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  • Periodic Report Summary 3 - ENVIGUARD (EnviGuard – Development of a biosensor technology for environmental monitoring and disease prevention in aquaculture ensuring food safety)

Periodic Report Summary 3 - ENVIGUARD (EnviGuard – Development of a biosensor technology for environmental monitoring and disease prevention in aquaculture ensuring food safety)

Project Context and Objectives:
EnviGuard is a response to the growing need for accurate real time monitoring of the seas/ocean and the aquaculture industry’s need for a reliable and cost-effective risk management tool.
Aquaculture operations are facing a multitude of challenges: Harmful algae blooms (HAB), which are constituted by microalgae able to synthesis toxins. HABs can have severe effects on fish and humans including mortalities; accumulation of enteric bacteria such as E.coli, which are related to water quality degradation. Food business operators must ensure that live bivalve molluscs placed on the market for human consumption must not exceed certain quantities of marine biotoxins or bacteria like E.coli.
The appearance of diseases of farmed fish in aquaculture is a direct consequence of the intensification of the sector. Every year, the industry faces disease outbreaks caused by long-known and emerging pathogens. Some have the capacity to heavily affect the sustainability of the business, while others can chronically affect the stocks, reducing the efficiency of farming operations.
EnviGuard’s objective is to develop a highly specific, precise, quantitative and qualitative in situ measurement device for currently hard to measure man-made chemical contaminants and biohazards (toxic microalgae, viruses & bacteria, biotoxins & PCBs) that can be used as an early warning system in aquaculture and as an environmental monitor to assess the good environmental status of the sea in compliance with the Marine Strategy Framework Directive (MSFD). It will be more cost-efficient than current monitoring devices leading to a clear marketing advantage for the European analytical and research equipment industry.
The modular system consists of three different sensor units (microalgae/pathogens/ toxins & chemicals) integrated into a single, portable device, which saves, displays and sends the collected data to a server by means of mobile data transmission and internet. EnviGuard will be able to accomplish this in real-time for a period of at least one week without maintenance in an offshore, marine surrounding. User of EnviGuard can access their data online any time they need to. Potential fields of use are marine environment pollution monitoring, marine research and quality control in seawater aquaculture, a sector in Europe highly occupied by SMEs. The biosensors developed in the project go far beyond the current state-of-the art in terms of accuracy, reliability and simplicity in operation by combining innovations in nanotechnology and molecular science leading to the development of cutting edge sensor technology putting European research and highly innovative SMEs in the forefront of quickly developing markets.
EnviGuard started 01/12/2013 and lasts for 60 months. The project S&T objectives are:
• highly specific, precise and reliable in situ measurements of biohazards and chemical contaminants in seawater
• multi-class, multi-analyte method for the simultaneous determination of harmful microalgae species, Betanodavirus, E. coli, Okadaic acid, and Saxitoxin, PCB 126 and PCB 169
• quantitative and qualitative analysis through combination of nanotechnologies with bioreceptors
• automatic sampling for a period of at least one week in the marine environment
• real-time results early warning system for aquaculture industry and beach surveillance & national park services
• easy access to data through internet database allowing environmental status /risk assessment online
• durable design for offshore use under multi-stressor conditions
• modular system (of up to 3 sensors) integrated in a single, portable device
• easily maintainable, user friendly device
• compatible with the FerryBox
• more cost-efficient than current monitoring practices
In order to reach the objectives, the work has been arranged in three types of activities organized in five work packages: RTD (WP1-6), Management (WP8) and Dissemination (WP7).

