Marine Biotoxin Management Plan

Victorian Shellfish Quality Assurance Program

January 2004

Fisheries Victoria Management Report Series No.11

Marine Biotoxin Management Plan © The State of Victoria, Department of Primary This publication was prepared for Fisheries Industries, 2004 Victoria by WATER ECOscience Pty Ltd. This publication is copyright. No part may be Authorised by the Victorian Government, produced by any process except in accordance 6/232 Victoria Parade, East Melbourne with the provisions of the Copyright Act 1968. Published by the Department of Primary Reproduction and the making available of this Industries. material for personal, in‐house, or non‐ Fisheries Victoria commercial purposes is authorised on the PO Box 500 condition that: East Melbourne Victoria 3002. • The copyright is acknowledged as the owner; Copies are available from the website: • No official connection is claimed; www.dpi.vic.gov.au/fishing • The material is made available without charge Follow the links Managing Fisheries, or cost; and Management Plans and Strategies • The material is not subject to inaccurate, Disclaimer: This publication may be of assistance misleading or derogatory comment. to you, but the State of Victoria and its employees Requests to reproduce or communicate this do not guarantee that the publication is without material in any way not permitted by this licence flaw or is wholly appropriate for your particular (or by the fair dealing provisions of the Copyright purposes and therefore disclaims all liability for Act 1968) should be directed to the Copyright an error, loss or other consequence which may Officer, 6/320 Victoria Parade, East Melbourne, arise from you relying on any information in this 3002. publication. ISSN: 1448‐1693 This research report cannot be used in court. Fishing laws change from time to time. It is your ISBN: 1 74146 059 X responsibility to ensure that you are acting within Preferred way to cite this publication: the law. If you are in doubt contact the Customer Department of Primary Industries (2004). Marine Service Centre (telephone 136 186 or Biotoxin Management Plan 2004. www.dpi.vic.gov.au/fishing/) Fisheries Victoria Management Report Series No.11.

Marine Biotoxin Management Plan ii Contents Acknowledgements 1

1 Amendments 2

1.1 Amendments 2 1.2 Amendments Record 2 2Introduction 4

2.1 Background 4 2.2 Aims and Objectives 4 2.3 Scope 5 2.4 Review 5 3 Administration 6

3.1 Legislation & Guidelines 6 3.1.1 Federal 6 3.1.2 State 6 3.2 Roles and Responsibilities 6 3.2.1 Department of Primary Industries (FV) 6 3.2.2 Department of Human Services (Victoria) 6 3.2.3 Local Government 6 3.2.4 PrimeSafe 7 3.2.5 Australian Quarantine and Inspection Service 7 3.2.6 Industry 7 3.2.7 WATER ECOscience P/L 7 3.2.8 Environment Protection Authority 8 3.2.9 Australian Shellfish Quality Assurance Advisory Committee 8 3.2.10 Victorian Shellfish Quality Assurance Advisory Committee 8 3.3 Local Marine Biotoxin Management 8 4 Monitoring 9

4.1 Monitoring Program Goals 9 4.2 Sampling Site Selection 9 4.3 Sampling Officers and Sample Collection 9 4.4 Sampling Safety 10 4.5 Phytoplankton Monitoring 10 4.5.1 Sampling Frequency 10 4.5.2 Methods 10 4.5.3 Phytoplankton Species Monitored 11 4.6 Tissue biotoxin monitoring 12 4.6.1 Sampling Frequency 12 4.6.2 Shellfish Species Sampled 12 4.6.3 Methods 12

Marine Biotoxin Management Plan iii 4.7 Environmental Information 14 4.8 Reporting and Notification 14 4.9 Data Storage 15 4.10 Contingency Plans for Marine Biotoxin Events 15 5Area Closure and Reopening 17

5.1 Closure Criteria 17 5.2 Mechanism for Closure 17 5.3 Industry Instigated Closure 17 5.4 Reopening Criteria 17 5.5 Mechanisms for Reopening 18 5.6 Surveillance of Harvesting Areas 18 6 Investigation of Illness due to Toxic Shellfish Poisoning 19

6.1 Notification 19 6.2 Investigation 19 6.3 Immediate Action for Suspected Toxic Shellfish Poisoning Cases 19 6.3.1 Closures of commercial harvesting areas 19 6.3.2 Control of movement of harvested shellfish 19 6.3.3 Notification 20 6.3.4 Communication 20 6.3.5 Sampling 20 6.3.6 Funding 20 7Product Control 21

7.1 Product Recall 21 7.2 Objectives 21 7.3 Responsibilities 21 7.4 Notification 21 8 References 22

Marine Biotoxin Management Plan iv List of Tables

Table 1: Marine Biotoxin Management Plan amendment record. 2 Table 2: Approximate schedule for receiving routine sampling results 15 Table 3: Phytoplankton Action Levels 45 Table 4: Biotoxin regulatory limits for the VSQAP. 47

Appendices

Appendix 1: Agency & Personnel Contacts 23 Appendix 2 Communication Network Diagram 25 Appendix 3: Approved Laboratories and Contacts for Phytoplankton Enumeration and Identification 26 Appendix 4: Approved Laboratories and Contacts for Marine Biotoxin Analysis of Shellfish Flesh 27 Appendix 5: Sampling Officers 28 Appendix 6: Marine Biotoxin Analytical Methods 29 Appendix 7: MBMP Sampling Collection Form and Proformas for External Laboratories 30 Appendix 8: Phytoplankton Sampling Procedures 35 Appendix 9: Phytoplankton Species Lists 37 Appendix 10: Toxic Shellfish Poisoning Case Definitions 42 Appendix 11: Phytoplankton Action Levels 45 Appendix 12: Marine Biotoxin Regulatory Levels 47 Appendix 13: Nuisance Phytoplankton Management Protocols 49 Alexandrium spp. 49 Dinophysis acuminata, Dinophysis spp. and Prorocentrum lima 52 Pseudo‐nitzschia spp. 56 Rhizosolenia cf chunii 60 Gymnodinium catenatum 61 Karenia brevis, Karenia cf brevis and Karenia mikimotoi 64

Acronyms 69 Glossary 70

Marine Biotoxin Management Plan v Acknowledgements

This project was funded by the Fisheries Division of the Department of Primary Industries (Victoria) and the Victorian aquaculture industry. The project team would like to thank the following people for their valuable input which facilitated the completion of this project:

The Department of Primary Industries Peter Appleford Fisheries Victoria (Aquaculture Planning Officer) Primary Industries Research, Victoria Neil Hickman Marine & Freshwater Systems Platform (Senior Scientist, Aquaculture Program) (Formerly: Marine and Freshwater Resources Institute) The University of Tasmania Prof Gustaaf Hallegraeff (Associate Professor in Aquatic Botany) Queensland Health Scientific Services Dr Geoff Eaglesham (Senior Scientist) Cawthron Institute Paul McNabb (Senior Scientist) State Chemistry Laboratories, Victoria Dr Craige Trenerry Food Chemistry Unit

Medvet Sciences, IMVS Chris Murray Laboratory Manager) Marlborough Shellfish Quality Helen Smale Programme (Program Manager)

Marine Biotoxin Management Plan 1 1 Amendments

1.1 Amendments 1.2 Amendments Record Amendments can be made to this Plan by It is important this Plan is kept up to date by the contacting the coordinator with the suggested prompt incorporation of amendments. changes and reasons for them. To update the Plan, remove the appropriate The co‐ordinator of this Plan is the Aquaculture pages, destroy them and replace with the newly Planning Officer, Fisheries Victoria. issued pages. Instructions will be included in the covering letter when amendments are issued and To become part of this Plan, amendments need to sent. File the covering letter at the back of the Plan be issued with a covering letter. Amendments are and sign off and date this page. identified by the issue number in the page header, by a vertical line in the left margin adjacent to the line(s) that has been changed and in the amendments record in Table 1 below. Amendments will be numbered in sequence.

Table 1: Marine Biotoxin Management Plan amendment record.

Issue No. Date Initials

Marine Biotoxin Management Plan 2 Issue No. Date Initials

Marine Biotoxin Management Plan 3 2 Introduction

the Marine and Freshwater Resources Institute 2.1 Background (MAFRI)) for Fisheries Victoria, and was funded Some species of marine microalgae entirely by the latter until it was discontinued at (phytoplankton) produce natural toxins which the end of 1996. The program collected surface may accumulate in the tissues of filter feeding water samples and tissue samples on a regular shellfish. Toxic shellfish poisoning (TSP) may basis, analysing the samples for phytoplankton result in humans that have consumed and biotoxin respectively. This sampling regime contaminated shellfish. provided for the monitoring of toxic Within Victoria, four shellfish poisoning phytoplankton species and biotoxins in syndromes are potentially of concern: commercial shellfish harvest areas. WES performed phytoplankton monitoring from • Paralytic Shellfish Poisoning (PSP) July 1997 until August 1999. During the absence • Amnesic Shellfish Poisoning (ASP) of a formal VSQAP, the mussel industry, either as the Victorian Mussel Growers Association or as • Diarrhetic Shellfish Poisoning (DSP) individual growers, contracted WES to continue • Neurotoxic Shellfish Poisoning (NSP) phytoplankton and biotoxin monitoring. The potentially causative organisms of these Currently, WES perform all of the monitoring and poisoning syndromes are provided in Section 5.6. reporting components of the VSQAP, and have prepared the Biotoxin Management Plan, The presence of biotoxins in shellfish not only operating under contract to Fisheries Victoria. poses a health risk to consumers but may also PIRVic continues to provide management, adversely impact on the aquaculture industry by technical and research services on behalf of lowering consumer confidence in the harvested Fisheries Victoria. The VSQAP is 67 per cent shellfish product. These risks can be managed by funded by Fisheries Victoria and 33 per cent by marine biotoxin management plans. the industry through a levy for all Aquaculture The Marine Biotoxin Management Plan for the (Crown land) Type A Licences. Shellfish Victorian Shellfish Quality Assurance Program harvesting at Beaumaris, and consequently (VSQAP) has been developed by WATER monitoring, ceased in March 2001 and this area is ECOscience P/L (WES) in conjunction with the now used solely for spat collection and bait Department of Primary Industries (DPI), Fisheries production. Victoria. Within the VSQAP, from 1990 until 2003, History of Biotoxin Surveillance phytoplankton and tissue PSP testing has been A history of VSQAP phytoplankton and biotoxin performed fortnightly at each of the five surveillance is presented in Section 5.7 of the harvesting areas in PPB and WP, except at Australian Marine Biotoxin Management Plan for Beaumaris where harvesting discontinued in 2001. Shellfish Farming (Todd, 2001). ASP testing has also been performed each fortnight at the Clifton Springs and Flinders In summary, the VSQAP was established in 1987 harvesting areas. From 2004, the frequency of to ensure commercially harvested blue mussels routine biotoxin testing has been reduced to were safe for human consumption. At that time, it monthly. As a result, the VSQAP has a significant serviced four aquaculture zones in Port Phillip database to support decisions in regard to Bay (PPB) (Clifton Springs, Grassy Point, biotoxin management. Dromana and Beaumaris) and another in Western Port (WP) (Flinders Bight). 2.2 Aims and Objectives Wildstock mussels from the Gippsland Lakes and The principal aim of the Marine Biotoxin scallops from PPB and Bass Strait have, in the Management Plan is to ensure the protection of past, been included in the VSQAP program. The consumers against the hazards of marine toxic program was operated by the Marine & shellfish poisoning (TSP) from the commercial Freshwater Systems Platform (MFSP), Primary harvest of bivalve shellfish within Victoria’s PPB Industries Research Victoria (PIRVic) (formerly and WP aquaculture fisheries reserves.

Marine Biotoxin Management Plan 4 The following objectives have been established to meet this aim: 2.3 Scope The Marine Biotoxin Management Plan is

• The maintenance of a monitoring program designed primarily for the commercial using phytoplankton monitoring in aquaculture harvesting of bivalve shellfish from conjunction with biotoxin testing of bivalve PPB and WP, areas for which extensive shellfish tissue. Phytoplankton monitoring is phytoplankton records exist. The Plan, with some used to provide early warning of the modifications, may be adopted for commercial presence of phytoplankton with the potential wild shellfish harvesting if appropriate. There is to contaminate shellfish with marine evidence that various shellfish species may not biotoxins. The results of this monitoring may bioconcentrate and metabolise particular be used to initiate biotoxin testing, and in biotoxins in the same manner. Hence, some some cases, harvesting closures. Shellfish review of biotoxin monitoring protocols may be tissue biotoxin levels are used to make required should additional species be grown and harvesting reopening and regulatory commercially harvested within PPB and WP decisions. aquaculture fisheries reserves. • To document all procedures and contacts required to effectively manage incidents of 2.4 Review shellfish biotoxin contamination. The Marine Biotoxin Management Plan will be • To facilitate the harvesting of shellfish which fully reviewed on a triennial basis and an are free from marine biotoxins. updated, numbered ‘version’ issued, incorporating all amendments made since the • To provide an effective and coordinated previous review. Reviews shall be undertaken by response to marine biotoxin events, an agency with good knowledge of the VSQAP minimising the risk of human illness. Marine Biotoxin Management Program and its • Ensure public awareness of shellfish biotoxin application in Victoria. events while minimising potential adverse Upon issue of an updated version of the Plan, all publicity to the shellfish industry. previous versions are to be destroyed or stored in • Maintain updated management protocols such a manner that superseded documentation (contingency plans) to allow rapid and will not be available for use. The Aquaculture effective responses to marine biotoxin events. Planning Officer will request written advice, verifying that this has been carried out. Amendments may be made at any time and will be circulated by the Aquaculture Planning Officer.

Marine Biotoxin Management Plan 5 3 Administration

• Retain records of aquaculture licences and 3.1 Legislation & Guidelines conditions, closure and reopening notices for A list of Commonwealth and State legislation and harvesting areas. guidelines relevant to biotoxin management are • Ensure the completion of sanitary surveys provided below. For further detail, refer to the and the classification of shellfish harvesting relevant document. areas by qualified personnel. 3.1.1 Commonwealth • Contribute 67 per cent of the total funding for • FSANZ Food Standards Code (2002). the VSQAP • • Export Control Act (1982). Provide representation at the national Australian Shellfish Quality Assurance • Export Control (Processed Food) Orders Advisory Committee (ASQAAC) (through (1992). service agreement with PIRVic). • Australian Shellfish Quality Assurance • Chair and provide expert advice to the Program (ASQAP) Operations Manual (2002). Victorian Shellfish Quality Assurance Advisory Committee (VSQAAC). • Australian and New Zealand Guidelines for Fresh and Marine Water Quality (2000). • Provision of the VSQAP hotline to advise industry and other stakeholders of the 3.1.2 State biotoxin and phytoplankton status of all • Health Act 1958. harvesting areas (through service agreement with PIRVic). • Fisheries Act 1995. • Provision of expert scientific opinion to • Food Act 1984. stakeholders concerning shellfish. • Seafood Safety Act 2003. • Manage all data and reports generated by the VSQAP (through service agreement with • Environment Protection Act 1970, including PIRVic). the relevant State Environment Protection

Policies. • Through an Authorised Officer, advise the appropriate DHS, AQIS, PrimeSafe and EPA personnel of harvesting closures due to the 3.2 Roles and Responsibilities presence of biotoxins in shellfish tissue. 3.2.1 Department of Primary Industries (Fisheries Victoria) 3.2.2 Department of Human Services (Victoria) The following are the responsibilities of Fisheries Victoria. Assistance is provided in undertaking The following are the responsibilities of the the responsibilities by PIRVic, Queenscliff Centre Department of Human Services (DHS). through a service agreement (as indicated). • Detain and recall product considered unfit • State Shellfish Control Agency (SSCA) for for human consumption. Victoria. • Provide expert advice to the VSQAAC.

• Issue licences authorising aquaculture • Licence food transport vehicles (subject to the activity under the Fisheries Act 1995. Seafood Safety Act 2003). • Control the harvesting of shellfish based on • Maintain epidemiological data for notifiable sanitary conditions. diseases (including TSP cases). • Oversee the sampling program. • Sub‐contract components of the program to 3.2.3 Local Government the private sector. The following are responsibilities of Local • Ensure no illegal harvesting takes place when Government through the Food Act 1984 but will pass to PrimeSafe on I July 2004. a closure is in place.

Marine Biotoxin Management Plan 6 • Licence food processing premises (subject to establishment in accordance with the compliance the Seafood Safety Act 2003). history of the establishment and food safety risk associated with the food being prepared for • Enforce necessary sanitary controls for export. The Export Control (Processed Food) Orders processing plants and vehicles handling 1992 also regulate the application controls for seafood. shellfish handling, processing, purification, 3.2.4 PrimeSafe packing, storage, shipping and labelling of shellstock to enable source identification, as well PrimeSafe is the new statutory authority as the recall, detention, seizure or destruction of responsible for maintaining Victoriaʹs reputation shellfish unfit for human consumption. as a producer of clean, safe and fresh meat and seafood. 3.2.6 Industry The creation of PrimeSafe follows the introduction The following are the responsibilities of the of the Seafood Safety Act 2003 to incorporate industry who harvest bivalve shellfish for human seafood safety regulation by expanding the role of consumption. the Victorian Meat Authority. The new seafood • Comply with the requirements of the safety legislation provides for all seafood ASQAP. industry businesses specified by the Minister, including those based on wildcatch and • Ensure no harvesting takes place when a aquaculture, to operate approved seafood safety closure is in place. management programs. • Provide representation to the ASQAAC. PrimeSafe will be responsible for red meat, • Provide expert advice to VSQAAC. chicken and seafood. A new Board of Directors has been appointed for PrimeSafe, including • Contribute 33 per cent of the total funding to expertise from the seafood industry. the VSQAP. Aquaculture and wildcatch harvesters and • Undertake a notification process, when aquaculture businesses will come under the new required, for the recall of contaminated scheme from 1 July 2004 and may be required to shellfish. have an approved Hazard Analysis Critical Control Point (HACCP) based Food Safety Plan. 3.2.7 WATER ECOscience P/L WES has been contracted by Fisheries Victoria, 3.2.5 Australian Quarantine and Inspection DPI (SSCA for the VSQAP) to carry out the Service majority of the tasks set out in Part B of the AQIS is the Commonwealth government agency ASQAP Operations Manual (2002). These include: responsible for the administration of the export • Comprehensive sanitary surveys/reports. controls for seafood. The agency administers the export inspection system and provides • Shoreline surveys. certification for shellfish exports. • Triennial update sanitary surveys/reports. AQIS administers the export inspection program, which includes provision for: • Annual update sanitary surveys/reports.

• The registration of premises, including • Phytoplankton monitoring. vehicles, which prepare shellfish intended for • Biotoxin monitoring. export. • Monitoring program evaluation and design. • The inspection of registered establishments for implementation of good food processing • Growing area classifications and re‐ practises. evaluations. • Conducting HACCP‐based food processing • Develop and maintain WES VSQAP controls. Operations Manual. • Auditing state shellfish quality assurance • VSQAP Marine Biotoxin Management Plans programs for compliance with the ASQAP. and reviews. AQIS staff conduct compliance inspections and audits of land‐based shellfish processing

Marine Biotoxin Management Plan 7 3.2.8 Environment Protection Authority 3.3 Local Marine Biotoxin • Provide expert advice to the VSQAAC. Management 3.2.9 Australian Shellfish Quality Assurance Section 3.4.4 of the current ASQAP Operations Advisory Committee Manual (2002), recommends that a marine • Provide a national overview of shellfish biotoxin control contingency plan (marine safety and quality. biotoxin management plan) should be developed for each shellfish growing area potentially • Provide a set of minimum requirements affected by toxic phytoplankton blooms, agreed to by all states and territories (refer to containing: the ASQAAC Terms of Reference). • Updated agency and personnel contact • Be responsible for the formulation and details at local and state levels. regular updating of the national ASQAP Operations Manual. • The marine biotoxin sampling sites for each commercial shellfish growing area and the 3.2.10 Victorian Shellfish Quality Assurance rationale for their location. Advisory Committee • The frequency of shellfish and phytoplankton VSQAAC is a Victorian inter‐agency and industry monitoring for each growing area. forum that represents shellfish quality assurance issues. • Procedures for phytoplankton and shellfish sample collection and dispatch. • Early warning indicators such as fish kills or the presence of potentially toxic phytoplankton. • Communication channels with other SSCAs and stakeholders concerning biotoxin‐related information. • Contingency plans. • Procedures for notification of phytoplankton and biotoxin results to industry and others. • Procedures and criteria for harvesting area closure and reopening. • Procedures for detention and recall of harvested product. • The ISO17025 accredited laboratory used for sample analysis. The biotoxin section of the ASQAP Operations Manual is soon to be updated incorporating elements of the Australian Marine Biotoxin Management Plan for Shellfish Farming (Todd, 2001).

