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Development of sustainable and cost effective water quality management technology for the aquaculture industry

Final Report Summary - RAZONE (Development of sustainable and cost effective water quality management technology for the aquaculture industry)

Executive Summary:
RAZONE is a project within Research for the Benefit of SMEs in which the research work is funded by the European Union’s FP7 administered by the Research Executive Directorate (REA). The project was officially started on December 1 2012 and has a duration of 27 months. The project was initiated by the Norwegian company, Normex. The consortium consists of four SMEs, three RTD performers and three large end users.

The project goal is to develop a technology that enhances cost efficiency and safety of ozone in its application for the management of water quality in the aquaculture sector, especially in RAS. RAZone addresses application of ozone in farms of both fresh and sea water species. To maximise production and cost efficiency in RAS, it is common for farmers to increase stocking densities and levels of water re-use. This can result in rapid accumulation of waste products making control of the environment within the fish production system more complex with sudden peaks of contaminants and potential disease outbreak. While nitrogenous compounds are removed efficiently with biofilters, ozone introduced to RAS via protein skimmers is a major tool for removing fine particles and dissolved organics. In this regard, many farms have significantly improved the quality of water in RAS through application of ozone that requires significant technical development. Thus RAZone is intended primarily for removing of fine particles and degrading dissolved organics.

The RAZone technology is intended to redress the current limitations in application of ozone technology in the aquaculture sector through development of improved ozone delivery and dispersion technique, floc separation, control of the ozonation process for optimum ozone dosage and cost effective application of the technology. To attain these objectives, first a novel ozone delivery system has been developed for maximizing the efficiency ozone dispersion in the effluent. To enhance floc separation results and improve clarity of the water entering the production system, innovative separation system has been designed and constructed. To manage the cleaning process, an intelligent process control system has been developed. The developed components have been integrated into the RAZone prototype and functionality tests have been performed after integrating to a trout RAS. During the trials two identical systems have been used, one ozonated and the other none ozonated. Fish have been sampled from the 2 systems for physiological data, and water quality has been monitored for a number of parameters.

The trial showed that ozone improved water quality by removing particulate and dissolved organics through improved skimming. The removal of heavy metals from RAS is dependent on the binding capacity of the metals to organic molecules which in turn are subsequently skimmed off from the systems.

The fish in both the ozonated and control tanks fed well and showed no abnormal behaviours. There was no reduced feeding response, “gasping” at the surface, spending long periods immobile on the bottom of the tank, or “flashing”. There were no abnormal behaviours observed in the treated fish. Indeed, while no significant differences were measured, the technical staff feeding the fish believed they observed a slightly elevated feeding response.

The external appearance of the fish was observed looking for evidence of skin, eye, gill and fin damage. There were no obvious signs of damage in either the control nor the ozone exposed fish. There were no signs of damage from the observation made at the end of the 30 day test: no erosion, erythema or hyperplasia, which are the most common signs of exposure to irritants. The fish were then dissected by making a midline incision. The gross appearance of the organs was normal without any signs of hyperplasia, inflammation or damage to the tissues. The liver, spleen and guts were removed and weighed and hepato-somatic, spleno-somatic and entero-somatic ratios obtained. These revealed no signs of abnormality between the control and treatment. The histology of all the tissues, both control and ozone-exposed was found to be normal.

Project Context and Objectives:
Land based aquaculture comprises hatchery, nursery and on growing operations. In order to try and maintain good biosecurity, these facilities are largely dependent on best management practice including the ability to provide high quality water conditions. Water to the farm can either be supplied on a flow-through basis, being used just once before being discharged or recycled several times. Flow-through systems causes production problems as the ability to control disease is vastly reduced while any uneaten food and faeces is released directly to the environment. Recycling the water using RAS technology involves processing the effluent water from the fish tanks and returning the purified water back to the production units. In the hatchery and nursery sectors, application of proper water quality control has significant economic benefits to the producer.

RAS technology has gained increasing interest in Europe and internationally due to its recognised advantages. These include flexibility in site selection; reduced water usage; lower effluent volumes and most importantly reduced disease outbreaks and improved environmental control while also optimizing animal welfare. The EU wishes to increase and diversify aquaculture production while substituting seafood imports. RAS technology is considered to be critical in supporting this goal. However, to encourage its uptake, greater efficiencies in terms of the performance of different components of water treatment technology and their associated operational costs are required.

