united nations educational, scientific and cultural organization the abdus salam international centre for theoretical physics international atomic energy agency SMR 155-16 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, 11-3414 trieste italy - tel. +39 4 224111 fax +39 4 224163 - sci_info@ictp.trieste.it - www.ictp.trieste.it
Marine Biotoxins - Occurrence in Scottish Waters
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
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)
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
Occurrence of shellfish toxins? Summary of available data Qualitative assessment Conclusions for monitoring programmes Requirement for alternative tests
Summary of available data
Toxins - Numbers of analyses Scottish Shellfish Toxin Database 422 PSP Bioassay DSP Bioassay DSP Chemical ASP Chemical 19434 6364 1513 1289 Scallop - Gonad 338 Scallop - Whole Animal 1514 Mussel - Whole Animal 968 Scallop - Gonad 4588 Scallop - Whole Animal 2875 Mussel - Whole Animal 293
Phytoplankton - Numbers of analyses Scottish Phytoplankton Database 595 Alexandrium spp. Pseudonitzschia spp. 162 329
Sea Temperatures - Numbers of analyses Scottish Monitoring At Toxin Sites Reynolds Global Data Set Monthly 2 degree resolution Satellite and in-situ
PSP Toxin (Mussel) Samples - Geographical distribution
Phytoplankton samples- Geographical distribution
Orientation Scottish waters NE Atlantic North Sea
Spatial Averaging Same zones used for: Shellfish Phytoplankton Temperatures Latitude 61. 6.5 6. 59.5 59. 58.5 58. 57.5 57. 56.5 56. 55.5 55. 54.5 54. -8. -7. -6. -5. -4. -3. -2. -1.. Longitude
Qualitative assessment
West coast + no sample 5 1 2 x no sample 1 2 3 cells l -1 STX eq 1 g -1 6 59 Latitude 58 57 56 55 1J F2 M3 4A M5 6J J7 A8 S9 1 O N11 12 D Month
East coast + no sample 3 5 1 x no sample 3 5 1 cells l -1 STX eq 1 g -1 6 59 Latitude 58 57 56 55 1J 2F 3M 4A 5M J6 7J 8A S9 1 O 11 N 12 D Month
3 5 1 3 5 1 6 59 East coast Latitude 58 57 56 55 1 2 3 4 5 6 7 8 9 1 11 12 Month 5 1 2 1 2 3 6 59 West coast Latitude 58 57 56 55 1 2 3 4 5 6 7 8 9 1 11 12 Month
East coast 32 13 27 146 82 24 27 211 181 118 758 44 22 27 381 79 156 29 27 2 27 29 787 594 253 115 38 18 54 34 1851 435 112 93 16 14 62 276 167 56 27 12 27 61 56 27 27 1 5 27 146 192 624 143 828 136 316 97 27 11 27 27 27 27 27 27 27 8 6 4-4 -2 2 Longitude (Degrees) J F M A M J J A S O N D 2 Time (Month)
Scapa Bay, Orkney Alexandrium spp. cells l -1 PSP (µg STX eq 1g -1 ) in mussels 21 21 2 2 Year 1999 Year 1999 1998 1998 1997 1997 1 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 25-25 µg STX eq/1g)
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
Conclusions for monitoring programmes
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
Requirement for alternative tests
Alexandrium spp.. Analyses Alexandrium spp. 16 Analyses Per Mo nth 12 8 4 199 1991 1992 1993 1994 1995 1996 1997 1998 1999 2 21 22 23 Year > 2 cells l -1-2 Not detected
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
Microscopy Labour intensive Traditional method of identifying and counting algal cells Day 1 Day 2 Day 3 Day 4 Day 5 Day...
Use of molecular probes No Probe Probe Applied Alexandrium tamarense (NA1) Alexandrium tamarense (NA1) and Pseudo-nitzschia spp.
PSP Bioassays 6 PSP Bioassay 5 Analyse sper Month 4 3 2 1 199 1991 1992 1993 1994 1995 1996 1997 1998 1999 2 21 22 23 Year > 8 µg STX eq. g -1 shellfish flesh >ND - 8 Not detected (ND)
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
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
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
Positive samples by MIST Alert TM compared to MBA 12 % Samples positive 1 8 6 4 2 n=139 1 n=157 98.1 n=8 91.2 n=427 28.1 8 4 - < 8 < 4 ND STX equiv. µg 1g -1 shellfish flesh by MBA
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
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
Acknowledgement Godfrey Howard, Eileen Bresnan, Bill Turrell