the abdus salam international centre for theoretical physics

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1 united nations educational, scientific and cultural organization the abdus salam international centre for theoretical physics international atomic energy agency SMR WORKSHOP ON THE USE OF RECEPTOR BINDING ASSAY (RBA) 1-5 September 23 Co-organized by the International Atomic Energy Agency (I.A.E.A.) MARINE BIOTOXINS - OCCURRENCE IN SCOTTISH WATERS Elizabeth A. SMITH Scottish Executive Environment & Rural Affairs Department Fisheries Research Services Marine Laboratory, Aberdeen, U.K. These are preliminary lecture notes, intended only for distribution to participants. strada costiera, trieste italy - tel fax sci_info@ictp.trieste.it -

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3 Marine Biotoxins - Occurrence in Scottish Waters

4 Shellfish Toxins and Industry In Scotland 29 million Euros of wild shellfish are landed (scallops) 6 million Euros shellfish are farmed (mussels, oysters) 64 million Euros processed market

5 Three Main Shellfish Toxins Pseudo-nitzchia spp. Dinophysis spp. Diarrhetic Shellfish Poisoning Toxins (DSP) Alexandrium spp. Paralytic Shellfish Poisoning Toxins (PSP) Amnesic Shellfish Poisoning Toxins (ASP)

6 Monitoring Since 199 PSP and DSP toxin monitoring extended in 199 Requirements of EC Directive 91/492/EEC ASP toxin monitoring commenced in 1998 EC legislation (Council Directive 97/61/EC) Sampling for potential toxin producing species began in1996

7 Occurrence of shellfish toxins? Summary of available data Qualitative assessment Conclusions for monitoring programmes Requirement for alternative tests

8 Summary of available data

9 Toxins - Numbers of analyses Scottish Shellfish Toxin Database 422 PSP Bioassay DSP Bioassay DSP Chemical ASP Chemical Scallop - Gonad 338 Scallop - Whole Animal 1514 Mussel - Whole Animal 968 Scallop - Gonad 4588 Scallop - Whole Animal 2875 Mussel - Whole Animal 293

10 Phytoplankton - Numbers of analyses Scottish Phytoplankton Database 595 Alexandrium spp. Pseudonitzschia spp

11 Sea Temperatures - Numbers of analyses Scottish Monitoring At Toxin Sites Reynolds Global Data Set Monthly 2 degree resolution Satellite and in-situ

12 PSP Toxin (Mussel) Samples - Geographical distribution

13 Phytoplankton samples- Geographical distribution

14 Orientation Scottish waters NE Atlantic North Sea

15 Spatial Averaging Same zones used for: Shellfish Phytoplankton Temperatures Latitude Longitude

16 Qualitative assessment

17 West coast + no sample x no sample cells l -1 STX eq 1 g Latitude J F2 M3 4A M5 6J J7 A8 S9 1 O N11 12 D Month

18 East coast + no sample x no sample cells l -1 STX eq 1 g Latitude J 2F 3M 4A 5M J6 7J 8A S9 1 O 11 N 12 D Month

19 East coast Latitude Month West coast Latitude Month

20 East coast Longitude (Degrees) J F M A M J J A S O N D 2 Time (Month)

21 Scapa Bay, Orkney Alexandrium spp. cells l -1 PSP (µg STX eq 1g -1 ) in mussels Year 1999 Year Jan 1 Apr 1 Jul 1 Oct Time 1 Jan 1 Apr 1 Jul 1 Oct Time Zero Alexandrium spp. Alexandrium spp. (values range from 2-5,7 cells/l) Zero PSP recorded PSP recorded (values range from µg STX eq/1g)

22 North Sea Primary Production Index Continuos Plankton Recorder Before 1985: Spring Autumn blooms After 1985: Continuos bloom Climate change altering plankton cycles J F M A M J J A S O N D Month Reid et. al., 21

23 Conclusions for monitoring programmes

24 Occurrence of shellfish toxins in Scotland? > 1 years of monitoring data Periodicity of sampling is important Consistency of sampling (toxin-phyto-env) Phytoplankton composition General trends can be observed Little is known about species which are routinely present

25 Requirement for alternative tests

26 Alexandrium spp.. Analyses Alexandrium spp. 16 Analyses Per Mo nth Year > 2 cells l -1-2 Not detected

27 Alexandrium spp. in Scottish Waters Samples are preserved in Lugol s iodine and identified to genus level only 1) Shape 2) Presence/absence of pores

28 Microscopy Labour intensive Traditional method of identifying and counting algal cells Day 1 Day 2 Day 3 Day 4 Day 5 Day...

29 Use of molecular probes No Probe Probe Applied Alexandrium tamarense (NA1) Alexandrium tamarense (NA1) and Pseudo-nitzschia spp.

30 PSP Bioassays 6 PSP Bioassay 5 Analyse sper Month Year > 8 µg STX eq. g -1 shellfish flesh >ND - 8 Not detected (ND)

31 Methods for monitoring PSP Toxins Standard AOAC procedure for PSP toxins analysis is the bioassay Expensive, requires mice, not sensitive (2 % error) Various alternative methods MNB tissue culture assay HPLC ELISA MIST (Jellet Rapid tests for PSP) Receptor binding assays

32 Jellet Rapid Test C T S % Quick and easy Lateral-flow immunogromatography PSP positive PSP negative 25 % 5 % 1 % Decreasing toxin concentration Qualitative (yes/no) indication Result in < 2 min No specialist equipment or training Monitoring and shellfish harvest management

33 Evaluation of MIST Alert TM Kit > 8 shellfish samples tested in 2 / 1 King Scallop, Pecten maximus Queen scallop, Aquipecten opercularis Mussels, Mytilus edulis Pacific Oyster, Crassostrea gigas Northern Oyster, Ostrea edulis Cockles, Cerastoderma edule Razor fish, Ensis ensis All samples tested blind Categories - Not detected, < 4, 4-8, > 8 µg -1 STX equiv. 1 g -1 shellfish flesh

34 Positive samples by MIST Alert TM compared to MBA 12 % Samples positive n=139 1 n= n= n= < 8 < 4 ND STX equiv. µg 1g -1 shellfish flesh by MBA

35 MIST Alert TM Kit Results show good correlation with MBA positive results Journal of AOAC International, Mackintosh et al. 22 Toxicon, Jellet et al. 22 Journal of Shellfish Research, Mackintosh and Smith 23 MIST Alert TM used in monitoring to eliminate negative and low toxicity PSP toxin samples Use of kit could reduce number of MBAs required by 5 % Method for quantitative analysis required

36 Conclusion > 1 years of monitoring data (for food safety) Question remains: What is the link between environment, climate, phytoplankton and the occurrence of shellfish toxins in Scotland? Periodicity and integration of sampling is important Monitoring Programme for food standards and environmental issues (ie, Marine Stewardship, WFD, OSPAR, aquaculture management, etc) Require simple, cheap, quick, reliable and accurate detection methods for phytoplankton and toxins

37 Acknowledgement Godfrey Howard, Eileen Bresnan, Bill Turrell