As stocking densities and levels of water re-use increase in RAS, waste products can accumulate rapidly and control of the environment within the production system becomes more complex. Conventional means of solids removal, such as microscreen filters and sedimentation tanks address the removal of coarse solids, but not the removal of fine particulates that can make up 50-70% of suspended solids in the water, whereas membrane filtration such as ultrafiltration and reverse osmosis are too costly to operate. Similarly, bacterial nitrification in biofilters removes dissolved ammonia and nitrite, but not other dissolved organic wastes which can reduce the quality of farmed fish for market while also destroying the stability of water, bacterial populations that are required for optimum growth conditions in the production system. Many operators apply submerged biofilters to mineralise fine particulates but this approach results in the build-up of even finer particles and increases dissolved organics in the water resulting in a characteristic brown "tea staining" effect. If the organic loading in a RAS farm increases, optimum biofilter operation declines, resulting in system instability, increased exposure of stock to elevated bacterial numbers and eventually elevated ammonia and nitrite levels. The result is reduced stock performance and an increasing potential for disease outbreaks.

Today, ozone introduced to RAS via protein skimmers is a key tool in processing the farm water to remove impurities and has been demonstrated to positively impact fish production in RAS. Ozone significantly reduced total suspended solids, colour, and biochemical oxygen demand. It has also resulted in a significant increase in ultraviolet transmittance necessary for controlling bacteria in RAS. Additionally, accumulating dissolved copper, zinc and iron were significantly lower within RAS operated with ozone. Overall, ozone has created an improved water quality environment within near­zero exchange RAS that resulted in enhanced rainbow trout growth rates, survival, feed conversion, and condition factor.

Application of ozone technology has, however, become a costly component of RAS operation due to poorly designed equipment for ozone feeding, inefficient configuration of skimmers and lack of controlled ozone dosage combined with poor understanding of the ozonation process. This may result in either avoidance of ozone technology altogether or limited application to achieve desired aims. Furthermore, the installation of poorly designed ozonation technology can affect the health of farm staff. One of the persistent obstacles to increased uptake of ozone technology usage is the lack of relevant risk assessment in culture conditions and a clear understanding of the basis of ozone toxicity. Inefficient dispersion of ozone in water for optimum contact with contaminants combined with poor understanding of the application of ozone results in ozone overdoses. This can occur as short-term intentional events, to cope with sudden peaks of nitrogenous compounds, or accidentally due to inexperience. The initiators of the RAZone project have discussed application of ozone with experienced users of protein skimmers who emphasise that up to 2/3 of ozone produced may be lost in ill-designed ozone feeding systems resulting in a financial loss of €10-12K per month in some RAS farms producing up to 1000 tonnes per year. Some large-scale commercial fish farms in the US also use ozone but few are ozonating at levels sufficient to achieve desired results. In addition, there have been several high profile commercial failures of RAS both in EU and other parts of the globe due to sub-standard ozone technology. Exposure to high concentration of ozone is harmful to fish and crustacea as it causes histopathological changes in gill tissue resulting in oxidative damage of key biological molecules.

Thus, ozonation or protein skimmer systems are often designed with little attention to the nature or size range of contaminating organic particles that must be removed from the farm water, or the impact of ozonation on the quality of the treated water. This can inadvertently lead to a concentration of fine particles in the system or depletion of useful minerals for the fish as a result of which RAS failures occur. This issue is a major problem within the EU aquaculture industry which slows attempts to improve sustainability and expansion of seafood production.

The RAZone technology will redress the current limitations in application of ozone technology in the aquaculture sector through development of improved ozone delivery and dispersion technique, floc separation, control of the ozonation process for optimum ozone dosage and cost effective application of the technology while also improving our understanding of the impact of ozone on water chemistry and livestock. This will be achieved through development of a novel ozone delivery system that maximizes efficiency of mass gas transfer to water with efficient and even dispersion. The RAZone ozone delivery device will substitute the universally applied Venturi (based on vacuum pressurized sucking) system. The Venturi approach gives inefficient generation of microbubbles and poor mixing of ozone with the water resulting in high concentration of ozone in only part of the water stream while other regions remain untreated. Moreover, RAZone provides an innovatively configured separation unit that significantly enhances floc separation resulting in improved clarity of the water entering the production system, which SOA devices cannot attain; and a process control unit that enables intelligent optimization of the ozonation process to avoid insufficient separation of pollutants or overdosing of ozone that is harmful to the stock. The RAZone project will thus provide a solution to a major bottleneck to the expansion of RAS technology both in Europe and globally.