Marine Biotoxin Management Plan 8 4 Monitoring

• Environmental factors likely to influence 4.1 Monitoring Program Goals sampling, such as: The VSQAP phytoplankton and biotoxin monitoring program has been designed with the o Major currents. following goals in mind: o Retention zones and circular patterns. • Provide early warning of potential marine o Areas where algal blooms and fish kills biotoxin contamination by detecting changes were regularly observed, or had been in the presence and abundance of potentially regularly observed in the past. toxic phytoplankton species. o Impact of rivers. • Increased knowledge and a wider understanding of those species that pose a o Impact of drains. potential marine biotoxin threat to o Any other factors that may have commercial harvesters of shellfish. influenced sampling. • Establish a long‐term data set of • Sites have been chosen so that the water phytoplankton abundance, marine biotoxin being sampled for phytoplankton is levels and events, and associated ecological representative of the water being filtered by factors. This dataset may be used to improve the shellfish within the harvesting area. risk assessment, facilitate the analysis of trends in phytoplankton abundance and aid • For line culture, the water samples are the prediction of marine biotoxin events. collected so that the entire depth of the lines bearing shellfish are sampled, to account for • Establish toxic phytoplankton levels in a the possibility of uneven vertical distribution timely manner to permit harvesting closures of phytoplankton. before biotoxins reach levels that may threaten human health. The aquaculture harvesting areas within PPB and WP are all in open, well circulated and vertically • Establish biotoxin levels in a timely manner mixed waters. In general, shellfish and to support harvesting closure and phytoplankton are sampled in the areas of greater reopenings. risk of contamination, where suitable shellfish are • Validate that phytoplankton monitoring does available, and where harvesting is due to occur. capture all toxic events. Consequently, sampling may be carried out at different sites within each harvesting area over • Monitor an up‐to‐date list of local, national consecutive sampling events. In the past, and international potentially toxic sampling at a single site within each area has phytoplankton species. proven to be adequate to detect nuisance phytoplankton. 4.2 Sampling Site Selection When sampling sites for toxic phytoplankton and 4.3 Sampling Officers and shellfish are established, the following general Sample Collection factors were considered: Sampling is carried out by experienced West • The history of phytoplankton and marine Coast Diving personnel, who have undergone biotoxin levels in PPB and WP. appropriate training by WES. In addition, every three months, a suitably qualified WES consultant • The need to monitor effectively the entirety of accompanies West Coast Diving on a field trip to all aquaculture shellfish harvesting areas. carry out Quality Assurance/Quality Control • Location of bivalve shellfish being harvested (QA/QC) checks on all sampling procedures and at various times. to carry out any additional training deemed necessary. • Accessibility of sample sites in various weather conditions.

Marine Biotoxin Management Plan 9 All sampling is performed in accordance with • Vessel check (boat equipment, radio, safety WES sampling protocols that are documented in equipment). the regularly updated WES VSQAP Operations • Work equipment secured to prevent Manual. movement around deck space and damage to Sample collection proformas are completed with personnel and craft. each sample event. These are used as chains of WES personnel accompany West Coast Diving on custody (COC) and include the documentation of a number of sampling events each year for any observations made within the harvesting QA/QC and other purposes. All such personnel areas, such as weather conditions, or anything are under the direction of the boat master while else that may be relevant to the sample collection on board and follow all relevant safety measures, process and sample integrity. West Coast Diving including those set out in the WES Occupational retains a copy of the COC and WES retains the Health and Safety Manual, SM 12 Safety in the original on the project file. All COCs are made Field. available to the SSCA upon request. Where samples cannot be collected during any 4.5 Phytoplankton Monitoring sampling event, this is noted on the COC (site and 4.5.1 Sampling Frequency reason). WES notifies the nominated SSCA Authorised Officer within 24 hours of any such Since 1987, phytoplankton sampling has been occurrence. carried out at all harvesting areas within PPB and WP, usually on a fortnightly basis. Consequently, a considerable body of data exists concerning 4.4 Sampling Safety phytoplankton blooms in these waters. In relation to the vessel used for sampling, the subcontractor, West Coast Diving, is required to Phytoplankton sampling is carried out at all meet the following requirements: harvesting areas on a fortnightly, routine basis all year round. This frequency of sampling has been • The operator is certified by the Marine Board found to be adequate in providing early warning of Victoria for competency as a coxswain and of the potential for biotoxin contamination of holds the appropriate Manning Certificate. bivalve shellfish tissue, and as a trigger to initiate • The vessel is currently surveyed by Marine tissue sampling for biotoxin analysis. Safety Victoria in accordance with the Where routine phytoplankton monitoring reveals Uniform Shipping Laws Code. the presence of potentially toxic phytoplankton • The vessel is manned with suitably qualified species, in numbers approaching the trigger for personnel. biotoxin testing or in rising numbers, the routine fortnightly sampling frequency may be increased to weekly or more often depending on the state of The Marine Safety Victoria On Board Code of the bloom. Once the bloom has degenerated, the Practice must be followed at all times. sampling frequency is again reduced to fortnightly. West Coast Diving performs routine safety checks prior to each trip. These include, but are not Phytoplankton sampling occurs whenever tissue limited to: sampling is carried out for biotoxin analysis. • Weather Analysis – Port of Melbourne 4.5.2 Methods Corporation. Sampling • Boats not operated in adverse weather Detailed instructions for the use of appropriate conditions phytoplankton sampling equipment is presented in Appendix 8. o Wind speed in excess of 25 knots o Wave height in excess of 1.5 m o All boating will cease and return to shore if any person on board feels unsafe and the decision will be final.

Marine Biotoxin Management Plan 10 Two samples, a concentrated plankton net haul • For some phytoplankton, definitive species and a depth‐integrated hosepipe sample, are level identifications can only be performed collected for phytoplankton analysis: using sophisticated equipment such as electron microscopes (e.g. Pseudo‐nitzschia • A concentrated sample using a 6m vertical spp). After preliminary identification at WES haul with a 20µm mesh plankton net; this using light microscopy, subsamples of these sample is used to identify any potentially are sent to University of Tasmania, School of toxic or nuisance species present. Although Plant Sciences for definitive identification. there is the potential for fragile algae such as the non‐armoured gymnodinioids to be • Each month, one sample is sent to the damaged by the use of these nets, experience University of Tasmania School of Plant has shown that these cells are sampled intact Science, for identification and enumeration. when the nets are used appropriately. These results are used as an inter‐laboratory comparison for QA/QC purposes. • The concentrated net sample is collected in a 75mL polycarbonate vial, transferred to a 4.5.3 Phytoplankton Species Monitored separate storage bottle leaving a 20–30mm air Appendix 9 contains lists of phytoplankton space, and capped tightly. The sample is species present or likely to be present in labelled appropriately with the date and time Australian waters, categorised on the basis of of sampling, sample type and harvesting their likelihood of occurrence and potential for area. toxicity: • A depth‐integrated sample is collected using • Category A1 ‐ Species known to be present in a 6m long, 25mm internal diameter hosepipe southern Australian waters including PPB sampler and placed into a clean bucket on and WP, and proven or suspected toxin board the sampling vessel. This is mixed producers in Australia. thoroughly taking care to avoid damage to any phytoplankton present and a 1 L sub‐ • Category A2 ‐ Species known to be present in sample collected for enumeration. A 20‐ Australian waters and proven to produce 30mm air space left prior to capping. The toxins in Australia or overseas. sample is labelled with the date and time of • sampling, sample type and harvesting area. Category B ‐ Potential toxin producing species (i.e. toxicity untested/unclear) known • All samples are collected so that foreign to be present in Australian coastal waters. inclusions are avoided (e.g. outboard motor • oil). Category C ‐ Other potential toxin producing species worldwide that may be present in • All samples are stored in an upright position Australian waters. in an esky containing a small ice pack that does not come in contact with the samples. The VSQAP must at all times be able to identify potentially toxic species on these lists, particularly The purpose of the ice‐pack is solely to prevent the interior of the esky from heating those in Categories A & B. In some cases, where up and not to cool the sample(s). species identification is difficult, or the taxonomy is unclear, similar species may be managed as a • Excess shaking of the samples during single group. For example, despite the fact that transport is avoided as this may damage only two species encountered by the VSQAP have some phytoplankton. a record of toxicity, all Pseudo‐nitzschia spp. are initially assumed to be as toxic as the most toxic Laboratory Testing member of the group. This allows for conservative Appendix 3 lists the name and contact details of management until definitive identification is the organisations that provide analytical services made. This principle is also applied to any case for the identification and enumeration of where the identification of a potentially toxic phytoplankton in water samples. For species is uncertain. phytoplankton sampling: • Routine identification and enumeration is carried out in the NATA registered WES, Mount Waverley Botany Laboratory. On completion, samples are preserved in Lugolʹs Solution and retained for at least one month.

Marine Biotoxin Management Plan 11 Appendix 11 lists the trigger levels for required. These are frozen and retained phytoplankton species within PPB and WP. These where necessary. relate to an integrated phytoplankton sample • Shellfish are transported to the WES collected with a hosepipe sampler or a surface laboratory after collection in eskies sample. These triggers are used to initiate tissue containing ice packs to keep them cool. biotoxin testing and precautionary harvesting Mussels are not to be frozen or cooled closures, pending biotoxin results. excessively. Mussels are to be shucked within 24 hours of collection and the tissue frozen 4.6 Tissue biotoxin monitoring prior to delivery by overnight courier to the 4.6.1 Sampling Frequency testing laboratories. • PSP biotoxin tissue analysis is carried out routinely every four weeks at each harvesting Laboratory Testing area, or when phytoplankton abundance No single Australian laboratory can perform the levels indicate this is necessary. full range of biotoxin analyses required by the VSQAP which includes PSP, ASP, DSP, NSP, YTX • ASP (domoic acid) biotoxin tissue analysis is and AZA (the latter two have not been detected, carried out routinely every four weeks at the nor are they regulated in Australia but qualitative Clifton Springs and Flinders harvesting areas, testing is proposed where appropriate). In to provide background data for the two major addition, NSP analyses are not currently bays where aquaculture harvesting areas performed routinely in Australia. As a result, four exist. It may be performed at any harvesting separate laboratories perform the testing area where phytoplankton abundance levels including three from Australia and one from New indicate this is necessary. Domoic acid has Zealand. A variety of methods are used. not been recorded in any harvesting area. Appendix 4 lists the organisations providing • Other biotoxin analyses (DSP, NSP) are analytical services for biotoxin analysis of performed when phytoplankton abundance shellfish tissue samples. Details of the levels indicate this is necessary. methodologies used are provided in Appendix 6. 4.6.2 Shellfish Species Sampled There are four main groups of toxins of concern in Currently, only blue mussels, Mytilus edulis Australia that may accumulate in shellfish tissue planulatus, are tested for biotoxins. and cause illness in humans. These are named after the poisoning syndrome they cause. The With some commercial interest being shown in regulatory limits noted are from the FSANZ Food scallop aquaculture, background domoic acid Standards Code (2002). monitoring will be recommended for this bivalve at the appropriate harvesting areas. Paralytic Shellfish Poisons (PSPs) A range of PSPs such as STX, C toxins and 4.6.3 Methods gonyautoxins are produced by several Sampling dinoflagellate species including Alexandrium • At the Clifton Springs and Flinders catenella, A. minutum, A. tamarense and harvesting areas, mussels are collected Gymnodinium catenatum. These toxins may be fatal routinely for PSP and ASP analysis. At the to human consumers of contaminated shellfish Grassy Point and Dromana harvesting areas, through respiratory paralysis, although this is mussels are routinely collected for PSP rare and there have been no fatal cases in analysis. For shellfish biotoxin analysis, 30‐40 Australia. PSP was detected in PPB mussels in large mussels are collected at each of these 1993 and 1994 at the Clifton Springs and Grassy sites. Point harvesting areas; the most likely source was A. tamarense (Arnott et al, 1999). The maximum • Mussels are shucked in the laboratory and PSP concentration detected was 276 µg/100g at 150–180g of flesh is prepared for each Clifton Springs. biotoxin analysis. Testing Agency: IMVS in Adelaide, South Australia. • A sufficient number of mussels are also collected at each harvesting area to provide Method: Mouse bioassay enough tissue to perform other biotoxin Units: µg/100g analyses (e.g. DSP or NSP) where phytoplankton monitoring indicates they are Regulatory Limit: 80 µg/100g (0.8 mg/kg)

Marine Biotoxin Management Plan 12 Amnesic Shellfish Poisons (ASPs) from this group of toxins (Aune in MacKenzie, Amnesic shellfish poisoning is caused by domoic 2002). The later group suggested a limit for PTX of acid produced by several species of diatoms 15 µg/100g. PTX is still regarded as toxic and, belonging to the genus Pseudo‐nitzschia, such as P. although currently included as a DSP for the australe and P. multiseries. ASP may cause VSQAP, should be regulated separately. PTX 2 symptoms from nausea, vomiting and abdominal and OA have been detected in PPB mussels but in cramps to dizziness, hallucinations, short‐term very low levels only. memory loss and seizures. Although most species The FSANZ Food Standards Code Regulatory of Pseudo‐nitzschia are non‐toxic, they are very Limit for DSP is retained for the VSQAP. difficult to separate definitively using only light microscopy. Hence, all Pseudo‐nitzschia are Testing Agency: Queensland Health Scientific initially assumed to be toxic until definitive Services. identification is made. There are no documented cases of amnesic shellfish poisoning in Australia. Method: HPLC Electrospray MS Domoic acid has not been detected in Victorian Units: µg/100g (ppm) mussels but has been detected in scallops from µ Bass Strait (Arnott et al, 1999). Regulatory Limit: 20 g/100g (total of all DSP toxins)(0.2 mg/kg) Testing Agency: State Chemistry Laboratories, Victoria. Neurotoxic Shellfish Poisons (NSPs) Neurotoxic shellfish poisoning is caused by Method: HPLC brevetoxins produced by the dinoflagellates Units: µg/g (ppm) Karenia brevis, K. cf brevis and perhaps K. mikimotoi. NSP symptoms vary from headaches, µ Regulatory Limit: 20 g/g (20 mg/kg) diarrhoea, muscle and joint pain, and vomiting in mild cases, to paraesthesia, altered perception of Diarrhetic Shellfish Poisons (DSPs) hot and cold and breathing and swallowing A range of DSP toxins such as OA, DTX 1–3 and difficulties in extreme cases. Which species PTX are produced by several species of produce BTX at levels sufficient to cause human dinoflagellate including Dinophysis acuminata, D. intoxication is confounded somewhat by a lack of acuta, D. fortii and Prorocentrum lima. Diarrhetic knowledge of the taxonomy of this group. The shellfish poisons may cause gastrointestinal only NSP incident in Australia was reported in problems including diarrhoea, vomiting and 1994 and resulted from the consumption of abdominal pain; recovery occurs within three wildstock mussels from the Tamboon Inlet in days irrespective of medical treatment Gippsland, Victoria. K. cf brevis was identified as (Hallegraeff, 1997). There have been no reported the organism responsible (Arnott, 1998). cases of diarrhetic shellfish poisoning within the areas covered by the VSQAP. There is no laboratory within Australia which tests routinely for NSPs. Until recently, the only In the past, PTX seco‐acids have been included as test for NSPs was by mouse bioassay which had a DSP toxin. However, recent work in New several limitations for the management of Zealand (MacKenzie 2002) for the Marlborough shellfish harvesting (Holland et al, 2002). LC/MS Sounds Shellfish Quality Program and within methods have been recently developed, validated Australia (Burgess 2002) has shown these and implemented by the Cawthron Institute compounds are not toxic to humans. which provide greater and more reliable Consequently, they are no longer regulated as a information facilitating harvesting management. DSP toxin. Most of the ‘DSP’ found in mussels tested from PPB during Dinophysis acuminata Testing Agency: Cawthron Institute, New Zealand blooms, was PTX‐2‐SA. Method: LC/MS The Commission of European Communities Mouse Bioassay (CEC) recently published draft regulations Units: MU/100g covering DSP toxins in shellfish where it is proposed that a limit of 16 µg/100g total DSP Regulatory Limit: 20 MU/100g content, including OA, DTXs and PTXs (CEC, 2001; Holland et al, 2002), be adopted. This is despite the fact that the EU expert working group on all fat‐soluble marine algal toxins (2001) removed the PTXs and YTXs (discussed later)

Marine Biotoxin Management Plan 13 Yessotoxins (YTXs) Testing Agency: Queensland Health Scientific YTXs and their derivatives have a structure Services. similar to that of brevetoxins but do not have the Method: HPLC Electrospray MS same neurological effects. YTXs and their analogues appear to be produced by a number of Units: Qualitative until standards dinoflagellates including Protoceratium reticulatum are available. and Coolia monotis (Hallegraeff, 2002). YTX has not been detected in Australia to date but P. Regulatory Limit: Not regulated in Australia. reticulatum is found in Australian waters. 4.7 Environmental Information YTX is not regulated in Australia and although it At the same time as phytoplankton/biotoxin is toxic to mice when applied intraperitoneally, its sampling is carried out, salinity and water oral toxicity is questionable (Cawthron Institute, temperature are also recorded and other 2001). Nonetheless, within the VSQAP, qualitative information on meteorological conditions (river analysis is carried out by HPLC/MS by runoff, rainfall, wind speed and direction, Queensland Health Scientific Services using the irradiance) and tidal movement DSP LC/MS method. The CEC draft regulations recommend a limit of 4.8 Reporting and Notification µ 100 g/100g for YTX equivalents (CEC, 2001; • Results from the phytoplankton analyses are Holland et al, 2002). provided to the WES VSQAP Project Testing Agency: Queensland Health Scientific Manager within 24 hours of sample receipt in Services. both hard copy (faxed at the completion of analysis) and electronic form (email). Method: HPLC Electrospray MS • Biotoxin results are faxed to the WES VSQAP Units: Qualitative until standards Project Manager when analysis is complete are available. (two to five days depending on the analysis Regulatory Limit: Not regulated in Australia. required). This is followed by a hard copy sent by mail. Azapiracids (AZA) • If analytical results reveal the presence of Azaspiracid Shellfish Poisoning (AZP) is caused toxic phytoplankton species in significant by a group of toxins with a novel chemical numbers, the DPI Authorised Officer must be structure, called azaspiracids. AZP has occurred informed immediately via phone and email. in Ireland and the symptoms include nausea, vomiting, diarrhoea and stomach cramps. The • If biotoxins are detected within mussel tissue, causative agent appears to be some strains of the the relevant laboratory contacts the WES dinoflagellate Protoperidinium crassipes VSQAP Project Manager immediately by (Hallegraeff, 2002). AZPs have not been detected phone to inform them of the result allowing in Australia or New Zealand. appropriate management action to be taken promptly. DPI VSQAP Authorised Officers AZA is not regulated in Australia. As part of the are contacted and advised immediately by VSQAP, qualitative AZA analysis is carried out telephone. Written confirmation by email is by the Queensland Health Scientific Services made as soon as practicable. using the DSP HPLC/MS method. • The approximate schedule for receiving The CEC draft regulations (CEC, 2001) laboratory results is displayed in Table 2. recommended a limit of 16 µg/100g for AZA equivalents. .

