Evaluation of multitrophic biofiltration system with new algae species and the sea cucumber "Holothuria sanctori"

EVALUATION OF A MULTITROPHIC BIOFILTRATION SYSTEM WITH NEW ALGAE SPECIES AND THE SEA CUCUMBER Holothuria sanctori. Luis, Felaco Duran TESIS PRESENTADA Y DEFENDIDA PUBLICAMENTE PARA LA OBTENCIÓN DEL TÍTULO DE MASTER OFICIAL EN CULTIVOS MARINOS Las Palmas de Gran Canaria a 23 de Junio de 2014 III MASTER OFICIAL EN CULTIVOS MARINOS Las Palmas de Gran Canaria, España 2012-2014 EVALUATION OF A MULTITROPHIC BIOFILTRATION SYSTEM WITH NEW ALGAE SPECIES AND THE SEA CUCUMBER Holothuria sanctori. Luis, Felaco Duran Trabajo realizado en las instalaciones del Parque Cientifico Tecnologico de Taliarte, el ICCM y el Centro de Biotecnologia marina de Las Palmas de Gran Canaria, España, bajo la dirección del Dr. Ricardo Haroun Tabaue y el Dr. Juan Luis Gomez Pinchetti. Presentado como requisito parcial para la obtención del Título oficial de Máster Universitario en Cultivos Marinos otorgado por la Universidad de Las Palmas de Gran Canaria y del Diploma de Master of Science en Acuicultura otorgado por el Centro Internacional de Altos Estudios Agronómicos Mediterráneos (CIHEAM). Director Director Autor Ricardo Haroun Tabraue Juan Luis Gómez Pinchetti Luis Felaco Duran III MASTER OFICIAL EN CULTIVOS MARINOS Organizado conjuntamente por la Universidad de Las Palmas de Gran Canaria (ULPGC), el Instituto Canario de Ciencias Marinas (Gobierno de Canarias) y el Centro Internacional de Altos Estudios Agronómicos Mediterráneos (CIHEAM), a través del Instituto Agronómico Mediterráneo de Zaragoza (IAMZ) Hay sol bueno y mar de espuma… A mi abuela. ACKNOWLEDGMENTS A Dios. A Venezuela que me ha hecho quien soy. A mi familia por ser siempre la más grande motivación. A Ana, por seguirme soportando. A BIOGES y su gente, especialmente a Javier por las tertulias sobre cambiar el mundo. Al GIA y toda su plantilla de investigadores y técnicos por su disponibilidad y profesionalismo. Al CBM-BEA y sus investigadores y técnicos por la disposición de sus instalaciones y equipos. A mis compañeros de master, por que más que unos colegas somos una familia. A Fernando Tuya por el apoyo estadístico y los consejos. A Emilio Soler y Susy por el apoyo en taxonomía. A Gercende por los consejos y por estar siempre disponible. A Dani y Paula por su amistad y los buenos momentos. A Ricardo por la tutoría de este trabajo y su cercanía y amistad. iv CONTENTS Page ABREVIATIONS LIST V TABLES INDEX Vi FIGURES INDEX Vii ANNEXES INDEX Viii ABSTRACT IX 1. INTRODUCTION 1 2. OBJECTIVES 12 3. METHODS 13 3.1. Study area 13 3.2. Initial state of the biofiltration facilities 15 3.3. Assessment of associated organisms 15 3.4. Species selection for culture trials 16 3.5. Culture care and tank design 16 3.6. Ammonia biofiltration capacity 19 3.7.Integrated culture of Holothuria sanctori and Hydropuntia cornea 20 3.7.1. Physiological status of the algae 22 3.7.2. Sea cucumber sediment biofiltration capacity 22 3.8. Holothuria sanctori growth 23 3.9. Algae culture in ropes 23 3.10. Statistical analysis 24 4. RESULTS 25 4.1Initial state of the biofiltration facilities and associated organisms 25 4.2. Algae growth rate and production 29 4.3. Algae biofiltration 30 4.3.1. Biofiltration relative to biomass 33 4.4. Integrated culture of Holothuria sanctori and Hydropuntia cornea 34 4.4.1. Growth and production rate 34 4.4.2. Biofiltration, ammonia and physiological state 35 4.4.3. Sediment filtration by Holothuria sanctori 38 4.4.4 Sea cucumber growth and survival 40 4.5. Rope culture experiment 41 5.DISCUSSION 43 5.1. Initial stat of the biofiltration facilities 43 5.2. Algae growth and biofiltration 44 5.3. Integrated culture of Holothuria sanctori and Hydropuntia cornea 46 5.4. Algae rope culture at the biofiltration facility 50 6. CONCLUSSIONS 51 7. RECOMMENDATIONS 52 8. REFERENCES 53 9. ANNEXES 61 v ABREVIATIONS LIST BCM Botanica Ciencias del Mar FAO Food and Agriculture Organization GIA Grupo de Investigacion en Acuicultura ICCM Instituto Canario de Ciencias Marinas IMTA Integrated Multi-Trophic Aquaculture NUE Nitrogen Uptake Efficiency NUR Nitrogen Uptake Rate OM Organic Matter P Production rate in dry weight PCT Parque Cientifico Tecnológico de Taliarte SA Surface Area SD Standar deviation TAN Total Ammonia Nitrogen TOM Total Organic Matter ULPGC Universidad de Las Palmas de Gran Canaria UV Ultra violet µ Average daily growth in percentage vi TABLES INDEX Page Table 1. Table showing the ammonia concentrations in µM found at the facilities of the biofilter of the PCT. 26 Table 2. Macroalgae species associated to the effluents from both of the aquaculture installations located in Taliarte. 26 Table 3. Fauna associated to the effluents from both of the aquaculture installations located in Taliarte. 27 Table 4. Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rate for Ulva rigida during the experimentation period with a density of 2g/L and a renovation rate of 10 Vol/day. 31 Table 5. Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rate for Colpomenia sinuosa during the experimentation period with an average density of 5.76g/L and a renovation rate of 10 Vol/day. 31 Table 6. Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rate for Schizimenia dubyi during the experimentation period with an average density of 2,64g/L and a renovation rate of 10 Vol/day. 32 Table 7. Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rate for Valonia utricularis during the experimentation period with an average density of 4.47g/L and a renovation rate of 10 Vol/day. 32 Table 8. Statistical analysis for the average of each parameter for each species. 33 Table 9. Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rate s for Hydropuntia cornea cultured with the effluents from Holothuria sanctori tanks during the experimentation period with a density of 4g/L and a renovation rate of 10 Vol/day. 36 Table 10. Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rates for Hydropuntia cornea cultured without the effluents from Holothuria sanctori tanks during the experimentation period with a density of 4g/L and a renovation rate of 10 Vol/day. 36 Table 11. Statistical analysis for each parameter evaluated for H. cornea with or without the effluents from the sea cucumber tanks. 37 Table 12. Survival of Holothuria sanctori in the experimentation tanks and the control tank. 40 vii FIGURES INDEX Page Figure 1. Conceptual model of the ideal IMTA system (from Fisheries and Oceans Canada 2013). 3 Figure 2. Facilities for the study A) Greenhouse where the culture tanks were located B) Sedimentation tank of the facilities of the complimentary building 1 of the PCT C) Biofilter facility of the PCT. 14 Figure 3. Species of algae tested under tank conditions: A) Ulva rigida, B) Codium tomentosum, C) Valonia utricularis, D) Colpomenia sinuosa, E) Dictyota menstrualis, F) Jania rubens, G and H) Schizymenia dubyi and I) Hydropuntia cornea. 