The problems highlighted here and the existing European and global markets demonstrate that the SMEs in the RAZone project have identified a significant market opportunity with the following commercial objectives. The main initiator of the project, Normex benefit from the developed technology as integrator of the RAZone system. Statiflo, as a global supplier of ststic mixers will be the manufacturer of the the oozone-water mixer, whereas Edur will manufacture and supply the multiphase pump with the developed impeller to be used as component of the ozone feeding system. Asio will have the right to manufacture and supply the reaction and separation units of the RAZone system.

Further to this, two major Europea producers of sea bass and smolt have taken part in the project with the objective improving their practice of water quality management for mitigating disease, improve the quality and size of their production.

Project Results:
Through literature study and database search as well as laboratory investigations the scientific basis for development of the RAZone technology has been established. This includes new knowledge on the kinetics of gas-liquid dispersion and impact of residual ozone in RAS, formation and level of possible intermediate ozonation products including methods of mitigation or removal of these. The investigation included also extent of mineral removal in marine RAS as a result of ozonation, the impact of water quality used for rearing tanks on the performance of ozonation process as well as ozone resistance of materials that can be used in RAZone, especially in sea water media. The literature review has been supplemented with laboratory investigations at the sites of the three RTDS in close cooperation with the industrial partners. The literature study related to kinetics of ozone dispersion in water showed that mass gas transfer and gas solubility are the main processes that determine dispersion of gas into liquid. These processes have a direct impact on the transfer of ozone into water and they depend on generated bubble size, ionic strength, temperature, pressure, turbulence and viscosity. The most important parameter is the size of the generated microbubbles. The study also made a mapping of methods for generation of micro- bubbles and dispersion of ozone in seawater on the basis of which devices for application in RAZone have been identified. The Laboratory trials at TI have established the optimum microbubble size to be generated for efficient dispersion of ozone in fresh and seawater with safe level of residual ozone.
The literature survey and laboratory experiments relating to the ozonation of freshwater and seawater undertaken by Liverpool University revealed that the half-life of O3 in water containing oxidisable substances is short but it is relatively long in pure water containing carbonate. Bromine and chlorine together are the main constituents of total residual oxidants that are formed by ozonation and often determined using standardized techniques. Regarding loss of minerals under ozonation, the literature and laboratory studies revealed the extent of mineral removal as a result of oxidation that in some cases the oxidation reactions as a result of ozonation can lead to losses of minerals. Other losses occur if specific organic molecules are broken up, leading to losses of vitamins and metal complexing agents . Specifically, the review showed that iodide has the fastest reduction kinetics, and this occurs at low levels in seawater. Investigation related to impact of fresh and seawater used for rearing tanks on performance of ozonation revealed that ozonation of the seawater in the presence of organic matter and nitrite significantly suppressed the formation of TROs indicating higher ozone-nitrite/organics reaction rate than that for ozone-Br ions. In freshwater, the presence of Cl/Br ions enhanced redox potential profile while bicarbonate ions suppressed the redox potential profile. Ozonation of freshwater in the presents of bromide ions at levels reported in seawater significantly enhanced the formation of TROs when compared to Cl ions (Cl ions are significantly high in seawater). Bicarbonate ions suppressed the formation of TROs.

The literature review and own experiments showed that measuring particle concentrations and particle size distributions is a difficult task in aquaculture waters. It turned out that the foreseen measuring principle of static light scattering is not reliably working on samples that have a particle concentration of only several milligrams per litre. Experiments were done to concentrate samples with different methods (centrifugation, cross-flow membrane filtration), however these methods were discarded as it was suspected that they alter the original sample too much, e.g. irreversible aggregation, loss of particles on membrane surfaces and equipment etc.
One trial was done with a scanning electron microscop (SEM), where a defined volume of a sample was first filtered through a nucleopore filter, the filter was then dryed and platinated and then analysed under the SEM. Particles could be seen very well, but particle counting turned out to be too labour-intensive.
Further research led to a measuring device that employs the so called time of transition laser technique that has successfully been applied for many years on aquaculture water by the research institute Fischereiforschungsstelle Langenargen at Lake Constance, Germany. This expensive device is going to be lend out to Fraunhofer by Fisc hereiforschungsstelle to perform the experiments with the lab scale unit.

