A REVIEW OF THE MARINE BIOTOXIN MONITORING PROGRAMME FOR

NON-COMMERCIALLY HARVESTED SHELLFISH

PART 1: TECHNICAL REPORT

A Report for the NZ Ministry of Health

Brenda E. Hay Coral M. Grant

Dorothy-Jean McCoubrey

AquaBio Consultants Ltd P. O. Box 560 Shortland St P.O. Auckland 1 New Zealand Hay@AquaBio.co.nz December 2000

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DISCLAIMER

This technical resources document was prepared under contract to the New Zealand Ministry of Health. The copyright in the report is owned by the Crown and administered by the Ministry. The views of the author do not necessarily represent the views or policy of the New Zealand Ministry of Health. The Ministry makes no warranty, express or implied, nor assumes any liability or responsibility for the use of or reliance on the contents of this report. Neither AquaBio Consultants Limited, nor any of its employees makes any warranty, express or implied, or assumes any liability or responsibility for use of the technical resource document or its contents by any other person or organisation. For bibliographic purposes, this document should be cited as follows: Hay, B. E., Grant, C. M. & McCoubrey, D-J. (2000) A Review of the marine biotoxin monitoring programme for non-commercially harvested shellfish. Part 1: Technical Report. A report prepared for the NZ Ministry of Health by AquaBio Consultants Ltd. NZ Ministry of Health. This document is available on the NZ Ministry of Health’s Web site: http://www.moh.govt.nz ISBN (Book) 0-478-24348-0 ISBN (Internet) 0-478-24349-9

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ACKNOWLEDGEMENTS

We wish to thank all those people, scientists and regulators, who have provided invaluable assistance in the form of information and discussion in the preparation of this report. Special thanks to Penny Truman and Yvonne Galloway from ESR, Kirsten Todd, Lesley Rhodes, Lincoln Mackenzie, and Alison Haywood from Cawthron Institute, Hoe Chang from NIWA, Paul Roberts (Ministry of Health) and the local Health Protection Officers in each area. We are very grateful to Beatriz Reguera (Instituto Español de Oceanografia), Paul Anderson (University of Maine) and Don Richard (Canadian Food Inspection Agency), who spent what must have been many hours reviewing our first draft. Particular thanks to Phil Busby (MAF), who, in addition to commenting on our drafts, provided us with access to many papers and books, and waited patiently for us to return them. Lastly, thanks to Janet Young, of the Ministry of Health, who efficiently provided us with information and feedback, and smoothed the process of this review throughout its course.

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FOREWORD Any review of an on-going monitoring programme runs the risk of being superseded by events subsequent to the period for which data analysis has been undertaken. This review is based on analysis of phytoplankton and shellfish data up to the end of June 1999. Since that time, there has been a major bloom of Gymnodinium catenatum off the western coast of the North Island. In addition, David Stirling of the Institute of Environmental and Scientific Research (ESR), is currently undertaking analysis of archived shellfish samples using LC-MS, which may throw some light on the identity of some of the unexplained acetone screen positive test results. The implications of these additional sets of data have not been considered in this review. Brenda Hay December 2000

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EXECUTIVE SUMMARY

Prior to a major biotoxins event in the summer of 1992-93, New Zealand had no recorded incidence of marine biotoxins of public health significance. A marine biotoxin monitoring programme, covering both commercial and non-commercial shellfish harvesting, has been operating since 1993. The current non-commercial marine biotoxin monitoring programme involves regular sampling at 30 phytoplankton sampling sites and 57 shellfish sampling sites. Data from the commercial biotoxin monitoring programme are also purchased. The programme is designed to monitor for potentially toxic phytoplankton, and for the presence of Neurotoxic Shellfish Poisoning (NSP), Paralytic Shellfish Poisoning (PSP), Amnesic Shellfish Poisoning (ASP) and Diarrhetic Shellfish Poisoning (DSP) (Okadaic acid and dinophysistoxins) toxins at levels that present a risk to human health. Internationally, New Zealand is unusual in having detected a wide range of different biotoxins. In addition to those producing PSP, ASP, NSP and “classic” DSP, pectenotoxin and yessotoxin have been detected in shellfish, and palytoxin in phytoplankton. A range of non-toxic compounds, or compounds of unknown toxicity, have also been found, including gymnodimine, and “Wellington Harbour toxin” (oral toxicity unknown). Compounds that are inferred to have caused Respiratory Irritation Syndrome (RIS) in New Zealand include brevetoxin and the “Wellington Harbour toxin”. The data collected in the marine biotoxin monitoring programme since 1993 presents some challenges to analysis: the data are not independent, and are stratified both spatially and temporally over several different scales. Analysis is further complicated by changes in toxin test methods, and differences in biotoxin accumulation and retention between different shellfish species. Effectively only a small proportion of the data can be used for meaningful quantitative analysis. In very broad overview, the situation in New Zealand with respect to marine biotoxins is characterised by: • Wide distribution of potentially toxin-producing phytoplankton throughout New

Zealand. • Periods of low frequency of biotoxin occurrence followed by periods of higher

biotoxin occurrence, generally in relatively localised areas. Biotoxins may then persist in a localised area for a period of time, sometimes in one shellfish species/at one location.

• Possible seasonal patterns in the occurrence of some biotoxins in shellfish (for example, NSP and ASP), and not in others.

• In periods of low biotoxin activity, some toxins are present very rarely (e.g. NSP toxins) and others are common at low levels in some areas (e.g. Domoic acid).

• Possible differences in the accumulation and retention of biotoxins by different New Zealand shellfish species. These differences are potentially significant in terms of the risk of Toxic Shellfish Poisoning (TSP) to consumers.

Currently there is a poor understanding, both here and internationally, of the factors influencing the occurrence of toxic phytoplankton blooms. There is insufficient

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information to be able to predict the future occurrence of marine biotoxins in New Zealand with confidence. The potential risks presented by marine biotoxins in New Zealand are not distributed evenly across the population. There is a disproportionate potential impact on sectors of the population that consume more non-commercially harvested shellfish (for example, Maori, and possibly Pacific and Asian peoples), on older people or people in poor health, and on asthmatics. The potential risks of TSP are distributed geographically with availability of desirable shellfish for harvest. There have been few studies undertaken on non-commercial shellfish harvesting in New Zealand, and there are significant discrepancies between the results of the studies. There are limited good quality epidemiological data for TSP in New Zealand: although 457 cases potentially related to TSP have been reported between January 1993 and June 1999, only 9 (1.97%) have been classed as probable cases of TSP. There are no confirmed cases under the current case definitions. The oral toxicity of some of the marine biotoxins found in New Zealand, including the effect of long-term ingestion of low levels of toxin, are still unknown. The current outcome surveillance may not detect these impacts. Lack of robust data severely limits quantitative assessment of the risks associated with biotoxins in New Zealand. There are sufficient data to suggest that in the absence of a non-commercial biotoxin monitoring programme, a significant number of people could become ill as a result of TSP in some years. Some of these people would be severely ill, with the risk of death. For example, one scenario suggests that 50% of the PSP cases (amounting to nearly 2000 people in a scenario relating to the Bay of Plenty) would have moderate to severe symptoms. The long-term effects of ingestion of “DSP” toxins, including Okadaic acid, dinophysistoxins, yessotoxin and pectenotoxin, are unknown, but potentially present a risk. The incidences of these two latter toxins are unknown because they are not currently included in the monitoring programme. The levels of Domoic acid detected in shellfish to date would only produce relatively mild symptoms of ASP in adults (although one scenario suggests that up to 750 people could be affected). The potential impact of NSP remains somewhat unknown due to a lack of robust data on shellfish toxicity levels from the 1993 event, but has been very low in subsequent years. RIS events have occurred in two of the last six years, and depending on wind strength and direction, affect anyone within a few kilometres of the coast. Asthmatic people are the most seriously affected, and data suggest that approximately 9.8% of people exposed to RIS toxins in New Zealand could potentially suffer an asthma attack as a result. Within the strategic framework for public health, and the global context of developments relating to marine biotoxins, a range of broad issues relating to the management of marine biotoxins in New Zealand require consideration. These encompass both strategic and technical issues. It is suggested that a more proactive, strategic approach to the collection and use of data would result in substantial improvements in cost effectiveness in the future. This would involve:

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• Improved management of the data collected in the marine biotoxin monitoring programme.

• A sampling strategy to ensure that robust, long-term data are available to detect patterns in the geographic and temporal distribution of biotoxins.

• A co-ordinated management strategy that ensures that sampling and analytical techniques used in the monitoring programme are scientifically validated, and that changes in the programme are consistent with the results of risk analysis and long-term strategies to increase cost-effectiveness.

An increased emphasis on advocacy and facilitation of relevant research utilising strategic alliances with organisations with common interests, is suggested to increase the cost-effectiveness of the marine biotoxin monitoring programme in the future. Research is required to: • Determine inter-specific differences between shellfish with respect to biotoxin

uptake, accumulation and detoxification processes. • Determine the oral toxicity, including the impacts of long-term ingestion of low

levels of toxin, for those compounds currently known to be toxic to mice by IP injection, but for which oral toxicity is unknown.

• Relate environmental parameters and processes to the occurrence of toxic phytoplankton, and toxin levels within species.

• Validate new toxin testing methods for biotoxins found in New Zealand (such as the PSP MISTTM Alert Kit).

• Rigorously determine non-commercial shellfish harvest patterns. • Evaluate the effectiveness of education and communication strategies with respect

to the management of the risk of marine biotoxins in New Zealand. A variety of changes to the marine biotoxin monitoring programme are discussed in response to both the results of data analysis, and to interest expressed by the Ministry of Health:

• Consideration is given as to whether costs could be reduced by broad reduction in temporal or spatial components of the monitoring programme in the light of a re-evaluation of risk arising from analysis of historical data. Based on consideration of risks to public health in the absence on monitoring, the value of the available historical data in the prediction of future events, and the lack of definitive data on non-commercial harvesting patterns, it is concluded that broad reductions in monitoring could not be recommended unless an increased level of risk to the public were acceptable under the current strategy for public health.

• It is suggested that species-specific public warnings be considered in areas

where consumption of non-commercially harvested shellfish is very important culturally or economically, and that are subject to public warnings for a long period of time due to biotoxin persistence in one shellfish species only. This would help to maintain the credibility of the marine biotoxin monitoring programme, which is compromised when people consistently harvest shellfish when a public health warning is in place but do not become ill. A telephone

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“hotline” with regularly up-dated recorded messages containing information about the biotoxin status of each area, is suggested as a useful method of conveying more complex information that the public may not remember from media messages.

• It is noted that there is currently no specific surveillance or management of

risk with respect to RIS, although this does occur informally to some extent. Formalisation of protocols for RIS, based on utilisation of the current phytoplankton monitoring programme, is suggested as an option.

• The development of new biotoxin test methods, changing social attitudes

toward the use of animals in testing, and moves by the shellfish industry to adopt new test methods, provide opportunities and impetus for consideration of new test methods. Testing that focuses more specifically on identifying particular toxins, or, in the case of functional assays, specific modes of toxic activity, has advantages in the reduction of “false positive” toxicity results that may occur in mouse bioassays. However, a move to testing for specific biotoxins or types of toxin activity would remove the hazard surveillance for new biotoxins currently provided by the mouse bioassays. In this situation, robust outcome surveillance would be of increased importance. In some instances, new biotoxin test methods may provide cost advantages over the current mouse bioassays. Specific options with respect to the potential use of new test methods are discussed in Part 2 of this report under separate cover.