Marine Biotoxin Management Plan 14 Table 2: Approximate schedule for receiving routine sampling results Results Day Methods Sampling 0 Phytoplankton Identification/enumeration 1 Email/fax ASP and DSP Analyses 3 Fax, later by mail

PSP 4 ‐ 5Fax, later by mail

• As individual sets of results are received, monitoring data from that date until the end they are entered into a results report and of 1996. PIRVIC also retain copies of all checked by a second member of the WES reports and monitoring data supplied by VSQAP team. The report is then emailed to WES from 1998 to the present. relevant DPI and WES personnel (all DPI and • A backup copy of all electronic, emailed and WES personnel listed in Appendix 1). This faxed results is stored at the WATER occurs as soon as possible after the receipt of ECOscience Hobart Water Quality Centre. the results (within two hours). • Every six months the WES database is copied • All reports contain comments explaining the to the DPI database. significance of any ‘positive’ results obtained and recommend management actions, where appropriate. 4.10 Contingency Plans for • Should biotoxins be detected in shellfish Marine Biotoxin Events tissue, it is the responsibility of the Contingency plans (management protocols) for appropriate DPI Authorised Officer to notify each of the known nuisance/toxic species the relevant DHS, AQIS and EPA personnel encountered or likely to be encountered in PPB or (see Appendix 2). WP have been formulated. These are attached in Appendix 13. The relevant agency contacts are presented in Appendix 1. Each protocol contains the following: • Title noting to which phytoplankton species 4.9 Data Storage it refers. • A hardcopy of all analytical results (mail, • Background information about the facsimile, email) is stored at the WES Mount phytoplankton concerned, including toxicity. Waverley Water Quality Centre together with the chain of custody/field sampling sheet. • Rationale for the protocol. • Any results received in electronic format are • Step‐by‐step contingency plan. also stored electronically on the WES file • Details of the relevant abundance triggers for storage system. tissue testing and harvest suspension. • Once all analytical results relating to a • Details of the regulatory limits for the sampling event are received, the data are relevant toxins. entered into the WES VSQAP database (Access) and checked by another team member. The data is then extracted as a ‘.csv file’ and emailed to the DPI personnel at the same time as the final report. • The WES VSQAP database contains all testing results from August 1998. • The DPI VSQAP database has been maintained by PIRVIC since the inception of the VSQAP in 1987 and contains all

Marine Biotoxin Management Plan 15 Management protocols have been prepared for: These contingency plans will be implemented in any of the following events: • Alexandrium spp. • If abundance triggers are exceeded for the • Pseudo‐nitzschia spp. relevant phytoplankton listed in Appendix • Dinophysis acuminata, Dinophysis spp., 11. Prorocentrum lima • If any phytoplankton species are present at • Gymnodinium catenatum levels known to be toxic overseas but of unknown toxicity in Australian waters. • Karenia brevis, Karenia cf brevis, Karenia mikimotoi • If biotoxins are present in shellfish flesh. • Rhizosolenia cf chunii • For any other reason as determined by the Authorised Officer. Contingency plans will be reviewed and updated annually, or immediately if any new information or regulation relating to marine biotoxins in shellfish becomes available.

Marine Biotoxin Management Plan 16 5 Area Closure and Reopening

• Where harvesting is suspended in a 5.1 Closure Criteria commercial harvesting area, a closure notice The following criteria determine whether a will be issued within 24 hours by Fisheries closure needs to be implemented: Victoria and communicated (fax, post, email or phone) to the following: • The abundance of potentially toxic phytoplankton species exceeds the trigger for o All Aquaculture (Crown Land) Type A harvest suspension pending toxin analysis (as Licence holders with water specified in well as that for the initiation of biotoxin the affected harvesting area. analysis) as noted in Appendix 11. o All VSQAP personnel on contact lists at • The abundance of potentially toxic DPI and WES. phytoplankton species has not yet exceeded

the trigger for biotoxin testing but is rising o The WES VSQAP Project Manager or rapidly and is likely to exceed the trigger their delegate. prior to the next sampling event. o The Victorian Aquaculture Council. • Biotoxins are present in shellfish at levels o AQIS Market Maintenance Group. over the regulatory limits noted in Appendix 12. o Relevant Department of Human Services personnel. • Confirmed cases of human illness consistent with the case definitions for PSP, NSP, DSP o PrimeSafe. and ASP (Appendix 10) have resulted from o Environment Protection Authority the consumption of shellfish from a particular (Victoria). harvesting area. • Where the presence of biotoxins in shellfish • The Authorised Officer determines a closure tissue is confirmed, the public will need to be is necessary for any other reasons (e.g. informed. Public warnings will be issued by potential toxin producing phytoplankton the Public Health Division, DHS based on species which have not previously been advice provided by Fisheries Victoria, DPI. recorded are present). • A recall of commercial product will be made 5.2 Mechanism for Closure where necessary (Refer to Section 8). The following procedure is useful for the closure 5.3 Industry Instigated Closure of a harvesting area: Industry may choose to instigate a voluntary • The Authorised Officer will close a shellfish closure based on criteria such as pending biotoxin harvesting area to harvesting and embargo testing results, toxins present in neighbouring the movement of all shellfish immediately, if harvesting areas, rising levels of toxic any of the criteria in Section 6.1 above are phytoplankton, the presence of Rhizosolenia cf met. chunii (bitter taste alga) or any other criterion • The closure area will extend to all of the deemed important enough to necessitate a harvesting areas concerned. closure. • Closures should be made on a shellfish 5.4 Reopening Criteria species‐specific basis due to differences in the • abilities of various shellfish to accumulate A shellfish harvesting area closed due to the presence of potentially toxic or unknown toxins. Where several species are involved, each should be tested to determine tissue phytoplankton, and pending biotoxin toxin levels. Within the VSQAP, only blue analysis, may be reopened by the Authorised Officer immediately if the results of biotoxin mussels Mytilus edulis planulatus are currently harvested. testing prove negative.

Marine Biotoxin Management Plan 17 • A shellfish harvesting area closed due to o Once below the regulatory limit, toxin marine biotoxins shall not be reopened until levels are decreasing or static in the the Authorised Officer has determined that required number of consecutive samples each of the following requirements for (dependent on the biotoxin type) in order reopening have been adequately addressed: for the area to be reopened. o The edible portion of each molluscan • Other conditions or limitations may be species harvested from the closed imposed if considered necessary by the harvesting area shall meet the following designated Authorised Officer. criteria: ƒ PSP levels are less than the 5.5 Mechanisms for Reopening regulatory limit of 80 μg saxitoxin The Authorised Officer will reopen a shellfish equivalent /100g edible shellfish harvesting area to harvesting and movement of flesh (80μg/g) as determined by shellfish only when each of the reopening criteria mouse bioassay in three consecutive have been meet: samples from the same site taken • The Authorised Officer shall, on each over a minimum period of 14 days. reopening event, prepare documents ƒ ASP levels are lower than the including the data, environmental conditions regulatory limit of 20 μg domoic and factors leading to that decision. acid/g edible shellfish flesh (20 μg/g • Resumption of harvesting may be or 20ppm), by high performance accompanied by increased monitoring where liquid chromatography (HPLC), in there is a risk of a secondary bloom or low three consecutive samples from the tissue biotoxin levels (less than the regulatory same site taken over a minimum limit) persist. period of 14 days. When harvesting is recommenced in a commercial ƒ DSP levels (not including harvesting area, a reopening notice will be issued pectenotoxin 2 seco‐acids and their by DPI and communicated (fax, post, email or derivatives in mussels) are less than phone) to the following: 20 μg/100g edible shellfish flesh (20 μg/100g) by HPLC/MS, in two o All Aquaculture (Crown Land) Type A consecutive samples from the same Licence holders with water specified in site taken not less than seven days the affected harvesting area. apart. o All VSQAP personnel on contact lists at ƒ NSP levels are less than 20 mouse DPI and WES. units/100g edible shellfish flesh, by o The WES VSQAP Project Manager or ether extraction and mouse bioassay their delegate. with a maximum observation time of six hours, in two consecutive o The Victorian Aquaculture Council. samples from the same site, taken o AQIS Market Maintenance Group. not less than two days apart. o Relevant Department of Human Services o The abundance of toxic phytoplankton personnel. relating to the toxin has shown a clear downward trend and the cell counts are o PrimeSafe. below the threshold level used to initiate

closure (Appendix 11). The Authorised o Environment Protection Authority (Victoria). Officer should consider and judge if the level of other potentially toxic phytoplankton species are increasing and 5.6 Surveillance of Harvesting therefore will not necessitate another Areas closure within a short timeframe. The Compliance Unit of Fisheries Victoria, DPI is responsible for ensuring harvesting does not occur during closure events.

Marine Biotoxin Management Plan 18 6 Investigation of Illness due to Toxic Shellfish Poisoning

Case definitions provide a detailed description of 6.1 Notification the effects of the various TSP syndromes and are Unlike food or water borne pathogens, suspected presented in Appendix 10. cases of toxic shellfish poisoning are not notifiable. 6.3 Immediate Action for 6.2 Investigation Suspected Toxic Shellfish Where there is evidence that TSP are the cause of Poisoning Cases an illness, it is the responsibility of the DHS to 6.3.1 Closures of commercial harvesting areas investigate potential sources of Where investigation indicates toxic shellfish from contamination/illness. PPB or WP shellfish harvesting areas have been the cause of illness, an immediate closure would Toxic shellfish poisoning investigations should be be placed on all of the relevant harvesting areas. undertaken in a timely manner and using sound epidemiological principles. This would ensure Knowledge that victims had consumed shellfish valuable information is gained so that TSP events harvested from one or more of these areas and in Australia can be better understood. As is the were suffering symptoms consistent with those case with any epidemiological investigation the from TSP, together with the presence of toxic aim is the control and prevention of further TSP phytoplankton species above threshold episodes. abundance trigger levels or the presence of biotoxins in shellfish tissue, would constitute All suspected cases of TSP should be investigated. evidence that the consumption of contaminated The investigation should include the following shellfish may be the cause of the incident. preliminary steps (not necessarily in the order below): Public warnings should be issued pending the results of more detailed investigations. The Public • Verify the diagnosis of reported cases and Health Division, DHS, should issue these in identify the specific etiological agent collaboration with Fisheries Victoria, DPI. responsible. • Confirm an incident exists. Check for other 6.3.2 Control of movement of harvested cases at appropriate points such as medical shellfish practices in the relevant area. It is the responsibility of DHS to undertake a product recall/detention, where appropriate, as • Describe the cases in the epidemic or detailed in Section 8, with the cooperation of the outbreak according to the variables of time, appropriate responsible agencies including: place and person. • Office of the Chief Health Officer, Public • Identify the source of the agent and its mode Health Division, DHS (Victoria). of transmission, including the specific vehicles, vectors and routes that may have • PrimeSafe been involved. • Fisheries Victoria, DPI. • Identify the populations that are at an • increased risk of exposure to the agent. All Aquaculture (Crown Land) Type A Licence holders in the relevant harvesting • Plan and implement control measures such as area(s). harvesting suspension, the issue of warnings • and the implementation of recalls. Victorian Aquaculture Council. • • Evaluate the control measures. EPA (Victoria).

Marine Biotoxin Management Plan 19 6.3.3 Notification 6.3.5 Sampling Notices shall be placed in prominent places near A suite of shellfish tissue sampling may be harvesting areas advising the public of the closure necessary to facilitate the investigation of a and to advise against consuming shellfish suspected TSP incident. purchased from growers in the area between the • Shellfish tissue samples should be taken, dates indicated. This notification will be where available, along the distribution undertaken by the SCCA in consultation with pathway from harvesting area to the Food Safety Victoria, Public Health Division, DHS suspected TSP sufferer. These may include (Victoria). remains of meals, samples of commercial product from the same batches of product as 6.3.4 Communication consumed and samples taken from the Liaison between all appropriate organisations and suspected harvesting areas. individuals will be established to ensure that investigations are well coordinated. The • Biotoxin levels in shellfish from each organisations and individuals may include: harvesting area will be available through the VSQAP. Additional sampling and analysis • Office of the Chief Health Officer, Public can be performed, as required. Health Division, DHS (Victoria). • These samples need to be of sufficient size to • Food Safety Victoria, Public Health Division, allow analysis for non‐marine biotoxin DHS (Victoria). sources of illness (such as bacterial, viral or • PrimeSafe chemical contamination) so that these sources can be eliminated as the primary cause of the • Fisheries Victoria , DPI suspected TSP incident. • EPA (Victoria) • If microbiological testing is required, the • All Aquaculture (Crown Land) Type A sample shall be transported in such a way as Licence holders in the relevant harvesting to prevent contamination, and area(s). identified/labelled appropriately. • Victorian Aquaculture Council • For cases showing gastro‐intestinal symptoms, faecal samples should be • AQIS Market Maintenance Group requested to eliminate bacterial/viral causes • WES of illness. 6.3.6 Funding Investigation of toxic shellfish poisoning incidents and the associated sampling and testing is funded by the investigating agency.

Marine Biotoxin Management Plan 20 7 Product Control

Product recall is the responsibility of growers, 7.1 Product Recall manufacturers, processors, distributors and When harvesting areas are closed due to the retailers of affected product, in conjunction with presence of marine biotoxins, and potentially regulators. contaminated shellfish have been harvested prior to closure, product will need to be recalled or Clause 12 of the Food Safety Standard 3.2.2 notes detained. However, VSQAP phytoplankton that: sampling and routine biotoxin testing will usually A food business engaged in the wholesale supply, provide advance warning of any potential risk of manufacture or importation of food must: shellfish biotoxin poisoning, allowing harvesting restrictions to be implemented before potentially (a) have in place a system to ensure the recall of contaminated shellfish are harvested. The recall unsafe food; will include any product harvested since the last (b) set out this system in a written document and satisfactory biotoxin/phytoplankton sampling make this document available to an authorised event and should be initiated within 24 hours of officer on request; and harvest area closure. (c) comply with this system when recalling A food product recall is carried out to protect unsafe food. public health and safety. A food withdrawal may also occur as a precautionary measure prior to an Aquaculture (Crown Land) Type A licence official recall, or for quality or similar reasons holders must comply with the labelling conditions (FSANZ, 2002) of their licences allowing product recall to proceed in an efficient and timely manner. 7.2 Objectives Licence holders must also prepare food recall The Food Industry Recall Protocol – A Guide to plans in accordance with Food Industry Recall Writing A Food Recall Plan and Conducting a Food Protocol (FSANZ, 2002), again permitting efficient Recall (FSANZ, 2002) notes there are three and effective product recall. primary objectives in any food recall: 7.4 Notification • Stop the distribution and sale of an affected product. Notification of food recalls is the responsibility of the business/es concerned. Guidance can be • Inform the statutory authorities (all recalls) provided by FSANZ, DHS or Fisheries Victoria and the public (consumer recalls only) of the (DPI) during the notification process. problem. Notification should include statutory authorities, • Effectively and efficiently remove from the Aquaculture (Crown Land) Type A Licence marketplace any product that is potentially holders in the relevant harvesting area(s), the unsafe. product distribution network, Victorian Aquaculture Council, AQIS, WES and the public 7.3 Responsibilities (should potentially contaminated product reach Product detention and recall will be instigated by the community). Food Safety Victoria, Public Health Division, DHS in accordance with the current Food Industry Recall Protocol (FSANZ, 2002). This process details the domestic and international recall processes, consumer notification, product detainment and disposal. PrimeSafe will also have the power to instigate product detention and recall in the future.

Marine Biotoxin Management Plan 21 8 References

Arnott, G.H., Reilly, D.J. and Werner, G.F. (1999). Holland, P.T., McNabb, P., Selwood, A., Page, T., Victorian Shellfish Quality Assurance Program. 7. Bell, K. and MacKenzie, L. (In Press). Marine Sanitary Survey Update: Clifton Springs and Grassy Biotoxin Monitoring of New Zealand Shellfish – A Point Portarlington) Aquaculture Zones. Marine and New Management Programme Based on LC‐MS. Freshwater Resources Institute, Report No. 13, In: Proc. 2nd Int. Conference on Harmful Algae 1 ‐ 36 Management and Mitigation. Nov 2001, Qingdao, China. Hall, S. & Zou, YL (Ed.) ASQAAC (2001). Australian Shellfish Quality Assurance Program Operations Manual MacKenzie, L. (2002). An Evaluation of the Risk to Consumers of Pectenotoxin 2 seco acid (PTX2‐SA) Commonwealth of Australia (1992). Export Contamination of Greenshell™ Mussels. Prepared Control (processed Food) Orders. No. 9 of 1992 for Marlborough Sounds Shellfish Quality Commission of European Communities (2001). Programme. Cawthron Institute Report 750, 1‐ 50 Draft Commission Decision of Establishing the MacKenzie, L., Holland, P., McNabb, P., Methods of Analysis and the Maximum Limits for Beuzenberg, V., Selwood, A. and Suzuki, T. Certain Marine Biotoxins in Bivalve Molluscs, (2002). Complex Toxin Profiles in Phytoplankton Echinoderms, Tunicates and Marine Gastropods. and Greenshell Mussels (Perna canaliculus), Brussels, SANCO/2227/2001 Rev 3 Revealed by LC‐MS/MS Analysis. Toxicon, 40: FSANZ (2002a). Food Standards Code. Food 1321 – 1330 Standards Australia New Zealand Todd, K. (2001). Australian Marine Biotoxin FSANZ (2002b). Food Industry Recall Protocol. A Management Plan for Shellfish Farming. Prepared Guide to Writing a Food Recall Plan and Conducting a for the Australian Shellfish Quality Assurance Food Recall. 5th Edition. Food Standards Australia Committee (ASQAAC) by the Cawthron Institute. and New Zealand. Cawthron Report No. 645 Hallegraeff, G. (1997). Algal toxins in Australian Todd, K. (2002). A Review of NSP Monitoring in Shellfish. In: Foodborne Microorganisms of Public New Zealand In Support of a New Programme. Health Significance. Fifth Edition. AIFST (NSW Prepared for Marine Biotoxin Technical Branch), Food Microbiology Group. Committee. Cawthron Report No. 660, 1 ‐ 30 Hallegraeff, G. (2002). Aquaculturistsʹ Guide to WATER ECOscience (2002). WATER ECOscience Harmful Australian Microalgae. The Print Centre, VSQAP Operations Manual. Hobart.

