Our research

The official EU reference method for the analysis of PSP in shellfish is the AOAC 959.08 mouse bioassay (MBA). In 2006, the EU approved the use of an alternative instrumental method (AOAC 2005.06), which involves the analysis of shellfish extracts by high-performance liquid chromatography with fluorescence detection (HPLC-FLD).

Whilst this method had been validated by both single and inter-laboratory schemes, work was required to assess its fitness for purpose when specifically applied to UK shellfish samples. With FSA funding, we conducted a programme of in-house validation of

• a qualitative HPLC-FLD screening method, and then
• the quantitative AOAC 2005.06 method (also referred to as the Lawrence method).

The methods have been refined where required for certain shellfish species and have since been implemented in the UK PSP monitoring programmes, resulting in a full replacement of the MBA for all species currently monitored under the scope of Great Britain statutory programmes.

Contract reports

• FSA contract report (2011): Refinement and validation of HPLC method for whole scallops (PDF, 2.8 MB)
• FSA contract report (2011): Method performance checks for minor clam species (PDF, 2.1 MB)
• FSA contract report (2010): Validation of AOAC method 2005.06 for scallops (PDF, 2.4 MB)
• FSA contract report (2010): Validation of AOAC method 2005.06 for clams (PDF, 2.6 MB)
• FSA contract report (2010): Validation of AOAC method 2005.06 for oysters and cockles (PDF, 4.3 MB)
• FSA contract report (2008): Validation of AOAC method 2005.06 for mussels (PDF, 580 KB)
• FSA contract report (2007): Validation of a qualitative HPLC screening method for PSP toxins (PDF, 498 KB)

Other publications

• Cefas report (2011): Method performance checks for processed scallops (PDF, 1.4 MB)
• Turner, A D and Hatfield, R G (2012) Refinement of AOAC 2005.06 LC-FLD method to improve the method performance characteristics for the analysis of king and queen scallops for Paralytic Shellfish Toxins. Journal of AOAC International. 95(1): 129-142.
• Turner, A D, et al. (2012) Investigations into matrix components affecting the performance of the official bioassay reference method for quantitation of paralytic shellfish poisoning toxins in oysters. Toxicon, 59: 215-230.
• DeGrasse, S L, et al. (2012) Pre-versus Post-column oxidation liquid chromatography fluorescence detection of paralytic shellfish toxins. Toxicon, 57: 619-624.
• Turner, A D, et al (2011). Comparison of AOAC 2005.06 LC official method with other methodologies for the quantitation of paralytic shellfish poisoning toxins in UK shellfish species. Analytical and Bioanalytical Chemistry. Vol 399: 1257-1270.
• Algoet, M, et al. (2009). Application of the AOAC HPLC official method for the qualitative screening of PSP toxins in shellfish. In Proceedings of the 6th International Conference on Molluscan Shellfish Safety (ICMSS 07).18-23 March 2007, Blenheim, New Zealand. pp 141-146.
• Turner, A D, et al. (2009). Single laboratory validation of the AOAC HPLC method (2005.06) for mussels: refinement and extension of the method to additional toxins. Journal of AOAC International. 92(1): 190-207.
• Turner, A D, et al. (2010). Single laboratory validation of a refined AOAC HPLC method (2005.06) for oysters, cockles and clams in UK shellfish. Journal of AOAC International  93(5): 1482-1493.

• Poster (NC3R event, 2009): Replacing mice in UK shellfish tests. (PDF, 2 MB)

• Poster (AOAC Dallas, 2008): Refinement and in-house validation of the AOAC 2005.06 HPLC method: the quantitative analysis of mussel samples for PSP toxins (PDF, 6 MB)
• Poster (Trieste, 2007): A practical and alternative use of the AOAC HPLC method: the qualitative screening of shellfish samples for Paralytic Poisoning toxins (PDF, 483 KB)

Recent amendments to EC Regulations have specified that the biological assay (MBA) is no longer the EU reference method for the determination of lipophic toxin content of shellfish flesh. This assay has been replaced by the liquid chromatographic, tandem mass-spectrometric method (LC-MS) (EC Regulation 15/2011).