• Further data are required to confirm the robustness of the current

phytoplankton monitoring programme in predicting shellfish toxicity. With respect to some phytoplankton species, the impact of the low level of precision in the phytoplankton methods currently used, and whether any additional assurance gained by improving this precision justifies additional cost, needs to be considered. In addition, the impact of succession of Pseudo-nitzschia species within a Pseudo-nitzschia bloom on the protocols for use of gene probes within the monitoring programme, requires further investigation before being formally incorporated into the marine biotoxin monitoring programme.

Many of the technical issues that require consideration in marine biotoxin monitoring are complex and specialised. It is suggested that an increased level of advice, from appropriately qualified technical specialists independent of organisations that may provide monitoring services, should be sought to peer review technical proposals when considering major changes to the marine biotoxin monitoring programme.

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TABLE OF CONTENTS FOREWORD iii EXECUTIVE SUMMARY iv TABLE OF CONTENTS viii LIST OF FIGURES xiii LIST OF TABLES xv SECTION 1: INTRODUCTION 1 1.1 BACKGROUND 1 1.2 THE CURRENT MONITORING PROGRAMME 4 1.3 BIOTOXINS IN NEW ZEALAND 7 1.3.1 Paralytic Shellfish Poisoning (PSP) 7 1.3.2 Amnesic Shellfish Poisoning (ASP) 9 1.3.3 Diarrhetic Shellfish Poisoning (DSP) 10 1.3.4 Neurotoxic Shellfish Poisoning (NSP) 11 1.3.5 Respiratory Irritation Syndrome 12 1.3.6 Other Marine Biotoxins in New Zealand 12

1.4 THE STRATEGIC FRAMEWORK AND SCOPE OF THIS REVIEW 13

SECTION 2: ANALYSIS OF BIOTOXIN MONITORING DATA 18 2.1 INTRODUCTION 18 2.2 GENERAL METHODOLOGY 19 2.2.1 Identification of a Valid Dataset 20 2.2.2 Determination of Areas for Analysis 21 2.2.3 Analysis of Phytoplankton Occurrence 24 a) Geographical Distribution 24 b) Temporal Distribution 25

2.2.4 Reliability of Phytoplankton Monitoring as a Predictor of Biotoxins in Shellfish 26 2.2.5 Differences in Biotoxin Accumulation between Shellfish Species 26 2.2.6 Summary of Cumulative Monitoring Results- Shellfish and Phytoplankton 26 2.2.7 Analysis of Occurrence of Biotoxins in Shellfish 27 a) Geographical Distribution 27 b) Temporal Distribution 28

2.3 CUMULATIVE MONITORING RESULTS 30 2.4 RESULTS OF ANALYSIS – PSP 32 2.4.1 Geographic Distribution 32 2.4.2 Temporal Distribution 41 2.4.3 Phytoplankton as a Predictor of PSP in Shellfish 44 2.4.4 Conclusions 46

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2.5 RESULTS OF ANALYSIS – ASP 48 2.5.1 Geographic Distribution 48 2.5.2 Temporal Distribution 59 2.5.3 Phytoplankton as a Predictor of ASP in Shellfish 62

2.5.4 The use of Whole Cell DNA Probes for Pseudo-nitzschia Species as a Predictor of Risk of Shellfish Toxicity 63

2.5.5 Conclusions 66 2.6 RESULTS OF ANALYSIS – DSP 68 2.6.1 Geographic Distribution 68 2.6.2 Temporal Distribution 79 2.6.3 Reliability of Acetone Screen 81

2.6.4 Phytoplankton as a Predictor of DSP in Shellfish 84 2.6.5 Conclusions 85 2.7 RESULTS OF ANALYSIS – NSP AND RESPIRATORY IRRITATION SYNDROME 86 2.7.1 Introduction 86 2.7.2 Geographic Distribution 86 2.7.3 Temporal Distribution 96 2.7.4 Conclusions 97 SECTION 3: NON-COMMERCIAL SHELLFISH GATHERING

AND CONSUMPTION IN NEW ZEALAND 98 3.1 INTRODUCTION 98 3.2 DISTRIBUTION OF SHELLFISH IN NEW ZEALAND 98 3.3 SHELLFISH GATHERING 104

3.4 POPULATION STRUCTURE AND SHELLFISH CONSUMPTION 109

3.5 TEMPORAL PATTERNS 112 3.6 CONCLUSION 112

SECTION 4: ANALYSIS OF EPIDEMIOLOGICAL DATA 113 4.1 INTRODUCTION 113 4.2 METHODOLOGY AND ASSUMPTIONS IN ANALYSIS 114 4.3 RESULTS 116 4.4 DISCUSSION 122 SECTION 5: RISK ASSESSMENT 125 5.1 INTRODUCTION 125 5.2 PARALYTIC SHELLFISH POISONING 125 5.2.1 Hazard Identification 125

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5.2.2 Dose-Response Assessment 127 5.2.3 Exposure Assessment 127 5.2.4 Risk Characterisation 128 5.3 AMNESIC SHELLFISH POISONING 130 5.3.1 Hazard Identification 130 5.3.2 Dose-Response Assessment 131 5.3.3 Exposure Assessment 131 5.3.4 Risk Characterisation 132 5.4 DIARRHETIC SHELLFISH POISONING 134 5.4.1 Hazard Identification 134 5.4.2 Dose-Response Assessment 136 5.4.3 Exposure Assessment 136 5.4.4 Risk Characterisation 137 5.5 NEUROTOXIC SHELLFISH POISONING 139 5.5.1 Hazard Identification 139 5.5.2 Dose-Response Assessment 139 5.5.3 Exposure Assessment 140 5.5.4 Risk Characterisation 141 5.6 RESPIRATORY IRRITATION SYNDROME 142 5.6.1 Hazard Identification 142 5.6.2 Dose-Response Assessment 143 5.6.3 Exposure Assessment 143 5.6.4 Risk Characterisation 144 SECTION 6: SUMMARY BY AREA 146 6.1 ZONE A 146 6.2 ZONE B 148 6.3 ZONE C 149 6.4 ZONE D 150 6.5 ZONE E 151 6.6 ZONE F 152 6.7 ZONE G 153 6.8 ZONE H 155 6.9 ZONE I 156 6.10 ZONE J 157 6.11 ZONE K 158 SECTION 7: BIOTOXIN MANAGEMENT IN NEW ZEALAND

AND OVERSEAS 161 7.1 EDUCATION AND COMMUNICATION STRATEGIES 161

7.2 OVERSEAS BIOTOXIN MONITORING PROGRAMMES

AND TECHNOLOGY DEVELOPMENTS 164

7.3 NEW ZEALAND BIOTOXIN MANAGEMENT IN A GLOBAL CONTEXT 168

SECTION 8: DISCUSSION AND CONCLUSION 170

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LITERATURE CITED 178 APPENDICES 193 APPENDIX I(A) PHYTOPLANKTON TRIGGER LEVELS 194 APPENDIX I(B) THE COMMERCIAL AND NON-COMMERCIAL

MONITORING PROGRAMME SAMPLING REGIMES 195

APPENDIX I(C) FLOW DIAGRAM ILLUSTRATING SHELLFISH TISSUE TESTING FOR NSP AND DSP 202

APPENDIX I(D) MAP SHOWING LOCATION OF BIOTOXIN

ZONES 203

APPENDIX II PHYTOPLANKON SITES INCLUDED IN ANALYSIS OVER THE “IDENTIFIED TIME INTERVAL” 204

APPENDIX III TEMPORAL PERIODICITY OF EL NINO/LA NINA WEATHER CONDITIONS 206

APPENDIX IV(A) MAP SHOWING THE LOCATION OF SAMPLING

SITES IN THE MARLBOROUGH SOUNDS (ZONE G) 207

APPENDIX IV(B) SITE COMPARISONS OF PSEUDO-NITZSCHIA OCCURRENCE IN THE HAURAKI GULF AND MARLBOROUGH SOUNDS/COLLINGWOOD 209

APPENDIX IV(C) SITE COMPARISONS OF DINOPHYSIS OCCURRENCE

IN THE HAURAKI GULF AND MARLBOROUGH SOUNDS/COLLINGWOOD 212

APPENDIX IV(D) SITE COMPARISONS OF GYMNODINIUM c.f. MIKIMOTOI OCCURRENCE IN THE HAURAKI GULF AND MARLBOROUGH SOUNDS /COLLINGWOOD 215

APPENDIX V RESULTS FROM WHOLE CELL DNA PROBES FOR PSEUDO-NITZSCHIA SPECIES 218

APPENDIX VI PSEUDO-NITZSCHIA SPECIES COMPOSITION AT THE SAME SITE OVER CONSECUTIVE WEEKS, DETERMINED BY WHOLE CELL GENE PROBES 223

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LIST OF FIGURES SECTION 1: INTRODUCTION 1

Figure 1.1 Current Phytoplankton Monitoring Sites 6 SECTION 2: ANALYSIS OF BIOTOXIN MONITORING DATA 18 Figure 2.1 Distribution of Biotoxin Zones and relevant

hydrographical features associated with the New Zealand coastline 23

Figure 2.2 Distribution of potentially toxic species of Alexandrium throughout New Zealand 33

Figure 2.3 Distribution of PSP at shellfish sample sites throughout New Zealand 34 Figure 2.4 Box and whisker plots showing the frequency

distribution of potentially toxic Alexandrium sp. above the regulatory levels 36

Figure 2.5 Comparison of levels of PSP in Greenshell™ mussels and tuatua from Ohope Beach 39

Figure 2.6 Comparison of levels of PSP in mussels, tuatua, and scallops from Waihi Beach 39

Figure 2.7 Comparison of levels of PSP in mussels and scallops from Rangaunu Bay 40 Figure 2.8 Cumulative incidence of PSP toxins above the

detectable level within each zone by month 42 Figure 2.9 Distribution of detectable levels of PSP toxin in

shellfish from consistently monitored sites 42 Figure 2.10 Distribution of Pseudo-nitzschia sp. in

New Zealand 49 Figure 2.11 Distribution of ASP at shellfish sampling sites throughout New Zealand 50 Figure 2.12 Box and whisker plots showing the frequency

distribution of Pseudo-nitzschia sp. above the regulatory levels 52

Figure 2.13 Comparison of levels of ASP in Greenshell™ mussels and scallops from Takaka River 57

Figure 2.14 Comparison of levels of ASP in Greenshell™ mussels, scallops and cockles from Four Fatham Bay 58

Figure 2.15 Cumulative incidence of ASP toxins above the detectable level within each zone by month 61

Figure 2.16 Distribution of detectable levels of ASP toxin in shellfish from consistently monitored sites 61

Figure 2.17 Distribution of potentially toxic species of Dinophysis throughout New Zealand 69

Figure 2.18 Distribution of potentially toxic Prorocentrum lima throughout New Zealand 70 Figure 2.19 Distribution of DSP at shellfish sample sites throughout New Zealand 71

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Figure 2.20 Box and whisker plots showing the frequency distribution of Dinophysis acuminata above the regulatory levels 73

Figure 2.21 Box and whisker plots showing the frequency distribution of Dinophysis acuta above the regulatory levels 75 Figure 2.22 Cumulative incidence of DSP toxins above the

detectable level within each zone 80 Figure 2.23 Distribution of detectable levels of DSP toxin in

shellfish from consistently monitored sites 81 Figure 2.24 Distribution of potentially toxic Gymnodinium species throughout New Zealand 88 Figure 2.25 Box and whisker plots showing the frequency

distribution of potentially toxic Gymnodinium c.f. mikimotoi above the regulatory levels 91