Marine Biotoxin Management Plan 22 Appendix 1: Agency & Personnel Contacts

Agency / Contact Responsibility Contact Details DPI, Fisheries Victoria State Shellfish Control Authority PO Box 500, East Melbourne, VIC 3002 Level 6, 232 Victoria Parade, East Melbourne, VIC 3002 (03) 9412 4011 (Phone) (03) 9412 5731 (Fax)

Dr Peter Appleford (1) Aquaculture Planning Officer 03 9412 5703 Authorised Officer 0412 797 720 03 9412 5770 (F) [email protected] Anthony Forster (2) Manager Aquaculture 03 9412 5710 SQAP Manager 0419 871 096 Authorised Officer 03 9412 5770 (F) [email protected] John Mercer (3) Aquaculture Extension Officer 03 5258 0218 03 5258 0270 (F) 0407 540 562 [email protected] DPI, PIRVic Aquaculture Research PO Box 114, Queenscliff, VIC 3225 (03) 5258 0111 (Phone) (03) 5258 0270 (Fax)

Neil Hickman (1) Senior Scientist (Aquaculture) 03 5258 0336 Authorised Officer 03 5258 0270 (F) [email protected] Richard Gasior (2) Scientist 03 5258 0336 03 5258 0270 (F) 0407 307 075 [email protected] DPI, Fisheries Victoria, Port Phillip Bay Region Cnr Little Malop and Fenwick St, Geelong, Port Phillip Region VIC 3215

Peter Lawson Port Phillip Region Aquaculture 03 5226 4510 Project Manager 03 5226 4725 (F) 0419 875 840 [email protected]

WATER ECOscience VSQAP Monitoring, Reporting, Private Bag 1, Mount Waverley, VIC 3149 Growing Area Assessment 68 Rickettʹs Rd., Mount Waverley, VIC 3149 (03) 9550 1000 (Phone) (03) 9543 7372 (Fax)

Rebecca Coello (1) WES VSQAP Project Manager 03 9550 1046 03 9543 7372 (F) [email protected] Chris Wood (2) Group Manager, Water Quality 03 9550 1050 Centres 03 9543 7372 (F) 0407 823 229 [email protected] Dr Terry Walker (E) WES VSQAP Technical Director 03 6233 2143 Phytoplankton/Biotoxins 03 6272 1908 (F) 0407 863 590 [email protected] Address: PO Box 34, New Town, TAS 7008 2 ‐ 4 Negara Crescent, Goodwood, TAS 7010

Marine Biotoxin Management Plan 23 Agency / Contact Responsibility Contact Details Office of Chief Health Department of Human Services Officer, DHS GPO Box 1670N Melbourne VIC 3001 Dr Robert Hall 03 9637 4204 03 9637 4250 (F) [email protected] Food Safety Unit, DHS Department of Human Services Food Unit, GPO Box 4057, Melbourne, VIC 3000 (03) 9637 4094 (Phone) (03) 9637 5212 (Fax) Website: www.foodsafety.vic.gov.au

Victor Di Paola State Coordinator, Food Recall 03 9637 4893 03 9637 5212 [email protected] AQIS Export Market Maintenance Group, AQIS Dept. of Agriculture, Fisheries and Forestry Australia GPO Box 858, Canberra, ACT 2601 Edmund Barton Building, Barton, ACT 2601 02 6272 3933 (Phone) 02 6272 5161 (Fax) Website: www.aqis.gov.au

Les Johns Senior Food Export Inspector 02 6272 4978 02 6272 3238 (F) [email protected] Mark Kelly Senior Policy Officer 02 6271 6537 02 6271 6522 (F) [email protected] Andrew Buckley Senior Policy Officer 02 6272 4017 02 6271 6522 (F) [email protected] EPA Victoria Marine Science ‐ Environmental Centre for Environmental Sciences monitoring & policy Ernest Jones Drive La trobe University Research & Development Park Macleod, VIC, 3085 03 8458 2300 (Phone) 03 8458 2301 (Fax) Dr Brett Light Environmental Chemist 03 8458 2453 03 8458 2444 (F) VAC Aquaculture industry peak body The Victorian Aquaculture Council Inc 366 The Esplanade Indented Head, VIC 3223

Steve Rodis President 03 5257 2033 03 5257 1544 (F) 0407 311 551 [email protected] (1) Primary Contact (2) Secondary Contact (E) Emergency Contact

Marine Biotoxin Management Plan 24 Appendix 2 Communication Network Diagram

WATER ECOscience ROUTINE FORTNIGHTLY MONITORING - Phytoplankton identification/enumeration - Tissue biotoxin analysis (PSP)

PSP

WATER ECOscience State Shellfish Control Authority Growers YES NO Phytoplankton abundance trigger Immediate notification and report Harvesting suspension exceeded to Authorised Officer(s) Pending biotoxin analysis

State Shellfish WATER ECOscience Growers Control Authority Tissue biotoxin testing Harvesting suspension Report to protocols initiated lifted immediately Authorized Officer(s) State Shellfish Control Authority Immediate notification and report Testing Laboratories to Authorised Officer(s) Samples to testing labs for analysis Department of Human Services Environment Protection Authority - Victorian Aquaculture Council WATER ECOscience Aust. Quarantine Insp. Service Reported to WES Notified immediately Immediate notification if + State Shellfish Control Authority Immediate notification and report Growers + to Authorised Officer(s) Harvesting suspension

Growers Testing Laboratories WATER ECOscience Harvesting suspension Samples to testing Biotoxin/phytoplankton monitoring Continued labs for analysis continued at appropriate frequency Advised of –ve result while result +ve and likely re-opening dates

WATER ECOscience State Shellfish Control Authority Department of Human Services Monitoring continues Immediate notification and report Environment Protection Authority Result - ve to Authorised Officer(s) Victorian Aquaculture Council But re-opening criteria NOT met Aust. Quarantine Insp. Service Notified immediately

WATER ECOscience State Shellfish Control Authority Growers Result remains negative Immediate notification and report Harvesting suspension Re-opening criteria met to Authorised Officer(s) lifted

Marine Biotoxin Management Plan 25 Appendix 3: Approved Laboratories and Contacts for Phytoplankton Enumeration and Identification

Agency / Contact Capability/Position Contact Details WATER ECOscience Phytoplankton Identification and Private Bag 1, Mount Waverley, VIC 3149 Botany Enumeration 68 Rickettʹs Rd., Mount Waverley, VIC 3149 (NATA Accreditation No. 922) (03) 9550 1000 (Phone) (03) 9543 7372 (Fax) Dr Kumar Eliezer (1) Senior Botanist 03 9550 1059/1061 03 9543 7372 (F) [email protected] Lisa Stephenson (2) Biological Analyst 03 9550 1061 03 9543 7372 (F) [email protected]

University of Tasmania Phytoplankton Identification, GPO Box 252‐255, Hobart, TAS, 7001 School of Plant Sciences Electron‐ microscopy, 03 6226 2999 (Phone) 03 6226 2603 (Plant Phytoplankton Culture, DNA Science Phone) Probes Prof Gustaaf Hallegraeff Assoc. Prof. Aquatic Botany 03 6226 2623 (1) 03 6226 2698 (F) [email protected]

Miguel de Salas (2) Algal Taxonomist 03 6226 2603 03 6226 2698 (F) Miguel [email protected]

(1) Primary Contact (2) Secondary Contact (E) Emergency Contact

Marine Biotoxin Management Plan 26 Appendix 4: Approved Laboratories and Contacts for Marine Biotoxin Analysis of Shellfish Flesh

Agency / Contact Responsibility/Position Contact Details State Chemistry Labs, ASP (Domoic Acid) Analysis Cnr Sneyde & South Roads, Werribee, VIC Victoria (HPLC) 3030 Food Chemistry Unit (NATA Accreditation No. 36) (03) 9742 8755 (Phone) (03) 9742 8700 (Fax) Email: [email protected] Dr Craige Trenerry (1) Food Chemistry Unit Manager 03 9742 8715 03 9742 8700 (F) 0407 050 313 [email protected] Paul Lawicki (2) Senior Chemist 03 9742 8752 03 9742 8700 (F) [email protected]

Medvet Sciences, IMVS PSP Analysis PO Box 14, Rundle Mall, SA 5000 Food & Environmental (Mouse Bioassay) Frome Road, Adelaide, SA 5000 Laboratory (NATA Accreditation No. 1521) (08) 8222 3194 (Phone) (08) 8222 3695 (Fax) Email: [email protected] Chris Murray (1) Laboratory Manager 08 8222 3194 08 8222 3695 (F) 0401 120 724 [email protected] Peter Cameron (2) Laboratory Supervisor 08 8222 3363 08 8222 3695 (F) [email protected]

Queensland Health DSP Analysis PO Box 594, Archerfield, QLD 4108 Scientific Services (HPLC Electrospray MS) 39 Kesselʹs Road, Cooperʹs Plains, QLD 4108 07 3274 9111 (Phone) 07 3274 9119 (Fax) Dr Geoff Eaglesham (1) Senior Scientist 07 3274 9085 07 3274 9186 (F) [email protected] Brad Davis (2) Scientist 07 3274 9085 07 3274 9186 (F) [email protected] Cawthron Institute NSP Analyses Private Bag 2 Nelson, New Zealand Biotoxin Laboratory (LC‐MS, Mouse Bioassay) 98 Halifax Street East, Nelson, New Zealand +64 3 548 2319 (Phone) +64 3 546 9464 (Fax) Paul McNabb (1) Section Head, Biotoxin Laboratory +64 3 548 2319 +64 3 546 9464 (F) [email protected] Tracey Neil (3) Laboratory Analyst +64 3 548 2319 +64 3 546 9464 (F) [email protected] Nico van Loon (E) Laboratory Services Manager +64 3 548 2319 +64 3 546 9464 (F) [email protected] (1) Primary Contact (2) Secondary Contact (E) Emergency Contact

Marine Biotoxin Management Plan 27 Appendix 5: Sampling Officers

Agency / Contact Responsibility Contact Details West Coast Diving VSQAP Field Sampling 25 Rodney Road, North Geelong, VIC 3215 03 5278 5426 (Phone/Fax) Lyall Mills (1) Field Sampling 03 5278 5426 (Ph/Fax) 0417 364 998 [email protected] Daniel Peters (2) Field Sampling 03 5285 2307 Tony Patterson (3) Field Sampling 0407050606 03 5278 4098 Max Ritchards (4) Field Sampling 03 5278 3868 (1) Primary Contact (2) Secondary Contact (E) Emergency Contact

Marine Biotoxin Management Plan 28 Appendix 6: Marine Biotoxin Analytical Methods

Paralytic Shellfish Poison (PSP) Draisci, R., Palleschi, L., Giannetti, L., James, K.J., Bishop, A.G., Satake, M., Yasumoto, T. New Association of Official Analytical Chemists Official approach to the direct detection of known and Method 959.08 Paralytic Shellfish Poison. 17th edition new diarrhoeic shellfish toxins in mussels and 2000. phytoplankton by liquid chromatography‐mass Amnesic Shellfish Poison (ASP) spectrometry. Journal of Chromatography A, 847 (1999), 213‐221 SCL method number 20113. Based on Association of Official Analytical Chemists Official Method 991.26 Cawthron Institute, Nelson, New Zealand, Domoic acid in mussels. Changes in official methods of Method 40.105, Determination of ASP and DSP analysis of the AOAC Second Supplement p.103 1991. toxins in shellfish by LC‐MS.

Diarrhetic Shellfish Poison (DSP) Mackenzie, L., Holland, P., McNabb, P., Beuzenberg, V., Selwood, A., Suzuki, T. 2002: Diarrhetic Shellfish Poisons are analysed using a Complex toxin profiles in phytoplankton and methanol extraction of macerated shellfish. The Greenshell mussels (Perna canaliculus), revealed extracted toxins are determined using HPLC/Mass by LC‐MS/MS analysis. Toxicon 40: 1321‐1330. Spectrometry with an electrospray source run in both positive and negative ion mode. Esters of okadaic acid Neurotoxic Shellfish Poison (NSP) (DTX3) are hydrolysed to okadaic acid using base Mouse bioassay based on ether extraction of hydrolysis based on the method of Mountford et al (see shellfish. below). Some references used in developing this method are listed below. APHA recommended procedures for the examination of seawater and shellfish 4th ed. 1970, Part V B Method Mountford, D.O., Suzuki, T., Truman, P. Protein for the Bioassay of Gymnodinium beve toxin (s) in phosphatase inhibition assay adapted for shellfish determination of total DSP in contaminated mussels. Toxicon 39 (2001), 383‐390 LC‐MS method developed in‐house at the Cawthron Institute. Goto, H., Igarashi,T., Yamamoto M., Yasuada, M., Sekiguchi, R., Watai, M., Tanno, K., Yasumoto, T. Cawthron Institute, Nelson, New Zealand, Method Quantitative determination of marine toxins 40.106, Determination of NSP toxins in shellfish by associated with diarrhetic shellfish poisoning by LC‐MS. liquid chromatography coupled with mass spectrometry. Journal of Chromatography A, 907 (2001), 181‐189

Marine Biotoxin Management Plan 29 Appendix 7: MBMP Sampling Collection Form and Proformas for External Laboratories CHAIN OF CUSTODY

(VSQAP) VICTORIAN SHELLFISH QUALITY ASSURANCE PROGRAM Date Sampled:______/______/______Sampled By:______

LABORATORY : WATER ECOscience Pty Ltd ROUTINE ANALYSIS REQUIRED ADDRESS : 68 Ricketts Road (L) Mt Waverley 3149

TELEPHONE : 03 9550 1000

FACSIMILE : 03 9543 7372 OLLECTED SAMPLED

C ATITUDE ONGITUDE

L L CONTACT : Rebecca Hayman ATER

SAMPLE TYPE/METHOD: MARTOXPP AMPLED W

S

AMPLED S

SAMPLE SITE LAB REFERENCE (LAB USE USSEL

IDENTIFICATION ONLY) OLUME IME EMPERATURE HYTOPLANKTON ALINITY C P V M T T o S Clifton Springs (VSQCLIFTON)

Grassy Point (VSQGRASSY)

Dromana (VSQDROMANA)

Flinders (VSQFLINDER)

RELINQUISHED BY : RECEIVED BY :

SIGNED BY : DATE : TIME : SIGNED BY : DATE : TIME :

Weather Conditions/Observations

Nuisance Algae ID within 24 hours Nuisance Algae Enumeration within 48 hours (if required) Temperature/Salinity at mussel site within Harvest Area

Marine Biotoxin Management Plan 30 Appendix 7 continued

Paralytic Shellfish Poisoning (PSP)

Direct Dial: (03) 9550 1046 WATER ECOscience Pty Ltd 11 June 2003 ACN 064 477 989

Private Bag 1 Mount Waverley Victoria 3149 Australia

Attn: Chris Murray Telephone 61 3 9550 1000 MEDVET Food & Environmental Laboratory Facsimile 61 3 9543 7372 Frome Road Email [email protected] ADELAIDE SA 5000

Dear Chris

RE: VICTORIAN SHELLFISH QUALITY ASSURANCE PROGRAM

Please find enclosed four mussel tissue samples for Paralytic Shellfish Poison (PSP) analysis taken on the 28th January 2003. The purchase order number for this work is *****. Please quote this order number on the invoice for this work. Our client code is ****** The mussel tissue is from: Clifton Springs Grassy Point Dromana Flinders

I look forward to the results.

Yours sincerely

VSQAP team member

Marine Biotoxin Management Plan 31 Amnesic Shellfish Poisoning (ASP)

Direct Dial: (03) 9550 1046

5 March 2004 WATER ECOscience Pty Ltd ACN 064 477 989

Attn: Craige Trenerry STATE CHEMISTRY LABORATORY Private Bag 1 Mount Waverley Food Chemistry Unit Victoria 3149 Australia Corner Sneyde and South Roads WERRIBEE VIC 3030 Telephone 61 3 9550 1000 Facsimile 61 3 9543 7372 Dear Craige Email [email protected]

RE: VICTORIAN SHELLFISH QUALITY ASSURANCE PROGRAM

Please find enclosed two mussel tissue samples for Amnesic Shellfish Poison (ASP) (domoic acid) analysis taken on the 28th January 2003. The purchase order number for this work is *****. Please quote this order number on the invoice for this work. I understand our client code is ******* The mussel tissue samples are from:

Clifton Springs Flinders

I look forward to the results.

Yours sincerely

VSQAP team member

Marine Biotoxin Management Plan 32 Diarrhetic Shellfish Poisoning (DSP)

Direct Dial: (03) 9550 1046

11 June 2003 WATER ECOscience Pty Ltd ACN 064 477 989

Attn: Dr Geoff Eaglesham Private Bag 1 Mount Waverley Victoria 3149 Australia Queensland Health Scientific Services 39 Kessel’s Road Cooper’s Plains Telephone 61 3 9550 1000 Queensland….4108 Facsimile 61 3 9543 7372 Email [email protected]

Dear Geoff

RE: VICTORIAN SHELLFISH QAULITY ASSURANCE PROGRAM

Please find enclosed one mussel tissue sample for Diarrhetic Shellfish Poison (DSP) analysis taken on 16/10/00 at Grassy Point.

I look forward to the results.

Yours sincerely

VSQAP team member

Marine Biotoxin Management Plan 33 Neurotoxic Shellfish Poisoning (NSP)

Direct Dial: (03) 9550 1046

11 June 2003 WATER ECOscience Pty Ltd ACN 064 477 989

Attn: Paul McNab or Karyn Bell Biotoxin Laboratory Private Bag 1 Mount Waverley Cawthron Institute Victoria 3149 Australia 98 Halifax Street East Nelson NEW ZEALAND Telephone 61 3 9550 1000 Facsimile 61 3 9543 7372 Email [email protected] Dear Paul/Karyn

RE: VICTORIAN SHELLFISH QUALITY ASSURANCE PROGRAM

Please find enclosed one mussel tissue sample for Neurotoxic Shellfish Poison (NSP) analysis taken on the 16/10/00 at Grassy Point (Sample No. ******). Our client code is ******.

I look forward to the results.

Yours sincerely

VSQAP team member

Marine Biotoxin Management Plan 34 Appendix 8: Phytoplankton Sampling Procedures Collecting Phytoplankton samples for enumeration using the hosepipe sampler

Purpose: Collect sample • Lower weighted (bottom) end very slowly to To collect a depth integrated sample of appropriate depth, to avoid disturbing any phytoplankton for enumeration over the entire layers of phytoplankton in the water column depth of the mussel lines appropriate to industry practice. This is preferred over a surface sample • Take care not to hit the bottom, particularly at due to variation in the vertical distribution of low tide and/or where there is a swell phytoplankton. • If the bottom is hit, discard the sample, clean Equipment: the sampler and retake the sample at a lesser depth. • 25mm internal diameter hosepipe sampler of appropriate length (marked at one metre Retrieve sample intervals and weighted at bottom end) • Replace bung securely in top of tube and pull the bottle and the sample up into the boat; • Strong line attached to bottom of sampler at make sure the bung remains firmly in place. the weight • Insert the bottom end of hosepipe into the • Spare bungs for hosepipe sampler bucket, remove the bung and empty sample • Clean bucket (>12L volume) into the bucket. • 1L sample bottles ‐ one for each sample taken Fill sample bottles plus spares • Label sample bottle(s) with time and date of sampling, sample type and harvesting area • Labels (and preservatives if required) • Gently mix sample in bucket • Eskies for transporting samples • Subsample by lowering a plastic, labelled 1L Care is to be taken that ALL equipment is attached bottle into bucket and fill, leaving a 10cm air securely to the boat. space at top; cap bottle firmly Method: • Fill required number of plastic bottles with Prepare hosepipe sampler sample water • Make sure top end is firmly attached to the • Store samples in an esky with one icepack to boat. keep cool (icepack NOT in contact with • Ensure bottom line is attached firmly both to samples – the purpose is merely to keep esky the bottom of the hosepipe and the other end cool while samples are transported to the to the boat laboratory) • Remove bung from end • Generally samples are returned to the laboratory live but some can be preserved with Lugolʹs iodine or other preservatives in the field. If samples are preserved, note on the label clearly.

Marine Biotoxin Management Plan 35 Collecting phytoplankton using the plankton net Purpose: Collect sample To collect a concentrated phytoplankton sample • Lower net to an appropriate depth along the entire depth of the mussel lines for the • Do not allow the net weight to hit the bottom purposes of detecting and identifying nuisance (clean net and repeat sample if it does) species, including those that may be present in low numbers. • Slowly but steadily pull the net up to the boat Equipment: • Wash material adhering to inside of net • 20µm mesh plankton net of 300mm diameter towards the net bottle end by gently dipping and one metre length and shaking the net. • 75mL polycarbonate net bottles fitting the Fill and store sample bottles plankton net end • Carefully remove 75mL bottle from the net • 125mL plastic sample storage vials end • Labels • Transfer sample to the appropriate, labelled 125mL storage vials leaving a 10 ‐ 20mm air • Lugolʹs iodine or other preservative if space. required • Cap tightly and store with other algal • Weight to attach to plankton net to facilitate samples in an esky with a single ice pack (ice sinking pack NOT in contact with samples – the purpose is merely to keep esky cool while Method: samples are transported to the laboratory) Check equipment • Ensure net line is firmly attached both to the • If further samples are required, wash net and net and the boat repeat as above • Wash net and bottle prior to use • Generally samples are returned to the laboratory live but some can be preserved • Attach plastic bottle onto net with Lugolʹs iodine or other preservatives in • Label and prepare 125mL sample storage the field. If samples are preserved, note on vials. the label clearly. • Wash the plankton net and net bottle prior to leaving the site or taking additional samples.

Marine Biotoxin Management Plan 36 Appendix 9: Phytoplankton Species Lists The following lists are presented to summarise the same area. In addition, there are records of the the phytoplankton species that potentially introduction of new forms in recent years through produce biotoxins and present a potential risk of agents such as ballast water e.g. Gymnodinium human illness resulting from the consumption of catenatum into South East Tasmanian waters. The shellfish contaminated with these toxins. potential therefore exists for the introduction of toxic species or strains not seen in an area It must be stressed the tables are ‘all inclusive’ previously. and there is great variability in the level of evidence resulting in the inclusion of species as Categories A2 – C are essentially reproduced from potentially toxic. This evidence varies from the the Australian Marine Biotoxin Management Plan for circumstantial (e.g. the species was present during Shellfish Farming (2001). In addition, detailed a single incident at one locality which had several records of phytoplankton occurrence and biotoxin potential causes, one of which was biotoxins) to presence in shellfish have been collected as part of very powerful evidence of widespread toxicity the VSQAP extending back to 1987. These records, supported by detailed biotoxin studies. The tables together with other information presented in the are presented as a guide and it is crucial they be literature, has enabled an additional list to be modified to incorporate local and international compiled of potentially toxic phytoplankton information as it comes to hand, and that specific to the areas covered by the VSQAP. This management decisions are made with full is presented as Category A1 and is modified from awareness of why a species was listed as Category A as presented in the Australian Marine potentially toxic. Biotoxin Management Plan for Shellfish Farming (2001). It must be stressed this list should only be Nonetheless, all records of toxicity should be used in relation to those harvesting areas covered examined carefully as the toxicity of specific algal by the VSQAP to date, PPB and WP. species may vary substantially between different Phytoplankton listed as Category A1 may also be geographical areas and even from time to time at included in the Category A2 list.