16 Figure 4. Details of the tanks and some of the cultures tested in them. A) Ulva rigida, B) Colpomenia sinuosa, C) coculture of Dyctiota menstrualis, Codium tomentosum and Valonia utricularis, D) culture of Codium tomentosum, E) Schyzimenia dubyi, F) general aspect of the tanks. 18 Figure 5. Holothuria sanctori. 20 Figure 6. Details of the sea cucumber setup A) the three tanks covered in dark plastic bags to stimulate feeding behavior and avoid growth of unwanted algae B) detail of the sediment adding procedure where the water level is lowered C) detail of the pure sediment added as feed D) relative measures of the sea cucumbers in one tank notice the different sizes F) observed reproductive behavior. 21 Figure 7. Experimental setting to evaluate the interaction between sea cucumber tanks and algae. 22 Figure 8. Algae species and setup used for the rope culture experiment A) Grateloupia turuturu, B) G. imbricata, C) Hydropuntia cornea, D) frame with ropes and entwined algae, the first rope have G. turuturu, the second G. imbricata and the third H. cornea. 24 Figure 9. Water temperatures during the study 25 Figure 10. Fauna associated with the effluents at the PCT and complimentary building 1 of the PCT sedimentation tank. A) Osilinus atratus, B) Siphonaria pectinata, C) Cerithium vulgatum, D) Patella aspera, E) Aplysia dactilomela, F) Aiptasia mutabilis, G) Tunicates, H) Myxicola infundibulum, I) Eupolymnia nebulosa, J) example of an aggregation of various organisms K) Example of escaped fish in this case, Seriola dumerilii. 28 Figure 11. Growth rate presented by the 4 species of algae evaluated during three weeks, standard deviation and average are presented 29 Figure 12. Production rate presented by the 4 species of algae evaluated during three weeks, standard deviation and average are presented. 30 Figure 13. Biofiltration in relation to biomass comparing the four algae species. 33 Figure 14. Specific growth rate of Hydropuntia cornea submitted to the two treatments showing the standard deviation. 34 Figure 15. Production rate of Hydropuntia cornea submitted to the two treatments 35 Figure 16. Physiological state in Fv/Fm of Hydropuntia cornea submitted to both treatments (with water from the effluents of sea cucumber or directly from the sedimentation tank ) during the period of study. 37 Figure 17. Average amount of ammonia at the entrance and exit for each week measured at the entrance and exit of the sea cucumber tanks and its difference. 38 Figure 18. Amount of organic matter (g/100g) present at the beginning and the end of each week during the study. 39 Figure 19. Total nitrogen (mgN/100g) present in the sediments at the beginning and 39 viii the end of each week of treatment. Figure 20. Shows the difference in the weight of Holothuria sanctori for each tank after 100 days of feeding with sediment from the sedimentation tank. 40 Figure 21. Average weight of Hydropuntia cornea, Grateloupia turuturu and G. imbricata at the beginning of the rope experiment and after 40 days. 41 Figure 22. Average growth rate of Hydropuntia cornea, Grateloupia turuturu and G. imbricata at the beginning of the rope experiment and after 40 days. 41 Figure 23. initial and final state after 40 days of the ropes stocked with Gracillaria cornea, Grateloupia imbricata and Grateloupia turuturu at the biofilter of the PCT. 42 ANNEXES INDEX Page Annex 1. Example of the identification process of Schizymenia dubyi using its macroscopic and microscopic morphology. A) morphology of the thallus with the very small stipe B) Morphology of thallus in natural conditions, C) Cross section of the blade showing epiphytes (right arrow) and medular filaments (left arrow), D) Cross section of the blade showing early development of the gonimoblast (left arrow), E) cross section showing the ostiole in a well-developed gonimoblast and the expulsion of spores (left arrow), F) detail of the spores (left arrow). 61 Annex 2. Proposed mechanism to increase the surface available for algae growth at the biofilter of the PCT A) initial state, B) condition after a month viewed from the surface and C) condition after a month filled with Colpomenia sinuosa at the continuously submerged side. 62 6 carbohydrates in brown seaweeds act as soil conditioners improving aeration and soil structure, especially in clay soils, and have good moisture retention properties. Some studies also show that extracts of brown algae can improve growth rates and reduce pests, consequently increasing crop yields as well as improving the overall quality of the product (Blunden et al. 1996; Leach et al. 1999). A more recent approach to seaweed use is as source of fuel and as carbon sinks (Packer. 2009), considering the possible use of large areas of cultivated seaweed, as a tool to mitigate global warming (Muraoka. 2004). CO2 acquisition by marine macroalgae can represent a considerable sink for anthropogenic CO2 emissions and harvesting and appropriate use of macroalgal primary production could play a significant role in carbon sequestration and amelioration of greenhouse gas emissions (Chung et al. 2011). At present there are approximately 200 species of seaweeds used worldwide (Zemke - White and Ohno 1999), of which the following species or genera are intensively cultivated: Laminaria japonica, Undaria pinnatifida, Porphyra spp., Eucheuma spp., Kappaphicus alvarezii, Hydropuntia spp., Monostroma spp. and Enteromorpha spp. (Lüning and Pang 2003). The inability of traditionally high value genera such as Hydropuntia, Porphyra and more recently targeted high yield species such as Asparagopsis (Dawes et al. 1999; Matos et al. 2006 ) to survive the broad environmental fluctuations of tropical pond - based systems provides an impetus for the selection of alternative bioremediation species (Muñoz et al. 2011). Most of the technology for integrated multi - trophic aquaculture (IMTA) strategies already exists, however it has not been properly implemented in a large, marketable, commercial scale (Buschmann et al. 2009 ). Furthermore, seaweed biofiltration of fish farm effluents has not been adopted by the aquaculture industry. Since most seaweeds are commercially less valuable than cultured marine animals, and considering that one of the most costly component of diets used in aquaculture is protein (Neori et al. 1998), One proposed way to increase the economic viability of seaweed biofilters has been to feed the biomass produced to commercially 7 valuable macroalgivores which can convert large quantities of the low-valued seaweed into highly valued commodities (Shpigel and Neori, 1996) such as sea urchins and abalone. The nutritional value of seaweeds differs according to the species and the family considered. For example, the red seaweeds contain a high level of proteins (Fleurence, 1999; Fujiwara-Arasaki et al. 1984) while the green seaweeds, such as Ulva lactuca, show a lower protein level and brown algae, such as L. japonica, have the lowest protein content. However, seaweed reared in fishpond wastewater effluents regularly increase their protein content and present a significantly higher dietary value compared to algae reared in regular saltwater. This translates to better growth