SECTION 3: NON-COMMERCIAL SHELLFISH GATHERING AND CONSUMPTION IN NEW ZEALAND 98

Figure 3.1 Population distribution by ethnic origin for each regional

authority within New Zealand 110

SECTION 4: ANALYSIS OF EPIDEMIOLOGICAL DATA 113

Figure 4.1 Distribution of suspected and confirmed cases of TSP arising from non-commercially harvested shellfish 120 Figure 4.2 Number of suspected and confirmed cases of TSP from

consumption of different seafood species 121

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LIST OF TABLES SECTION 1: INTRODUCTION 1

Table 1.1 Specific toxicity of species of Alexandrium found in New Zealand waters. 8

Table 1.2 Maximum level of Domoic Acid found in New Zealand isolates of Pseudo-nitzschia. 10

SECTION 2: ANALYSIS OF BIOTOXIN MONITORING DATA 18 Table 2.1 Summary of the cumulative results of the

monitoring for PSP, ASP, DSP and NSP toxins in shellfish (Jan 1993-Jun 1999). Current regulatory levels for each toxin group are also given 30

Table 2.2 Cumulative results of phytoplankton monitoring for all Biotoxin Zones 31

Table 2.3 Percentage occurrence of potentially toxic Alexandrium sp. and percentage above the regulatory levels 35

Table 2.4 Summary by zone of the total occurrence of PSP toxins detected in shellfish and PSP toxins above the regulatory levels 37

Table 2.5 Summary by zone of the occurrence of PSP toxins in Greenshell™ mussels from consistently monitored sites 38

Table 2.6 Summary of the occurrence of PSP toxins in the major shellfish species sampled 38

Table 2.7 Percentage occurrence of Pseudo-nitzschia sp. and percentage occurrence above the regulatory levels 51

Table 2.8 Summary by zone of the total occurrence of ASP toxins detected in shellfish and ASP toxins above the regulatory level 53

Table 2.9 Results of analysis for ASP in whole, and portions, of scallops sampled concurrently 54 Table 2.10 Summary by zone of the occurrence of ASP toxins in

Greenshell™ mussels from consistently monitored sites 55

Table 2.11 Summary by zone of the occurrence of ASP toxins in scallops from consistently monitored sites 55

Table 2.12 Summary of the occurrence of ASP toxins in the major shellfish species sampled 56

Table 2.13 Comparison of Domoic Acid levels in scallops and mussels sampled concurrently 58

Table 2.14 Risk assessment guidelines for toxin flesh testing in shellfish for various Pseudo-nitzschia sp. 64

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Table 2.15 Percentage occurrence of Dinophysis acuminata and percentage above the regulatory levels 72

Table 2.16 Percentage occurrence of Dinophysis acuta and percentage above the regulatory levels 74

Table 2.17 Percentage occurrence of Prorocentrum lima and percentage above the regulatory levels 76

Table 2.18 Summary by zone of the occurrence of DSP toxins in shellfish samples 77

Table 2.19 Summary by zone of the occurrence of DSP toxins in Greenshell™ mussels from consistently monitored sites 78

Table 2.20 Summary of the occurrence of DSP toxins in the major shellfish species 78 Table 2.21 The corrected results for samples recorded in FoodNet as having negative Acetone Screen Results, but positive DSP ELISA results 83 Table.2.22 Percentage occurrence of potentially toxic

Gymnodinium c.f. breve and percentage above the regulatory levels 89

Table 2.23 Percentage occurrence of Gymnodinium c.f. mikimotoi and percentage above the regulatory levels 90

Table 2.24 Summary of the occurrence of lipid soluble toxins in shellfish samples 93

SECTION 3: NON-COMMERCIAL SHELLFISH GATHERING AND CONSUMPTION IN NEW ZEALAND 98

Table 3.1 Number of trips by survey respondents targeting the main shellfish species in each Biotoxin Zone 106

Table 3.2 Comparison of the percentage of trips targeting each bivalve species within each zone 106

Table 3.3 The number of each species taken from each Biotoxin Zone by respondents from the survey 108

Table 3.4 Estimated percentage of non-commercial shellfish harvesting by ethnicity, per year, throughout New Zealand 109

Table 3.5 Numbers and percentages of total weight of cockles harvested, and harvesting population structure by ethnic group at Lews Bay, Whangateau 111

SECTION 4: ANALYSIS OF EPIDEMIOLOGICAL DATA 113 Table 4.1 Summary of reported cases of TSP by year as assessed

with respect to the causative agents 116 Table 4.2 Comparison of the number of cases and rates per

100, 000 people in New Zealand between TSP, and illness caused by Campylobacter and Vibro parahaemolyticus 117

Table 4.3 Age distribution of the “suspected” and “probable” cases of TSP 118

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Table 4.4 Analysis of “suspected” and “probable” cases of TSP from non-commercially and commercially harvested seafood by ethnic origin 118

Table 4.5 Analysis of source of seafood, by ethnic origin, for “suspected” and “probable” cases of TSP 119

Table 4.6 Geographical distribution of sites from which shellfish were gathered in “suspected” and “probable” cases of TSP 119

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SECTION 1: INTRODUCTION 1.1 BACKGROUND Awareness of marine biotoxins with respect to public health in New Zealand is relatively short: prior to a major biotoxin event in the summer of 1992-1993, New Zealand had no recorded incidence of shellfish biotoxins of public health significance. Preceding this, most concern focussed on the effects of harmful algal blooms with respect to potential impacts on fish farming, shellfish farming and wild marine species (e.g. Taylor et al., 1988; Chang et al., 1990; Mackenzie, 1992; Mackenzie et al., 1992; Rhodes et al., 1993). A basic monitoring programme for Paralytic Shellfish Poisoning (PSP) had been established in April 1992, based on monthly mouse bioassays from shellfish sampled from four sites: Bay of Islands, Coromandel, Marlborough Sounds, and Southland (Till, 1993). Although it was known that phytoplankton potentially able to produce Diarrhetic Shellfish Poisoning (DSP) toxins were present in New Zealand waters (e.g. Burns & Mitchell, 1982), there was no routine monitoring for these toxins. However, a limited number of commercial samples of mussels from Coromandel and Marlborough destined for the Japanese market had been tested for PSP and DSP since July 1989. No toxins had been detected in any of these samples (Till, 1993). In January 1993, authorities became aware of the presence of biotoxins in shellfish when a veterinarian in Northland reported poisoning in cats that had been fed with shellfish. Owners of the animals were also found to have experienced toxic symptoms after eating shellfish (Bates et al., 1993). Publicity led to further notifications of human illness following shellfish consumption and the coastline was progressively closed for shellfish harvesting. Consequently, by January 23rd the entire coastline was closed. About 186 people were affected with symptoms that resembled a case definition that had been used in an outbreak of Neurotoxic Shellfish Poisoning (NSP) in North Carolina (Bates et al., 1993). This case definition was sufficiently broad not to exclude other known shellfish toxins and narrow enough to exclude most gastrointestinal illnesses associated with infectious agents (Bates et al., 1993). In addition to symptoms related to consumption of shellfish, the NZ Communicable Disease Centre (CDC) also received reports of dry cough and eye irritation affecting large numbers of people visiting or working near the beach at Orewa, north of Auckland. These symptoms were consistent with reports of “respiratory irritation syndrome” (RIS) associated with blooms of Gymnodinium breve in Florida caused by brevetoxin aerosols (Bates et al., 1993; Steidinger & Joyce, 1973; Pierce, 1986). An epidemiological investigation, which included consideration of data regarding phytoplankton species present at coastal sites, subsequently suggested that: “The major toxicity involved in the shellfish poisoning outbreak was Neurotoxic Shellfish Poisoning (NSP), which affected mainly the north-eastern coastline of the North Island from the Bay of Islands to the Bay of Plenty. It is likely that some cases were of Diarrhetic Shellfish Poisoning (DSP). It is likely that Paralytic Shellfish

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Poisoning (PSP) played no more than a minor role at the most in the outbreak, and that Amnesic Shellfish Poisoning (ASP) probably played no role at all” (Bates et al., 1993). However, while only NSP and possibly DSP, played a role in the epidemiological events, a range of shellfish toxins was detected in shellfish testing (Hannah et al., 1993). These included a minimum of toxins related to ASP, DSP, NSP, PSP, and non-specific 24-hour mouse toxicity. While the toxin event of 1992-93 caught everyone somewhat by surprise, biotoxin management structures were quickly brought into place to provide a coordinated response. By February 1993, the Ministry of Agriculture and Fisheries (MAF) had formulated a National Management Plan for Marine Biotoxin Control. A National Marine Biotoxin Coordination Committee comprised of representatives from regulatory bodies (Ministry of Agriculture & Fisheries, Ministry of Health, Public Health Commission), research organisations, and the Fishing Industry Board was established (Ministry of Agriculture & Fisheries, 1993a). The plan outlined the guidelines for reporting and shared decision-making, the objectives for the management programme, an overview of a multi-tiered management plan, operation standards, and the responsibilities of regional agencies. A comprehensive “National Management Plan for Marine Biotoxin Control in New Zealand” was completed in March 1993 (Ministry of Agriculture & Fisheries, 1993b). This included a Marine Biotoxin Management Plan for each regional area. Management within the National Management Plan was based on: • “Assay and analytical results demonstrating the presence of marine biotoxins in

shellfish; and • Reports of marine biotoxins being involved in food-borne illness; and • Phytoplankton analysis; and • General environmental information including aerosol-borne illness and evidence

from bathing, kills or other evidence of the possible detrimental effects of toxin producing algae on sea-birds, fish or other marine life; and

• The flexibility to review and modify the programme as the database develops.” The operational standards in the Plan specified 106 shellfish sampling sites (reduced from the initial 244 sites) sampled weekly. These sites included 60 sites within commercial shellfish farming/harvesting areas, and 46 sites where there was no commercial shellfish activity (recreational areas). In practice however, sampling continued at approximately 165 sites. In November 1993 the Toxic Shellfish Coordination Committee was replaced by a Marine Biotoxin Management Board, comprised of senior representatives from MAF, Ministry of Health (now restructured to incorporate the functions of the Public Health Commission) and NZ Fishing Industry Board. The Marine Biotoxin Management Board established a Marine Biotoxin Surveillance Unit in January 1994 to manage the operation of the biotoxin and phytoplankton monitoring programmes. Several committees reported to the Board: a Financial Committee, which prepared advice on contracts for sampling and analysis, a Technical Committee, which set standards and specifications, and a Communications Committee, which handled media coordination,

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press releases, Board communications and publications. Each committee comprised representatives from each of the three member organisations (NZ Marine Biotoxin Management Board, 1995). The Marine Biotoxin Surveillance Unit was comprised of a Technical Coordinator from MAF, and two National Advisors, one from MAF with expertise in marine science, and one from Ministry of Health with expertise in public health protection (Trusewich et al., 1996). Health Protection Officers in each of the 21 Crown Health Enterprises were responsible for shellfish sampling, and decisions on closure and re-opening of areas were made jointly between the Surveillance Unit and the local Health Protection Officer. The programme was jointly funded by industry (23%) through a Fishing Industry Board levy, and vote Health (77%). Following a review of data collected, including the correlation of data between sites, and the patterns of accumulation of toxins in different species, the number of sample sites was reduced down to 120 sites per week in the 1994-95 year (Trusewich et al., 1996). The cost-effectiveness of this monitoring was of some concern both to the shellfish industry and the Ministry of Health. This prompted reviews of the programme by both parties (NZ Fishing Industry Board, 1995; Wilson, 1996; Wilson & Sim, 1996). The moves for change resulted in the establishment of weekly phytoplankton monitoring in 1995 in a few areas where biotoxin activity had occurred. The purpose of this was to gather the data necessary to establish a robust phytoplankton monitoring programme. A major restructuring of the marine biotoxin monitoring programme followed in 1997. This restructuring involved changes to the monitoring programme itself, and to the funding and administration of the programme. The commercial marine biotoxin monitoring programme is now managed by MAF and administered at a local level by Health Protection Officers from the local Hospital and Health Services as part of a Shellfish Quality Assurance Programme. Each commercial shellfish farming/harvesting area is now responsible for the funding of the monitoring programme in its area. Based on the 1993-97 data on biotoxins in each area, the monitoring programmes were modified in consultation with the Marine Biotoxin Technical Committee. In most areas these modifications included the introduction of weekly phytoplankton monitoring as an early warning system (undertaken by trained personnel), and a reduction in the frequency of shellfish samples tested. Specified numbers of potentially toxin-producing phytoplankton in the water trigger shellfish flesh testing, or at a higher number, closure to harvest pending the results of shellfish flesh testing. The frequency of routine shellfish testing for each of the four toxins (PSP, NSP, DSP, and ASP) is related to the toxin activity in each area. The Ministry of Health manages routine monitoring in non-commercial sites, through Health Protection Officers in each Hospital & Health Services organisation. The Ministry of Health also purchases information from commercial biotoxin programmes to supplement their own monitoring programme. The non-commercial marine biotoxin monitoring programme was revised to include phytoplankton monitoring where environmental conditions allow, with a reduction of shellfish testing in areas of low biotoxin activity.