Marine Biotoxin Management Plan 37 Category A1: Species occurring in south‐eastern Australian waters, which are known or suspected toxin producers in Australia. Species Toxins/Comments Bacillariophyceae (Diatoms) Pseudo‐nitzschia australis ASP (domoic acid) Pseudo‐nitzschia multiseries ASP (domoic acid) Pseudo‐nitzschia delicatissima NT in PPB & Tas ASP (domoic acid) overseas Pseudo‐nitzschia pungens NT in PPB and Bass Strait Toxic strains elsewhere ‐ ASP (domoic acid) Pseudo‐nitzschia pseudodelicatissima NT in PPB, Vic, NSW One of main bloom species in PPB, Vic and Tas Toxic strains elsewhere? ‐ ASP (domoic acid) Pseudo‐nitzschia multistriata NT in Aust? Very common. ASP (domoic acid) New Zealand (weakly toxic) Rhizosolenia cf chunii NT but produces a bitter taste in mussels, oysters and scallops in PPB. Dinophyceae (dinoflagellates)

Alexandrium catenella PSP (Saxitoxins, C1 ‐ C4, gonyautoxins) Alexandrium tamarense NT in all Australian isolates so far – some toxic strains?

PSP (Saxitoxins, C1 ‐ C4, gonyautoxins) Alexandrium fundyense A. fundyense from PPB shown to be A. catenella Alexandrium minutum PSP (Saxitoxins, mostly gonyautoxins) Alexandrium ostenfeldii Not linked to toxicity in Aust Sometimes toxic NZ – saxitoxins and derivatives Canada ‐ spirolides Dinophysis acuminata NT in PPB? OA (traces only), ?DTX 3 (not tested yet) Dinophysis caudata ?DSP (?OA, ?DTX 1 – 3) Dinophysis fortii ?DSP (?OA, ?DTX 1 – 3) Dinophysis acuta ?DSP (?OA, ?DTX 1 – 3) Dinophysis miles ?DSP (?OA, ?DTX 1 – 3) Dinophysis tripos ?DSP (?OA, ?DTX 1 – 3) Prorocentrum lima ?DSP (?OA, ?DTX 1 – 3) Gymnodinium catenatum PSP (sulphamate saxitoxins) Karenia cf brevis ?NSP (BTX) Karenia mikimotoi ?NSP (?BTX) – NR of toxicity in Aust to date Low BTX levels in NZ; Gymnocin in Japan NT = Non Toxic PPB = Port Phillip Bay Vic = Victoria Tas = Tasmania NZ = New Zealand Aust = Australia OA = Okadaic acid DTX = Dinophysis toxins DTX3 = diol esters BTX = brevetoxins ? Indicates this toxin has not been confirmed in Australian strains of this species, at the time of this report.

Marine Biotoxin Management Plan 38 Category A2: Species known to be present in south‐eastern Australian waters and proven to produce toxins either in Australia or internationally. (Modified from Australian Marine Biotoxin Management Plan for Shellfish Farming (2001) Species Toxins/Comments Pseudo‐nitzschia australis ASP (domoic acid) Pseudo‐nitzschia delicatissima ASP (domoic acid) Pseudo‐nitzschia fraudulenta ASP (domoic acid); NT Australia, weakly toxic NZ Pseudo‐nitzschia multiseries ASP (domoic acid) Pseudo‐nitzschia ASP (domoic acid) pseudodelicatissima Pseudo‐nitzschia pungens ASP (domoic acid) Usually NT but some strains produce high ASP levels ASP (domoic acid) Pseudo‐nitzschia turgidula ASP (domoic acid); NT Australia, weakly toxic NZ Alexandrium catenella PSP (saxitoxin and derivatives) Alexandrium minutum PSP (saxitoxin and derivatives) Alexandrium ostenfeldii PSP (saxitoxin and derivatives) Spirolides in Canada Alexandrium tamarense PSP (saxitoxin and derivatives) Also has non‐toxic strains Dinophysis acuminata DSP (OA?, DTX 1 – 3?) Dinophysis acuta DSP (OA?, DTX 1 – 3?) Dinophysis caudata DSP (OA?, DTX 1 – 3?) Dinophysis fortii DSP (OA?, DTX 1 – 3?) Dinophysis hastata DSP (OA?, DTX 1 – 3?) Dinophysis mitra DSP (OA?, DTX 1 – 3?) Dinophysis rotundata DSP (OA?, DTX 1 – 3?) Dinophysis tripos (DSP (OA?, DTX 1 – 3?) Some strains only Gymnodinium catenatum PSP (saxitoxin and derivatives) Karenia cf brevis NSP (brevetoxins) Prorocentrum lima DSP (OA?, DTX 1 – 3?) Pyrodinium bahamense var. Tropical habitats compressum PSP (saxitoxin and derivatives) NT = Non Toxic ? DTX 3 = OA esters Indicates this toxin has not been confirmed in Australian strains of this species, at the time of this report .

Marine Biotoxin Management Plan 39 Category B: Potential toxin producing species (i.e. toxicity untested/unclear) known to be present in Australian coastal waters including species known/suspected to be toxic overseas (Modified from Australian Marine Biotoxin Management Plan for Shellfish Farming (2001). Species Toxins/Comments Pseudo‐nitzschia cuspidata Possibly ASP (domoic acid) Pseudo‐nitzschia heimii Possibly ASP (domoic acid) Non‐toxic in New Zealand; toxicity unknown elsewhere Pseudo‐nitzschia lineola Possibly ASP (domoic acid) Pseudo‐nitzschia multistriata Possibly ASP (domoic acid) Non‐toxic in New Zealand Pseudo‐nitzschia subfraudulenta Possibly ASP (domoic acid)

Pseudo‐nitzschia subpacifica Possibly ASP (domoic acid) Alexandrium pseudogonyaulax Possibly PSP (STX and derivatives, goniodomin) Chattonella marina/antiqu Possibly NSP (brevetoxins) Fibrocapsa japonic Possibly NSP (brevetoxins) Heterosigma akashiwo Possibly NSP (brevetoxins)

NT = Non Toxic STX = saxitoxin

Marine Biotoxin Management Plan 40 Category C: Other potential toxin producing species world‐wide that may be present in Australian waters (Modified from Australian Marine Biotoxin Management Plan for Shellfish Farming (2001). Species Toxins/Comments Alexandrium angustitabulatum Possibly PSP (saxitoxin and derivatives) Present in New Zealand Alexandrium acatenella Possibly PSP (saxitoxin and derivatives) Alexandrium cohorticula Possibly PSP (saxitoxin and derivatives) Alexandrium fraterculus Possibly PSP (saxitoxin and derivatives) Alexandrium fundyense Possibly PSP (saxitoxin and derivatives) Alexandrium lusitanicum Possibly PSP (saxitoxin and derivatives) Alexandrium tamiyavanichi Possibly PSP (saxitoxin and derivatives) Coolia monotis Cooliatoxin Dinophysis norvegica Major DSP producer in Europe Gymnodinium aureolum Possibly NSP (brevetoxins) Low levels of BTX in New Zealand; NT in Aust? Gymnodinium impudicum Possibly NSP (brevetoxins) Low levels of BTX in New Zealand? Gymnodinium pulchellum Possibly NSP (brevetoxins) NT in PPB and Aust? Karenia bidigitata Possibly NSP (brevetoxins) Low levels of BTX in New Zealand? Karenia brevisulcata Wellington Harbour Toxin (WHT) Low levels of BTX in New Zealand? Karenia papilionacea Possibly NSP (brevetoxins) Karenia selliformis Gymnodimine and low BTX levels ‐ New Zealand Karlodinium micrum Possibly NSP (brevetoxins) – low BTX levels in NZ Lingulodinium polyedra Yessotoxins in Japan Nitzschia navis‐varingica ASP(domoic acid) in brackish Vietnamese waters Ostreopsis siamensis Ostreotocin Pfiesteria piscicida Toxin being characterised Prorocentrum concavum DSP (OA?, DTX 1 – 3?) Prorocentrum elegans DSP (OA?, DTX 1 – 3?) Prorocentrum hoffmannianum DSP (OA?, DTX 1 – 3?) Prorocentrum maculosum Prorocentrolides Prorocentrum minimum The toxin linked to this organism (185 fatalities in Japan) has not yet been elucidated, and the role of P. minimum is still in question Protoceratium reticulatum Yessotoxin producer in New Zealand NT = Non Toxic PPB = Port Phillip Bay BTX = brevetoxin DTX 3 = OA esters ? Indicates this toxin has not been confirmed in Australian strains of this species, at the time of this report .

Marine Biotoxin Management Plan 41 Appendix 10: Toxic Shellfish Poisoning Case Definitions Surveillance Case Definition for all Forms of Paralytic Shellfish Poisoning (PSP) Case Toxic Shellfish Poisoning Definition Suspected case (general clinical case definition) Suspected case (clinical case definition) • Vomiting or diarrhoea occurring within 24 The following neurological symptoms occurring hours of consuming shellfish. within 12 hours of consuming shellfish: • Any of the following neurological symptoms • Neurosensory paraesthesia (i.e. numbness or occurring within 24 hours of consuming tingling around the mouth, face or shellfish. extremities). Neurosensory symptoms: • And one of the following neuromotor/neuro‐ cerebellar symptoms: o Paraesthesia (i.e. numbness or tingling around the mouth, face or extremities). o Weakness such as trouble rising from seat or bed o Alternation of temperature sensations such as a prickly feeling on the skin o Difficulty in swallowing during a bath/shower or exposure to sun, o Difficulty in breathing or difficulty distinguishing hot or cold objects. o Paralysis Neuromotor/neurocerebellar symptoms: o Clumsiness o Weakness such as trouble rising from o Unsteady walking seat or bed o Dizziness/vertigo o Difficulty swallowing o Slurred/unclear speech o Difficulty breathing o Double vision. o Paralysis Probable case o Clumsiness • Meets the case definition. o Unsteady walking • And within seven days of the collection of shellfish consumed by the case, PSP biotoxins o Dizziness/vertigo are detected at or above the regulatory limit o Slurred/unclear speech (currently 80 μg/100g tissue) in shellfish obtained from near or at the same site (not o Double vision. leftovers). • One or more of the following neurological Confirmed case signs/symptoms occurring within 48 hours of • Meets the clinical case definition. consuming shellfish: • AND PSP biotoxins are detected in leftover o Confusion shellfish at a level that meant the case o Memory loss consumed a dose likely to cause illness (current level: 10 MU/kg body weight, about Disorientation o 2 μg/kg body weight). o Seizure • o Coma.

Marine Biotoxin Management Plan 42 Amnesic Shellfish Poisoning (ASP) Case Diarrhetic Shellfish Poisoning (DSP) Case Definition Definition Suspected case (clinical case definition) Suspected case (clinical case definition) • Vomiting or diarrhoea or abdominal cramps, • Vomiting or diarrhoea occurring within 24 occurring within 24 hours of consuming hours of consuming shellfish. shellfish. • And no other probable cause identified by • And no other probable cause identified by microbiological examination of a faecal microbiological examination of a faecal specimen from the case or microbiological specimen from the case or microbiological testing of left‐over food. testing of left‐over food. Probable case • And/or one or more of the following • Meets the clinical case definition. neurological signs/symptoms occurring • And within seven days of collection of within 48 hours of the consumption of the shellfish consumed by the case, DSP shellfish: biotoxins are detected at or above the o Confusion regulatory limit (currently 20 μg/100g shellfish or 5 MU/100g) in shellfish obtained o Memory loss from near or at the same site (not leftovers). o Disorientation Confirmed case o Seizure • Meets the clinical case definition o Coma. • And detection of DSP biotoxins in leftover shellfish at a level resulting in the case Probable case consuming a dose likely to cause illness • Meets the clinical case definition. (current level: ingestion of 48μg or 12MU). • And within seven days of the collection of shellfish consumed by the case ASP biotoxins are detected at or above the regulatory limit (currently 20ppm domoic acid/100g tissue) in shellfish obtained from near or at the same site (not leftovers). Confirmed case • Meets the clinical case definition. • And ASP biotoxins detected in leftover shellfish at a level resulting in the case consuming a dose likely to cause illness (current level: 0.05 mg/kg body weight).

Marine Biotoxin Management Plan 43 Neurotoxic Shellfish Poisoning (NSP) Case Definition Suspected case (clinical case definition) Two or more of the following neurological symptoms occurring within 24 hours of consuming shellfish: Neurosensory: o Paraesthesia (i.e. numbness or tingling around the mouth, face or extremities). o Alternation of temperature sensations such as a prickly feeling on the skin during a bath/shower or exposure to sun, or difficulty distinguishing hot or cold objects. Neuromotor/neurocerebellar: o Weakness such as trouble rising from seat or bed o Difficulty in swallowing o Difficulty in breathing o Paralysis o Clumsiness o Unsteady walking o Dizziness/vertigo o Slurred/unclear speech o Double vision. Probable case • Meets the clinical case definition. • And within seven days of collection of shellfish consumed by the case, NSP biotoxins detected at or above the regulatory limit (currently 20 MU/100g shellfish) in shellfish obtained from near or at the same site (not leftovers). Confirmed case • Meets the clinical case definition • Detection of NSP toxins in leftover shellfish at a level resulting in the case consuming a dose likely to cause illness (current level: 0.3 MU/kg body weight).

Marine Biotoxin Management Plan 44 Appendix 11: Phytoplankton Action Levels The following table summarises the phytoplankton presence/abundance and biotoxin phytoplankton levels (in cells/litre) that are used levels in shellfish tissue as part of the VSQAP, in to trigger the sampling of shellfish flesh for PPB and WP. They should be further revised as biotoxin analysis and harvesting suspensions. additional monitoring and research is undertaken These levels are derived from levels used and supports a change. internationally and in various states in Australia. Note: For Pseudo‐nitzschia spp risk remains high They have been modified in accordance with for a minimum of two weeks post bloom crash. specific information obtained pertaining to

Table 3: Phytoplankton Action Levels WATER ECOscience Phytoplankton Abundance Triggers for the VSQAP (cells/L) Alga / Algal Group Toxin Warning Tissue Harvest Harvest Resumption Issued Testing Suspension to Growers Pending Toxin Analysis Bacillariophyceae Pseudo‐nitzschia spp. ASP 100,000 300,000 500,000 <20 µg/g domoic acid for 3 successive samples over 14 (<50% total phytoplankton) (domoic days; phytoplankton acid) abundance not rising.

Pseudo‐nitzschia spp. (>50% total phytoplankton) ASP 50,000 100,000 200,000 As Above Rhizosolenia cf chunii Bitter 10,000 N/A 20,000 Harvesting suspended/resumed by Taste Level 2 growers depending on taste Warning of mussels.

Dinophyceae Alexandrium catenella PSP 100 Routine *500 <80 µg/100g PSP for 3 successive samples over 14 or 100 days; phytoplankton abundance not rising. Alexandrium minutum PSP 100 Routine *500 As Above or 100 Alexandrium tamarense ?PSP 100 Routine *500 As Above or 100 Alexandrium spp. ?PSP 100 Routine *500 As Above (unknown or in doubt) or 100

♦Dinophysis acuminata DSP 1,000 1,000 2,000 <20 µg/100g DSP for 2 successive samples taken not < 7 days apart; phytoplankton abundance not rising.

Marine Biotoxin Management Plan 45 Dinophysis caudata DSP 1,000 1,000 2,000 As Above Dinophysis fortii Dinophysis spp. ?DSP 1,000 1,000 2,000 As Above – precautionary only till further information available

Gymnodinium catenatum PSP 100 Routine *500 <80 µg/100g PSP for 3 successive samples over 14 or 100 days; phytoplankton abundance not rising.

Karenia mikimotoi NSP 1,000 2,000 5,000 < 20 MU/100g for 2 successive samples taken Karenia brevis brevetoxin not <2 days apart; Karenia cf brevis (Flat, (BTX) phytoplankton abundance Australian species not rising. morphologically similar to K. brevis). (K. brevis probably not present in Aust)

Prorocentrum lima ?DSP 1,000 1,000 2,000 <20 µg/100g DSP for 2 successive samples taken not < 7 days apart; phytoplankton abundance not rising.

* Draft Model National Marine Biotoxin Management Plan trigger adopted for now until more information on DTX 3 (OA esters) is available for PPB; PTX‐2‐SA are no longer included as a toxin.

NOTE: Harvest suspension pending biotoxin analysis is precautionary; resumption of harvesting will be determined by toxin levels.

Marine Biotoxin Management Plan 46 Appendix 12: Marine Biotoxin Regulatory Levels The following are the regulatory limits for marine biotoxins in the edible portions of shellfish.

Table 4: Biotoxin regulatory limits for the VSQAP.

Toxin Class Units Regulatory Method Limit of Laboratory Detection Limit Utilised

PSP µg/100g 80 Bioassay 26 Medvet (IMVS)

ASP µg/g 20 HPLC 0.5 – 1.0 State Chem Labs (Vic)

(domoic acid) (ppm)

DSP µg/100g 20 HPLC/MS 0.3 Qʹld Health Scientific Services

NSP µg/100g N/A LC/MS ‐ Cawthron Institute (NZ)

MU/100g 20 Bioassay 10 Cawthron Institute (NZ)

*Yessotoxins µg/100g *100 HPLC/MS Qualitative Qʹld Health Scientific Services (YTX)

*Axaspiracids µg/100g *16 HPLC/MS Qualitative Qʹld Health Scientific Services (AZA)

MU = mouse units PSP, ASP, DSP & NSP regulatory limits from FSANZ Food Standards Code (2002).

* Not regulated or detected in Australia to date. CEC draft regulation recommended level. Presence of both monitored as part of VSQAP

Paralytic Shellfish Poisoning (PSP) Diarrhetic Shellfish Poisoning (DSP) PSP toxins greater than or equal to 80μg of Greater than or equal to 20 μg/100g of edible saxitoxin equivalent/100g of edible shellfish flesh shellfish flesh (∼5 mouse units) by 24‐hour mouse (∼ 400 mouse units), by mouse bioassay with a bioassay or HPLC Electrospray Mass maximum observation time of one hour. Spectrometry. DSP toxins include OA, DTX1, DTX2, DTX3 (OA esters) and PTX. Amnesic Shellfish Poisoning (ASP) PTX is included as a DSP toxin for now, as the Greater than or equal to 20 μg/g (20ppm) of Commission of European Communities has domoic acid in the edible shellfish flesh by high recently published draft regulations proposing performance liquid chromatography (HPLC). that a limit of 16 µg/100g total DSP content Neurotoxic Shellfish Poisoning (NSP) including OA, DTXs and PTXs be adopted, despite the fact that the 2001 EU expert working NSP toxins greater than or equal to 20 mouse group on all fat‐soluble marine algal toxins units/100g of edible shellfish flesh, by ether removed the PTXs from the DSP group of toxins extraction and mouse bioassay with a maximum (Aune in MacKenzie, 2002). observation time of six hours. The FSANZ Food Standards Code Regulatory Limit for DSP is retained for the VSQAP.

Marine Biotoxin Management Plan 47 Yessotoxins (YTX) Azaspiracid Shellfish Poisoning (AZP) YTX is not regulated in Australia and although it AZAs are not regulated in Australia and have not is toxic to mice when applied intraperitoneally, its been detected in Australia or New Zealand. As oral toxicity is questionable (Cawthron Institute, part of the VSQAP, qualitative AZA analysis is 2001). Within the VSQAP, qualitative analysis is carried out by the Queensland Health Scientific carried out by HPLC/MS by Queensland Health Services using the DSP HPLC/MS method. Scientific Services using the DSP LC/MS method. The EU draft regulations recommend a limit of 16 The CEC draft regulations recommend a limit of µg/100g for AZA equivalents. Azaspiracids 100 µg/100g for YTX equivalents (Holland et al, include AZA1, AZA2 and AZA3. 2002). The yessotoxins include YTX, 45OH‐YTX, homo‐YTX and 45 OH Homo YTX.