In order to be able to measure TOC in a seawater matrix, a special high salt kit and a halogen trap was bought and installed for the existing TOC analyser. Afterwards a complete calibration of the TOC analyser was required.
It was tried to get an idea of the status quo water quality at the end-user's fish farms. Therefore, water samples from Follafos and from Anglesey were sent by express sh ipment to Fraunhofer for chemical analyses and for preliminary assessment of the required ozone dosage. The TOC in Follafos was approximately 5 mg/l and at least 12 mg/l in Anglesey. The TOC in Anglesey might be much higher, because samples from Anglesey arrived unchilled. Therefore it is unknown how much of the original T OC was biologically degraded.
An preliminary jar test with ozonation (doasage: 0.4to 0.6 mg Ozon/mg TOC) of the water from Follafos was performed and a particle size distribution was done. Resul ts indicate a slight shift towards larger particles and would therefore be consistent with results published by other researchers.

In the development of the ozone delivery unit, different configurations of mixing elements and multiphase pump impellers have been tested both separately and in combination to evaluate the best configuration for generation of microbubblease and efficient dispersion of the gas in the effluent.
In addition, Computational fluid dynamic analysis was undertaken based on the design criteria of both the ozone delivery system and the flotation unit. The results obtained were examined with respect to 3 main distributions:
1. Streamlines structure
2. Volume fraction of air/ozone mixture
3. Ozone mass fraction in water/ozone mixture

Streamlines structure shows the flotation effectiveness based on absolute velocity of water/ozone mixture.
Air/ozone volume fraction shows how well the inlet jet is dispersed inside the flotation chamber
Ozone mass fraction shows the level of uniformity of ozone mass fraction in water (“quality” of ozone solution).

Conclusions drawn from the analysis include:
• Ozone mass fraction changes in relation to inlet ozone mass fraction.
• Portion of ozone dissolved in the water varies non-monotonically with mass fraction of ozone in supplied air – the values, however suggest that Henry’s constant taken into consideration was still too conservative as it is not likely that only 15-20% of ozone will be solved in the water.
• Based on the 2D analysis it is favourable to design a cylinder that has rather high than low H/D ratios (height to diameter) by means of the dimensions proposed in the study – the reason for that is the same as the reason for the choice of cases 1 and 8 – uniformity of ozone distribution in the upper cylinder + proper pattern of flotation
• Based on the streamlines distribution it is recommended that more than 2 outflows should be tried to assure better axis symmetry of the flow
• An approach to reduce the vertical distance between the outlets and the bottom is worth examining as the part of the cylinder between them does not take any active part in the flotation process itself – special care should be taken before such a step is taken on real design.

Based on the laboratory investigations and the CFD analysis, the mixing element and pump impeller have been selected for manufacturing at Statiflo and Edur respectively.
Further to this, it has been the objective of the project to select appropriate materials with resistance to corrosion under ozonation. As a strong oxidant ozone advances corrosion of metals and fatigue in plastic materials, especially in seawater environment. Whereas there is sufficient documentation about corrosion in ozonated fresh water, there is very limited knowledge on the effect of dissolved ozone in seawater on the corrosion behaviour of materials, although ozone based oxidation has been used for a considerable length of time. The material selection to most ozonization processes in seawater are done without major detailed corrosion investigations. The decomposition mechanism of ozone in aqueous solutions is also a matter of uncertainty as views on the mechanism differ widely because of the variety of ways in which decomposition can take place under different conditions.
Depending on the pH of the solution in which it is dissolved, molecular ozone will either react directly with components in solutions or decompose into hydroxyl radical, oxygen and hydroxide. At pH values above 7.5 the decomposition of ozone to hydroxyl radicals increases. Below this pH value molecular ozone is stabilized and only a small fraction converts to radicals.
As a basis for selection of materials results of tests carried out at TI’s Laboratory of Material Technology have been used to verify that the materials identified from the literature study are promising for the ozone delivery unit, piping, gaskets and joints of the RAZone system; and if they indeed have the required resistance when exposed to the ozone concentration that will be used in the system and at the anticipated water temperatures. As the literature review revealed no corrosion risk in fresh water of the materials subjected to laboratory trials, the investigation are undertaken only in seawater.
The materials subjected to ozonation for a period of 11 weeks are:
Polymers: Teflon , EPDM Rubber, Neoprene Rubber and PVC
Metals: SS 316, Copper Nickel and Duplex. Titanium has been excluded due to its high cost.
All materials have been subjected to the same ozone concentration in artificial seawater. The ozone concentration is slightly higher than what is considered to be the upper limit in seawater based RAS
The condition of the metallic materials in relation to corrosion has been monitored with electron microscope during the test period, whereas the polymers were subjected to tensile tests and analysed in relation to Tensile modulus (measure of sifness), % at break point and Peak value (maximum force applied at breakage) after the end of the test period.