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With the restructuring of the marine biotoxin monitoring programme in 1997, the Marine Biotoxin Management Board, and the Marine Biotoxin Surveillance Unit were disbanded, and their functions decentralised. However, the Marine Biotoxin Technical Committee has continued to function in an informal capacity, and is an example of the continuing cooperation in marine biotoxin issues between MAF (now the Ministry of Agriculture & Forestry), Ministry of Health, and the shellfish industry. Awareness of the potential problems of marine biotoxins in shellfish has resulted in the establishment of significant research capability in the field of marine biotoxins in New Zealand. Research has focussed primarily on identification of biotoxins and their sources, and the development of new test methods. Every six months researchers, regulators, public health officials and shellfish industry representatives meet at a “Marine Biotoxin Science Workshop”, facilitated by MAF. These workshops provide an opportunity for the presentation of research results in the field of marine biotoxins, and for discussion about issues of concern. A Marine Biotoxin Science Strategy is being developed through this group, to ensure that adequate research funding is directed to marine biotoxin research from the Foundation for Research, Science and Technology. 1.2 THE CURRENT MONITORING PROGRAMME The Health Act (1956) provides the legislative framework for the management of public health with respect to marine biotoxins in New Zealand. Section 3A defines the function of the Ministry in relation to public health: “Without limiting any other enactment or rule of law, and without limiting any other functions of the Ministry or of any other person or body, the Ministry shall have the function of improving, promoting and protecting public health”. Section 117 of the Act provides for the ability of the Director General to make such regulations as necessary to conserve public health. In addition, Section 37 of the Food Act (1980) provides for statements to be issued by the Director General of Health, and this provision is used occasionally to issue national press statements from the Director General warning the public not to eat shellfish from defined areas. The management of public health with respect to marine biotoxins is set out in the Ministry of Health’s Food Administration Manual: Section 27: Marine Biotoxin Control (Ministry of Health, 1997a). Health Protection staff in Hospital and Health Services are responsible for the public health management of marine biotoxins in commercial and non-commercial shellfish harvesting areas. This includes sample collection, opening and closing commercial growing areas, domestic market product recall, and warning the public when non-commercial growing areas should not be harvested for shellfish. Generally, sample collection is contracted out to locally based private individuals. Each Hospital and Health Services Public Health Unit is responsible for maintaining a local marine biotoxin management plan. The local marine biotoxin management plans cover both non-commercial and commercial harvesting of shellfish. This is because in some areas non-commercial sample sites may be used to close commercial growing areas, and vice versa.

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The non-commercial marine biotoxin monitoring programme is designed to match the levels of biotoxin activity and shellfish availability in each area, and utilizes phytoplankton monitoring where possible. In areas where testing of shellfish samples has shown persistent or recurrent PSP, ASP, NSP or DSP toxins, weekly sampling and testing of shellfish for the specific biotoxins is undertaken. In areas where shellfish testing has shown a low risk of toxin accumulation, and that are not suitable for phytoplankton monitoring, fortnightly shellfish samples are taken for analysis of all toxins. In areas of low risk where phytoplankton testing is possible, weekly phytoplankton samples are taken, and monthly shellfish samples for analysis for all four toxins. Phytoplankton results are used to trigger shellfish testing and closure at the same levels as in the commercial programme. Details of these levels are provided in Appendix I(A). There are currently 61 routine phytoplankton sampling sites throughout New Zealand. Thirty of these sites are either non-commercial sites, or sampled under the non-commercial marine biotoxin monitoring programme when the shellfish industry is not sampling there. All phytoplankton sites are sampled weekly. The distribution of phytoplankton sampling sites is illustrated in Figure 1.1 on the following page. (Note that where sites are too close together, only one site is marked due to the limitations of scale). At sites where phytoplankton monitoring occurs, phytoplankton numbers provide an early warning of toxicity in shellfish. For each type of potentially toxic phytoplankton, there are trigger levels at which shellfish monitoring is instigated, and closures to harvest implemented (Refer to Appendix I(A)). The non-commercial marine biotoxin monitoring programme includes 57 sites from which shellfish samples are taken. Some of these sites are monitored as part of the commercial monitoring programme and only monitored as part of the non-commercial monitoring programme when the shellfish industry is not sampling there. Details of the monitoring regime for both the commercial and non-commercial programmes, including frequency of sampling and testing for each type of toxin, are provided in Appendix I(B). Shellfish samples are tested for PSP, ASP, NSP and DSP. Samples are tested for PSP using acid extraction techniques followed by mouse bioassay (Delaney, 1985). HPLC techniques are used for the detection of ASP (Wright et al., 1989; Lawrence & Menard, 1991; Lawrence et al., 1991). Detection of NSP and DSP is a two-step procedure (a flow diagram to illustrate this process is provided in Appendix I(C)). Initially shellfish samples are screened using an acetone extraction technique and mouse bioassay (Hannah et al., 1995; Yasumoto et al., 1978). If the results of the screening step are positive, this is followed by a DSP ELISA (DSP-Check Kit, Panapharm Laboratories) for DSP, and a mouse bioassay using ether extraction techniques for NSP (Delaney, 1985; Yasumoto et al., 1978).

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2 non-commercial and 24commercial sites in theMarlborough Sounds

Non-Commercial site

Commercial site

Shared site

Current PhytoplanktonMonitoring Sites

2 non-commercial sites and 1shared site

2 commercialsites atCoromandel

1 non-commercial site and 1shared site (Tamaki Strait andWaimangu Point)

Figure 1.1: Current phytoplankton monitoring sites along the New Zealand coastline.

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1.3 BIOTOXINS IN NEW ZEALAND New Zealand is unusual in having four of the five biotoxin groups, i.e. PSP, ASP, NSP and DSP. No AZP (Azaspiracid Poisoning, now recognised as a separate toxin group) has been found in New Zealand to date. Several additional lipophilic compounds, some of them toxic, are also present, and these complicate the detection of NSP. Few other countries have such a range of toxins present in one place (Andersen, 1996). The major marine biotoxin groups found accumulated in New Zealand shellfish are discussed below. 1.3.1 Paralytic Shellfish Poisoning (PSP) Paralytic Shellfish Poisoning (PSP) may be caused by several species of phytoplankton, including a range of species of the genus Alexandrium (Halim), Gymnodinium catenatum, and most commonly in tropical areas, Pyrodinium bahamense (Hallegraeff, 1995). Several Alexandrium species have been found in New Zealand, with varying levels of toxicity. These include Alexandrium angustitabulatum, A. catenella, A. minutum, A. ostenfeldii, A. tamarense, A. margalefii, A. pseudogonyaulax, A. concavum, A. c.f. fraterculus, and an isolate from Marsden Point (Northland) tentatively ascribed to A. cohorticula or A. tamiyavanichi (Mackenzie et al., 1994; Mackenzie et al., 1996a; Mackenzie et al., 1997; Mackenzie et al., 1998a). (Note that subsequent to the preparation of this report, Gymnodinium catenatum has also been found in New Zealand waters). The toxins that cause PSP comprise a suite of at least 24 naturally occurring neurotoxic tetrahydropurine analogues. They act as sodium channel blocking agents in vertebrate nervous systems. The PSP toxin analogues can be classified according to their chemical structure and specific potency in mammals: The carbamate toxins (GTX1-GTX4, Neo, STX) are the most potent, whereas the N-sulfocarbamoyl derivatives (B1, B2, C1-C4) have much lower specific toxicity. The decarbamoyl (dc-) analogues are of intermediate toxicity (Cembella et al., 1993). In the absence of environmental stress and in the exponential growth phase, different strains of Alexandrium have characteristic toxin profiles (Cembella et al., 1987). Since different toxin derivatives have different toxicity, the toxin profile impacts on the overall toxicity of the strain. The detoxification processes within different species of shellfish also alters the toxin profile of PSP toxins within the shellfish, and this may vary from species to species (Cembella et al., 1993; Chang et al., 1997). The toxicity of New Zealand Alexandrium species is summarized in Table 1.1 (summarized from Chang et al., 1997; Mackenzie et al., 1998a).

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Species Specific Toxicity (pg STX eq./cell)

A. angustitabulatum (Bream Bay) 1.1 A. catenella (Bay of Plenty) 3.4 A. minutum (Anakoha A) 2.2 A. minutum (Anakoha B) 1.9 A. minutum (Croiselles 1) 1.8 A. minutum (Bay of Plenty) 11.6 A. minutum (Bay of Plenty) 6.0 A. minutum (Whangarei) 0.9 A. ostenfeldii (Kaitaia) 217.0 A. ostenfeldii (Taharoa) 21.4 A. ostenfeldii (Wellington) 0 A. ostenfeldii (Timaru) 13.4 A. tamarense (Tasman Bay) Unknown A. margalefii (Bream Bay) 0 A. pseudogonyaulax (Hauraki Gulf) 0 A. concavum (Northland/Hauraki Gulf) 0 A. c.f. fraterculus (Coromandel/Hauraki Gulf) 0 A. sp. (Marsden Point) 3.2

Table 1.1: Specific toxicity of species of Alexandrium found in New Zealand

waters. STX eq./cell= Saxitoxin equivalent per cell. It can be seen from Table 1.1 that there is some variation in specific toxicity within the same species, most notably in A. ostenfeldii, in which specific toxicity of isolates ranges from 0 to 217 pg STX eq./cell. The phytoplankton monitoring that occurs as part of the marine biotoxin monitoring programme is based on the detection of significant numbers of potentially toxic species using morphological features (size and shape) visible under a light microscope. In the case of Alexandrium species, Calcofluor stain may be used to assist in identification. However, distinguishing between toxic and non-toxic species using these methods is not always possible. For example, A. c.f. fraterculus (which has so far exhibited no toxicity in New Zealand, although it has been associated with toxicity in shellfish in Uruguay (Balech, 1995)), has been found to exist in three morphophytes in culture: in chains, and in large and small morphophytes as single cells. The general size and form of chains of A. c.f. fraterculus is superficially like A. catenella, a toxic species found in the Bay of Plenty. In its single cell form, the smaller morphophytes resemble the toxic Alexandrium species isolated from Marsden Point (possibly A. cohorticula or A. tamiyavanichi), while the larger morphophytes closely resemble A. concavum (also non-toxic) (Mackenzie et al., 1998a). The difficulties in distinguishing between toxic and non-toxic species on a routine basis in the monitoring programme are overcome by the initiation of toxicity testing in shellfish whenever significant numbers of apparent A. c.f. fraterculus occur in the plankton. Molecular probes are being developed to distinguish between toxic and non-toxic species (Rhodes et al., 1998a). Like some other dinoflagellates, Alexandrium species produce resting cysts. Research on toxic Alexandrium blooms overseas suggests that the cyclical development of