Project Results:
In WP1 the system’s requirements were specified in detail. Potential test sites were visited, and prevailing outdoor conditions assessed. A market survey on biosensors in finfish operations was performed and published on major aquaculture conferences. A new survey on the opinion of northern aquaculture stakeholders, national/environmental authorities and shellfish farmers was prepared.
In WP2 a nucleic acid biosensor unit and a sensor chip for highly sensitive detection of toxic algae were developed. The application of ultrasound for sample preparation to generate cell extracts ready for analyses was incorporated. Several approaches to increase the sensitivity such as reducing the height of the hybridisation chamber were pursued. A first prototype was evaluated. Three feasibility studies with an automated filtration system for water sample collection (AUTOFIM) on board ships and land-based sites for subsequent molecular genetic analyses were carried out. During a large field campaign samples for testing and evaluating the nucleic acid biosensor were collected.
In WP3 an immobilization method for aptamers on suitable polymeric surfaces and a biological activation method were developed. A subcontractor provided aptamers specific to Betanodavirus. An RNA extraction method for virus particles bound to the surface of sensor chips and a Betanodavirus specific RT-qPCR system were developed. An optical detection system and microfluidic flow cells were designed and optimized. A stand-alone and a version for later integration were build. A full Modbus slave server for intersystem communication was developed and successfully tested. Based on the results an aptamer-based biosensor for Betanodavirus and an antibody-based biosensor for E. coli were developed. A SOP for this pathogen detection unit was developed.
In WP4 antibodies for PCB 128, PCB 169 and Okadaic Acid, a new photo spectrometer as well as fabrication process for resonant nanopillars and their biofunctionalization were successfully developed. A microfluidic system for holding the newly developed BiCells chips with twelve BiCells distributed in two channels was evaluated. A stand-alone prototype and a version for the implementation in the EnviGuard Port were build. An analysis algorithm for the data treatment and SOPs for the sensor were developed.
In WP5 the design philosophy and architecture of platform for the sensors, the EnviGuard Port, was developed and constructed. A first prototype was build and tested including several tests for communication. A remote-control functionality was implemented. A rechargeable battery stack was designed with an interface to renewable energies. The integration of the detection units was accomplished with some delay. The prototype was optimized into a final system designed to be more robust. EnviGuard.NET, an online platform accessible via browser to show the recorded data was developed.
In WP6 the AUTOFIM was tested in a number of field campaigns. For the algae sensor unit trials were carried out under controlled conditions. The device is modular and involves filtration, nucleic acid preparation and detection. The AUTOFIM unit provides the free nucleic acid from water samples. Subsequently, the free nucleic acids can be detected with the semi- automated nucleic acid biosensor using sensor chip technology. The tests revealed the unexpected need for optimization of various components of this module. This optimization was far more time-consuming than expected. The development in the pathogen sensor was delayed and no practical tests have occurred yet. In a series of experiments, ABT was able to detect the minimum dose required to produce a VNN infection in European sea bass juveniles though, a vital information for an early warning system. For the chemical detection unit sensitivity test were carried out.
In WP7, and twitter account (@ENVIGUARD_OCEAN) were launched. Nine project newsletters were released. Project logos, brochure, poster and a training handbook were developed. The planning of the workshops and for the international conference (held in November 2018) started. TTZ has participated in several meetings of Ocean of Tomorrow projects to intensify the cooperation. BAZ has developed three versions of the PUDF and an Exploitation Strategy Seminar took place the 08/11/17.
In WP8, internal communication, troubleshooting with partners and communication with the EC/PO including amendments took place. TTZ was prove reading and submitting all deliverables according to the DoW. The CA as well as three progress reports and three PRs were prepared.
Potential Impact:
The multidisciplinary RTD approach of the EnviGuard project brings together the best scientists in Europe in the fields of nanotechnology, microbiology, molecular genetics, chemistry, marine research, process control, automation engineering and other related sectors. They act together with highly specialized and experienced technological SMEs and endusers from the aquaculture and environmental monitoring sector in order to fulfill the goals set within EnviGuard.
The strong involvement of SMEs on the technology development and the enduser level in EnviGuard ensures the translation and the transfer of cutting-edge research results into market applications. The European society will benefit from the application of new monitoring techniques for the marine environment (quality of life) and aquaculture sites (improved food safety). Early detection of epizootic diseases in marine aquaculture and fisheries as well as manmade pollution of marine environment in general and in coastal zones in particular will lead to prevention of damages caused to the marine environment and will support the production and supply of healthy high quality seafood. Implementation of the EnviGuard sensor technology will significantly contribute to a sustainably thriving maritime economy, one of the EU’s integrated maritime policy’s objectives, and thus, to a maximization of the impact of research and innovation on European society and economy.
EnviGuard aims at the detection of compounds or organisms which are potentially hazardous for the human health such as, Saxitoxin, Okadaic acid, and human pathogens such as E.coli and will aid in the compliance and enforcement of current EU food safety regulations such as Regulation (EC) No 853/2004, which establishes the maximum levels for marine biotoxins in bivalves and Regulation (EC) No 2073/2005, which established the microbiological criteria for foodstuffs.
EnviGuard has the potential to become a standard for toxin and pathogen testing in the future. As EnviGuard works as an on-site diagnostic tool, it lowers current surveillance costs which include manual water sampling, lab analysis and bio-assays using mice and rats.
By the end of the project EnviGuard will be able to detect and quantify in situ
• Relevant toxic algae in European waters (e.g. Alexandrium minutum, Alexandrium tamarense, Alexandrium ostenfeldii and Pseudonitzschia sp.),
• Pathogens relevant to European aquaculture (specifically Betanodavirus and E .coli)
• Emerging pollutants i.e. toxins (specifically Okadaic acid and Saxitoxin) and persistent, manmade pollutants (PCBs) in the marine environment.
Furthermore, the potential for developing EnviGuard into a wider range of environmental sensors could have important implications for the aquaculture sector worldwide:
• Array of sensors could be deployed for every important aquaculture disease. This could bring dramatic benefits for some of the deadliest diseases aquaculture producers are facing worldwide.
• Sensors could help farmers fend-off algal blooms in high risk areas.
• Collections of different sensors could also provide comprehensive and up-to-date information on water quality profiles for a given location.
• The sensors could also be designed to sample tissues instead of water, allowing their use in processing facilities for food safety purposes.
• The combined data sets of EnviGuard and FerryBox allow scientists to get a deeper understanding of the correlation between harmful algae blooms, toxin production and classical abiotic and biotic parameters.

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