Marine Biotoxin Management Plan 48 Appendix 13: Nuisance Phytoplankton Management Protocols Alexandrium spp. Background mussels) in Port Phillip Bay (PPB) in the past. This Alexandrium spp. are small, armoured situation, coupled with the nature of the toxin, dinoflagellates. The latter are golden‐brown algae means this group of algae present a substantially with a large nucleus. They have two flagella, one greater potential threat to human health than all protruding from a horizontal girdle groove and other potentially toxic species in these waters. the other from a vertical sulcus groove However, it should be noted that most of the (Hallegraeff, 2002). previous blooms of Alexandrium did not occur in the vicinity of any of the shellfish growing areas. A number of species of Alexandrium have been The most susceptible area has been Hobsonʹs Bay found to produce a range of toxins grouped as near the mouth of the Yarra River, and the main Paralytic Shellfish Poisons (PSP) that may be public health threat was from the recreational accumulated in the flesh of shellfish. PSP may be harvesting of mussels. Past monitoring included fatal to human consumers of contaminated both the VSQAP and additional bay‐wide shellfish through respiratory paralysis, although monitoring funded by the former Department of this is rare and there have been no fatal cases in Health and Community Services. This later Australia. It should be noted that toxicity within a monitoring no longer occurs, and due to the species may be variable both with locality and separation between the mussel harvesting areas time. It is stressed that some Alexandrium species and the more susceptible recreational areas nearer are difficult to identify definitively and expert to the Yarra River, it is unlikely that the VSQAP assistance should be sought where doubt exists. will provide any warning of the presence of Until definitive identification is obtained, it Alexandrium in the latter. PSP was detected in should be assumed that all the forms of mussels from the Clifton Springs and Grassy Alexandrium present are toxic. Point harvesting areas in 1993 and 1994. A. The symptoms of paralytic shellfish poisoning tamarense was considered the most likely source of include numbness, dizziness, nausea, tingling in PSP in the winter of 1993 (Arnott et al, 1999). the extremities, vomiting and diarrhoea in mild The Alexandrium spp. known from PPB and cases (within 30 minutes), to choking sensations, Western Port (WP) in Victoria include the breathing difficulties and death from respiratory following. Those of major concern are the three paralysis two to 24 hours after ingestion in severe PSP producing species. Several of these are new cases (Hallegraeff, 1997). records for these areas, having been detected for Very high levels of Alexandrium catenella have the first time by the VSQAP phytoplankton resulted in highly toxic shellfish (including wild monitoring.

Alexandrium catenella PSP (C1 – C4, gonyautoxins); present in PPB esp. Hobsons Bay

Alexandrium tamarense Some strains PSP (C1 – C4, gonyautoxins); can be toxic but NT in all Australian isolates so far.

Alexandrium fundyense PSP (C1 – C4, gonyautoxins); PPB material has been shown to be A. catenella Alexandrium minutum PSP (mainly gonyautoxins); high PSP levels in SA; has bloomed in PPB in winter. Alexandrium ostenfeldii Sometimes toxic in NZ, probably non‐toxic in PPB & Aust Alexandrium pseudogonyaulax Non‐toxic Alexandrium concavum Non‐toxic, PPB, rare Alexandrium peruvianum Non‐toxic, PPB Alexandrium affine Non‐toxic, may occur in PPB Alexandrium margalefi Non‐toxic, may occur in PPB Additional information concerning toxigenic species of Alexandrium in Australia may be found in Hallegraeff et al (1991) and Hallegraeff (2002).

Marine Biotoxin Management Plan 49 Management Protocol 3. If sampling frequency is not increased, then The following management protocol has been harvesting should be suspended pending the designed to ensure that mussels harvested from results of the next routine sampling event. PPB and WP are safe for human consumption, Previous history shows the numbers of these and that harvesting does not occur when the phytoplankton can increase very rapidly. A mussels are affected by toxins. The protocol is live phytoplankton sample (preferably based on the following key factors: concentrated) is to be sent by overnight courier to Professor Gustaff Hallegraeff at the • A number of species of Alexandrium occur University of Tasmania for definitive naturally in PPB and WP. identification. Advise Professor Hallegraeff • Definitive identification of the various in advance the sample has been despatched. species of Alexandrium may be difficult. 4. Where doubt exists as to the identity of the • A. catenella has bloomed several times in the form of Alexandrium present, toxicity should past in Hobsonʹs Bay (northern PPB) be assumed until biotoxin levels are known. although not in the vicinity of shellfish harvesting areas. 5. Where Alexandrium species are detected in • A. tamarense may have been responsible for numbers >500cells/L, harvesting should be the presence of PSP in mussel tissue in PPB suspended pending the results of tissue in the past. testing. The relevant analytical laboratory should be advised of the phytoplankton • Extreme PSP intoxication is potentially lethal result and the urgency of the situation. to human beings. • The potentially toxic species A. catenella, A. 6. Where tissue is found to contain PSP at a minutum and A. tamarense have been detected level exceeding 80 µg/100g tissue (the in PPB. regulatory limit), harvesting is to be suspended and is to remain suspended until • Due to the status of PPB as a harbour, and three successive samples over a two‐week the presence of a substantial number of period reveal toxin levels < 80 µg/100g tissue. foreign species probably introduced through ballast water, there is a danger that other 7. Where lower levels of toxin are detected toxic forms will be introduced. during the growth phase of a bloom, • Routine fortnightly phytoplankton sampling harvesting should be suspended and and monthly biotoxin testing for PSP will sampling frequency increased to monitor the continue at all VSQAP harvesting areas. development of the bloom. 8. Where toxin levels have exceeded the 80 The following management protocol has been µg/100g tissue regulatory limit during a used successfully within the VSQAP since August bloom, but the bloom is clearly degenerating, 1999. It has been adopted for the current VSQAP harvesting may be resumed once toxin levels including the mussel growing areas in PPB and remain less than 80 µg/100g for three WP. It should be noted the principal trigger for successive samples taken over a two‐week harvest suspension is the biotoxin level in period. shellfish tissue; phytoplankton abundance forms an additional, early warning trigger allowing 9. If any toxin producing Alexandrium species precautionary closure pending biotoxin results. are present, and/or low PSP levels are detected, the frequency of sampling should 1. Phytoplankton and mussel tissue samples are be reviewed and amended to ensure it is taken routinely each fortnight at all VSQAP adequate to detect changes in either harvesting areas, and tissues are analysed phytoplankton or biotoxin levels in an routinely each month for PSP. effective and timely manner. 2. If potential toxin producing species (or 10. When harvesting is suspended during a toxic unknown species) of Alexandrium are bloom, the sampling frequency may be detected in a routine sample at an abundance reduced to the routine fortnightly monitoring of >100 cells/L (1 cell/mL), a warning should program to save resources and costs. be issued to the relevant growers and the However, three ‘clear’ biotoxin results (clear sampling frequency should be reviewed with = < 80 µg/100g tissue) over a two‐week period a view to increasing it to weekly. are required before harvesting can resume. 11. Once Alexandrium and PSP toxins are undetectable, the routine sampling regime may be resumed.

Marine Biotoxin Management Plan 50 VSQAP Phytoplankton Abundance Threshold Levels (cells/L) Phytoplankton Toxin Warning Tissue Harvest Harvest Resumption Species Issued Testing Suspension to Growers (Pending Toxin Analysis) Alexandrium PSP 100 Routine 500 <80 µg/100g PSP for 3 successive catenella or 100 samples over 14 days; phytoplankton abundance not rising. Alexandrium PSP 100 Routine 500 As Above minutum or 100 Alexandrium PSP 100 Routine 500 As Above tamarense Some or 100 strains Alexandrium PSP 100 Routine 500 As Above spp. Some or 100 (unknown/ in strains doubt) PSP REGULATORY LIMIT: 80 µg saxitoxin equivalents/100g tissue

General the variability in the toxicity of the various forms It must be stressed that this protocol is specifically of Alexandrium, it would be prudent to follow designed for use within PPB and WP where an more conservative threshold levels proposed in extensive record of the occurrence and toxicity of the Australian Marine Biotoxin Management Plan for Alexandrium exists, extending from 1987 to the Shellfish Farming (2001) detailed below. present. Where other regions are involved, due to

Phytoplankton Species Toxin Tissue Sampling Industry Voluntary Public Health Abundance Closure Level Warning Threshold Trigger (Pending Biotoxin Analysis) (cells/L) (cells/L) (cells/L) Alexandrium minutum PSP 100 500 5,000 Alexandrium catenella PSP 100 500 5,000 Alexandrium tamarense PSP 100 500 5,000 Alexandrium ostenfeldii PSP 100 500 5,000

References Arnott, G.H., Reilly, D.J. and Werner, G.F. (1999). Hallegraeff, G.M., Bolch, C.J., Blackburn, S.I. & Oshima, Victorian Shellfish Quality Assurance Program. 7. Sanitary Y (1991). Species of the Toxigenic Dinoflagellate Genus Survey Update: Clifton Springs and Grassy Point (port Alexandrium in Southeastern Australian Waters. Arlington) Aquaculture Zones. Marine and Freshwater Botanica Marina, 34: 575 – 587. Resources Institute, Report No. 13, 1 – 36 Hallegraeff, G. (1997). Algal toxins in Australian Todd, K. (2001). Australian Marine Biotoxin Management Shellfish. In: Foodborne Microorganisms of Public Health Plan for Shellfish Farming. Prepared for the Australian Significance. Fifth Edition. AIFST (NSW Branch), Food Shellfish Quality Assurance Committee (ASQAAC) by Microbiology Group. the Cawthron Institute. Cawthron Report No. 645 Hallegraeff, G. (2002). Aquaculturistsʹ Guide to Harmful Australian Microalgae. The Print Centre, Hobart.

Marine Biotoxin Management Plan 51 Dinophysis acuminata, Dinophysis spp. and Prorocentrum lima Dinophysis acuminata and will be in future. Despite the consumption of large Dinophysis spp. quantities of mussels from the aquaculture zones in PPB and WP, there has never been a report of a Background case of diarrhetic shellfish poisoning. Dinophysis are large, bag‐shaped dinoflagellates with well‐developed sulcal lists and reduced The Commission of European Communities has epitheca (Hallegraeff, 2002). They are common in recently published draft regulations covering DSP Australian waters but not often abundant. toxins in shellfish where it is proposed that a limit of 16 µg/100g total DSP content be adopted, Some Dinophysis species have been found to including OA, DTXs and PTXs ( CEC, 2001; produce fat‐soluble polyether compounds called Holland et al, 2002). This is despite the fact that Diarrhetic Shellfish Poisons (DSP) that may the EU expert working group on all fat‐soluble accumulate in the shellfish that consume them. marine algal toxins (2001) removed PTX and YTX DSP may cause illness in human consumers of from this group (Aune in MacKenzie, 2002). The contaminated shellfish. later group suggested a limit for PTX of 15 The major symptoms of DSP poisoning are µg/100g. PTX is still regarded as toxic and diarrhoea, vomiting, nausea and abdominal pain. although currently included as a DSP for the There is also some evidence of tumour formation VSQAP, should be regulated separately. The in the digestive system as a result of chronic FSANZ Food Standards Code regulatory limit for exposure. Recovery occurs after about three days DSP is adopted for the VSQAP. irrespective of medical treatment (Hallegraeff, YTX and azaspiracids (AZA), which are included 1997) and no human fatalities have been recorded. by some as DSP but are chemically distinct, are The first toxin characterised was okadaic acid not regulated in Australia. For the purposes of the (OA) and later, okadaic acid derivatives were VSQAP, these compounds are not considered isolated from shellfish including the dinophysis members of the DSP group of toxins. The oral toxins (DTX) incorporating the OA esters (DTX 3). toxicity of YTX is questionable and neither YTX or Subsequently, other toxins have been included in AZA has been detected in Australian shellfish to this group despite their different chemical date. Currently, these compounds are analysed structures and modes of action. These include the using the HPLC/MS method for DSP toxins. pectenotoxins (PTX), pectenotoxin seco acids Within the VSQAP, when DSP biotoxin testing is (PTX2‐SA) and yessotoxins (YTX). Recent work in performed, qualitative testing is also carried out Australia by Burgess (2002), and in New Zealand for YTX and AZA. Once standards are available, for the Marlborough Sounds Shellfish Quality and quantitative analysis is possible, regulation Program by MacKenzie (2002), has shown that within the VSQAP will be considered. At this PTX2‐SA compounds are not toxic to humans. time, the EU draft regulations have recommend Consequently, for the purposes of VSQAP, PTX2– limits for both YTX and AZA. SAs are no longer regulated as a DSP toxin. The species of Dinophysis known to be or likely to Diarrhoegenic effects have been demonstrated be recorded in PPB and WP are: only for OA and DTX; PTX 1‐4 have been found to Dinophysis acuminata (produces PTX‐2‐SA in cause liver damage and YTX damages cardiac PPB) muscle in mice (Hallegraeff, 1997). Dinophysis fortii (potential DSP producer in The main bloom species in Port Phillip Bay (PPB) PPB) and Western Port (WP) appears to be Dinophysis Dinophysis caudata (potential DSP producer acuminata. Dinophysis acuminata in Australian and but not recorded in PPB at New Zealand waters has not been found to this time) produce significant amounts of OA or DTX, although it does produce significant amounts of Dinophysis acuta (potential DSP producer PTX 2 that seems to be rapidly converted to non‐ but rare in Australia) toxic PTX2‐SA in mussels. Mussels from PPB and Dinophysis tripos (potential DSP producer, WP have been tested for DSP revealing that PTX2‐ widespread in Australia SA predominated with very small amounts of but rare) PTX2 and traces of OA. No DTX 1 or 2 was found. DTX 3 (OA esters) has not been tested to date but

Marine Biotoxin Management Plan 52 Dinophysis hastata (potential DSP producer, widespread in Australia 1. Phytoplankton monitoring is carried out but rare) routinely each fortnight at each Since August 1999, only Dinophysis acuminata has harvesting area. occurred in numbers sufficient to initiate biotoxin sampling/analysis within the VSQAP. 2. On each fortnightly routine monitoring sampling event, ~ 40 mussels are to be Management Protocol collected from each growing area in case This management protocol specifically relates to biotoxin analysis is indicated by the Dinophysis acuminata where abundance and phytoplankton sample analysis. biotoxin data has been collected over several years. However all other species are treated in the 3. Once the mussels are returned to the same way until additional information indicates WATER ECOscience Laboratories, they otherwise. Most species do not generally occur in are to be bagged, labelled clearly, frozen numbers sufficient to cause concern. The protocol and stored. Mussels from the previous is designed to ensure mussels harvested from PPB fortnight may be discarded, if necessary, and WP are safe for human consumption and but preferably each batch of mussels harvesting does not occur when the mussels are should be held for one month before affected by DSP toxins produced by various being discarded. Dinophysis species. It is based on the following 4. It is recommended more rapid key factors: sedimentation methods than gravity be • Various species of Dinophysis have been used to concentrate samples for counting detected in PPB and WP, notably D. acuminata Dinophysis due to the necessity for a rapid turnaround time. It is also recommended • A number of species are known to produce the concentration factor be X10 greater significant DSP levels in shellfish tissue at than the usual method for counting algae very low abundances. due to the very low threshold value for • Dinophysis acuminata in PPB produces PTX2‐ Dinophysis and the necessity for greater SA but little OA & PTX and no DTX. OA accuracy at low abundance levels. esters (DTX 3) have not been tested for to 5. If Dinophysis acuminata numbers exceed date. 1,000 cells/L, the relevant mussel sample • PTX2‐SA has been shown to be non‐toxic to should be thawed, the mussels shucked humans and is excluded as a DSP toxin. and sent to the Queensland Health Department (Dr Geoff Eaglesham) for • Due to its low abundance trigger, the DSP biotoxin analysis as per the VSQAP numbers of D. acuminata can vary quickly Operations Manual. Qualitative analysis between levels above and below the trigger for YTX and AZA will also be performed. level, making management difficult. 6. If Dinophysis acuminata numbers exceed • The relationship between tissue DSP levels 2,000 cells/L, growers are to be notified and D. acuminata numbers is poor. and a voluntary suspension of harvesting • The symptoms of DSP are relatively minor implemented pending the biotoxin and human deaths have never occurred. analysis.

Tissue testing is based on phytoplankton triggers 7. If the total DSP level in mussel tissue as DSP is not routinely tested for in the VSQAP. (OA, DTX and PTX but excluding PTX2‐ The following management protocol has been SA) exceeds the regulatory limit of 20 µ used successfully within the VSQAP since August g/100g tissue, harvesting should be 1999 for the management of Dinophysis acuminata suspended and sampling frequency blooms. It has been adopted for the current increased. The latter is very important as VSQAP including the mussel growing areas in the abundance of Dinophysis acuminata PPB and WP. It should be noted the principal can vary from problem to non‐problem trigger for harvest suspension is the biotoxin level levels within days. in shellfish tissue; phytoplankton abundance forms an additional, early warning trigger allowing precautionary closure pending biotoxin results.

Marine Biotoxin Management Plan 53 8. If phytoplankton monitoring indicates difficult to manage this situation once that a D. acuminata bloom is developing closure has been initiated. This situation (trend of increasing numbers), the is likely to be complicated by the fact that monitoring frequency should be abundance may not only vary rapidly increased and harvesting suspended if with time, but also between sites. DSP levels exceed 20 µg/100g tissue. 11. Other Dinophysis species should be dealt 9. Once harvesting has been suspended due with using the same thresholds as for D. to the presence of DSP in mussel tissue, acuminata, until more information is harvesting may not be resumed until two gathered on their toxin production and successive ‘clear’ biotoxin results are toxicity. obtained at least seven days apart. In this 12. The abundance threshold values and case, a clear biotoxin result means DSP biotoxin regulatory limits for Dinophysis levels less than 20 µg/100g tissue. spp. should be updated regularly as new 10. Because the abundance of Dinophysis spp. information becomes available. This is can rise and fall rapidly above and below particularly the case with DTX 3 and the threshold levels for tissue testing and Dinophysis acuminata. suspension of harvesting, it may be

VSQAP Phytoplankton Abundance Threshold Levels (cells/L) Phytoplankton Toxin Warning Tissue Harvest Harvest Resumption Species Issued Testing Suspension to Growers (Pending Toxin Analysis) Dinophysis DSP 1,000 1,000 2,000 <20 µg/100g DSP for 2 successive acuminata samples taken not < 7 days apart; phytoplankton abundance not rising. Dinophysis DSP 1,000 1,000 2,000 As Above caudata Dinophysis acuta DSP 1,000 1,000 2,000 As above Dinophysis fortii ?DSP 1,000 1,000 2,000 As Above Dinophysis spp. ?DSP 1,000 1,000 2,000 As Above – precautionary only till further information available Prorocentrum ?DSP 1,000 1,000 2,000 <20 µg/100g DSP for 2 successive lima samples taken not < 7 days apart; phytoplankton abundance not rising. DSP REGULATORY LIMIT: 20 µg OA equivalents/100g tissue

Prorocentrum lima This species has been found to produce DSP Prorocentrum lima is another dinoflagellate which overseas (specifically OA and DTX‐1). However, is oval in shape and bears a small anterior its toxicity status in Australia is uncertain and a indentation. It has been recorded widely over culture from WA was found to be non‐toxic. southern Australia including in PPB, the For the purposes of the VSQAP, it has been Gippsland Lakes and Tasmania. It is a benthic or assumed that this species is a DSP producer epibenthic species commonly found attached to similar to Dinophysis acuminata, and the seaweed and in shallow sand (Hallegraeff, 2002). abundance and biotoxin triggers utilised for Dinophysis acuminata have been adopted until more information becomes available. Hence the management protocol for Dinophysis should be utilised for this species as well.

Marine Biotoxin Management Plan 54 General (Dinophysis spp. & Prorocentrum regions are involved, due to the potential lima) variability in the toxicity of the various forms of It must be stressed that the protocol for Dinophysis Dinophysis and the uncertainty surrounding P. (and Prorocentrum lima) is specifically designed for lima, it would be prudent to follow the threshold use within PPB and WP where an extensive levels proposed in the Australian Marine Biotoxin record of the occurrence and toxicity of Dinophysis Management Plan for Shellfish Farming (2001) acuminata and related species exists. Where other detailed below.