performance as experiments in Haliotis tuberculata coccinea (Viera et al., 2011) and other commercially important species such as the sea urchins Psammechinus miliaris and Paracentrotus lividus (Cook and Kelly, 2006) have showed. These improvements are significant considering that a limiting factor for further expansion of abalone and other macroalgivores aquaculture is the restricted availability of an economically and environmentally sustainable feed, as these cultures frequently require large quantities of wild harvested macroalgae (Viera et al., 2005) and that animals fed mixed diets perform significantly better than those fed a single algal diet (Viera et al., 2011). The use of macroalgae as biofilters and as valuable mariculture products is not limited to land based installations, an alternative approach in seaweed pond and ditch culture, using stakes and ropes, has emerged from the technologies used for large-scale coastal seaweed farming of Southeast Asia. This approach, however, has not provided the nutrient removal rates and the areal yields necessary for intensive mariculture, for reasons probably related to insufficient water turbulence, high turbidity and low nutrient concentrations, however, with the increase of interest in offshore aquaculture, they have been shown to work even in harsh offshore conditions (Buck and Buchholz. 2004, Neori et al. 2004, Garcia 2012; Leonczek 2013) mostly using long line seaweed aquaculture techniques increasing its potential as integrated multi trophic aquaculture species. 8 Detritivores in IMTA Issues such as solid waste management, nutrient recycling and feed conversion enhancement are also more easily and profitably addressed on an industrial scale on land than in open-water fish farms (Neori et al., 2004), however, with the expansion of cage aquaculture, there is a need for treating these solid waste directly in open water conditions, that is the reason why other complementary integrated approach for the reduction of the environmental impact on the sea bottom by the net pen sludge has been the culture underneath them of scavengers like sea cucumbers as secondary crops (Ahlgren, 1998) using them as sort of animal biofilter for sediments. In this way, the integrated multitrophic aquaculture approach is not limited only to the production of macroalgae from the effluents of traditional feed aquaculture, it has also been tested as food source for suspension feeders and detritivores, this gains importance considering that the most significant polluting components of aquaculture effluents is not only ammonia, but also, suspended solids (Tovar et al. 2000). Organic fouling debris made up of fish feces, excess fish food, algae, invertebrates, and other particulate organic matter can accumulate along the sides and floor of the floating pens and on the sea floor below, clogging the nets and restricting water circulation and possibly depleting oxygen in the water and sediments which in turn can stress fish. The feeding habits of some species of sea cucumbers can alleviate fouling debris problems at salmon mariculture facilities this was demonstrated by Ahlgren in 1998 who conducted experiments in which red sea cucumbers Parastichopus californicus were allowed to feed inside floating net pens at a salmon rearing facility in Southeast Alaska. These experiments showed that sea cucumbers consumed fouling debris and cleared a significant amount of surface area on the nets. By doing this, sea cucumbers can take advantage of much of the fouling debris which is derived from protein-rich organic matter such as fish food, both before and after it has passed through a fish gut and even dead fish fry, turning harmful debris into a marketable product (Ahlgren. 1998). 9 These impacts do not happen only in fish aquaculture, it has been demonstrated that dense assemblages of filter-feeding bivalves enhance the vertical flow of organic matter towards the benthic environment. Palzat et al. (2008) gathered evidence that sea cucumbers will utilize, and therefore reduce, the benthic organic deposition from oysters given that when grown in co - culture showing both active selection of organic material from the sediments and digestion/assimilation of these organics in the gut. This suggests the feasibility of developing a commercial-scale co-culture system that would both reduce the amount of organic deposition underneath shellfish farms and produce a secondary cash crop (Paltzat et al. 2008). There have also been trials for the polyculture of shrimp and sea cucumbers (Holothuria scabra) but their success has been limited. In several trials, it seemed that shrimp caused the deaths of sea cucumbers, and in some cases it was clear that they did so (Pitt et al. 2004). In other trials, survival and growth of sandfish reared with shrimp for 3 weeks were significantly lower than for sandfish reared alone. Confirmed that coculture is not viable. All sandfish reared in co-culture were dead or moribund after a month (Bell et al. 2007). Some species of sea cucumbers have been reported to be able to eat up to 76 % of Total organic matter (TOM). Zamora and Jeffs in 2011 demonstrated the ability of the sea cucumber Australostichopus mollis to use different levels of TOM to generate similar growth rates mainly by changes in their feeding behavior and digestive physiology. The TOM, in the biodeposits sampled beneath mussel farms ranged from 2.8% to 18.2% which were within the range covered in the laboratory feeding experiments (Zamora and Jeffs, 2012). Other diets have been tested by Yuan and collaborators (2006), they reported that a mixed diet containing mostly mussel feces and 25 % powdered algae showed the best standard growth rate. In the Canary Islands the main representatives of Holothurians are Holothuria mammata, Holothuria arguinensis and Holothuria (Platyperona) sanctori Delle Chiaje 1823 the latter species is the most abundant in the archipelago and very conspicuous in the Mediterranean and adjacent eastern Atlantic where it has commercial value (Aydin, 2008). It shows a markedly nocturnal behavior, with larger distances and fastest 10 displacements during the end of the nighttime, remaining hidden in cracks and crevices during daylight and moving at night to feed (Pérez-Ruzafa et al. 1984; Pérez-Ruzafa and Marcos 1987) H. sanctori is one of the most selective species when it comes to the selection of organic matter from the sediments (Mezali and Soualili. 