Main findings of the laboratory study:
• Duplex is found to be not affected by the ozonation and can be recommended for seawater application.
• CuNi has suffered from general corrosion. Below or along the gasket, green verdigris were formed. On the surface in general, also under the gasket, some oxide is visible. However, alloys with higher Ni content than the sample tested in the project (> 20% Ni) should have higher resistance to corrosion in ozonated seawater
• In general, the changes seen for the four polymers are small. The changes are smaller than the standard deviation for two of the materials: Neoprene rubber and Teflon.
• EPDM rubber becomes however stiffer and more brittle.

In the development of the separation system, the data from the particle size analysis have been correlated with crucial parameters that impact both on the ozonation, flocculation and flotation processes. These include particle size, particle-bubble attachment efficiency, residence time, bubble concentration, and bubble size. Particle size and efficiency of particle attachment to bubbles influences the ozonation and flocculation processes, whereas the other three parameters impact on the flotation process.
To enhance the ozonation process and subsequently the particle separation, a flocculation unit has been designed using the principle of Around-the-end-flocculation. The flocculator is designed to be adaptable with the possibility of water depth and inlet position.
Based on the results of the particle size analysis that revealed dominance of micro and nano particles in effluent, the flotation process should be enhanced by increasing the concentration of microbubbles in the flotation unit. For this, a saturation unit was included controlled by a pressure sensor. High saturation of microbubbles is attained by dissolving air in fresh water under high pressure, and releasing in the flotation chamber under atmospheric pressure

The separation unit and flocculator were manufactured according to the design specification for a capacity of 1000L/hr. The flocculation unit and the flotation chamber were manufactured by Asio in the Czech Republic, whereas the saturator was built in Germany. The manufactured units were first tested separately and then subjected to hydraulics test after integration to the RAZone system at Fraunhofer in Stuttgart. The integration of the RAZone was carried out from mid May until mid July 2014 after some delay due to delayed delivery of some components including sensors and special valves specially ordered for manufacturing for the RAZone prototype. To undertake functionality tests close to a real situation, it was decided to undertake the ozone flotation trials after the RAZone system was integrated with the experimental RAS in Liverpool. Pictures and 3D-drawings are attached in PDF format.

At the Mid Term Management Meeting, the consortium decided that the RAZone prototype be integrated to the RAS at LU after necessary modifications rather than building a test setup at the premises of the end users in the project. After preliminary optimization of parameters at LU, the RAZone system has been tested in relation to separation of suspended particles, dissolved organics. The separation system has performed efficiently along with the other components of the RAZone prototype.

