1. Jo King:
SPIES-DETOX EC Collective Research Project ‘Early warning and detection of algal toxins’
I’d like to kick-off by providing an overview of a project that is funded by the EC – it is a three year Framework Six project
It’s a Collective research project – which means that research providers (like Fisheries Research Services and the Scottish Association of Marine Science (SAMS)), try to provide the research that Industry Associations and SMEs (small to medium enterprises) request.
Overall the project has 13 partners consisting of leading national Universities, Government Agencies, research laboratories, SMEs and Industry Associations from five countries
Guillaume Hermann:

2. Funding… This project is supported and funded by the …
Sixth Framework Programme of the European Community
SPIES-DETOX Collective Research Project
Scottish Government

3. Algal Toxin Groups Diarrhetic Shellfish
Amnesic Shellfish
Poisoning Toxins
(ASP)
Pseudo-nitzchia
spp.
Diarrhetic Shellfish
Poisoning Toxins
(DSP)
Dinophysis spp.
Paralytic Shellfish Poisoning Toxins
(PSP)
Alexandrium
spp.
In the EU, there are four main groups of algal toxins which can cause problems (many of you will be familiar with some of these toxin groups)
In particular Scottish shellfish can be affected by the four main groups of algal toxins.
Amnesic shellfish poisoning produced by diatoms (Pseudo-nitzchia sp.) which has previously been responsible for harvesting bans on king scallop (Pecten maximus) fishing grounds over much (14,400 square miles) of the Scottish west coast
PSP toxins, produced by some species of Alexandrium (the dinoflagellate) have been detected in shellfish at concentrations far exceeding the regulatory limit at some Scottish sites. And concentrations above the regulatory limit are detected in most years (Hot-spots appear to be around Orkney and Shetland)
Periodically, we also detect the DSP toxins mainly OA and DTXs, although other LSTs (AZP, YTX, PTX) have been detected in shellfish from Scottish waters.
The legislation stipulates the levels of shellfish toxins permitted in the shellfish placed on the market.
Periodically shellfish become toxic to humans because they ingest toxins produced by algae or phytoplankton (microscopic cells in the water columns)
The shellfish feed on these phytoplankton and the toxins become accumulated in the shellfish flesh which can present a serious hazard to the shellfish consumer
Lipophilic Shellfish Toxins (LSTs)
(AZP, YTX, PTX)
Protoperidinium spp.
Dinophysis spp.
Protoceratium spp.
Dinophysis spp.

4. MOLLUSCS CONTAMINATION
Transfer of the toxin
HARMFUL ALGAL EVENT
MOLLUSCS CONTAMINATION
So to start with a little bit of background just to set the scene and show some of the problems that the shellfish/aquacultrue industry face in relation to harmul algal events

5. Algal Toxins and Industry
Present a serious hazard to human
EC legislation require member states to:
Monitor shellfish for toxins
Monitor water for toxin producing algae
Closure of Harvesting areas
EC legislation requires shellfish industry to:
Implement HACCP
Undertake ‘End-Product’ testing

6. - SHELLFISH MONITORING
Current Monitoring Methods
- SHELLFISH MONITORING
Sampling of shellfish at low frequency
Sample transport to labs often difficult
Sample turnaround is slow (up to 7 days)
False positive/negative results possible with Mouse Bioassay

7. Phytoplankton identified to genus level only
Current Monitoring Methods
- WATER MONITORING
Phytoplankton identified to genus level only
Speciation not easily possible
Toxic and non-toxic species exist

8. Industry Requirements
Currently...
General trends
Occurrence of toxins in shellfish is UNPREDICTABLE
Industry requires…
PREDICTION of harvesting closure
Methods to REMOVE TOXINS from shellfish
Better scheduling of operational and marketing activities

9. The SPIES-DETOX Project
The PROJECT…
The SPIES-DETOX Project

10. SPIES-DETOX Project Team
Participant Role
Participant Type
Participant organisation name
Participant short name
Country
PROJECT CO-ORDINATOR
IAG
1) Orkney Fisheries Association
OFA UK
PARTNER
2) Association of Scottish Shellfish Growers
ASSG
3) Biomarin Vekst
BMV
NORWAY
4) ANFACO –CECOPESCA
ANFACO
SPAIN
5) Greek Federation of Shellfish Producers
SEMYO
GREECE
6) Clew Bay Marine Forum
CBMF
IRELAND
SME
7) Havbruksompaniet A S HK
8) Hvalerskjell A S HV
9) Killary Fjord Shellfish Ltd
KFS
10) Applied Enzyme Technology Ltd
AET
11) Kvitsoy Edelskjell KE
12) Biosense Laboratories A.S.
BSL
SCIENTIFIC CO-ORDINATOR
RTD
13) Fisheries Research Services
FRS
14) National University of Ireland, Galway
NUIG
15) Aristotle University of Thessalonika
AUT
16) Scottish Association for Marine Science
SAMS
17) University of Santiago de Compostela
USDC
18) Central Science Laboratory
CSL