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blooms can be dependent upon the presence of seedbeds of cysts (Anderson & Wall, 1978). High numbers (up to 70,000 cysts per square meter) of Alexandrium ostenfeldii have been found in sediments around New Zealand, but cysts of other species are less evident (L. Mackenzie, Cawthron Institute, pers. comm.). The number of Alexandrium present in the plankton in order to trigger toxicity testing in shellfish is relatively low: 100 cells/L of species known to be toxic (including Alexandrium c.f. fraterculus because of gross morphological similarities to toxic species). This is set at the level of detection for the phytoplankton methodology used. An immediate public health warning is issued if numbers in the phytoplankton reach 5,000 cells/L. 1.3.2 Amnesic Shellfish Poisoning (ASP) Amnesic Shellfish Poisoning (ASP) is caused by Domoic acid (Wright et al., 1989). Species within the genus Pseudo-nitzschia produce Domoic acid. This toxin is unusual in being produced by diatoms rather than dinoflagellates. A range of Pseudo-nitzschia species occur in New Zealand. Domoic acid has been confirmed in some but not all New Zealand isolates of Pseudo-nitzschia australis, P. pungens, P. turgidula (Rhodes et al., 1996), P. delicatissima, and P. pseudodelicatissima (Rhodes et al., 1998b). Two other species are frequent components of Pseudo-nitzschia blooms: P. heimii and P. multistriata (L. Rhodes, Cawthron Institute, pers. comm.), but to date neither of these species have been found to be toxic. Discrimination between some species of Pseudo-nitzschia is virtually impossible under a light microscope because of morphological similarity between species – some species differ in details that can only be detected under an electron microscope. However, whole cell DNA probes have been developed to distinguish between species (Rhodes et al., 1997), and these are utilised by industry in risk management when deciding whether to implement voluntary closures to harvesting, pending the results of shellfish toxicity testing. Although not part of the formal biotoxin management plan, they also appear to be used at non-commercial monitoring sites in the event of a Pseudo-nitzschia bloom, as a means of deciding whether shellfish samples should be taken for toxicity testing. (This is discussed in more detail in Section 2.5.4). Pseudo-nitzschia blooms differ from blooms of most other toxic phytoplankton in that high numbers of cells (50,000-100,000 cells/L) are required to reach a significant toxin level in shellfish. The toxicity of Pseudo-nitzschia species varies widely between and within species, and temporally. Table 1.2 summarises the maximum level of Domoic acid per cell found in New Zealand isolates.

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Species Domoic Acid Maximum Level (pg/cell) P. australis 35.0 P. pungens 0.47 P. fraudulenta 0.03 P. delicatissima* 0.12 P. turgidula* 0.03 P. pseudodelicatissima 0.12 P. heimii 0 P. multistriata 0 *There is some evidence to suggest that P. delicatissima and P. turgidula are the same species (L. Rhodes et al., 1998b).

Table 1.2: Maximum level of Domoic acid found in New Zealand isolates of

Pseudo-nitzschia. (Summarised from Rhodes et al., 1998b). It should be noted that some species are not always toxic (for example, P. pungens), so an accurate measure of the risk of ASP cannot be gained by identification of the phytoplankton species alone. Domoic acid production varies with the stage of the Pseudo-nitzschia bloom, with maximum production in the stationary or senescent phase (Bates et al., 1996; Bates et al., 1998). Consistent with this is the appearance of toxins in shellfish several days after the peak of a bloom of toxic Pseudo-nitzschia. Because of this, the monitoring of phytoplankton levels can provide an effective early warning system for Domoic acid in shellfish. It appears that different species of shellfish may accumulate Domoic acid at different rates, although few controlled experiments have been undertaken on New Zealand species. It is thus difficult to distinguish environmental factors from differences between species. However, feeding experiments have shown that Domoic acid is very rapidly eliminated from the GreenshellTM mussel, Perna canaliculus – under some conditions the rate of excretion is equivalent to the rate of ingestion, and accumulation within the tissue does not take place (Mackenzie et al., 1993). 1.3.3 Diarrhetic Shellfish Poisoning (DSP)

Diarrhetic Shellfish Poisoning (DSP) is produced by dinoflagellates in the genera Dinophysis, and Prorocentrum (Murakami et al., 1982; Lee et al., 1989; Jackson et al., 1993). Species that are potentially toxic include Dinophysis acuminata, D. acuta, D. fortii, D. mitra, D. norvegica, D. rotundata, D. tripos, Prorocentrum lima, and P. concavum. The species in New Zealand that have been found to contain DSP toxins include Dinophysis acuta and Prorocentrum lima (Rhodes & Syhre, 1995). Low levels of DSP toxins have also been found associated with Dinophysis acuminata (L. Mackenzie, Cawthron Institute, pers. comm.). Prorocentrum lima is an epibenthic dinoflagellate, and thus is not commonly found in plankton samples from the water column. DSP toxins are lipophilic. There are several toxins in the “DSP group”. They are sub-divided into three groups: Okadaic acid (OA) and the closely related

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dinophysistoxins (DTX); the pectenotoxins, which are polyether lactones consisting of three compounds with known structure (PTX 1-3) and at least two additional compounds with presumed slightly modified skeletons; and thirdly, yessotoxin (YTX), with two sulphate esters, which resemble the brevetoxins from Gymnodinium breve (Aune & Yndestad, 1993). Of these toxins, only Okadaic acid, DTX1 and DTX3 are generally associated with diarrhoea. However because they are frequently found together and are all extracted by the acetone extraction method, they tend to be grouped together as the “DSP group”. The pectenotoxins and yessotoxin are acutely toxic to mice. Both these toxins are present in New Zealand: the causative agent of yessotoxin in the Marlborough Sounds was identified as Protoceratium reticulatum (Satake et al., 1997; Mackenzie et al., 1998b), and new analogues of pectenotoxin have been found in Dinophysis acuta (Daiguji et al., 1998). 1.3.4 Neurotoxic Shellfish Poisoning (NSP) Neurotoxic Shellfish Poisoning (NSP) is caused by toxins produced predominantly by Gymnodinium species. Several species of phytoplankton in New Zealand have been found to produce NSP toxins. These include: Gymnodinium c.f. breve, Gymnodinium c.f. mikimotoi (which may include three separate species), Gyrodinium galatheanum and a species of Heterosigma (Mackenzie et al., 1995a; Haywood, 1998). The identity of the causative agent in the 1993 NSP event in Northland is uncertain: both Gymnodinium c.f. breve and Gymnodinium c.f. mikimotoi were present in elevated numbers at the time (Chang, 1996; Mackenzie et al., 1995b). Recently, further work on elucidating the New Zealand species of Gymnodinium has been undertaken by Alison Haywood of Cawthron Institute. This has resulted in the proposal of two new species: Gymnodinium papilonaceum (recorded as Gymnodinium c.f. breve in the biotoxin monitoring data) and Gymnodinium selliforme (recorded as Gymnodinium c.f. mikimotoi in the biotoxin monitoring data). Gymnodinium mikimotoi is also present in New Zealand waters (A. Haywood, Cawthron Institute, pers. comm.). Lipophilic, polycyclic ether compounds, known as brevetoxins, cause NSP. The brevetoxins have been classified under several nomenclatures since toxicity was first detected in the causative organism, and this is somewhat confusing (Baden, 1989). According to the structure of the backbone skeleton, they can be classified into three categories: brevetoxin A (BTX-A), brevetoxin B (BTX-B) and hemi-brevetoxin (HemiBTX). Within each category, several analogues are known. Brevetoxins act as sodium channel activators. Different analogues of the toxins have different potencies (Baden, 1989). The toxin profiles of brevetoxin-producing dinoflagellates vary between species, and with the growth stage of the algae (Roszell et al., 1990). NSP ELISA has detected brevetoxins in cultures of Gymnodinium papilonaceum, G. selliforme, G. mikimotoi and Gyrodinium galatheanum from New Zealand waters (Ian Garthwaite, AgResearch, pers. comm.). Several known brevetoxin analogues were isolated from shellfish collected during the 1992-93 biotoxin event in New Zealand.

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These included brevetoxins PbTX-2 and PbTX-3 from oysters in the Coromandel area (Ishida et al., 1994). Several new brevetoxin analogues were also isolated: Brevetoxin B1 (Ishida et al., 1995), Brevetoxin B2 (Murata et al., 1998) and Brevetoxin B3 (Morohashi et al., 1995). As a result of this work, it was suggested that the detoxification mechanism for brevetoxin differed between cockles (Austrovenus stutchburyi) and green-lipped (GreenshellTM) mussels (Perna canaliculus). 1.3.5 Respiratory Irritation Syndrome Two Respiratory Irritation Syndrome (RIS) events have occurred in New Zealand – one that was associated with the 1993 biotoxin event, in which residents of Orewa, a coastal settlement north of Auckland, complained of sore throats, eye and nose irritation and dry coughing (Bates et al., 1993). The second event occurred in the summer of 1998, and affected residents and visitors of Hawkes Bay and the Wairarapa coast. The symptoms reported during this event were very similar to those in the earlier event (Chang et al., 1998a). Similar symptoms have been reported for some years in association with the Florida (USA) “red tides” that are caused by blooms of Gymnodinium breve (Pierce, 1986). The respiratory irritation reported in Florida has been attributed to aerosolised brevetoxins produced by Gymnodinium breve. The mechanism by which the toxins become aerosolised follows the process of bursting bubbles caused primarily by wind-generated whitecaps and breaking waves (Pierce, 1986). The 1998 respiratory irritation event in New Zealand was associated with a dense bloom of Gymnodinium c.f. mikimotoi that was also responsible for fish kills off Wairarapa, Kaikoura, and in Wellington Harbour (Chang et al., 1998b). While the respiratory symptoms were very similar to those caused by brevetoxin, analysis by neuroblastoma assay indicated that the toxin was not brevetoxin (F. H. Chang, NIWA, pers. comm.). Chemical analysis is currently being undertaken to determine the structure of the new toxin. The Gymnodinium species responsible for the toxin has been identified as a new species, and named Gymnodinium brevisulcatum (F. H. Chang, NIWA, pers. comm.). 1.3.6 Other Marine Biotoxins in New Zealand Several other marine biotoxins have been identified in New Zealand since 1993. In 1994 a new toxic imine (named Gymnodimine) was isolated from Foveaux Strait oysters and a Gymnodinium species similar in appearance to Gymnodinium mikimotoi (Mackenzie, 1994; Seki et al., 1995; Seki et al., 1996; Mackenzie et al., 1996b) was identified as the causative agent. (This has subsequently been named Gymnodinium selliforme (A. Haywood, Cawthron Institute, pers. comm.)). A limited short-term rat feeding trial was undertaken to ascertain the oral toxicity of gymnodimine (Towers, 1994). The results of this trial and epidemiological evidence indicated that gymnodimine does not produce Toxic Shellfish Poisoning when consumed in contaminated shellfish. Long-term feeding trials are currently being undertaken by AgResearch, using gymnodimine extracted from Gymnodinium species cultured by Cawthron Institute. This will determine whether the consumption of gymnodimine over a long period of time has any impact on health (I. Garthwaite, AgResearch, pers. comm.). In the meantime, the lack of any epidemiological evidence to suggest that