2013).Its consumption of OM has been shown to increase with OM availability, particularly during formation of the gonads (Navarro et al. 2013a). Economic value and uses of sea cucumbers Sea cucumbers, can be very marketable specifically on their dried form also called beche-de-mer they have been a dietary delicacy and medicine for Asians over many centuries. The collection of sea cucumbers to supply the market has seen a depletion of this resource in the traditional fishing grounds close to Asia and more recently the expansion of this activity to new and more distant fishing grounds. Currently, there are fisheries harvesting sea cucumbers across most of the resource range, including remote parts of the Pacific, the Galapagos Islands, Chile and the Russian Federation. Sea cucumber stocks are under intense fishing pressure in many parts of the world and require effective conservation measures. Sea cucumbers provide an important contribution to economies and livelihoods of coastal communities, being the most economically important fishery and non-finfish export in many countries. A multitude of sea cucumber species are being exploited worldwide, with new species being brought to market as established species become scarcer and more difficult to find. Little is known about the ecology, biology and population status of most commercial species. (ToralGranda et al., 2008) The majority of sea cucumbers are exported for the beche-de-mer market and few species for the live trade (aquarium) market. In many countries, particularly in the Western Pacific region, some sea cucumbers or their organs are consumed as delicacies or as a protein component to traditional diets (Toral-Granda et al. 2008). They have high commercial value coupled with increasing global production and trade. Sea cucumbers have long been used for food and medicine in the communities of Asia and Middle East. Nutritionally, sea cucumbers have an impressive profile of valuable nutrients such as Vitamin A, Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), and 11 minerals, especially calcium, magnesium, iron and zinc. A number of unique biological and pharmacological activities including anti-angiogenic, anticancer, anticoagulant, antihypertension, anti-inflammatory, antimicrobial, antioxidant, antithrombotic, antitumor and wound healing have been described for various species (Bordbar et al. 2011) Aquaculture, sea ranching and restocking have been evaluated as possible solutions to wild sea cucumber overexploitation, and some countries have started such ventures (e.g. Australia, China, Kiribati, Philippines, Viet Nam and Madagascar) China is successfully producing an estimate of 10,000 tonnes dry weight of Apostichopus japonicus from aquaculture, mainly to supply local demand (Toral-Granda et al., 2008). In the last years, the research group in Aquaculture of the ULPGC has been working on biofiltering processes of the effluents from the aquaculture production unit in the Complementary Module 1 of the Scientific and Technological Park. The main focus has been few seaweed species as nutrient stripers and as source of biomass for abalone production. To our knowledge, there has not been any study where the effluents collected at the sedimentation tanks of in land aquaculture facilities are used to feed detritivores, furthermore, the interaction between sediment feeders, water column and cultured algae has not been thoroughly studied. This study was started to enhance the potential use of new macroalgae to be incorporated in the biofiltering system as well as to determine the role of sea cucumber as a sediment organic matter consumer. The combination of these two types of nutrient remover organisms (biofilter organisms) has not been evaluated yet and the need to understand the possible synergy between sea cucumber and seaweed aquaculture are the main relevant new factors in this work. In this sense, 3 main research lines have been opened around the concept of integrated aquaculture systems to allow environmental friendly and more profitable land-based aquaculture production. There is a great need to diversify cultivated seaweeds that could be used as biofilters with market viability and it is important to determine the efficiency of filtration of these seaweeds, these are key factors that are addressed in this work. 12 2. OBJECTIVES General objective: The general objective of this study was to evaluate and implement potential new marine species with sediment and water biofiltration capacities of the nutrients from a landbased aquaculture production unit. Specific objectives: In order to accomplish the main objective of this study several specific objectives where designed  Identify the main organisms present in effluents from the in land aquaculture facilities in the Scientific and technologic park of Taliarte (Parque Cientifico Tecnológico de Taliarte, PCT) focusing on the Biofiltration potential of the organisms.  Evaluate the initial state of the biofiltration facility in the Parque Científico Tecnológico de Taliarte PCT and make the possible and necessary modifications to improve it.  Determine the biofiltration capacity of the most abundant species of algae present in the effluents as well as their production and resistance to free floating culture systems.  Assess the viability of adding Holothuria sanctori as a way to biofiltrate the sediments from the sedimentation tank at the PCT and increase the nutrients available in the water for the growth of algae in free floating systems.  Determine the growth and survival of Holothuria sanctori fed with sediments from the sedimentation tank of the effluents of the PCT.  Test the potential use of algal rope culture to cultivate algae with commercial interest in the biofilter installation of the PCT. 13 3. MATERIALS AND METHODS 3.1. Biofiltration facilities: This study was made at the integrated culture facilities of the research group in aquaculture (GIA; ULPGC) and the Parque Cientifico Tecnológico de Taliarte (PCT) located in the eastern side of the Island of Gran Canaria, in Taliarte, Telde. The experimental tanks (description of tanks on 3.5) where located inside a greenhouse in the facilities of the complimentary building 1 of the PCT (Figure 2 A); the greenhouse received water through a pump located at an 11 m3 sedimentation tank that collects the suspended particles of all the effluents from the culture of various species of the facility (Figure 2 B). On the other hand, at the PCT´s facilities, the biofilter facility located outdoors consists on an elongated tank system with a staircase design that creates cascades of water on each level, the approximated total volume of this biofilter is of 160 m3 with approximately 30 renovations of its volume per day. The effluents from three fish production facilities enters trough 3 pipes located in the upper side of the biofilter at the center, it has a small sedimentation tank of approximately 1.60 m deep on one of the sides of the first level, from this first level the water falls by gravity to 8 other levels that are made of concrete tanks of approximately 30 cm wide, 40 cm deep by 14 m long. The biofilter ends in a cascade of approximately 2.3 m after which the water flows directly to the ocean (Figure 2 C). 