11. Study Areas
GREECE

12. Objectives of SPIES-DETOX
To work with the aquaculture industry to develop ‘early-warning’ tools for the detection of toxic algal blooms and toxins before shellfish are contaminated
DETOX
To develop procedures, for use by industry, to remove algal toxins from contaminated shellfish
6 research Work-Packages

13. Dinophysistoxins (DTXs)
Work-Package 1: SPATT Bags
Resin
Dinophysistoxins (DTXs)
Okadaic acid (OA)
At FRS we have been investigating the possibility of using passive adsorption of algal toxins onto synthetic resins which was first suggested by researchers from New Zealand.
The idea is that when low levels of toxic algae are in the water column significant amounts of toxins are dissolved in the seawater. Adsorption of the toxins onto the resins and their subsequent extraction and analysis may provide warning of forthcoming shellfish contamination events.
In addition it can also provides information on toxin profiles
Pectenotoxins (PTX)
Yessotoxins (YTXs)
Azaspiracids (AZA)

14. Work-Package 1 Deploy at shellfish farm Elute with solvent Wash
Pectenotoxins
Resin
Yessotoxins
Okadaic Acid
Azaspiracids
Dinophysistoxins
Deploy at shellfish farm
Elute
with
solvent
Wash
Concentrate
Immunoassay
(ELISA)
LC-MS or

15. Work-Package 2: Field Trial
Deployment
Early Warning

16. SPATT Field Trial at Loch Ewe
Loch Ewe, Scotland
Sampling week
D. acuminata 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99
101
103
105
107
109
111
113
113
115
117
119
121
123
125
127
129
131
133
135
137
3000
600
Loch Ewe
Position: 57°50.14'N, 005°36.61'W
Dinophysis spp.
OA (ng g-1 resin)
2500
500
PTX-2 (ng g-1 resin)
2000
400
D. norvegica
Cell count (cells L-1)
[Toxin] (ng g-1 resin)
1500
300
1000
200
D. acuminata
Unidentified Dinophysis
Unidentified
Dinophysis sp.
500
100
D. acuminata
0.0
04/04/2005
21/04/2005
02/05/2005
16/05/2005
30/05/2005
13/06/2005
27/06/2005
11/07/2005
25/07/2005
08/08/2005
22/08/2005
07/09/2005
19/09/2005
03/10/2005
17/10/2005
31/10/2005
14/11/2005
28/11/2005
12/12/2005
26/12/2005
09/01/2006
23/01/2006
06/02/2006
20/02/2006
06/03/2006
22/03/2006
03/04/2006
17/04/2006
01/05/2006
15/05/2006
29/05/2006
12/06/2006
26/06/2006
10/07/2006
24/07/2006
07/08/2006
21/08/2006
04/09/2006
18/09/2006
02/10/2006
16/10/2006
30/10/2006
13/11/2006
29/11/2006
11/12/2006
25/12/2006
08/01/2007
05/02/2007
19/02/2007
05/03/2007
19/03/2007
02/04/2007
16/04/2007
30/04/2007
14/05/2007
30/05/2007
11/06/2007
25/06/2007
09/07/2007
23/07/2007
06/08/2007
20/08/2007
03/09/2007
17/09/2007
01/10/2007
16/10/2007
29/10/2007
12/11/2007
26/11/2007
Apr, May, Jun, Jul, Aug, Sept, Oct, Nov, Dec, Jan, Feb, Mar, Apr, May, Jun, Jul, Aug, Sep, Oct, Nov, Dec, Jan, Feb, Mar, Apr, May, Jun, Jul, Aug, Sep, Oct, Nov, Dec
2005
2006
Date
2007