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gymnodimine is a threat to public health resulted in its exclusion from the regulatory marine biotoxin monitoring programme. The morphological similarities between the species grouped together as Gymnodinium c.f. mikimotoi, and the variations within one species, mean that these species/strains are difficult to separate under the light microscope. However, they may produce a variety of toxins (brevetoxins, gymnodimine, “Wellington Harbour toxin”), or no toxin at all. This means that the results of phytoplankton monitoring do not provide a clear indication of the risk of Toxic Shellfish Poisoning. As a result, phytoplankton trigger levels are set on the assumption that the species contain brevetoxins. Other toxins identified in New Zealand include a novel neurotoxin isolated from cockles (from the Bay of Plenty) following the 1993 biotoxin event ((4-methoxycarbonyl butyl) trimethylammonium chloride) (Ishida et al., 1994), a novel polyether compound from Coolia monotis from Rangaunu Harbour (Rhodes & Thomas, 1997), and palytoxin in Ostreopsis siamensis (also from the Rangaunu Harbour) (Briggs et al., 1998). Palytoxin is a sodium channel activator and potent tumour producer (Redondo et al., 1996; Yasumoto & Satake, 1998). 1.4 THE STRATEGIC FRAMEWORK AND SCOPE OF THIS REVIEW Options for the management of the risk of Toxic Shellfish Poisoning and Respiratory Irritation Syndrome can only be formulated and recommended if there are clear objectives to be achieved. The goal and objective of this review of the non-commercial marine biotoxin monitoring programme were stated in the project scope as follows: • To ensure a social and physical environment which improves, promotes and

protects public health and whanau public health.

• To optimise the safety of all food available for consumption in New Zealand.

This goal and objective are contained within a broader strategic framework for public health in New Zealand. This public health strategy is outlined in a paper produced by the Ministry of Health’s Public Health Group, entitled Strengthening Public Health Action: The strategic direction to improve, promote and protect public health (Ministry of Health, 1997b). This strategy sets out a vision for public health action, values to guide public health action, goals, objectives and targets, and criteria for determining priorities for action. The vision for public health action outlined in the strategy is:

We see New Zealand as a country in which Maori and non-Maori enjoy equitable health outcomes. Everyone lives longer in good health, disease is progressively reduced and people with disabilities are able to achieve independence. We see people empowered to realise their full potential through effective healthy public policy, supportive social, cultural and physical environments, strong communities, well-developed personal skills and a health system focused on health gain. We see fully informed and

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resourced people able to make healthy choices in the context of a healthy and sustainable environment.

The values to guide public health action include: • Appropriateness • Effectiveness • Efficiency • Empowerment • Equity • Partnership • Safety • Sustainability All these values are defined in more detail in the strategy document. The goal and objective outlined in the project scope (see above) are those from the strategy that primarily relate to the non-commercial marine biotoxin monitoring programme. There were no specific targets set for this particular objective in the Ministry of Health’s strategic document. There are several other potentially relevant objectives outlined in the strategy. These include the goals and relevant objectives relating to Maori and Pacific peoples’ health, the global environment, and sustainable management of natural and physical resources: • To improve, promote and protect Maori heath status so in the future Maori will

have the opportunity to enjoy at least the same level of health as non-Maori. • To ensure that all services funded are culturally appropriate and

compatible with gains in Maori health. • To show an understanding of, and commitment to the Treaty of Waitangi.

• To improve, promote and protect the health of Pacific people.

• To ensure that all services are culturally appropriate and relevant to Pacific people in structures, settings and languages that Pacific communities can identify with and use.

• To provide Pacific people with the opportunity to play a major role in the design, development, implementation and evaluation of public health services which affect their communities.

• To improve, promote and protect the health of children.

• To reduce disability and death rates from asthma. • To reduce the adverse health effects and optimise the positive health effects of the

global environment, including climate change, import control, international travel, ozone depletion, and vector control.

• To ensure public health issues are identified and addressed in decisions made on

sustainable management of natural and physical resources.

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(These are goals and objectives potentially relevant to the management of the risk of marine biotoxins in New Zealand. The full set of goals and objectives is provided in “Strengthening Public Health Action: The strategic direction to improve, promote and protect public health” (Ministry of Health, 1997b). The essential criteria outlined for use in determining public health priorities are: • Does the health issue have a significant impact on the current and future health

status of the total population or priority groups in terms of morbidity, mortality, quality of life, and/or potential years of life lost?

• Are there effective means, using population-based methods, to improve, promote or protect health, or prevent disease, in respect of the particular health issue? If not, are there potential innovative means that could be evaluated?

The criteria given high weighting are: • If the health issue is tackled, will this contribute to reducing inequalities in health

status, including reducing the inequalities between Maori and non-Maori? • Will tackling this issue provide the best health gain for the resources required? The criteria given medium weighting are: • Is there public support for tackling the issue? • If programmes are developed to address an issue, are they sustainable over time

and across sectors? • Is it possible to engage other sectors of government and the community, including

Maori and iwi, in efforts to address the issue? Cross-cutting themes identified as underpinning all public health goals, objectives and targets include (Ministry of Health, 1997b): • Focusing on the determinants of health. • Building strategic alliances. • Implementing comprehensive programmes. • Strengthening public health infrastructure. This broad strategic framework is utilised in the evaluation of management options arising from our review of the marine biotoxin monitoring programme. The Ministry of Health has defined the scope of this review in their project proposal. The purpose of this review is to: Analyse all shellfish flesh and phytoplankton data collected in the New Zealand marine biotoxin monitoring programme since January 1993, both non-commercial and commercial, assess the risk to the New Zealand public from consumption of toxic shellfish, and recommend options for cost effective management and mitigation.

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The scope of the review is summarised as follows: • Review and report on the data collected in the Ministry of Health database (and

other data sources if necessary) from shellfish flesh analysis by the marine biotoxin monitoring programme (non-commercial and commercial) for NSP, DSP, PSP and ASP for the period January 1993 to March 1999.

• Review the data collected in the Ministry of Health database from phytoplankton

analyses by the marine biotoxin monitoring programme on algae that can produce biotoxins capable of causing NSP, DSP, PSP, and ASP, and on algae which cause respiratory irritation syndrome for the period November 1996 to March 1999.

• Gather local information including environmental, climatic and oceanographic

variables that may influence development and demise of toxic algal blooms. • Review the epidemiological data on cases of Toxic Shellfish Poisoning and

respiratory irritation syndrome in New Zealand since January 1993. • Analyse, interpret and summarise all data on an area by area basis; relate this

information to shellfish gathering and consumption patterns in New Zealand. • Assess whether there is any relationship between toxic phytoplankton

concentrations and concentration of toxins in shellfish that could be used to predict the likelihood of risk to the consumer.

• Using the results of analysis and consultation:

• Assess the risk of Toxic Shellfish Poisoning to consumers of non-commercially gathered shellfish, in New Zealand as a whole, and on an area by area basis;

• Assess the risk of respiratory irritation syndrome to the public. • Examine environmental factors and educational strategies that already exist or

could be implemented to contribute towards effective management or mitigation of Toxic Shellfish Poisoning and toxic algae in New Zealand.

• Identify cost-effective options available to protect the New Zealand public from

Toxic Shellfish Poisoning from non-commercially gathered shellfish and from respiratory distress syndrome.

The analyses in this report are based on up to six years of epidemiological and shellfish toxin data, and, depending upon the area, up to two or three years of phytoplankton data. This is a comparatively short time period from which to gain some understanding of the risk of TSP in the future. While convening a session at the “Harmful Algal Bloom 2000” conference in Hobart in February 2000, Don Anderson commented that “even after 20 years of intensive study to determine the environmental factors impacting on the blooms of PSP-causing phytoplankton off the coast of Maine, prediction of blooms is still not possible”. The structure of this report reflects consideration of these limitations, with an emphasis being placed on broad trends across New Zealand rather than working with a possibly ill-founded initial

17

assumption that there are distinct differences in the risk of biotoxin occurrence in different geographical areas. The results of our review of the marine biotoxin monitoring programme for non-commercially harvested shellfish are reported in two parts, in separate documents. This report (Part 1), contains the results of analysis of available data, risk analysis, and a general discussion of the resulting conclusions with respect to management of marine biotoxins in non-commercially harvested shellfish in New Zealand. The second report (Part 2) provides a more specific discussion of the options and recommendations to the Ministry of Health for the cost effective management and mitigation of marine biotoxins in New Zealand.

18

SECTION 2: ANALYSIS OF BIOTOXIN MONITORING DATA 2.1 INTRODUCTION Since 1993, much has been learnt about the types of biotoxins present in phytoplankton in New Zealand waters. The marine biotoxin monitoring programme has evolved with the development of new, more precise methods to cope with the range of toxins present. This evolution has improved the quality of the data collected as part of the monitoring programme. Since the initiation of the marine biotoxin monitoring programme in 1993, a substantial amount of data has been collected. This includes the results of tests for biotoxins in shellfish, results of monitoring for toxic phytoplankton, some environmental data recorded at the time the shellfish or phytoplankton samples were collected, and epidemiological data. This section of the review covers the analysis of shellfish test results and phytoplankton monitoring results. The analysis of the epidemiological data is undertaken in Section 4 of this report. The collection of the shellfish toxin analysis data and phytoplankton data within the monitoring programme has not been planned with a view to facilitating meaningful data analysis in the long term. Rather, its primary function has been the immediate determination of the biotoxin status at specific sites. Consequently, a number of challenges are faced in the analysis of the monitoring data. These include the following factors: • Missing or incomplete data, and data that are incorrectly reported in the database. • Variations in the interpretation and reporting of results. • Changes in test methods for biotoxin detection, including changes in toxin

extraction procedures, and in overall testing procedures for biotoxins in shellfish. • Difficulties with and improvements made in the identification of toxin-producing

phytoplankton. • Changes in the number of sample sites and the frequency of sampling over time

(seasonally, and from year to year). • Clumping of sample sites both within and between zones (i.e. the sample sites are

not distributed evenly around the coastline). • Variations in sampling position within one “sample site”. • The relationship between the occurrence of positive results, and the frequency of

sampling and number of sites sampled (for example, under the current programme, shellfish sampling frequency may increase if positive results are returned, and in some places this also triggers sampling at additional sites).

19

• Differences in species of shellfish sampled within and between sample sites, coupled with the possible variation in the uptake and retention of toxins in different shellfish species.