To allow member states to adapt their methodology, the MBA will continue to be allowed for the monitoring of lipophilic toxins (LTs) in shellfish until December 2014. However, the MBA is qualitative and does not provide information on the profile of toxins present in samples.

With financial support from the FSA, we validated the LC-MS method as an alternative to the biological assay to determine the suite of EU-regulated marine lipophilic toxins in shellfish typically harvested in UK waters.

Following a thorough review of method validation and performance, the method was implemented in the UK LT-monitoring programme in July 2011, resulting in a reduction in use of MBA by more than 95%.

Contract reports

• FSA contract report (2011): In-house validation of a LC-MS/MS method for the determination of lipophilic toxins in shellfish species typically tested in the UK (PDF, 6 MB)
• FSA contract report (2007): Internal validation of a LC-MS method for the determination of selected lipophilic toxins (PDF, 630 KB)

Other publications

• Poster (ICMSS, 2009): Single laboratory validation of a LC-MS/MS method for the determination of regulated marine lipophilic phycotoxins in shellfish typically tested in the UK (PDF, 478 KB)

• Poster (ICMSS, 2011): McMillan, D. et al. (2011). Improving LC-MS/MS methodology for lipophilic toxins analysis using UPLC and novel MS techniques.8^th International conference on Molluscan Shellfish Safety, June 2011, Prince Edward Island, Canada. (PDF, 604 KB)

Assessment of the Jellett Rapid Test (JRT) for PSP testing
In an FSA-funded study, we undertook an assessment of the Jellett commercial antibody-based testing kit for PSP toxins. Shellfish samples were subjected to screening by the kit and results compared with those generated by the EU reference method. Issues having been identified with results generated by the Jellett kit, the method ceased to be used in the UK official control monitoring programmes in May 2006.

• FSA contract report (2006): Assessment of Jellett Rapid Test for PSP (PDF, 498 KB)

Assessment of the suitability of EU reference methods for the testing of toxins in periwinkles and sea-urchins
In an FSA-funded study, we undertook an assessment of several shucking methods and a preliminary assessment of the suitability of the HPLC method for ASP toxins, the biological assay for DSP toxins and the AOAC 959.08 and 2005.06 methods for periwinkles and sea-urchins.

Results indicated some issues with both PSP reference methods when used for periwinkles. Further work is also required before the reference methods may be used for the official control toxin-testing of sea-urchins and for ASP testing of periwinkles.

• FSA contract report (2010): Assessment of methods suitability (periwinkles & sea-urchins) (PDF, 1.4 MB)

Using analytical methods such as the liquid chromatographic, tandem mass-spectrometric method (LC-MS) or high-performance liquid chromatography (HPLC), we are able to describe the quantity and profiles of toxins found in UK shellfish and UK waters.

The results of such analyses are reported in our annual monitoring reports and in the specific publications listed below.

Analytical methods such as LC-MS and HPLC are also used to inform investigations performed in areas affected by specific harmful algae, unusual toxin events or large-scale algal bloom events in the UK and abroad. 

Contract reports

• FSA contract report (2010): Investigations of Karenia sp. bloom Cornwall, August 2009 (PDF, 861 KB)
• FSA contract report (2010): Profiling of lipophilic toxins in Common mussels and Pacific oysters - Scotland 2009 (PDF, 3.8 MB)
• FSA contract report (2009): Profiling of lipophilic toxins in Pacific oysters and phytoplankton assemblage: West Loch Tarbert: Loup Bay, Scotland 2008 (PDF, 1.1 MB)
• FSA contract report (2009): Profiling of lipophilic toxins - Scotland 2008 (PDF, 2.9 MB)
• FSA contract report (2008): Profiling of lipophilic toxins - Scotland 2007 (PDF, 2.5 MB)
• FSA contract report (2007): Profiling of lipophilic toxins - Scotland 2006 (PDF, 757 KB)

