_/m3_{(Moe and Irvine, 2000). The available} surface area expresses how many square meters per cubic meter bacteria will thrive on; each square meter of biologically active surface can metabolize nearly one gram of ammonia per day, dependent on temperature. The outlet pipe at the lower-middle of the tank carried the water to the pump and, thus, divided between the plant growing unit, the fish tank and the biofilter. An agricultural shading net provides the protection from direct sunlight.

Water pump and valves. A 0.55 kW (0.75 HP) centrifugal pump provided the water

(30)

29 fish tank whereas the remaining was distributed between the bio-filter and the plant growing tanks. The presence of valves allowed the flow control to the fish tank, the bio-filter, and the hydroponic units. The recirculation rate (turnover time) is the amount of water exchanged per unit of time. It can easily be determined by dividing the volume of water in the tank by the capacity of the pump. The average capacity was 1000 L/h toward the fish tank, which was roughly 50% of the total capacity of the pump.

Plant growing unit (floating system). In 70 L/each tank (35 cm depth, 50 cm length, 40

cm wide), polyethylene sheets enabled the proper mechanical support for plants. The total water volume was of 420 L and the total growing area of 1.2 m2. With a reciprocating flood/drain cycle, the pump let water flew into the tanks through independent pipes for each tank. A well as the inlet pipes, independent outlet pipes conducted the water to the bio-filter, where was again pumped to the fish tank.

Aeration system. Water was aerated through an air compressor that delivered, by means

thin polyethylene pipes, the air in the fish and plant growing tanks.

Water heating system. In order to ensure the optimal rearing condition for the organisms,

mainly fish and bacteria, the water was heated by mean of an electrical heater.

2.2. Small-scale hydroponic system design

In the same greenhouse, an identical floating system was realized as hydroponic control system. The total water amount was approximately 360 L of standard nutrient solution, oxygenated by bubbling air supplied from the same air compressor used for the aquaponic prototype.

2.3. Experiment description

2.3.1 Fish experiment

Forty-eight yellow European eels (6.4 kg of total biomass) were supplied by Cooperativa Pescatori la Peschereccia (Orbetello, GR) and stocked at low density (6.4 kg/m3_{) for 30} day of acclimation to the freshwater.

(31)

30 Fig. 7. Pit-tagging operation

A starting feed ratio of 128 g/day shared in two administrations per six days/week, was calculated and adjusted during the experimental period according to the water quality, the animal behaviour and the fishes' growth. The feed was represented by a 4.5 mm pellet (Performance ultra®, Naturalleva©, Italy) whose composition was 44% of crude protein, 22% of crude fats, 1.4% crude cellulose, 6.5% crude ash, 1.2% P, 0.3% Na and 1% Ca, was administered until the end of the experimental period. Every month, fishes were anesthetized, identified, weighed and measured in order to record the individual performance parameters, described the chapter dedicated to the measurements. The day before the weighing, an anaesthetic solution was prepared the day before as follow: in 800 ml of osmotic water 3 g of tricaine methanesulfonate (Sigma-Aldrich©) were diluted and stored in a fridge, at a conservation temperature of +4 °C. The day of weighing, fishes were not fed. By mean of a syphon, the water level was gradually reduce and rearing water was stored in plastic bins of about 100 L. Through that operation, solid wastes deposited sediment and the water could be reused later. Moreover, in that way catching operation, performed with small nets, was less stressful for fish. After that, fishes were rapidly stunned in a basket containing rearing water mixed with the prepared anaesthetic (Figure 8).

(32)

31 Fig. 8. Anaesthetic procedure

When signs of consciousness were not observed anymore, the body length and weight were recorded (Figure 9 and Figure 10).

Fig. 9. Body length recording