Development of the process control system included first the design of flow chart and process instrumentation diagram (P&ID) were developed. These were used as the basis for the development of the control cabinet and the software. Layout of the control cabinet has been continuously updated to accommodate changes in the RAZone process and associated sensors. As the main control unit for the prototype control system, the Saia PCD and PLC system was chosen. This is a standard, mainstream PLC with a wide range of standard and specified input and output modules which can be used with the PLC unit. The prototype control system consists of integrated inputs and outputs with a possibility for adding further extensions. The PLC gathers signals necessary for system loop control. It controls main areas and communication channels. It also has a Human Machine Interface for process supervising and control.
Further, a summary list for the PLC input and output (IO) signals was prepared. The IO list shows relationships or connecting methods between equipment and PLC IO modules. Together with P&I diagrams, it serves as the basis for wiring diagrams. In addition, instrument and aggregate (I&A) lists were prepared. The instruments include main line flowmeters for the main line and recirculation, alarm for empty pipe in the main and recirulation lines and level sensor in the saturator. The aggregate list includes the multiphase pump, recycling pump, on/of valves for recirculation, main line, air supply, ozone supply and foam spray.
Based on the IO list and equipment specifications, the PLC TAG list was created as a basis for PLC control software development. All physical and intern PLC signals are stated in the TAG list. PLC tag is automatically made from area name, object name and signal name. Object names correspond to the objects defined in the P&ID Instrument list. Main signal names correspond to the signals defined in the P&ID Instrument list. PLC TAG list was continuously updated with new tags during PLC control loops development.
The list includes several types of tags, which were used for the PLC software development. The tags are based on signal types which they are representing. Digital signal is named flag (F). Analogue signal can be represented as integer (R) or floating point (R Float) register. Alphanumerical based tags are named TEXT tag. Each tag has a unique PLC address which can be chosen manually in the TAG list or automatically in the PLC.
Physical signals connected to the IO modules are included as Input or Output with their specified IO module address. IO module addresses can be found in the IO List. TAG list is imported to the PLC symbol editor, which is a part of PLC PG5 programming tools.
In the selected Saia syste, PG5 v2.1.100 programming suite that is the latest version of the SAIA PLC programming interface has been used. PG5 has possibilities for both object-oriented, code programming, and has a built-in HMI and energy meter-programming interface. PG5 Program manager is a programming interface with all relevant information concerning PLC hardware and software. Bsed on this, the PG5 project structure description, device configuration and PLC communication settings were established.
In the developed control loops, all controlled system components are logically connected together since they influence each other in many different ways. PLC with operational control software bonds all hardware parts and software areas together into the one fully functional production system. PLC processes in real time gives all necessary information from the operator and signals from equipment. All important signals and information are illustrated on the touch screen or WEB interface.
Main control areas include:
• Water circulation cycle for Saturator
• Flow control for P101
• Gas production control
• ORP control
• HMI (WEB interface)
• Further, a touch screen panel that represents visual HMI has been developed. This is the supervising and controlling interface between the system operator and the PLC control unit. With LAN switch integrated, the touch screen panel has additional possibilities for supervision and controlling throughout LAN. Each PC or other device with WEB browsing capabilities can be connected to the PLC control unit WEB server through LAN or remotely via WWW.

WEB user pages are divided into several areas for easy and convenient navigation. Top field shows the Title of the project. Auto/Manual button always leads back to the start page. The main part of the screen is a system layout with interactive objects. All major system equipment presented on the page controlled directly by finger touch (touch panel) or mouse pointer (PC based internet explorer). The lower part of the screen functions as page navigation menu. Again, all major equipment information can be found on these pages, the alarm limits (default values), system status and user. Because of safety measures, all start functions can be protected with password. Without the correct password system, the operator could not be able at the process access. There are four password levels availe on the system, one is the lowest and four is the highest level. Password levels for all equipment / functions can be easily added through the PG5 Web Editor Interface as well as additional new pages.

During the building phase of the process control unit the wiring diagram and installation of the cabling in the control cabinet, configuration of the PLC, integration of the software and the control cabinet, and connection of the control cabinet with the aggregates and instruments has been undertaken.

The control cabinet was configured based on the control cabinet layout (part of P&I diagrams) and wiring diagrams. The control cabinet and all electrical equipment were hooked-up with appropriate wires after being assigned to their various places. Selection of sensors were based on the instrumentation list generated from the process and instrumentation diagram (P&ID). Selections of other electrical equipment are based on the control cabinet layout and the standard PLC control system setup. The control system functions for the system unit completely covers all functions of the system. New functions can be implemented in order to satisfy the needs of the specific sensors and equipment.
After the RAZone system has been commissioned, ORP guard has been installed to monitor and safeguard the stock from exposition to high ozone concentration. The probe consists of a sensor, transmission cable, a recorder and monitor with operation for manual or automatic temperature compensation and calibration of the measuring system. The measurement unit is integrated with the process control system, and ozone production will automatically stop if the ORP level exceeds the set value. In the functionality tests the set level of Redox used is to be 300 - 350mV.
After the control cabinet has been built, the software developed and controlled on the computerthe system was subjected to first round of validation at Fraunhofer in Stuttgart where the RAZone prototype was integrated. The validation involved testing of the control cabinet while the RAZone prototype was running using freshwater. During the tests functionality of the algorithms was checked in relation to the function of the different instruments and aggregates, both under automatic and manual modules. Based on the outcome of the tests, modifications were made in relation to the range of flow and pressure values in the system.
Further to this, final validation was undertaken after RAZone was integrated to the experimental RAS in Liverpool. This was done during the commissioning of the RAZone prototype in WP5, as a result of which the delay time for production of ozone after the oxygen generator is started has been changed in the software.