14 Figure 2: Facilities for the study A) Greenhouse where the culture tanks were located B) Sedimentation tank of the facilities of the complimentary building 1 of the PCT C) Biofilter facility of the PCT. A B C 15 3.2. Initial state of the biofiltration facilities The initial state of the biofiltration facility was determined via direct observation of both of the designated biofilter installations (the complimentary building 1 of the PCT and the main building) specially taking into account the sedimentation patterns and the direction of the water flow. In order to improve the direction of the water flow and the sedimentation of particles (which was too fast and in the center of the biofilter) a series of pipes where connected to the two main exits, redirecting their flow towards the sedimentation tank located on the right side of the entrance of the water to the biofilter. The first set of pipes was connected to the first entrance of the water to the system which was the one with the strongest water flow, this modification enabled the water to enter the biofilter next to the sedimentation tank, the second set of pipes was connected to the second entrance of water located approximately 30 cm to the left of the first entrance and 15 cm deeper, here the water flow was redirected also towards the sedimentation tank, but under the first set of pipes. Water samples were collected in the left, center and right area at the entrance and the exit of the water through the system to determine total ammonia of the biofiltration facility at the PCT. 3.3. Assessment of associated organisms A rapid census of the organisms associated with the facility was made by recording every macroscopic organism seen along each level of the biofilter at the PCT and the sedimentation tank of the complimentary building 1 of the PCT facility, pictures and samples were taken for each species with emphasis in algae species, which were collected and placed on the reference collection of the herbarium “Herbario BCM (Botanica Ciencias del Mar)”. Each species was identified using specialized taxonomic references based on the original descriptions and on a critical analysis of the literature and in the case of algae, stereo and compound microscopes were used to look and describe the morphological and anatomical characters for the proper identification of the species. 22 Figure 7: Experimental setting to evaluate the interaction between sea cucumber tanks and algae. 3.7.1. Physiological status of the algae: The physiological status of Hydropuntia cornea submitted to both treatments was verified by measurement of photosynthetic efficiency of chlorophyll (Fv/Fm) by the fluorescence emission using the Hansatech Pocket PEA Chlorophyll fluorimeter. 3.7.2. Sea cucumber sediment biofiltration capacity. Two surface sediment samples of approximately 100 ml were taken from each tank the day after sediment was poured in and after a week by introducing a urine sample bottle of 100ml at about 1 cm in the sediment at a random place of the tank, and then moving it for about 5 cm in a straight line, each sample was frozen until all the samples of the 3 days trial were collected, then they were dried in a stove at 100 ºC for approximately 24 hours until they reached constant weight, then each sample was homogenized manually for about 20 minutes, each analysis was made in triplicates, the methods used for the analysis are as follows. Percentage of organic matter was calculated by burning approximately 4 g of sediment sample in a Muffle furnace at 600 ªC during at least 4 hours (AOAC, 2005). 23 Total nitrogen in the sediments was analyzed using the method described by Kjeldahl (AOAC, 2005), which consists on the digestion of the samples at 420 ªC with concentrated sulfuric acid in presence of a mercuric catalyzer during an hour, after which the sample is distilled with 40% NaOH with boric acid as a receiver substance (using the P Selecta Bloc Digest for the digestion and the P Selecta Kjeldahl distiller pro nitro M for the distillation), in the end, the sample is titrated with HCl 0.1 N and the amount of nitrogen is calculated according to the formula N (mg/100g)=ml of titration – average pattern titration ml) x 0.1 (molarity of HCl) x 14.007 mg of sample x 100 3.8. Holothuria sanctori growth Tree tanks of sea cucumbers were fed every 15 days (except for the period of 3 weeks when the experiment with H. cornea took place, when they were fed weekly, also keeping a control tank with no feed) they were weighed at the beginning and then at the end of the 100 days experiment and monitored each week. 3.9. Algae culture in ropes. Three PVC frames of 35 cm by 1 m were created, three 1 m ropes were placed in each frame and each rope was stocked with either Grateloupia imbricata, Grateloupia turuturu or Hydropuntia cornea (Figure 5, A, B and C), so that each frame had one species per rope, the algae were put in the ropes by entwining the thallus into the rope (Figure 8 D). Each frame was placed in the same level of the biofilter installation at the PCT at the left, center, and middle section to ensure all possible combinations and to reduce the effect of the location inside the biofilter, they were harvested after 40 days under those conditions. 24 Figure 8: Algae species and setup used for the rope culture experiment A) Grateloupia turuturu, B) G. imbricata, C) Hydropuntia cornea, D) frame with ropes and entwined algae, the first rope have G. turuturu, the second G. imbricata and the third H. cornea. 3.10. Statistical analysis. Non parametric statistics was used because the data does not comply with the requisites for parametric statistics, the U of Mann Whitney Test and analysis of variance via permutations (PERMANOVA) where used to evaluate if there were significant differences between treatments and species. A C B D 25 4. RESULTS 4.1. Initial state of the effluents and associated organisms. The temperatures during the study are shown in figure 9, they varied between 24 to 20 ºC. The Total ammonia associated with the biofilter at the main building of the PCTT did not differ at the entrance and exit of the facility (table 1). The organisms associated with the effluents of both facilities diverged, in general, the sedimentation tank of the complimentary building 1 of the PCT had more species richness, 11 macroalgae (Table 2) and 16 macrofauna species (Table 3) while the biofilter at the PCT only presented 7 species of macroalgae (Table 2) and 14 species of macrofauna (Table 3). Regarding macroalgae groups, there was a dominance of species of red algae in the effluents from the complimentary building 1 of the PCT with 5 species, followed by representatives of green algae with 4 species, meanwhile, in the biofilter installation at the PCT, Brown algae dominated together with green algae, both with 3 species (Table 2). The associated fauna presented the same richness in both facilities, with 16 species. The most prevalent and dominant group were the mollusks with 5 species at the installation in the PCT and 7 at the sedimentation tank of the complimentary building 1 of the PCT facility (Table 3), (figure 10). Figure 9: Water temperatures during the study 15 17 19 21 23 25 27 10/7/2013 11/7/2013 12/7/2013 1/7/2014 2/7/2014 3/7/2014 4/7/2014 Temperature (ºC) Date 26 Table 1: Table showing the ammonia concentrations in µM found at the facilities of the biofilter of the PCT. Entrance Exit Left 8.485 8.345 Middle 9.467 8.626 Right 11.289 10.168 Average 9.747 9.046 Stdev 1.161 0.801 Table 