Basically the data are not independent, and are stratified both spatially and temporally over several different scales. This suggests that meaningful quantitative analysis is extremely difficult. Extreme care must therefore be taken to avoid misleading conclusions or unfounded extrapolation from this data set. Some of the initial analysis of data undertaken in this review was designed to provide a framework for further analysis. Overseas research and observations from the natural environment have indicated that different shellfish species may accumulate biotoxins to different levels and detoxify at different rates (e.g. Shumway et al., 1988; Gainey & Shumway, 1988; Shumway et al., 1990; Cembella et al., 1993; Bricelj et al., 1996). Similar observations have been made in New Zealand (Marsden, 1993; Chang et al., 1997; Mackenzie et al., 1998b), but comparatively little research has been undertaken here. These potential interspecific differences are significant in determining how the analysis of patterns of biotoxins is undertaken. If the differences are significant, then the composition of sample species needs to be taken into consideration when making comparisons both between and within sites. Species-specific differences are also of potential importance in assessing the risk of TSP to shellfish consumers. 2.2 GENERAL METHODOLOGY Shellfish test results from January 1993 to June 1999, and phytoplankton monitoring results from 1997 to June 1999 from the FoodNet database were downloaded into Microsoft Access. Analysis of data was undertaken using Microsoft Excel. Phytoplankton monitoring began in 1994 in the Marlborough Sounds, and in January 1995 in the Hauraki Gulf, and also occurred sporadically in some other areas prior to 1997. There are a number of problems associated with the analysis of phytoplankton data prior to 1997 on the FoodNet database: the naming of sites is extremely inconsistent (some sites are identified by up to four different names, and site codes are generally not used), and the data appears incomplete. The most comprehensive phytoplankton data from this time period were collected in the Hauraki Gulf and the Marlborough Sounds. Because of the difficulties associated with the data in FoodNet, phytoplankton data for these areas were sourced from elsewhere. A complete set of data from phytoplankton monitoring at the Marlborough Sounds was kindly provided to us for analysis by the Marlborough Sounds Shellfish Quality Assurance Programme, who keep their own database. Data relating to Pseudo-nitzschia, Dinophysis and Gymnodinium mikimotoi for the Hauraki Gulf phytoplankton sites was obtained from an AquaBio Consultants database. The Hauraki Gulf data were originally compiled from hard-copy monitoring results supplied to shellfish farmers in the area by Cawthron Institute. Before discussing methods for specific analyses, the generic assumptions in this data analysis are outlined.

20

2.2.1 Identification of a Valid Data Set The analysis of data presented in this report is based on data collected using appropriate test methods and from which valid conclusions can be drawn. Changes in test methods were in general not implemented simultaneously over samples received from all sample sites. When changes were made, it was usually necessary for the testing laboratory to phase in the new method over a period of several weeks, to allow time to train staff in the new methods. Where these changes are significant in terms of data analysis, the data set has been selected to ensure that all samples were analyzed using the same methods by the laboratory. The data sets used for each of the four toxins are thus as follows: • For PSP, only results of toxicity testing in shellfish after June 1993 are used in

data analysis. Prior to this, the method of extracting PSP toxins from shellfish tissue involved a modification of the standard APHA acid extraction method. This modification, which was recommended by the US FDA at the time, used a concentration of hydrochloric acid ten times above the standard method (Hall, 1991). Several test results above the regulatory level for PSP were an artifact of this modified method. Our analysis therefore only considers data produced from tests using the current extraction method (Delaney, 1985). All samples were being analyzed by the current extraction method by the end of June 1993 (Penny Truman, ESR, pers. comm.)

• Routine analysis for ASP toxins by HPLC was introduced in mid-May 1993, and

was being undertaken at all sites by the beginning of July 1993. Data from July 1993-June 1999 are thus included in our analysis.

• Testing for lipid soluble toxins has been somewhat problematical over the course

of the marine biotoxin monitoring programme. Initially, all lipid soluble toxins in shellfish samples were detected by mouse bioassay, using an acetone extraction method (Hannah et al., 1995). This method was developed early in 1993 to replace the standard APHA ether extraction method for detection of NSP toxins, since the testing laboratory was processing very large numbers of samples, and the volatility of diethyl ether was a hazard to laboratory workers. This acetone extraction method detected all lipid soluble compounds that can cause mouse deaths, including NSP toxins, DSP toxins, gymnodimine and possibly free fatty acids. However, all the toxin levels were calculated from the mouse bioassay results as if they were NSP toxins. These toxin levels (from January 1993 to September 1994) have thus been excluded from our data analysis.

• The dataset for NSP toxins includes the results of toxicity tests in shellfish using a

mouse bioassay following an extraction of the toxins using diethyl ether (Delaney, 1985; Yasumoto et al., 1978). This was introduced in conjunction with a DSP ELISA test for DSP toxins along with the introduction of a prior screening step using an acetone extraction method in September 1994. It is acknowledged that this test is not necessarily specific to NSP toxins (i.e. brevetoxins), but this is the test on which regulation is based. It is likely that some of the NSP positive results could be confounded by the presence of other lipid soluble toxins (such as

21

pectenotoxin, yessotoxin) and free fatty acids. Where possible these data are thus used in conjunction with data from the phytoplankton monitoring programme.

• Analysis for DSP using the DSP ELISA (DSP Check-Kit, Panapharm

Laboratories) began in September 1994 when the revised protocols for detection of lipid soluble toxins were introduced. All these data are included in our analysis. The DSP Check-Kit does not detect the presence of DTX-3 or Okadaic acid diol esters. Thus it is possible that the incidence of DSP toxins in shellfish tissue is under-reported in the biotoxin monitoring results.

With respect to the dataset for phytoplankton analysis, increasing knowledge of the phytoplankton species that cause shellfish toxicity has meant an increase in the number of species specifically identified in the phytoplankton counts. In our analysis, a species has only been assumed absent in a sample when it was noted as a zero count in the data. Where it is apparent that data may be missing, as distinct from species not present in the sample counts, these instances were noted. 2.2.2 Determination of Areas for Analysis Part of our brief was analysis of monitoring data on an “area by area” basis. From the beginning of the marine biotoxin monitoring programme in 1993, there has been a division of the coastline into eleven areas, known as Biotoxin Zones A-K (see Appendix I (D)). Initially in 1993 all the sites within each Zone were opened and closed for harvesting shellfish collectively. However, this quickly changed so that areas within each zone were regulated separately. Nonetheless, the naming of sample sites according to the zone in which they are located has persisted. Figure 2.1 illustrates the Biotoxin Zones in relation to basic hydrographic influences around the coastline. It has been assumed that the sizes of the areas required for analysis were similar in scale to the regulatory zones, and that the analysis should be designed to investigate broad differences comparing risk to shellfish consumers. The benefits of redefining the areas for the purpose of analysis were considered. The two very broad influences on the coastal marine environment in New Zealand are the surface circulation patterns (the currents shown in Figure 2.1) and prevailing south-westerly swells that arise in the storm belt between 40o-60o South. The differentiation of coastal areas between existing biotoxin zones is consistent with these very broad patterns. It is recognized that within these areas there may be significant variation between individual sites, due to differences in environmental conditions related to geography, wave exposure, flushing action, nutrient availability due to run-off from the land, etc. Variation in the same environmental conditions may also occur as a result of small-scale patches, such as differences in phytoplankton abundance at different depths, and horizontal differences due to small-scale concentration of phytoplankton, such as that caused by Langmuir cells. It thus seemed that a redistribution of existing biotoxin zones into areas considering more detailed environmental factors would achieve little. Conversely, consolidation of zones into larger areas (for example, merging Zones A and B into one area) would result in a loss of detail from which we have benefited in

22

previous analysis. Since the existing biotoxin zones are well recognised in the public health arena and in the shellfish industry, it seemed expedient to retain these as areas for comparison, while noting significant differences within zones. Our analysis of the monitoring data on a broad area by area basis does not presume to investigate causative relationships for any differences between areas, but merely provides a description of relative risk to shellfish consumers in each area. In this analysis an underlying assumption is made that the distribution of sample sites is representative of shellfish harvesting patterns. This appears a reasonable assumption given that the monitoring programme has been designed in this way. The methods and assumptions related to specific analyses undertaken are described in the following sections.

23

Zone A: Tauroa Point to Cape BrettZone B : Cape Brett to

Cape Rodney

Zone C : Cape Rodney toCape Colville

Zone D: Cape Colville toCape Runaway

Zone E : CapeRunaway to Cape

Palliser

Zone F : Tauroa Point to Cape Egmont

Zone G: CapeFarewell to Cape

Campbell

Zone H: Cape Egmont to CapePalliser

Zone I: Cape Campbell to Bluff

Zone K : ChathamIslands

Zone J : CapeFarewell to Bluff

TASMAN FRONT

WEST AUCKLANDCURRENT

NORTH CAPE EDDY

EAST AUCKLANDCURRENT

EASTCAPEEDDY

EASTCAPE

CURRENT

WAIRARAPAEDDY

SOUTHLANDCURRENT

WESTLAND CURRENT

D’URVILLE CURRENT

Prevailing South-Westerly Storm

Swells.

Figure 2.1: Distribution of Biotoxin Zones and relevant hydrographical

features associated with the New Zealand coastline. Hydrographical features summarised from Carter et al. 1998.

24

2.2.3 Analysis of Phytoplankton Occurrence a) Geographical Distribution To provide an indication of the extent of the geographic distribution of potentially toxic phytoplankton species, the occurrence of potentially toxic phytoplankton species was mapped for species related to all four toxin groups (PSP, ASP, NSP and DSP) as follows: Regularly monitored phytoplankton sample sites were mapped, and those where potentially toxic phytoplankton species had been detected were identified from the FoodNet database. Causative agents for PSP, ASP, NSP and DSP were included in this analysis. There were several limiting factors in this analysis: • Much of the phytoplankton data on the FoodNet database does not distinguish

between toxic and non-toxic species, since often this distinction cannot be made under the light microscope (for example, Pseudo-nitzschia species). In some cases, gene probes have been used to further distinguish toxic from non-toxic species, but unfortunately these results have not been recorded on the database.

• Knowledge of the phytoplankton causing toxicity in shellfish has grown over the

period the monitoring programme has been in operation. Thus in samples taken early in the phytoplankton monitoring programme some species may not have been specifically identified in the recorded data.

This analysis is not quantitative, but merely provides a visual representation of the areas in New Zealand in which potentially toxic phytoplankton have been detected. The occurrence of potentially toxic phytoplankton and the percentage of samples at elevated levels in each zone were analysed as follows: The occurrence of potentially toxic phytoplankton relating to each of the four toxin groups (PSP, ASP, NSP and DSP) was analysed. Analysis was undertaken using both the entire number of phytoplankton samples from each zone (=Total Samples Taken), and also for an “Identified Time Interval” (from the week of 19/12/97 to 25/5/99) to standardize for the different temporal sampling lengths at different sites. No phytoplankton samples were available from Zones F or K (there are no phytoplankton monitoring sites in these zones), and Zones B, H, and J were represented by one phytoplankton monitoring site only. In the analysis using the “Identified Time Interval”, some sites were not used because they started after the initial date (19/12/97) or because large amounts of data were missing or incomplete. Thus out of a possible 61 sites, only 48 sites were used for the “Identified Time Interval” analysis. A list of these sites is provided in Appendix II. Analysis included the following species: Pseudo-nitzschia species (all species were included, since individual species are not identified on the database), Dinophysis acuta, D. acuminata, Prorocentrum lima, Gymnodinium c.f. breve, Gymnodinium mikimotoi, Alexandrium species including Alexandrium minutum, A. ostenfeldii, A. angustitabulatum, A. catenella, A. tamarense, and A. cohorticula/tamiyavanichi.

