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  • Periodic Report Summary 2 - ENVIGUARD (EnviGuard – Development of a biosensor technology for environmental monitoring and disease prevention in aquaculture ensuring food safety)

ENVIGUARD Report Summary

Project ID: 614057
Funded under: FP7-KBBE
Country: Germany

Periodic Report Summary 2 - ENVIGUARD (EnviGuard – Development of a biosensor technology for environmental monitoring and disease prevention in aquaculture ensuring food safety)

Project Context and Objectives:
The objective of the EnviGuard project is to develop a highly specific and precise in-situ measurement device for currently hard-to-measure chemical contaminants and biohazards in sea water that can be used as an early warning system in aquaculture and as an environmental monitor. It is a direct response to the growing need for accurate real time monitoring to assess the environmental status of the sea and better risk management in aquaculture industry.
EnviGuard users will be able to detect
1) toxic microalgae – a common threat to shellfish and fish farmers as they frequently cause the closure of shellfish sites and fish loss,
2) enteric bacteria such as E.coli, which are related to water quality degradation and can equally lead to closures of shellfish sites,
3) Betanodavirus, a pathogen causing disease outbreaks in fish farms the Mediterranean that heavily affect the sustainability of the business,
4) Okadaic acid and Saxitoxin, two toxins synthesized by microalgae that have severe effects on fish and humans including mortalities,
5) Polychlorinated biphenyls (PCB) that can cause cancer in animals and are suspected to be carcinogenic to humans.
Therefore a consortium of 18 partners is going to develop the modular EnviGuard system that will consist of three different sensor modules (microalgae/pathogens/ toxins & chemicals) integrated into a single device that can be deployed in an offshore, marine surrounding and work autonomously for a period of at least one week before maintenance will be required. The device will be able to collect quantitative and qualitative data on the above mentioned targets Users can access their data any time online which will be more cost-efficient and faster than current analytical techniques. As EnviGuard will offer real-time-results it can be used as an early-warning system for the aquaculture industry, beach surveillance & national park services. EnviGuard will also be compatible with current offshore sampling platforms such as the FerryBox system to broaden its field of use. The sensors 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 cutting edge sensor technology putting European research and highly innovative SMEs in the forefront of quickly developing markets
The EnviGuard project started the 01/12/2013 and will last for 60 months. The project S&T objectives are:
• highly specific, precise and reliable in situ measurements of biohazards and chemical contaminants in seawater (main objective)
• 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
• their 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 from everywhere through internet database allowing environmental status /risk assessment online
• durable design for offshore use under multi-stressor conditions
• a modular system (of up to three 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 foreseen 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:
Within WP1 the system’s requirements were specified in detail. Potential future application areas were visited and the prevailing outdoor conditions assessed. The results of the detailed market survey performed in the 1st RP was summarised and published on major aquaculture conferences. The survey’s results were reviewed and a new survey to incorporate the opinion of northern aquaculture stakeholders and national/environmental authorities was prepared. The task continue until the end of the project.
In WP2 a nucleic acid biosensor unit and a reusable 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 assessed and promising results were achieved. For maximising the sensor sensitivity, several approaches were pursued. The concept of a reusable sensor chip was abandoned as trials showed that a concept for a mechanical exchange of sensor chips is way more cost efficient in practise. A mechanical exchange unit was build and a first prototype of the sample preparation module is currently being evaluated. Furthermore, a set of three different feasibility studies for the automated filtration device AUTOFIM for collection of particulate organic matter for molecular genetic analyses from water samples on board ships or land based observation sites have been carried out. In parallel, the impact of reducing the height of the hybridisation chamber on the signal intensity was assessed, as well as the calibration of molecular probe sets for detection of toxic algae. In addition, a large sampling campaign to collect field samples for testing and evaluating the nucleic acid biosensor has been carried out.
WP3 dealt with the development of an aptamer based biosensor. Aptamers are essentially a chemical equivalent of antibodies and can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets. As a first step an immobilization method for aptamers on polymeric surfaces was developed. Different surface materials were tested for optical properties and biological activation properties. Sensitivity tests within a first microfluidic chamber were done with E. coli in saltwater. Secondly, work on the development of the optical detection unit and the casing was carried out. Further work has been focused on the adaptation and optimization of the biological activation method to use it on PMMA. Alternative surface coating methods and three different aptamer sequences for E. coli taken from scientific literature were tested. Betanodavirus binding aptamers are available and will be tested soon. Furthermore, optimisations of the optical detection system were carried out. A new custom housing for the Pathogen sensor was developed containing all relevant fluid, power and data transfer connections at back capable to operate in standard 19” rack. A full Modbus slave server for intersystem communication was developed and successfully tested.
WP4 is dedicated to the production of antibodies for the detection of PCBs and toxins. In parallel, a fabrication process of resonant nanopillars has been developed and several chips have been fabricated to test their optical performance. Different activities for the biofunctionalization of these nanopillars have taken place and the development of protocols for this and for the detection of targets were carried out. A proposal of a preliminary microfluidic system for holding BiCells chips with eight BiCells distributed in two channels was evaluated and two chips and chipholders were fabricated. Two CDU prototypes have been successfully integrated. For these integrations, the development of both the fluidic systems and optical interrogation units has been completed and optimized within this reporting period. The first CDU prototype has been used for the adaptation of the PCBs and OA measurement protocols for the use of the final detection system using real oceanic water samples. These protocols had been previously developed using a preliminary set-up. In addition, the performance of resonant nanopillars is being optimized to redesigning their multilayer configurations and trying with new materials.
Within WP5 the design philosophy and architecture of the EnviGuard port was developed and the main elements, i.e. the control & storage unit, the sensor interfaces, the communication modules and the power supply unit were constructed. The initial tests of the full EnviGuard Prototype were accomplished. The web tool has been designed and developed as an online platform accessible via browser to show the data record by the EnviGuard Port.
WP6 work has been focused on the design of the procedures for the testing and repeated optimisation of the different EnviGuard modules in closed containment systems.
In order to showcase the project’s findings and bring attention to the new technology an official project website ( was launched. Seven project newsletters were released as well as the project brochure and poster. Both were showcased at different events by different partners. Several logos, for the project and for the EnviGuard port and the units were developed. The coordinator of the project has participated in several meetings of similar projects with the intention to intensify the cooperation and avoid double efforts. With regards to IPR issues, BAZ has developed the 1st and the 2nd version of Plan for Using and Disseminating the Foreground and an Exploitation Strategy Seminar took place the 8th of November 2016 alongside the General Meeting, given by an expert assigned by the EC.

Fig 3 - EnviGuard Logos
In WP8, the main objectives are internal communication, troubleshooting with partners, communication with the EC/PO as well as prove reading and submitting all deliverables according to the DoW. The CA as well as the 1st and 2nd progress reports and the 1st and 2nd PRs were prepared. In addition, the process of the internal working groups WG1 (positioning of the biosensors) and WG2 (threshold levels) was monitored.

Potential Impact:
The multidisciplinary research and development 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 end users from the aquaculture and environmental monitoring sector in order to fulfill the goals set within EnviGuard.
The 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.
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.
In general 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. 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 most deadly 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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