2: Macroalgae species associated to the effluents from both of the aquaculture installations located in Taliarte. Species Complimentary building 1 Biofilter PCT Phaeophyta Dyctiota menstrualis Dictyota menstrualis Colpomenia sinuosa Colpomenia sinuosa Platoma Sp. Rhodophyta Jania rubens Jania rubens Schizymenia dubyi Grateloupia imbricata Grateloupia turuturu Hydropuntia cornea Chlorophyta Ulva rigida Ulva rigida Valonia utricularis Valonia utricularis Codium tomentosum Codium tomentosum Caulerpa racemosa Total richness 11 7 27 Table 3: Fauna associated to the effluents from both of the aquaculture installations located in Taliarte. Species Complimentary building 1 Biofilter PCT Porifera Spongionella pulchella Cnidaria Aiptasia mutabilis Aiptasia mutabilis Chordata Ascidiacea Sp Ascidiacea Sp Echinodermata Holothuria sanctori Coscinasterias tenuispina Coscinasterias tenuispina Ophioderma sp Ophioderma sp Annelida Polychaeta sp. Eupolymnia nebulosa Sipunculus nudus Sipunculus nudus Myxicola infundibulum Molusca Aplysia dactylomela Aplysia dactylomela Patella aspera Patella aspera Cerithium vulgatum Cerithium vulgatum Osilinus atratus Osilinus atratus Siphonaria pectinata Siphonaria pectinata Haliotis tuberculata coccinea Aplysia depilans Arthropoda Pachygrapsus marmoratus Vertebrata Sparus aurata Sparus aurata Seriola rivoliana Dicentrarchus labrax Total richness 16 16 28 Figure 10: Fauna associated with the effluents at the PCT and complimentary building 1 of the PCT sedimentation tank. A) Osilinus atratus, B) Siphonaria pectinata, C) Cerithium vulgatum, D) Patella aspera, E) Aplysia dactilomela, F) Aiptasia mutabilis, G) Tunicates, H) Myxicola infundibulum, I) Eupolymnia nebulosa, J) example of an aggregation of various organisms K) Example of escaped fish in this case, Seriola dumerilii. C A E D F G B I H J K 29 4.2. Algae growth rate and production. The experiments were carried out between the 15/10/2013 and the 07/11/2013 for Colpomenia sinuosa and Ulva rigida and for Schizymenia dubyi and Valonia utricularis between the 27/01/2014 and the 18/02/2014. Every species of algae evaluated showed a tendency to decrease during each experimentation period, the species that showed the higher specific growth rate was Ulva rigida (11.03 %d-1) followed by Schizymenia dubyi with 1.58 %d-1 and Valonia utricularis with 1.31 %d-1 and, Colpomenia sinuosa decreased during the whole experimentation period with an average of –6.09 %d-1 (Figure 11). Figure 11: Growth rate presented by the 4 species of algae evaluated during three weeks, standard deviation and average are presented. 15.93 11.80 5.37 11.03 0 2 4 6 8 10 12 14 16 18 20 Week 1 Week 2 Week 3 average Growth rate (% d-1) Ulva rigida 2.32 1.60 0.83 1.58 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Week 1 Week 2 Week 3 average Growth rate (% d-1) Schizymenia dubyi 3.46 0.59 -0.12 1.31 -3 -2 -1 0 1 2 3 4 5 Week 1 Week 2 Week 3 average Growth rate (% d-1) Valonia utricularis -5.01 -7.44 -5.83 -6.09 -14 -12 -10 -8 -6 -4 -2 0 Week 1 Week 2 Week 3 average Growth rate (% d-1) Colpomenia sinuosa 30 Algae production presented a similar tendency as the one showed by the growth rate, with the highest production shown by U. rigida followed by S. dubyi and V. utricularis (figure 12) Figure 12: Production rate presented by the 4 species of algae evaluated during three weeks, standard deviation and average are presented. 4.3. Algae biofiltration: Tables are presented to summarize the algae biofiltration values of Nitrogen uptake rate and Nitrogen uptake efficiency, the Nitrogen uptake efficiency was the highest for Ulva rigida with 90% however, the highest nitrogen uptake ratio was shown by Schizymenia dubyi with 0.94 mmol NH4+ m-2d-1, on the other hand, the lowest of these parameters was shown by Valonia utricularis. Each value presented is an average of the triplicates for each week (tables 4 to 8). 57.23 35.75 14.78 35.92 0 10 20 30 40 50 60 70 80 Week 1 Week 2 Week 3 average Production rate (g PS m-2d-1) Ulva rigida -42.93 -63.79 -50.00 -52.24 -120 -100 -80 -60 -40 -20 0 Week 1 Week 2 Week 3 average Production rate (g PS m-2d-1) Colpomenia sinuosa 4.41 3.46 1.94 3.27 0 1 2 3 4 5 6 7 8 9 Week 1 Week 2 Week 3 average Production rate (g PS m-2d-1) Schizymenia dubyi 4.60 0.90 -0.19 1.77 -6 -4 -2 0 2 4 6 Week 1 Week 2 Week 3 average Production rate (g PS m-2d-1) Valonia utricularis 31 Table 4: Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rate for Ulva rigida during the experimentation period with a density of 2g/L and a renovation rate of 10 Vol/day. Ulva rígida ENTRANCE NH4+ NUE NUR GROWTH RATE PRODUCTION RATE WEEK μM % mmol NH4+ m-2d-1 % d-1 g PS m-2d-1 1 3.501 99.999 0.656 15.930 57.225 2 10.380 70.570 1.373 11.800 35.75 3 0.941 99.999 0.176 5.368 14.775 AVERAGE 4.941 90.190 0.735 11.032 35.916 SD 3.985 13.873 0.492 4.345 17.330 Table 5: Weekly results summarizing the average amount of ammonia at the entrance of the tanks, the NUE, the NUR, together with the growth and production rate for Colpomenia sinuosa during the experimentation period with an average density of 5.76g/L and a renovation rate of 10 Vol/day. Colpomenia sinuosa ENTRANCE NH4+ NUE NUR GROWTH RATE PRODUCTION RATE WEEK μM % mmol NH4+ m-2d-1 % d-1 g PS m-2d-1 1 4.434 87.931 0.731 -3.359 -42.928 2 7.571 74.842 1.062 -4.714 -63.785 3 0.747 99.999 0.140 -4.787 -50.000 AVERAGE 4.251 87.591 0.644 -4.287 -52.238 SD 2.789 10.273 0.381 0.656 8.660 38 Figure 17: Average amount of ammonia at the entrance and exit for each week measured at the entrance and exit of the sea cucumber tanks and its difference. 4.4.3. Sediment filtration by Holothuria sanctori: The amounts of organic matter and total nitrogen in the sediments are presented in the next figures, the results show an increase for both of the parameters at the beginning of each week, when the sediments from the sedimentation tank were added, and then, after a week of activity by the sea cucumbers, the quantities of both parameters showed a decrease, however, this decrease is not significant for Organic matter (U=25, P=0.18) but in the case of total nitrogen in the sediments the difference is significant (U= 3, P=0.001) and its amount is reduced to the control levels at the beginning of the experiment and does not differ significantly from them (U=38, P=0.85) (figures 18 and 19). 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 average Ammonia (µM) Week entrance exit difference 39 Figure 18: Amount of organic matter (g/100g) present at the beginning and the end of each week during the study. Figure 19: Total nitrogen (mgN/100g) present in the sediments at the beginning and the end of each week of treatment. 