Phytoplankton Species Toxin Tissue Sampling Industry Public Health Abundance Voluntary Closure Warning Threshold Trigger Level (cells/L) (cells/L) (Pending Biotoxin Analysis) (cells/L) Dinophysis acuminata DSP 1,000 2,000 N/A Dinophysis acuta DSP 500 1,000 N/A

Prorocentrum lima DSP 500 1,000 N/A

References ANZFA (2002). Food Standards Code. Australian and MacKenzie, L. (2002). An Evaluation of the Risk to New Zealand Food Authority Consumers of Pectenotoxin 2 seco acid (PTX2‐SA) Contamination of Greenshell™ Mussels. Prepared for Commission of European Communities (2001). Draft Marlborough Sounds Shellfish Quality Programme. Commission Decision of Establishing the Methods of Cawthron Institute Report 750, 1‐ 50 Analysis and the Maximum Limits fro Certain Marine Biotoxins in Bivalve Molluscs, Echinoderms, Tunicates MacKenzie, L., Holland, P., McNabb, P., Beuzenberg, and Marine Gastropods. Brussels, SANCO/2227/2001 V., Selwood, A. and Suzuki, T. (2002). Complex Toxin Rev 3 Profiles in Phytoplankton and Greenshell Mussels (Perna canaliculus), Revealed by LC‐MS/MS Analysis. Hallegraeff, G. (1997). Algal toxins in Australian Toxicon, 40: 1321 – 1330 Shellfish. In: Foodborne Micro‐organisms of Public Health Significance. Fifth Edition. AIFST (NSW Branch), Food Todd, K. (2001). Australian Marine Biotoxin Management Microbiology Group. Plan for Shellfish Farming. Prepared for the Australian Shellfish Quality Assurance Committee (ASQAAC) by Hallegraeff, G. (2002). Aquaculturistsʹ Guide to Harmful the Cawthron Institute. Cawthron Report No. 645 Australian Microalgae. The Print Centre, Hobart. Holland, P.T., McNabb, P., Selwood, A., Page, T., Bell, K and MacKenzie, L. (In Press). Marine Biotoxin Monitoring of New Zealand Shellfish – A New Management Programme Based on LC‐MS. In: Proc. 2nd Int. Conference on Harmful Algae Management and Mitigation. Nov 2001, Qingdao, China. Hall, S. & Zou, YL (Ed.)

Marine Biotoxin Management Plan 55 Pseudo‐nitzschia spp.

Background Pseudo‐nitzschia (non‐toxic in PPB, Pseudo‐nitzschia spp. are narrow, elongated pseudodelicatissima mildly toxic in diatoms that are difficult to identify to species Derwent R.) level using light microscopy; generally, electron microscopy is required. Pseudo‐nitzschia heimii (non‐toxic) A number of species, notably P. multiseries and P. Pseudo‐nitzschia fraudulenta (non‐toxic) australis, have been found to produce the Amnesic Shellfish Poison (ASP) domoic acid, that may be accumulated in the flesh of shellfish. ASP may Additional information concerning Australian cause illness in people consuming contaminated Pseudo‐nitzschia and their toxicity may be found in shellfish such as mussels, oysters and scallops. It Hallegraeff (1994) and Lapworth et al (2000). should be noted that toxicity within a species may ASP (domoic acid) biotoxin sampling and analysis be variable both with locality and time. Until is carried out on a routine, monthly basis at the definitive identification is obtained, it should be Clifton Springs and Flinders Bight harvesting assumed that all the forms of Pseudo‐nitzschia areas, but not at the other VSQAP harvesting present are toxic. areas. However, mussel samples are taken during A serious shellfish‐poisoning outbreak in humans every routine fortnightly VSQAP sampling event in Canada in 1987 resulted in memory loss in at all harvesting areas. These are frozen and used extreme cases of intoxication, and consequently for biotoxin testing should phytoplankton the syndrome was called Amnesic Shellfish monitoring indicate that this is necessary. Poisoning (ASP). The causative compound was Management Protocol found to be domoic acid. The symptoms of ASP The following management protocol has been are nausea, vomiting, diarrhoea and abdominal designed to ensure that mussels harvested from cramps after three to five hours. In extreme cases PPB and WP are safe for human consumption, there may be a decreased reaction to deep pain, and that harvesting does not occur when the dizziness, hallucinations, confusion, short‐term mussels are affected by ASP toxins. The protocol memory loss and seizures (Hallegraeff, 1997, is based on the following key factors: 2002). A small number of deaths have occurred in Canada with immuno‐depressed patients most at • Pseudo‐nitzschia spp. are present as a risk. There is evidence the concentration of component of the phytoplankton domoic acid in shellfish may be species communities in PPB and WP for much of the dependant, with scallops most at risk and mussels year. They rarely form the dominant algal much less so. There are no documented cases of group within these communities (i.e. rarely > amnesic shellfish poisoning in Australia. Domoic 50% of the total phytoplankton). acid has not been detected in Victorian mussels • The major blooms of this genus generally since the commencement of the VSQAP in 1987 consist of P. pseudodelicatissima, P. delicatissima but has been detected in scallops from Bass Strait and P. pungens all of which have been found (Arnott et al, 1999). to be non‐toxic in PPB. Pseudo‐nitzschia currently known to be present in • Definitive identification of the various species Port Phillip Bay (PPB) and Western Port (WP) in of Pseudo‐nitzschia may require electron Victoria include the following species. Several of microscopy, and they are therefore managed these are new records for these areas, having been as a genus (group of species). detected for the first time by VSQAP phytoplankton monitoring. • The management guidelines adopted in New Zealand appear to be unnecessarily Pseudo‐nitzschia multiseries (potentially toxic) conservative for PPB and WP where a long Pseudo‐nitzschia australis (potentially toxic) history of phytoplankton and biotoxin monitoring exists. Nonetheless, for situations Pseudo‐nitzschia pungens (non‐toxic in PPB) where uncertainty exists, these are adopted as Pseudo‐nitzschia (non‐toxic in PPB) a ‘fall back’ position. delicatissima

Marine Biotoxin Management Plan 56 • P. heimii and the potentially toxic species P. 3. If Pseudo‐nitzschia spp. are detected in australis and P. multiseries have been detected numbers greater than 300,000 cells/L (300 as minor components of Pseudo‐nitzschia cells/mL), institute ASP biotoxin testing as blooms in PPB. part of the routine fortnightly sampling program. This analysis is additional to the • There is a risk the potentially toxic species P. current routine VSQAP program. australis and/or P. multiseries may become a major component of blooms. 4. If numbers exceed 500,000 cells/L, suspend harvesting pending the biotoxin analysis • Due to the status of PPB and WP as harbours results. and the presence of a substantial number of foreign species in the former, probably 5. If ASP is not detected, harvesting may be introduced via ballast water, there is a danger resumed immediately. that other toxic forms or species of Pseudo‐ 6. Continue ASP analysis as part of the nitzschia may be introduced. fortnightly routine program at the affected • If environmental conditions alter in Port harvesting areas until Pseudo‐nitzschia spp. Phillip Bay there is a risk that current non‐ levels drop below 300,000 cells/L (300 toxic Pseudo‐nitzschia species may become cells/mL). toxic. 7. Continue to monitor phytoplankton levels, • Routine phytoplankton sampling on a and during each month of the bloom, send a fortnightly basis will continue at all VSQAP concentrated sample to the University of harvesting areas, as is currently the case. Tasmania for species level identification in case the species mix varies with time. • Biotoxin testing will be performed at other harvesting areas other than Clifton Springs 8. If the main components of a bloom are found and Flinders when phytoplankton to be species known to be non‐toxic, such as monitoring indicates this is necessary. P. pungens, P. delicatissima and P. pseudodelicatissima, and ASP analysis is The following management protocol has been negative, continue to repeat steps five and six used successfully within the VSQAP since August until the bloom degenerates. 1999 for the management of Pseudo‐nitzschia blooms where ASP biotoxin analysis was not 9. If any domoic acid is detected, it is carried out routinely. It has been adopted for the recommended an industry warning be current VSQAP including the mussel harvesting released and sampling frequency be increased areas in PPB and WP. It should be noted the to weekly. principal trigger for harvest suspension is the 10. If domoic acid is detected at levels > 20 µg biotoxin level in shellfish tissue. Phytoplankton domoic acid/g tissue (the regulatory limit), abundance forms an additional, early warning harvesting should be suspended and trigger allowing precautionary closure pending sampling frequency be amended with a view biotoxin results and as a trigger for biotoxin to increasing it to weekly. testing. 11. Harvesting areas remain closed until domoic 1. If Pseudo‐nitzschia spp. are detected in acid levels <20 µg/g tissue are found on three numbers less than 100,000 cells/L (100 successive sampling occasions over a two‐ cells/mL), no further action except monitoring week period. numbers is required. Report presence to growers. 12. It should be noted the risk from toxic Pseudo‐ nitzschia remains high for two weeks after the 2. If Pseudo‐nitzschia spp. are detected in post bloom crash. numbers greater than 100,000 cells/L (100 cells/mL) for the first time in a bloom, send 13. Once Pseudo‐nitzschia levels drop below the part of the concentrated phytoplankton relevant triggers and ASP is undetected in sample taken (or a duplicate sample) to shellfish, ASP sampling/analysis and Professor Gustaff Hallegraeff at the additional sampling can be discontinued. University of Tasmania for definitive species Routine fortnightly phytoplankton analysis identification. should continue as usual.

Marine Biotoxin Management Plan 57 14. If P. australis, P. multiseries or other known o Where Pseudo‐nitzschia spp. forms > 50% toxic species become a major component of a of the total phytoplankton bloom, it is recommended the Australian ƒ Instigate ASP tissue testing when Marine Biotoxin Management Plan for Shellfish Pseudo‐nitzschia spp. levels exceed Farming (2001) triggers be applied as below: 50,000 cells/L (50 cells/mL) o Where Pseudo‐nitzschia spp. forms < 50% ƒ Suspend harvesting when Pseudo‐ of the total phytoplankton nitzschia spp. levels exceed 200,000 ƒ Instigate ASP tissue testing when cells/L (200 cells/mL) Pseudo‐nitzschia spp. levels exceed 15. Re‐evaluate the Pseudo‐nitzschia trigger levels 100,000 cells/L (100 cells/mL) as more ASP testing is completed and related ƒ Suspend harvesting when Pseudo‐ to Pseudo‐nitzschia abundance over the period nitzschia spp. levels exceed 500,000 of the program. cells/L (500 cells/mL)

VSQAP Phytoplankton Abundance Threshold Levels (cells/L) Phytoplankton Toxin Warning Tissue Harvest Harvest Resumption Species to Testing Suspension Growers (Pending Toxin Analysis) Pseudo‐nitzschia ASP 100,000 300,000 500,000 <10 µg/g domoic acid for 3 spp. successive samples over 14 days; (domoic phytoplankton abundance not (<50% total acid) rising. phytoplankton) Pseudo‐nitzschia spp. ASP 50,000 100,000 200,000 As Above (>50% total phytoplankton) ASP REGULATORY LIMIT: 20 µg domoic acid /g tissue (=mg/kg)

General the more conservative threshold levels proposed It must be stressed that this protocol is specifically in the Australian Marine Biotoxin Management Plan designed for use within PPB and WP where an for Shellfish Farming (2001) detailed below. extensive record of the occurrence and toxicity of This would also be the case if scallops were Pseudo‐nitzschia exists, extending from 1987 to the harvested rather than mussels, as some evidence present. Where other regions are involved, due to exists suggesting scallops are more likely to the variability in the toxicity of the various forms accumulate domoic acid than mussels. of Pseudo‐nitzschia, it would be prudent to follow

Marine Biotoxin Management Plan 58 Phytoplankton Species Toxin Tissue Sampling Industry Voluntary Public Health Abundance Closure Level Warning Trigger (Pending Biotoxin Analysis) Threshold (cells/L) (cells/L) (cells/L) Pseudo‐nitzschia spp. (< 50% of total phytoplankton) ASP 100,000 500,000 N/A

Pseudo‐nitzschia spp. (> 50% of total phytoplankton) ASP 50,000 200,000 N/A

*Risk remains high for a minimum of two weeks post bloom crash.

References Hallegraeff, G. (1997). Algal toxins in Australian Arnott, G.H., Reilly, D.J. and Werner, G.F. (1999). Shellfish. In: Foodborne Microorganisms of Public Health Victorian Shellfish Quality Assurance Program. 7. Sanitary Significance. Fifth Edition. AIFST (NSW Branch), Food Survey Update: Clifton Springs and Grassy Point (port Microbiology Group. Arlington) Aquaculture Zones. Marine and Freshwater Hallegraeff, G. (2002). Aquaculturistsʹ Guide to Harmful Resources Institute, Report No. 13, 1 – 36 Australian Microalgae. The Print Centre, Hobart. Todd, K. (2001). Australian Marine Biotoxin Management Lapworth, C., Hallegraeff, G and Ajani, P.A. (2001). Plan for Shellfish Farming., Prepared for the Australian ʺIdentification of Domoic‐Acid‐producing Pseudo‐ Shellfish Quality Assurance Committee (ASQAAC) by nitzschia species in Australian Watersʺ In: Harmful Algal the Cawthron Institute. Cawthron Report No. 645 Blooms 2000. Hallegraeff, G.M., Blackburn, S.I., Bolch, Hallegraeff, G.M. (1994). Species of the Diatom Genus C.J. and Lewis, R.J. (Eds). Intergovernmental Psuedo‐nitzschia in Australian Waters. Botanica Marina, Oceanographic Commission of UNESCO 37: 397 ‐ 411.

Marine Biotoxin Management Plan 59 Rhizosolenia cf chunii Background • The sale of mussels with a bitter taste caused Rhizosolenia cf chunii is straight, cylindrical by R. cf chunii would be detrimental to the diatom, often found in chains. aquaculture mussel industry. Blooms of this species can impart a bitter taste to This management protocol has been used mussels and other shellfish and render them unfit successfully within the VSQAP since August 1999. for human consumption. The chemical nature of It has been adopted for the current VSQAP the bitter taste is unknown but the effect can including the mussel growing areas in PPB and persist for up to seven months (Hallegraeff, 2002). WP. Biotoxin is not carried out. Monitoring is In the 1987 Port Phillip Bay (PPB) bloom, the based on the abundance of Rhizosolenia cf chunii. digestive glands of exposed shellfish showed 1. Routine fortnightly monitoring of degeneration and significant mortality occurred phytoplankton numbers and diversity occurs three to eight months after the bloom (Parry et al, as part of the VSQAP. 1989; Hallegraeff, 2002). Consequently, although posing no threat of toxicity to humans, the 2. Growers are to be kept up to date as to the occurrence of blooms of this species constitutes a abundance of R. chunii in each harvesting major threat to the mussel industry. area as routine sampling events are carried out. Management Protocol 3. If Rhizosolenia cf chunii is detected in numbers The following management protocol is designed greater than 20,000 cells/L, a warning is to be to give growers warning of blooms of this species issued to growers that its abundance is to facilitate management of their mussel resource. approaching levels at which a bitter taste Potentially this could include the transportation of occurs in mussels. mussels from impacted areas (not currently possible) to areas with lower numbers of 4. Additional sampling/monitoring may be Rhizosolenia cf chunii, the suspension of harvesting requested by growers at any time. and the withdrawal of affected mussels from the 5. Growers are to be informed once the threat market. It is based on the following: has passed. • Rhizosolenia cf chunii is regularly detected 6. The mechanism used to inform growers within PPB harvesting areas. should be the VSQAP Hotline, maintained by • In 1987 this species was responsible for PIRVic on behalf of Fisheries Victoria. This is making mussels unpalatable by imparting a updated regularly with information supplied bitter taste to them. by WATER ECOscience as part of the VSQAP but additional sources of information • Since then, other instances of this have been may be incorporated in the future. recorded by the VSQAP. VSQAP Phytoplankton Abundance Threshold Levels (cells/L) Phytoplankton Toxin Warning Issued Tissue Harvest Harvest Resumption Species to Growers Testing Suspension (Pending Toxin Analysis) Rhizosolenia cf Non‐toxic 10,000 N/A N/A N/A chunii Bitter Taste Level 1 Warning 20,000 Level 2 Warning

Harvesting suspension is based on the presence of References a bitter taste in mussels and is invoked voluntarily Hallegraeff, G. (2002). Aquaculturistsʹ Guide to Harmful by growers as they see fit. The role of the VSQAP Australian Microalgae. The Print Centre, Hobart. in relation to R. chunii is to keep growers Parry, G.D., Langdon, J.S. and Huisman, J.M. (1989). informed concerning the presence of this species. Toxic Effects of a Bloom of the Diatom Rhizolsolenia chunii on Shellfish in Port Phillip Bay, South‐eastern Australia. Mar. Biol., 102:25 ‐ 41

Marine Biotoxin Management Plan 60 Gymnodinium catenatum Background Tasmania in 1973 and to Victoria at a later date, Gymnodinium spp. are small, unarmoured probably via ballast water. It has not been dinoflagellate phytoplankton (golden‐brown recorded in PPB or Western Port (WP) to date but algae with a large nucleus). They have two as both are ports, there is a risk of its introduction flagella, one protruding from a horizontal girdle and a watching brief is required. G. catenatum groove and the other from a vertical sulcus groove appears to be most common in estuaries and (Hallegraeff, 2002). hence if found in PPB, the most likely area would be near the Yarra River well away from the Many species belonging the genus Gymnodinium shellfish growing areas. At this stage there is no are common in Australian and New Zealand evidence of intoxication that may be attributable waters and over 21 species have been recorded to Gymnodinium catenatum in PPB or WP. from Port Phillip Bay (PPB) alone. When in bloom, a number of species within this group are Management Protocol fish killers, particularly where fish cannot avoid The following management protocol has been the blooms. Despite this, the vast majority of these designed to ensure that mussels harvested from species donʹt appear to cause any adverse PPB and WP are safe for human consumption, reactions in humans who consume shellfish from and that harvesting does not occur when the bloom areas in Australia or New Zealand. In mussels are affected by biotoxins. The protocol is south‐eastern Australia, only one species of based on the following key factors: Gymnodinium has found to be toxic to humans. • A number of species of Gymnodinium occur It must be stressed that the taxonomy of this naturally in PPB and WP. group is poorly understood, although this • Definitive identification of the various species situation has improved over the past two years of these genera may be difficult. with an increase in the research being carried out in New Zealand and Australia. G. catenatum is a • G. catenatum has NOT been recorded from chain forming dinoflagellate which can be PPB but should it be introduced, it is most recognised under the light microscope. Care likely to occur near the Yarra River some should be taken not to confuse it with Alexandrium distance from the shellfish harvesting areas, catenella, another chain forming dinoflagellate. in the same way as it is found in estuaries in Under suboptimal conditions, G. catenatum may Tasmania. be present as single cells in low numbers. These • may require expert assistance to identify. G. catenatum was introduced to south‐east Tasmania, probably in ballast water, and has Identification is best done with live material. become well established in the Huon and Gymnodinium catenatum produces sulphamate Derwent Estuaries. Shellfish farms have been saxitoxins that accumulate in shellfish such as adversely affected since 1986 although the mussels and oysters. This may cause Paralytic degree of impact varies from year to year. Shellfish Poisoning (PSP) in humans who • consume contaminated shellfish. Extreme cases of Gymnodinium catenatum produces PSP with the potential to cause serious human illness. toxication may result in death although this is rare. Within Australia, only mild cases of • Extreme PSP intoxication is potentially lethal poisoning have been reported in Tasmania to human beings (three children died in (Hallegraeff, 2002). Mexico), although only mild cases have The symptoms of PSP include numbness, occurred in Tasmania attributable to dizziness, nausea, tingling in the extremities, Gymnodinium catenatum. vomiting and diarrhoea in mild cases (within 30 • Due to the status of PPB and WP as harbours minutes), to choking sensations, breathing and the presence of a substantial number of difficulties and death from respiratory paralysis foreign species probably introduced through within two to 24 hours after ingestion in severe ballast water, there is a danger that other cases (Hallegraeff, 1997). toxic forms such as G. catenatum will be G. catenatum is a major problem in south‐east introduced. Tasmania, particularly the Huon Estuary where • The PSP toxin fractions produced by G. extensive salmonid aquaculture is carried out. catenatum are less toxic than those produced This species appears to have been introduced to by several PSP producing species of

Marine Biotoxin Management Plan 61 Alexandrium, but there is concern that cooking testing. The laboratory should be advised of may increase toxicity. the phytoplankton result and the urgency of • Routine phytoplankton sampling and the situation. biotoxin testing for PSP will continue at all 7. Where tissue is found to contain PSPs at a VSQAP harvesting areas. level exceeding 80 µg/100g tissue (the regulatory limit), harvesting is to be This management protocol has been devised for use within the VSQAP should Gymnodinium suspended and is to remain suspended until catenatum be found in the vicinity of the shellfish three successive samples over a two‐week µ growing areas in PPB or WP (NOT the case to period reveal toxin levels < 80 g/100g tissue. date). It adopts the conservative alert levels 8. Where lower levels of toxin are detected recommended by the Australian Marine Biotoxin during the growth phase of a bloom, Management Plan for Shellfish Farming (2001). harvesting should be suspended and sampling frequency increased to monitor the

1. Phytoplankton and mussel tissue samples are development of the bloom. taken routinely each fortnight at all VSQAP harvesting areas, and the tissue routinely 9. Where toxin levels have exceeded the 80 analysed monthly for PSP. µg/100g tissue regulatory limit during a bloom, but the bloom is clearly degenerating,

2. If what is suspected to be Gymnodinium harvesting may be resumed once toxin levels catenatum is detected in a routine sample at remain less than 80 µg/100g for three an abundance of >100 cells/L (1 cell/mL), a successive samples taken over a two‐week warning should be issued to the relevant period. growers and the sampling frequency reviewed with a view to increasing to 10. If Gymnodinium catenatum is present, and/or weekly. This is important as previous history low PSP levels are detected, phytoplankton shows that the numbers of many and biotoxin samples should be taken every phytoplankton can increase very rapidly. four days. 3. Notify the laboratory performing the testing 11. When harvesting is suspended during a toxic of the presence of this species. bloom, the sampling frequency should be reviewed and amended to ensure it is

4. A live phytoplankton sample (preferably adequate to detect changes in either concentrated) is to be sent by overnight phytoplankton or biotoxin levels in an courier to Professor Gustaff Hallegraeff at the effective and timely manner. University of Tasmania for definitive identification. Advise Professor Hallegraeff 12. When harvesting is suspended during a toxic in advance the sample has been despatched. bloom, the sampling frequency may be reduced to the routine fortnightly monitoring

5. If there is uncertainty concerning the program to save resources and costs. identification as Gymnodinium catenatum, PSP However, three ‘clear’ biotoxin results (clear toxicity should be assumed until biotoxin = < 80 µg/100g tissue) over a two‐week period levels are known. are required before harvesting can resume.