17. Higher sampling frequency
Work-Package 2
Higher sampling frequency

18. Work-Package 2

19. Work-Package 3: Algal Detection
Difficult to distinguish cells using light microscopy
Need advanced microscopy and molecular techniques
Intensive labour using Microscopy
WP3 - To develop molecular techniques to easily identify phytoplankton and distinguish between toxic and non-toxic species
Using routine light microscopy difficult to distinguish – samples are preserved in Lugols and identified to genus level only.
Here are some Alexandrium cells – some of which produce PSP toxins – Some species can be distinguished based on shape and absence of pores, but still does not tell us if they are toxin producing strains.
Might see high numbers of Alexndrium, but not necessarily a problem as they do not produce toxins… etc.
Need time and work force

20. Work-Package 3 Molecular probes Non-Toxic species Toxic species
DNA sequence specific to a species
Toxic species
Non-Toxic species
Using molecular techniques to assess the abundance of specific toxic algae (e.g., Dinophysis, Alexandrium, Pseudo-nitzschia)
Cells can be seen glowing under fluorescence microscope using molecular probes
Software for the calculation
QPCR offers, high specificity, high sensitivity, higher sample throughput and is easier to automate and requires less specialist skills
Probes for Alexandrium tamrense Nort-Am (toxic) and Alexandrium tamrense West-Eur (non-toxic)
Also for Alexandrium OStenfeldii (Produces SPX dixit JPL) and Alexandrium Minutum (toxic and non-toxic)
High specificity, high sensitivity, easier to automate
Probes for Alexandrium tamarense NA (toxic)
and Alexandrium tamarense WE (non-toxic)

21. Training Course at SAMS
Work-Package 4: Washing
Protocol for removal of ASP toxins from scallops
Extension of the Protocol for removal of PSP toxins
Instructional DVD
Training Course at SAMS
It has previously been demonstrated that the ASP toxin, domoic acid (DA), can be eliminated by washing to such an extent, that even in highly contaminated scallops, all edible parts should have DA toxin concentrations below the regulatory safety limit. These results suggest edible scallop tissues need not fail end-product testing for DA. The current legislation states then when whole scallop DA is greater 20 μg g-1 but less than 250 μg g-1 then scallops must only be landed to an approved processor for shucking. Dive caught and cultivated scallops represent quite a small portion of the scallop landings, but it is these sectors which most benefit from selling ‘live-in-shell’ scallops to the top end of the market. Currently shucked scallops from approved processors do not appear to be failing end product testing for DA. However protocols are required to cover all scales of the commercial sector, and to allow the ‘live-in-shell’ trade to commence.
SPIES-DETOX project participants were recruited for a training course in ‘shucking’ and washing of scallops. Rapid assays were used to analyse scallops before and after the washing process allowing protocols to be developed to permit the resumption of the ‘live-in-shell’ trade. This validation process should result in data of immediate relevance to the regulatory authorities.
Additionally washing protocols developed for the removal of DA were applied to PSP toxin contaminated scallops collected from Scottish coastal waters. The washing procedures were developed in a controlled laboratory based environment and, if successful, will be tested in a commercial setting during the next reporting period. The batch-size of the tissues washed and the scale of the processing activity and whether or not the product is soaked will affect the PSP toxin cleansing routine. Any protocol developed must allow the same rigorous standards to be met whether scallops are processed in small batches or in a mechanised large-scale facility.
Currently…
Artificial Toxification of Scallops with PSP
Evaluation of washing protocol

22. Work-Package 5: Degradation
Bacteria
Currently…
Micro spheres
Feeding has started
Probiotics are dietary supplements containing potentially beneficial bacteria.
A range of bacteria from the digestive system of a number of shellfish types were previously found to degrade marine biotoxins and it was suggested that these bacteria might contribute to toxin profiles observed in shellfish during depuration/purification. Possible practical applications are feasible in terms of manipulating the bacterial flora of shellfish to depurate shellfish of these toxins. Assays to determine bacterial utilisation of marine biotoxins have been developed and research on their potential to aid shellfish depuration will continue throughout the project.
Investigating the use of bacterial diets to remove algal toxins from contaminated shellfish
Bacteria capable of degrading algal toxins are microencapsulated to allow delivery of bacteria to shellfish
Alginate Microspheres containing bacteria

23. Work-Package 6 Training for the detection of algal toxins
Training at SAMS (Oban)
Training at the NAFC (Shetlands)
Training for the detection of algal toxins
A two day training course in the rapid detection and early warning of algal toxins in shellfish held in at SAMS in Oban and at the NAFC in Shetland.

24. Benefits to Scottish Industry
Scottish Industry Associations have the opportunity to meet and colalborate with other Shellfish Associations across Europe and Norway
Research providers can interact to try and provide the research that the industry requests.

26. Thanks for your attention