2.21 2.22 2.69 2.32 6.08 4.68 3.29 2.83 4.02 3.27 0 1 2 3 4 5 6 7 control tanks Initial Final Initial Final Initial Final Initial Final start Week 1 Week 2 Week 3 Average Organig matter (g/100g) 15.39 13.85 28.54 9.14 24.58 17.06 25.39 14.64 26.17 13.61 0 5 10 15 20 25 30 35 40 control tanks initial final initial final initial final initial final start Week 1 week 2 week 3 average Total nitrogen (mgN/100g) 40 4.4.4. Sea cucumber growth and survival: Regarding sea cucumber growth, after a period of 100 days of feeding with sediments from the sedimentation tank every 15 days in average, the average weight showed a tendency to decrease in all of the tanks including the control however, the differences are not significant (U=2, P=0.11) (Figure 20). Survival in each treatment tank was 100 % while in the control tank only 84.615 % (Table 12) Figure 20: Shows the difference in the weight of Holothuria sanctori for each tank after 100 days of feeding with sediment from the sedimentation tank. Table 12: Survival of Holothuria sanctori in the experimentation tanks and the control tank. Tank Survival (%) 1 100 2 100 3 100 control 84.6 Average 96.15 76.69 76.31 78.38 86.96 79.58 68.19 69.38 67.25 76.95 70.44 -10 10 30 50 70 90 110 130 t1 t2 t3 control average weight (g) Tank initial after 100 days 41 4.5. Rope culture experiment Regarding the experiment of rope culture in the biofilter installation at the PCT, after 40 days of placing the ropes in the biofilter the tendency showed a marked decrease for Hydropuntia cornea, a slight increase for Grateloupia turuturu and some decrease for G. imbricata (figures 21, 22 and 23). Figure 21: Average weight of Hydropuntia cornea, Grateloupia turuturu and G. imbricata at the beginning of the rope experiment and after 40 days. Figure 22: Average growth rate of Hydropuntia cornea, Grateloupia turuturu and G. imbricata at the beginning of the rope experiment and after 40 days. 100 7 20 12.27 8.45 12.40 0 20 40 60 80 100 120 Hydropuntia cornea Grateloupia turuturu Grateloupia imbricata Average weight of algae (g) Begining End -10 -8 -6 -4 -2 0 2 4 Hydropuntia cornea Grateloupia turuturu Grateloupia imbricata Growth rate (%d-1) 42 Figure 23: initial and final state after 40 days of the ropes stocked with Gracillaria cornea, Grateloupia imbricata and Grateloupia turuturu at the biofilter of the PCT. INITIAL FINAL 43 5. DISCUSSION 5.1. Initial state of the effluents and associated organisms. There was a difference in the main algae groups present at the facilities of the complimentary building 1 of the PCT where green and red algae dominated and the facilities of the PCT where brown algae dominated with occasional outburst of green algae, this is possibly due to the differences in the design of both installations, the complimentary building 1 of the PCT one is design as a large sedimentation tank, while the biofilter at the PCT was originally conceived with the aim of using it as a biofilter and also as an architectonic asset, this means that the water flow is considerably larger at the PCT and also that the surface available for algae that typically grow attached is larger, apart from this, the facilities at the complimentary building 1 of the PCT are older than the PCT ones so there has been more time there for the development of a more diverse community. The fauna assemblage at both of the facilities was dominated in apparent abundance by suspensivores such as Aiptasia mutabilis, and less abundant, but equally common, sponges and tunicates which indicate that the amount of nutritional particles in suspension is high at both areas. A very important faunistic component due to its diversity and abundance at both facilities were the gastropods and the fact that most of them were macroalguivores (with the exception of Cerithium vulgatum) creates important changes in possible management of these installations as algae biofilters because they are expected to eat the algae that was meant to be used as biofilters and could suppose a big problem for production, however, some of these naturally growing organisms are consumed and could have potential for aquaculture, Patella aspera and Osilinus atratus are examples of this. The presence of some species can be indicators that some practices in the aquaculture facilities could be improved, for example, in natural conditions it would be extremely rare to find Haliotis tuberculata coccinea at the effluents, more importantly, the presence of Dicentrarchus labrax, Sparus aurata and Seriola rivoliana at the effluents mean that further measures should be taken to prevent escapes. The modifications made to the direction of the flow of the effluents at the PCT is expected to change in some ways the fauna and flora composition in the biofilter, it would create a better disposal of the sediments thus making them easier to manage 44 either by adding organisms able to use them as feed or by taking them out of the biofilter to be disposed elsewhere. Some selections had to be made of all the algae and animals found at both facilities for further research, these organisms were selected based on various criteria, for example, in the case of Schizymenia dubyi the main interest was due to its morphology, secondly because it is a representative of red algae which has been cited as one of the preferred groups for abalone to eat in the Canary Island in wild conditions (Espino and Herrera, 2002 cited in Viera et al., 2005), and thirdly because of its abundance at the sedimentation tank of the complimentary building 1 of the PCT and for it being a species that has not been cited for the Canary Islands (Haroun et al. 2002), on the other hand, for Colpomenia sinuosa the main deciding factor was its abundance at the PCT, Ulva rigida was abundant and a proven biofilter organism, and Valonia utricularis presented relatively good growth. For the filtration of sediments, the sea cucumber Holothuria sanctori was the only species present that fit the criteria of having potential as a sediment feeder and as a marketable organism, and even though some polychaete worms were abundant, the broad distribution and abundance of H. sanctori (Navarro et al. 2013), its potential commercial value (Aydin. 2008) and its easy handling were the determinant factors for the selection of this species 5.2. Algae growth and biofiltration The growth rates differed between the species as expected, being Ulva rigida the species with the biggest growth rate and production, followed by Schizymenia dubyi this concurs with various references that mention that the genus Ulva is generally a very good biofilter (Neori. 2004) and that one of the most important characteristics to consider a species for biofiltration is the surface area, which increases the area available for the uptake of nutrients Growth rate of an algae is largely defined by morphology (Littler and Littler, 1980); generally speaking, the higher the ratio of surface area to volume (SA/Vol), the faster the specific growth rate. Phytoplankton have higher SA/Vol than seaweeds, similarly, the thin 45 sheet morphology has a higher growth rate than does the fleshy one that have a growth rate typically less than 10% day-1 (Marinho-Soriano et al., 2002; Nagler et al., 2003; Neori. 