6. Where Gymnodinium catenatum is detected in 13. Once Gymnodinium catenatum and PSP toxins numbers >500cells/L, harvesting should be are undetectable, the routine sampling suspended pending the results of tissue regime may be resumed. VSQAP Phytoplankton Abundance Threshold Levels (cells/L) Phytoplankton Toxin Warning Tissue Harvest Harvest Resumption Species Issued Testing Suspension to Growers (Pending Toxin Analysis) Gymnodinium PSP 100 Routine 500 <80 µg/100g PSP for 3 successive catenatum or 100 samples over 14 days; phytoplankton abundance not rising. PSP REGULATORY LIMIT: 80 µg saxitoxin equivalents/100g tissue

Marine Biotoxin Management Plan 62 General would be prudent to consult the Australian Marine It must be stressed that these protocols are Biotoxin Management Plan for Shellfish Farming specifically designed for use within PPB and WP (2001). For some phytoplankton, more where extensive phytoplankton records exist conservative phytoplankton triggers are extending from 1987 to the present. Where other recommended than those utilised as part of the regions are involved, due to the variability in the VSQAP. toxicity of the various forms of dinoflagellate, it

Phytoplankton Species Toxin Tissue Sampling Industry Public Health Abundance Voluntary Warning Threshold Trigger Closure Level (cells/L) (cells/L) (Pending Biotoxin Analysis) (cells/L) Gymnodinium catenatum PSP 100 N/A N/A

References Hallegraeff, G. (1997). Algal toxins in Australian Todd, K. (2001). Australian Marine Biotoxin Management Shellfish. In: Foodborne Microorganisms of Public Health Plan for Shellfish Farming. Prepared for the Australian Significance. Fifth Edition. AIFST (NSW Branch), Food Shellfish Quality Assurance Committee (ASQAAC) by Microbiology Group. the Cawthron Institute. Cawthron Report No. 645 Hallegraeff, G. (2002). Aquaculturistsʹ Guide to Harmful Australian Microalgae. The Print Centre, Hobart.

Marine Biotoxin Management Plan 63 Karenia brevis, Karenia cf brevis and Karenia mikimotoi Background that microbiological contamination may have Karenia spp. are small, unarmoured dinoflagellate played a role in the illness (Todd, 2000). It was phytoplankton (golden‐brown algae with a large later found that what was reported as Karenia cf nucleus). They have two flagella, one protruding brevis or Gymnodinium cf mikimotoi at that time from a horizontal girdle groove and the other may have contained four or more gymnodinioid from a vertical sulcus groove (Hallegraeff, 2002). species including what, in New Zealand, have been called K. brevisulcata and K. sellifromis as well Many species belonging to this and related genera as Karenia bidigitata, Gymnodinium aureolum and K. are common in Australian and New Zealand mikimotoi. The latter species is very common in waters. When in bloom, a significant number of Australia, including Victoria. At the 18th Marine species within this group are fish killers, Biotoxin Science Workshop (2001) in the New particularly where fish cannot avoid the blooms. Zealand, the consensus was that K. mikimotoi was Despite this, the vast majority donʹt appear to the dominant organism present although it is still cause any adverse reactions in humans who unclear exactly what species were present during consume shellfish from bloom areas in Australia the event and which was responsible for the or New Zealand. incident. It is stressed that the taxonomy of this group is Karenia mikimotoi is very common in Victorian poorly understood although this situation has waters and has been associated with fish kills. improved over the past two years with an Like other species resembling K. brevis, it is a increase in the research being carried out in New flattened species although much less so in extent. Zealand and Australia. Species of Karenia may be It has never been associated with human toxicity very difficult to identify definitively using light in Australia despite huge blooms of it including in microscopy and expert assistance should be the waters of shellfish harvesting areas, and large sought. Identification is best done with live quantities of mussels have been consumed from material. areas where it was present. Karenia brevis in Florida which is principally There has only been a single recorded NSP associated with fish kills, produces brevetoxins incident in Australia occurring in Gippsland, (BTX) that may cause non‐fatal but unpleasant Victoria in 1994, resulting from the consumption neurological symptoms in humans exposed to of wildstock mussels from the Tamboon Inlet. them by direct contact (eg. swimming through Karenia cf brevis was identified as the causative blooms), inhalation (eg. near fish kills or breaking organism (Arnott, 1998; Todd, 2001). Karenia cf waves containing blooms) or through the brevis has also been recorded twice in Port Phillip consumption of contaminated shellfish. For mild Bay (PPB) at the Clifton Springs aquaculture cases of intoxication, the symptoms include chills, harvesting area as part of VSQAP phytoplankton headache, diarrhoea, muscle weakness, muscle monitoring, in numbers up to 32,000 cells/L and joint pain and vomiting three to six hours (Arnott et al 1999). However, there have been no after exposure. In extreme cases, other symptoms reports of any type of shellfish poisoning over may occur including paraesthesia, altered that period. Whether this was the same species as perception of hot and cold, difficulty breathing, that implicated at Tamboon Inlet, or another double vision, and trouble talking and species resembling K. brevis, is not known. swallowing (Hallegraeff 1997, 2002). There have been no fatalities associated with NSP Based on ELISA, the main BTX producer in New intoxication. Zealand is considered to be Karenia mikimotoi but several other, dorsally flattened species were It is doubtful that Karenia brevis sensu strictu is identified as producing much lower BTX levels. present in Australia or New Zealand but a group The actual BTX levels produced by New Zealand of morphologically similar species identified as Karenia species are not known. However, in the Karenia cf brevis are present. Approximately 180 USA, K. mikimotoi produces only about one third cases of shellfish poisoning occurred in New as much BTX as K. brevis (Todd, 2002) and New Zealand in 1993. It was concluded the symptoms Zealand isolates produce much lower levels than experienced by these people after consuming these. In Florida, shellfish harvesting is shellfish, were most likely caused by an NSP suspended when K. brevis numbers reach 5,000 toxin. However, it was also noted that other toxins cells/L (Hallegraeff, 2002). were present apart from BTX including DSP, and

Marine Biotoxin Management Plan 64 Other species such as Karenia selliformis from New Karenia brevis (NSP – BTX; Unlikely to be Zealand are known to be associated with fish kills present in Aust.) and produce ichthyotoxins including Karenia cf brevis (?NSP; Flattened species gymnodimine. Gymnodimine is not a risk to like K. brevis; PPB, human health and does not produce NSP, Gippsland, NZ) although it can kill mice during bioassays. Although there is little evidence these species are Karenia mikimotoi (?NSP; Fish kills; toxic to human consumers of shellfish, the widespread incl. PPB, ichthyotoxins they produce are not well Gippsland Lakes; toxicity understood and further local information should questionable) be gathered. Karenia (low levels NSP ‐ New BTX testing by mouse bioassay is not as yet papilionaceum Zealand; fish kills) routinely carried out in Australia, but is in New Zealand. However, its interpretation is complex Karenia selliforme (NSP (gymnodimine) ‐ New Zealand; fish kills) and can be complicated by the effects of other marine toxins and related compounds such as Karenia bidigitatum (low levels NSP ‐ New gymnodimine, Wellington Harbour Toxin and Zealand; fish kills) fatty acids naturally found in shellfish that may kill mice during bioassay but NOT indicate Karenia digitata (fish kills; Hong Kong human toxicity from NSP (false +ve). The Harbour) Cawthron Institute in New Zealand has recently Karenia (fish kills, toxin?; Hong developed more definitive LC/MS methods for cf longicanalis Kong, Tasmania (similar NSP analysis that will improve the management sp.) of this biotoxin and eventually replace the current ether mouse bioassay (Todd, 2002). Karenia brevesulcata (fish kills, ʺWellington Harbourʺ Toxin; At this time, within PPB and Western (WP) only Wellington Harbour only, one or more flattened Karenia species similar to K. NZ) brevis and perhaps K. mikimotoi seem to offer any potential for human toxicity from the Management Protocol consumption of shellfish. The risk appears slight The following management protocol relates to but until more information is known, their Karenia brevis, Karenia cf brevis (flattened presence should be monitored and toxin testing Australian species similar to K. brevis) and Karenia performed when the threshold abundance levels mikimotoi. It has been designed to ensure that are exceeded. mussels harvested from PPB and WP are safe for human consumption, and that harvesting does The potentially toxic Karenia known from not occur when the mussels are affected by southern Australia, and in particular from PPB biotoxins. The protocols are based on the and WP in Victoria, include the following species. following key factors: Due to the uncertain state of the taxonomy of this group, some other fish kill species not yet found • A number of species of Karenia and related in PPB, WP or Australia have been listed. Out of genera occur naturally in PPB and WP. this group of dinoflagellates, the Australian Marine • Definitive identification of the various species Biotoxin Management Plan for Shellfish Farming of Karenia may be difficult. (2001) lists only Karenia cf brevis in its phytoplankton abundance trigger table. The • A form of Karenia in PPB has been identified FSANZ Food Standards Code regulatory limit for as Karenia cf brevis (=Gymnodinium cf breve) in NSP is adopted for the VSQAP. the past although the level of knowledge of the taxonomy of this group was incomplete at that time. • Large blooms of Karenia mikimotoi have been recorded in PPB, which were responsible for massive fish kills in 1950s.

Marine Biotoxin Management Plan 65 • There is no evidence of an incident of NSP 2. If the abundance of the above species rises to intoxication in PPB or WP despite blooms of 2,000 cells/L, then a mussel tissue sample Gymnodinium/Karenia occurring in the past, must be collected and sent to the relevant including Karenia mikimotoi and Karenia cf laboratory for biotoxin analysis. In most brevis. instances, mussel tissue samples will already have been collected during routine sampling • Based on New Zealand experiences, there in case phytoplankton monitoring indicated may be a risk of NSP from Karenia brevis (if biotoxin analysis was required. In this present in Australia), Karenia cf brevis (other, instance, the tissue should be prepared and flat Karenia species that resemble K. brevis then dispatched to the analytical laboratory which may produce BTX) and Karenia as quickly as possible. mikimotoi. 3. Sampling frequency should be reviewed to • In Florida, shellfish harvesting is banned ensure it is adequate to detect a rapid when K. brevis abundance reaches 5,000 increase in phytoplankton numbers. A live cells/L (Hallegraeff, 2002). K. mikimotoi phytoplankton sample (preferably produces only a third as much toxin in the concentrated) is to be sent by overnight USA as K. brevis, and even less in New courier to Professor Gustaff Hallegraeff at the Zealand. The threshold levels recommended University of Tasmania for definitive in the Australian Marine Biotoxin identification. Advise Professor Hallegraeff in Management Plan for Shellfish Farming advance the sample has been despatched. (based on the New Zealand limits) of 1,000 cells/L for tissue testing and 5,000 cells/L for 4. If there is uncertainty concerning the voluntary harvesting suspension and the identification as Karenia brevis, Karenia cf issue of public health warnings, seem very brevis or Karenia mikimotoi, NSP toxicity conservative but similar triggers have been should be assumed until biotoxin levels are adopted for the VSQAP pending more, known. locally derived information. 5. Where Karenia brevis, Karenia cf brevis or • There is no evidence that other fish killing Karenia mikimotoi are detected in combined species can cause human intoxication as a numbers >5,000cells/L, harvesting should be result of the consumption of shellfish. suspended pending the results of tissue testing. The laboratory should be advised of The following management protocol adopts the phytoplankton result and the urgency of threshold levels similar to those recommended by the situation. the Australian Marine Biotoxin Management Plan for Shellfish Farming (2001). Expert assistance may be 6. Where tissue is found to contain NSP (BTX) required to identify the relevant species. at a level exceeding 20 MU/100g tissue (the Phytoplankton and mussel samples are taken at regulatory limit), harvesting is to be all VSQAP harvesting areas every fortnight. suspended and is to remain suspended until Biotoxin testing is carried out when the two successive samples taken at least two phytoplankton abundance triggers indicate this is days apart reveal toxin levels < 20 MU/100g necessary. tissue. 1. If what is suspected to be Karenia mikimotoi, K. 7. Where lower levels of toxin are detected brevis or Karenia cf brevis is detected in a during the growth phase of a bloom, routine sample at an abundance of >1,000 harvesting should be suspended and cells/L (1 cell/mL), a warning should be sampling frequency increased to monitor the issued to all relevant growers. development of the bloom. 8. Where toxin levels have exceeded the 20 MU/100g tissue regulatory limit during a bloom, but the bloom is clearly degenerating, harvesting may be resumed once toxin levels remain less than 20MU/100g for two successive samples taken at least two days apart.

Marine Biotoxin Management Plan 66 9. If Karenia brevis, Karenia cf brevis and/or program to save resources and costs. Karenia mikimotoi are present in numbers However, two ‘clear’ biotoxin results (clear = approaching trigger levels and/or low NSP < 20 MU/100g tissue) at least seven days apart levels are detected, the phytoplankton and are required before harvesting can resume. biotoxin sampling frequency should be Once Karenia brevis, Karenia cf brevis and revised to ensure adequate monitoring of any 11. Karenia mikimotoi abundance is clearly less bloom that may develop. than the trigger values and NSP toxins are 10. When harvesting is suspended during a toxic undetectable, the routine fortnightly bloom, the sampling frequency may be sampling regime may be resumed. reduced to the routine fortnightly monitoring

VSQAP Phytoplankton Abundance Threshold Levels (cells/L) Phytoplankton Toxin Warning Tissue Harvest Harvest Resumption Species Issued Testing Suspension to Growers (Pending Toxin Analysis) Karenia brevis NSP 1,000 2,000 5,000 <20 MU/100g for two successive (Probably not present samples taken at least 2 days in Australia) apart; phytoplankton abundance not rising Karenia cf brevis ?NSP 1,000 2,000 5,000 As Above (Flattened Australian species resembling K. brevis)

Karenia mikimotoi ?NSP 1,000 2,000 5,000 As Above

NSP REGULATORY LIMIT: 20 MU/100g tissue

General due to the variability in the toxicity of the various It must be stressed that these protocols are forms of dinoflagellate, it would be prudent to specifically designed for use within PPB and WP consult the Australian Marine Biotoxin Management where extensive phytoplankton records exist Plan for Shellfish Farming (2001) – abundance through VSQAP monitoring, extending from 1987 triggers detailed below. to the present. Where other regions are involved,

Phytoplankton Species Toxin Tissue Sampling Industry Voluntary Public Health Abundance Closure Level Warning Threshold Trigger (Pending Biotoxin Analysis) (cells/L) (cells/L) (cells/L) Gymnodinium cf breve NSP 1,000 5,000 5,000

Marine Biotoxin Management Plan 67 References Significance. Fifth Edition. AIFST (NSW Branch), Food ANZFA (2002). Food Standards Code. Australian and Microbiology Group. New Zealand Food Authority Hallegraeff, G. (2002). Aquaculturistsʹ Guide to Harmful Arnott, G.H. (1998). Toxic Marine Microalgae: A Australian Microalgae. The Print Centre, Hobart. worldwide Problem With Major Implications for Todd, K. (2001). Australian Marine Biotoxin Management Seafood Safety. Advancing Food Safety 1: 24 ‐ 34 Plan for Shellfish Farming. Prepared for the Australian Arnott, G.H., Reilly, D.J. and Werner, G.F. (1999). Shellfish Quality Assurance Committee (ASQAAC) by Victorian Shellfish Quality Assurance Program. 7. Sanitary the Cawthron Institute. Cawthron Report No. 645 Survey Update: Clifton Springs and Grassy Point (port Todd, K. (2002). A Review of NSP Monitoring in New Arlington) Aquaculture Zones. Marine and Freshwater Zealand In Support of a New Program. Prepared for the Resources Institute, Report No. 13, 1 – 36 Marine Biotoxin Technical Committee, New Zealand. Hallegraeff, G. (1997). Algal toxins in Australian Cawthron Report 660: 1 – 30 Shellfish. In: Foodborne Microorganisms of Public Health

Marine Biotoxin Management Plan 68 Acronyms AQIS Australian Quarantine Inspection Service ASP Amnesic Shellfish Poison (Poisons – domoic acid) ASQAAC Australian Shellfish Quality Assurance Advisory Committee ASQAP Australian Shellfish Quality Assurance Program AZA Azaspiracids AZP Azaspiracid Shellfish Poison BTX Brevetoxins CC toxins CEC Commission of European Communities DHS Department of Human Services, Victoria DPI Department of Primary Industries, Victoria DSP Diarrhetic Shellfish Poison (Poisons – OA, DTX 1‐3, PTX ) DTX Dinophysis toxins ELISA Enzyme Linked Immuno‐Sorbent Assay EPA Environment Protection Agency, Victoria FSANZ Food Standards Australia & New Zealand GTX Gonyautoxins HPLC High Performance Liquid Chromatography LC/MS Liquid Chromatography – Mass Spectrometry MFSP Marine & Freshwater Systems Platform, PIRVic MS Mass spectrometry MU Mouse Units NATA National Association of Testing Authorities neoSTX Neosaxitoxins NSP Neurotoxic Shellfish Poison (Poisons – BTX) OA Okadaic acid PIRVic Primary Industries Research, Victoria PPB Port Phillip Bay PSP Paralytic Shellfish Poison (Poisons – STX, GTX, neoSTX, C toxins) PTX Pectenotoxins PTX‐2‐SA Pectenotoxin‐2‐seco acids SSCA State Shellfish Controlling Agency STX Saxitoxins TSP Toxic Shellfish Poison VAC Victorian Aquaculture Council VSQAP Victorian Shellfish Quality Assurance Program WES WATER ECOscience P/L WP Western Port YTX Yessotoxins ug/100g Micro‐grams per 100 grams

Marine Biotoxin Management Plan 69 Glossary Aquaculture Planning Officer An officer employed to facilitate growth in Victorian aquaculture through the development, coordination and implementation of policies and management plans relating to the aquaculture industry, including the VSQAP. Authorised Officer An officer employed to perform specified duties to ensure compliance with the requirements of the VSQAP. Growing Area A discrete or enclosed marine body of water (e.g. bay, harbour, inlet) in which commercial species of bivalve molluscs grow naturally or by means of aquaculture. Harvesting Area An area that has been designated within a growing area by the SSCA or other competent State authority for the purpose of growing and/or harvesting commercial quantities of shellfish; may include wildstock or shellstock grown by aquaculture. State Shellfish Control Authority State government agency with the legal authority to classify (SSCA) shellfish growing areas, control harvesting and handling of shellstock, and to seize shellstock that is contaminated or has been harvested from prohibited or closed shellfish harvesting areas.

Marine Biotoxin Management Plan 70