Integration of RAZone and Functionality Tests:
For efficiency of the integration process and optimum use of resources, the consortium did decide integration of the RAZone system to be carried out at Fraunhofer rather than at the premises of the end users. Further to this, there was a consensus that rather than establishing a new experimental RAS at Anglesey or Salmar due to logistical problems, it would be more beneficial to integrate the RAZone prototype with the existing RAS at Liverpool University with its fully equipped system including pretreatment, biofilter and rearing tanks. In this regard, the experimental RAS should include two systems, for ozonated and non ozonated (control) units.
The integration started in May 2014 and the system was to be placed in two separate racks. Rack no.1 accommodated the ozonation/flocculation system; whereas the saturator, flotation chamber and the process control unit were to be placed in rack no. 2. Installation of the components including the flocculator, the O3 & O2 generators and separation system as well as the piping has been undertaken by Fraunhofer, whereas TI has carried out installation of the control system and sensors. After the integration was completed, the system was subjected to hydraulic and leakage tests before shipping to Liverpool and integration with the experimental RAS. The installation and integration with the RAS was carried out in July and beginning of August 2014, and the system was commissioned during the Month 21 Consortium Meeting at LU on August 6 2014.

Functionality test of the developed RAZone system:
The activities in this reporting period commenced by installation of two identical recirculating aquaculture systems (RAS) which were stocked with sea bass at roughly the same stocking density of 6kg/m3. The first activity was to complete task 1.3 which had been postponed in the first reporting period. This involved running the two stocked systems for a sustained period (19 days) during which investigations on the effect of ozone on water quality were undertaken. It has to be noted that one of the system acted as a control (without ozone) while ozone was administered into the other system at a redox potential of 350mV.

The trial showed that ozone improved water quality by removing dissolved organics through improved skimming. There was no indication of the effect of ozone on ammonia but it reacted quickly with nitrite which is more toxic to fish than nitrate. The removal of heavy metals from RAS is dependent on the binding capacity of the metals to organic molecules which in turn are subsequently skimmed off from the systems. Ozone played a significant role in this process. It was clear that ozone rapidly oxidises iodide to iodate and when the conditions are right, iodate can be reduced to iodide (when RAS is not ozonated) through bacterial/phytoplankton activity.

While ozone plays a significant role in improving water quality in RAS, it has also the potential to strip off trace ‘elements’ such as iodide and trace metal ions that are essential for normal growth of fish. It was noted that ozonation will not remove all the heavy metals from the water matrix because some metals have a low binding capacity to organics and therefore are not removed through skimming. As such, a build-up of potentially toxic metals can accumulate in RAS.

The second set of activities involved testing of the RAZone system in which the effect of ozone on fish behaviour and physiological responses were investigated over a 30 day period using 2 RAS stocked with trout fish. In addition, water quality was closely monitored during the trial. The RAZone system was integrated into one of the RAS systems while the other system acted as control and as such the second system was non-ozonated. In relation to water quality, it was noted that the RAZone system removed about 50% of copper over 48hr period and there was no indication of the removal of iodide, nitrite and ammonia. However, the RAZone system showed enhanced TOC removal.

Throughout the experiment the fish fed well and showed no abnormal behaviours. Had they been stressed by the presence of ozone we would have expected them to show one or more of the following behaviours: reduced feeding response, “gasping” at the surface, spending long periods immobile on the bottom of the tank, “flashing” - i.e. making rapid turning movements as though to dislodge a parasite or other irritant. There were no abnormal behaviours observed in the treated fish. Indeed, while no significant differences were measured, the technical staff feeding the fish believed they observed a slightly elevated feeding response.