2004) this explains the fact that S. dubyi and U. rigida had a better growth rate than C. sinuosa or V. utricularis. For these reasons, further research should be done on the Schizymenia genus and specifically in S. dubyi since it represents a possibly viable new biofiltration species. Such large standard deviations indicate that the experimental setting could be improved, not necessarily an instability in the algae cultures, the greenhouse set for these experiments had different conditions of light for each tank and the building next to it casted a shadow upon one of the sets of tanks at some hours, to avoid these problems, each week tanks were rotated, however, further measures should be taken to ensure more replicable results. These problems could be solved increasing the amount of replicates while changing the disposition of the tanks ensuring a more even distribution of sunlight and using light sensors during the study at each tank if possible to include light and temperature as a covariate in the statistical analysis. The variability in the cultured species could also be explained by the variability of the nutrient content in the effluents which change according to the reared organisms at the facilities, the hour of their feeding, the day of the week (no feeding on Sundays) and other factors such as the cleaning of the facilities with formalin and chlorine. The growth rate and production of Colpomenia sinuosa was negative, probably because it could not adapt to the free floating conditions; the temperature and photoperiod are not expected to have an important role in these results because during the whole experiment it was growing naturally at the biofilter of the PCT, under tank conditions it usually changed color from green to brownish and then fragmented and started to get epiphytes Specifically in one of the weeks during the culture of S. dubyi and Valonia utricularis a broad cleaning with chlorine and formalin was done in one of the areas established for larval rearing, these chemicals got to the sedimentation tank and could have gotten to the algae culture tanks in high amounts. Biofiltration expressed by NUR and NUE also presented the expected results with U. rigida and S. dubyi performing good at both parameters, Colpomenia sinuosa also 46 presented a very high nitrogen uptake efficiency even though it did not perform well under the culture conditions, this could mean that the epiphytes that grew on the Colpomenia are the real responsible for the biofiltration or that some bacterial biofiltration could be occurring. To further improve these results and the overall health of the algae cultures some options for preventing and combat epiphytes can be applied such as filtered and even UVtreated inflow water which has been cited to greatly reduce the potential for epiphyte contamination of a system, also, the control over environmental conditions in the tanks may be used strategically to influence competition between maricultured seaweeds and epiphytes, using nutrients pulses and a high stocking density have also been successfully proven anti epiphytes techniques (Friedlander et al., 1987, 1991; Neori. 2004). The proposed calculation of NUR/ Kg indicates that, apart from Ulva (that may be greatly underestimated due to the little amount of ammonia present during the weeks of study for this species), the species with more potential for biofiltration and production is S.dubyi. however, this calculation has some limitations because it depends on the amount of ammonia at the entrance and the exit of the biofilter which varied each week and varies even according to the hour of the day; to really evaluate the potential of different species at the same time the amount of ammonia should be controlled and the stocking density of each species should be the same, as well as the amount of light and its physiological state. Another important parameter to be controlled is light, during the weeks of study, it varied due to cloudiness and logically, It is important to mention that the maximal biofiltration of any nutrient occurs, in daytime, yet some TAN is also taken up at night, particularly when the TAN load to the algae is low (Cohen and Neori, 1991; Schuenhoff et al., 2003) such as the conditions of the complimentary building 1 of the PCT. 5.3. Integrated culture of Holothuria sanctori and Hydropuntia cornea The effluents from the sea cucumber tanks presented a significantly larger concentration of total ammonia this means that sea cucumbers have the ability of releasing nutrients from the sediment into the water column, the amount of ammonia 47 from these effluents was also less variable than the effluents from the sedimentation tank which would make it easier to estimate the production of the whole system. The growth and production rate of H. cornea had a tendency to increase when sediments and sea cucumbers were added to the setup and, even though this difference was not significative, it is expected, and the magnitude of the effect may increase with increased amounts of nutrients being released by the sea cucumbers from the sediments, the large variability between tanks can be explained by differences in light because of the orientation of the tank sets at the greenhouse. As for the NUE and the NUR the results had no significant differences, this means that both parameters depend more on the species of algae than on the amount of ammonia being added and thus, being the same species these parameters do not change. On the other hand, the Fv/Fm indicates that the treatment is not interfering with the physiological state of the algae and they seem to be well adapted for both cases. The results of organic matter at the beginning of each week of experimentation where very similar to those encountered by Lucia Molina in 2000 directly under the aquaculture cages located at Melenara bay, in Gran Canaria. Organic matter presented high variations between weeks and no big differences at the beginning and the end of each week; this could be associated with the error of the method for estimating this parameter, which may vary if the sample was not properly dried to a constant weight due to the humidity in it or if different scales where used to weigh it. For total nitrogen in the sediments there was a marked difference between the beginning and the end of each week, especially at the first week of the experiment the difference between both dates was larger, probably because of an increased feeding activity by H. sanctori due to previous starvation during its time in quarantine. 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A) morphology of the thallus with the very small stipe B) Morphology of thallus in natural conditions, C) Cross section of the blade showing epiphytes (right arrow) and medular filaments (left arrow), D) Cross section of the blade showing early development of the gonimoblast (left arrow), E) cross section showing the ostiole in a well-developed gonimoblast and the expulsion of spores (left arrow), F) detail of the spores (left arrow). A D B E C F 62 Annex 2: Proposed mechanism to increase the surface available for algae growth at the biofilter of the PCT A) initial state, B) condition after a month viewed from the surface and C) condition after a month filled with Colpomenia sinuosa at the continuously submerged side. A C B