Other publications

• Poster (MASTS, 2011): The widespread exposure of Scottish harbour seals (Phoca vitulina) to biotoxins from harmful algae. (PDF, 194 KB)
• Poster (14th International Conference on Harmful Algae, 2010): Lacaze, J P, et al. (2010). Investigations into the toxin profile of Alexandrium ostenfeldii isolated from Scottish waters. 14th International Conference on Harmful Algae, Crete, November 2010.
• Poster (14^th International Conference on Harmful Algae, 2010): Nascimento, S M, et al. (2010). Molecular taxonomy and toxin profile of Ostreopsis cf. ovata (Dinophyta) from Rio de Janeiro, Brazil. 14th International Conference on Harmful Algae, Crete, November 2010.
• Poster (14th International Conference on Harmful Algae, 2010): Nascimento, S. M, et al. (2010). Toxin profiles from Prorocentrum lima and Prorocentrum sp. isolates from Brazilian coastal waters. 14th International Conference on Harmful Algae, Crete, November 2010
• Poster (Marine and Freshwater Toxins Analysis: Second Joint Symposium and AOAC Task Force Meeting, 2011): Burrell, S, et al. (2011). First reports of Paralytic Shellfish Poisoning Toxins in blue mussels (Mytilus edulis) from Breidafjordur and Eyjafjordue, Iceland. Marine and Freshwater Toxins Analysis: Second Joint Symposium and AOAC Task Force Meeting. May 2011, Spain.
• Poster (ICMSS, 2009): Detection and quantitation of lipophilic phycotoxins using LC-MS/MS in pacific oyster samples to investigate the causes of the prolonged closure of a Scottish harvesting site (PDF, 219 KB)
• Poster (RAFA, 2007): LC-MS profiling of lipid soluble biotoxins in Scottish shellfish during summer 2006 (PDF, 1.4 MB)

Access to contaminated shellfish material is essential for method-validation programmes. Such material is also used to control the quality of routine analyses.

It can be difficult, however, to acquire naturally contaminated material as toxic events are uncommon and unpredictable.

We have developed protocols to artificially contaminate shellfish through feeding experiments with high concentrations of toxic algal species such as Alexandrium. This species is responsible for the accumulation of PSP toxins in bivalves. Such protocols are used to generate PSP-contaminated samples of cockles, Pacific oysters, native oysters and king scallops - containing a variety of different PSP toxin profiles.

Our work in this area has paved the way for the in-house production of reference materials, with recent work developing the use of a large-scale photobioreactor for the generation of larger volumes of contaminated materials.This equipment is now routinely used to produce matrix-specific reference material.

It has also enabled the recent production of a large batch of a candidate Pacific oyster reference material for PSP toxins. This material is currently under characterisation and should be available commercially as a PSP certified reference material in summer 2012. This material is also being utilised in an ongoing collaboration with the Marine Institute in Ireland to produce certified freeze-dried reference materials for PSP toxins.

For further information on our marine biotoxin reference materials please visit Our services and advice or contact referencematerials@cefas.co.uk.

Publications

• Turner, A D, et al. (2010). Potential use of gamma irradiation in the production of mussel and oyster reference materials for paralytic shellfish poisoning toxins. Anal Bioanal Chem. 397, 743-749.
• Higman, W A and Turner, A D (2010). A feasibility study into the provision of paralytic shellfish toxins laboratory reference materials by mass culture of Alexandrium and shellfish feeding experiments. Toxicon 56: 497-501.

• Poster (ICMSS 2011 and Second Joint Symposium and AOAC Task Force Meeting. 2011): Production of PSP laboratory and certified reference materials by Cefas. (PDF, 8.5 MB)
• Poster (124th AOAC Conference, 2010): Investigations into various sample preparation techniques for the production of shellfish laboratory reference materials for Paralytic Shellfish Poisoning toxins. (PDF, 1.3 MB)
• Poster (Berm, 2009): Mass culture of toxic algae and shellfish feeding experiments for production of PSP reference materials (PDF, 465 KB)