At the end of the trials the fish were killed using the approved schedule one (Home Office approved) technique of stunning with a blow to the head then destruction of the brain and spinal cord. The external appearance of the fish was observed looking for evidence of skin, eye, gill and fin damage. There were no obvious signs of damage in either the control not the treatment (ozone exposed) fish. Photographs were taken for reference for the external signs and at all stages of the following comprehensive postmortem. The operculum cover was then reflected to expose the gills, there were no signs of damage: erosion, erythema (redness) or hyperplasia, which are the most common signs of exposure to irritants. The fish were then dissected by making a midline incision. The gross appearance of the organs was normal without any signs of hyperplasia, inflammation or damage to the liver, spleen, gut and other tissues. The liver, spleen and guts were removed and weighed and hepato-somatic (liver mass: body mass ratio), spleno-somatic and entero-somatic ratios obtained. These revealed no signs of abnormality between the control and treatment. The histology of all the tissues, both control and ozone-exposed was also found to be normal. The Experiments were subject to review by the UK Home Office at the start of the Project period. However, the inspector concluded that the experiments did not require regulation as the Work was deemed to be an agricultural practices and practices normally undertaken for the purpose of recognised animal husbandry and not reasonably expected to cause pain, suffering, distress or lasting harm. Moreover, the work was conducted under strict compliance with local rules of fish welfare and the fish were observed four times a day for any abnormal behaviour such as reduced feeding response, “gasping” at the surface, spending long periods immobile on the bottom of the tank, “flashing” - i.e. making rapid turning movements as though to dislodge a parasite or other irritant. The external appearance of the fish was also observed throughout the experiment to check for abnormal growth, evidence of sores or damage.

Potential Impact:
The innovations of RAZone will contribute to reduced foot print of devices currently used in RAS system as well as energy efficiency due to efficient dispersion of ozone in water, and cost associated for elevation of water to skimmer towers. The intelligently controlled ozonation process in RAZone will contribute to significantly improved water quality management , reduced disease outbreak and increased production in aquaculture, particularly RAS. This will contribute to improved production of protein rich fish for health conscious European consumers as well as globally.
Ozonation has proven to be useful in recirculating aquaculture systems (RAS) promoting the stabilization of water quality and disease control. However, there are persistent obstacles to increased uptake of ozone technology usage. One of the main obstacles is the lack of relevant risk assessment in culture conditions and a clear understanding of the basis of ozone toxicity. Poor understanding of the application of ozonation technology leads to overdose of ozone in fish farms which can occur as short-term events either intentionally to cope with sudden peaks of nitrogenous compounds or accidentally due to inexperience or technical problems related to the technology. Furthermore, ozonation or protein skimmer systems are often designed with little attention to the nature, size range of contaminating organic particles that must be removed from the farm water. This can
inadvertently lead to a concentration of fine particles in the system, reduced production and RAS failure. Another important obstacle is related to inefficient equipment for feeding and evenly dispersion of ozone in the water, and poorly designed floc separation chamber which cause insufficient control of the water quality. These issues are major problems within the EU aquaculture industry which slows attempts to improve sustainability and expansion of seafood production.
RAZone will contribute to development of improved industrial scale marine and freshwater RAS for on-growing market size fish that is required to meet an ever increasing demand for seafood. Significant opportunities for new job creation can also be realized through RAS development in rural areas, where coastal fisheries offer decreasing employment opportunities. Improper management of water quality will however result in losses due to diseases in hatcheries, nurseries and ponds estimated in billions of Euros per year. This plays a significant role if one takes into account additional costs including unemployment and social upheaval, reduced requirements for feed, chemicals and other supplies, reduced requirements for packing, processing, export and shipment of final products and reduced investor confidence. The results of RAZone are expected to contribute in redressing this problem and impact on improving quality of product, reduced farm failures and increased employment in the aquaculture sector.

Today disposal of fish waste including dead fish is a significant cost for the aquaculture industry. This can be redressed with improved water quality management and reduced cases of disease. RAZone is expected to contribute to this benefit. Further to this, the best practice of ozonation can contribute to standards of ozonation that would enhance water quality management.

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website address:

Contact details: Mr. Stig Johansen, Normex AS, Phone: +47 91708023,