25

For each species, the percentage occurrence in total samples, the percentage of samples above the level to trigger flesh testing, the percentage above the level to trigger voluntary industry closure, and (where they occurred) the percentage above the level to trigger a public health warning, were analysed. Analysis was by zone for both the “Total Samples” taken, and the samples in the “Identified Time Interval”. These results were recorded in tabular form. Where five or more data points exist, data above the levels to trigger flesh testing or voluntary closure by industry were represented graphically as “Box and Whisker Plots”. Where there were less than five data points, the numbers were simply stated. This analysis provided information on the distribution of potentially toxic phytoplankton by zone, including relative abundance by zone. “Box and Whisker Plots” are a simple graphical way of representing the range of a data set. Interpretation of the “Box and Whisker Plots” is as follows: 5% of the data lies below the bottom whisker line, and 25% of the data lies below the bottom of the box. The median (middle value) dissects the box, and 75% of the data lies below the top of the box. 95% of the data lies below the top whisker. Any outlying points are marked as single points. b) Temporal Distribution The phytoplankton monitoring data available on the FoodNet database represents an insufficient time period for meaningful analysis of temporal patterns. However, some analysis of temporal patterns of potentially toxic phytoplankton was possible for Zone G using data from the database supplied by the Marlborough Sounds Shellfish Quality Assurance Programme, and for Zone C using data from AquaBio Consultants Ltd. database. For some toxins, seasonal patterns in the occurrence of potentially toxic phytoplankton were investigated by determining the percentage of the total samples for each season (summed over five years, i.e. 1994-1999), that were above the level to trigger flesh testing. The seasons analysed were spring (September-November), summer (December-February), autumn (March-May), and winter (June-August). In addition, the phytoplankton counts of potentially toxic species for selected sites with continuous data from 1995 to 1999 were graphed to investigate seasonality of occurrence. For each selected site, the phytoplankton counts for each week of the year were plotted on the same graph to determine whether consistent seasonal patterns occurred. This also allowed qualitative comparison of seasonal distribution patterns between sites.

Median

26

2.2.4 Reliability of Phytoplankton Monitoring as a Predictor of Biotoxins in Shellfish

Analysis was undertaken to investigate the probability that phytoplankton monitoring at all phytoplankton monitoring sites might fail to predict the occurrence of significant levels of toxin in shellfish at the same site. All phytoplankton monitoring sites with associated shellfish monitoring sites were used in this analysis. Analysis was undertaken for each of the four major toxin groups. For each toxin, the biotoxin events at each shellfish site were identified from the FoodNet database. An event was defined as a single or consecutive series of shellfish samples with toxin levels above the regulatory level. In some instances where there were no samples above the regulatory level, a lower detectable level was used to investigate this relationship. For each event, the number of cells of the relevant potentially toxic phytoplankton in the concurrent, and previous water samples (1-3 weeks earlier, depending on the species) were identified from the database. The percentage of shellfish samples above the regulatory levels for closure due to biotoxin that were not associated with levels of the appropriate potentially toxic phytoplankton was calculated. Similarly, in cases where there were no shellfish samples with toxins above the regulatory level, the percentage of shellfish samples with biotoxin above the detectable level but no corresponding occurrence of toxic phytoplankton was calculated. 2.2.5 Differences in Biotoxin Accumulation between Shellfish Species For each of the four toxins (PSP, ASP, NSP and DSP), the number of samples with detectable levels of biotoxin, and the number of samples with biotoxin levels above the regulatory level, were determined for each of the major shellfish species sampled. The percentage of the total tests for each species was then calculated for each toxin level. It is recognized that this analysis has major limitations: there is no account taken of the impact of seasonal sampling (for example, scallops), clumping of sites with respect to shellfish species, or the increase in frequency of shellfish sampling, and in some cases, the number of sample sites, in the event of positive biotoxin results. However, as a small part of a larger picture, it was decided that this analysis could still be useful if viewed cautiously and in conjunction with other data. The shellfish monitoring data in the FoodNet database was examined to determine where possible, the differences in accumulation and depuration of biotoxins between New Zealand species of shellfish. Instances in which different shellfish species had been sampled concurrently from the same site were identified, and graphed. Where there were more than 30 data points, differences in biotoxin levels were analysed using a single factor ANOVA, and in some instances, two sample t-tests. Our ability to rigorously investigate these relationships was greatly limited by lack of good data. 2.2.6 Summary of Cumulative Monitoring Results – Shellfish and

Phytoplankton A summary of the cumulative results of the monitoring for PSP, ASP, NSP and DSP was developed using all valid results recorded on the FoodNet database from shellfish toxicity testing (see Section 2.2.1 for criteria in selection of datasets). This summary

27

provides a description of the scale of the marine biotoxin monitoring programme in comparison to the detection of biotoxins in shellfish over the last six years. The total number of samples using valid testing techniques, the number of these samples in which toxins were detected, the number of samples exceeding the relevant regulatory level for that toxin, and the percentage of the total samples that this represents, were calculated. The maximum toxin level for any sample over the same time period was also recorded. Similarly, a summary of the cumulative results of the phytoplankton associated with PSP, ASP, NSP and DSP toxins was developed using all the phytoplankton results recorded on the FoodNet database, plus data from the Marlborough Sounds Quality Assurance Programme. The data were analysed to determine the percentage of total samples in which toxic phytoplankton were detected, and the percentages of total samples that were above the level to trigger shellfish sampling, above the level to trigger voluntary closure by industry, and above the level to trigger a public health warning. 2.2.7 Analysis of Occurrence of Biotoxins in Shellfish A comparison of the occurrence of biotoxins in shellfish by zone and through time was undertaken using data from shellfish monitoring on FoodNet. This analysis was made difficult by differences in toxin accumulation characteristics between shellfish species, the clumping of sample sites, changes in test methods, and the variations in sampling regimes both through time and between sample sites. By using only regularly monitored sites over the same time period subsequent to the introduction of valid toxin test methods, and accounting for changes in sampling frequency, it was possible to identify a data set that could be used to analyze the occurrence of biotoxins through time. However, data able to be used in this analysis represented a small proportion of the total data, and thus significant information with respect to the geographical distribution of biotoxins was lost. For this reason, some analysis of the total data set was also included. a) Geographical Distribution To provide an indication of the extent of the geographic distribution of biotoxins in shellfish throughout New Zealand, the occurrence of PSP, ASP, NSP and DSP in shellfish samples was mapped. Using all valid results from shellfish toxicity tests for each toxin group recorded on the FoodNet database, all sample sites at which levels of toxin above the regulatory level were recorded were marked on a map of New Zealand in red. Similarly, sites at which detectable levels of toxin were recorded were mapped in yellow. From the remaining sites, only those sites that had been frequently monitored over the 1993-99 period were selected, and these were mapped in blue. Where sample sites were too close together to mark separately, the area in which the sites are situated was marked with the colour representing the highest level of toxin found in the area. This analysis is not quantitative, but merely provides a visual representation of the areas in New Zealand in which biotoxins have been detected in shellfish.

28

Using the complete data set for each of the four biotoxin groups (PSP, ASP, DSP and NSP), a summary by zone of the occurrence of biotoxins was undertaken. The percentage of samples containing biotoxins above the level of detection, and percentage of samples containing biotoxin above the regulatory level, were calculated. The maximum toxin levels were also noted by zone for the samples with biotoxin levels above the regulatory level. For all four toxins (PSP, ASP, NSP and DSP), a set of sample sites that had been regularly and consistently monitored since the initiation of valid toxin test method was identified. The species of shellfish sampled at each site was identified, and single-species sites were chosen (i.e. sites where the shellfish species sampled had been consistent). Using the sites for the most widely sampled shellfish species (and in some cases, shellfish species in which biotoxins had been frequently recorded), the percentages of biotoxin levels above the regulatory level, and above the level of detection, were calculated by zone. Several assumptions were made in this analysis in order to standardize the data to cope with changes in shellfish sampling since the introduction of phytoplankton sampling:

• Since 1997, the frequency of shellfish sampling has been reduced to fortnightly or

monthly samples at many sites. In order to accommodate differences in the frequencies of sampling between different sites, it was assumed that phytoplankton monitoring would reliably trigger shellfish sampling (i.e. that between fortnightly or monthly shellfish samples that were recorded as having an undetectable level of toxin, any intervening weekly shellfish samples, had they been taken, would have had no detectable levels of toxin). Sites that were not monitored weekly following detectable levels of toxin were not used in this analysis.

• At sites where there was only fortnightly shellfish sampling (except when toxins were detected, at which time the frequency of sampling increased to weekly) it was assumed that toxin levels in shellfish in the intervening weeks between samples with no detectable toxin levels would have been zero had they been tested.

b) Temporal Distribution The seasonal distribution of biotoxins in shellfish was investigated for all four toxins (PSP, ASP, NSP and DSP), using a set of sample sites that had been regularly and consistently monitored since the initiation of a valid toxin test method. The species of shellfish sampled at each site were identified, and single-species sites were chosen. Several assumptions were made in this analysis in order to standardize the data to cope with changes in shellfish sampling since the introduction of phytoplankton sampling:

• Since 1997, the frequency of shellfish sampling has been reduced to fortnightly or

monthly samples at many sites. In order to accommodate differences in the frequencies of sampling between different sites, it was assumed that phytoplankton monitoring would reliably trigger shellfish sampling (i.e. that

29

between fortnightly or monthly shellfish samples that were recorded as having an undetectable level of toxin, any intervening weekly shellfish samples, had they been taken, would have had no detectable levels of toxin).

• At sites where there was only fortnightly shellfish sampling (except when toxins were detected, at which time the frequency of sampling increased to weekly) it was assumed that toxin levels in shellfish in the intervening weeks between samples with no detectable toxin levels would have been zero had they been tested.

Within each zone, the number of samples above the detectable level for each month were determined, and summed for each month across years (i.e. the number for June 1994 plus the number for June 1995 etc.). To account for differing numbers of sites per zone, the data were standardized, by dividing the total for each zone by the number of sites in the zone. The results were graphed cumulatively for each month, to obtain the distribution of detectable levels of toxin by month. The same data set was used to examine the temporal distribution of biotoxin occurrence between years. The toxin level for each sample was graphed as a point in time, by zone, on a scatter plot. This showed the comparative frequency of toxin occurrence year by year. It should be noted that the relative frequency of toxin occurrence between zones within one year is influenced by the number of sites within each zone included in the data set, so comparisons between zones cannot be made from the data presented in this particular analysis.

30

2.3 CUMULATIVE MONITORING RESULTS The following table presents an overall summary of the cumulative results of the monitoring for PSP, ASP, NSP and DSP toxins in shellfish from January 1993 until the end of June 1999 for Biotoxin Zones A-J.

Samples exceeding regulatory levels

Biotoxin (time period)

Regulatory levels

Total No. of samples

taken

No. of samples

with positive

toxin results

No.

% of total

samples

Maximum toxin level (per 100g shellfish

tissue from whole

shellfish) NSP toxins (Sept 1994 - June 1999)

20 mu/100g

18,326(a)

117

26

0.14

44 mu

DSP toxins (Sept 1994 – June 1999)

20 µg/100g

18,572(b)

229

82

0.44

96 µg

PSP (May 1993 – June 1999)

80 µg/100g

29,367

1,002

94

0.32

1,007 µg

ASP (Jan 1993 - June 1999)

20 µg/g

18,814

933

36

0.19

210 µg

Table 2.1: Summary of the cumulative results of the monitoring for PSP,

ASP, NSP and DSP toxins in shellfish from January 1993 until the end of June 1999. Current regulatory levels for each toxin group are also given. (a) Total samples = total number of acetone screen tests, plus the ether extraction assays that were undertaken without prior acetone screen tests. (b) Total samples = total number of acetone screen tests, plus ELISA tests that were undertaken without prior acetone screen tests.

Table 2.1 shows the magnitude of the number of shellfish samples tested for each toxin over the duration of the monitoring programme in comparison to the relatively low number of samples that represent a potential health hazard to shellfish consumers. The cumulative results of phytoplankton monitoring in all biotoxin zones are presented in Table 2.2. These data are drawn both from the FoodNet database (late 1997 to June 1999), and the data for the Marlborough Sounds area supplied from the Marlborough Sounds Shellfish Quality Assurance Programme (1994-June 1999). The data are therefore biased toward the results of Zone G, both because of differences in temporal length (sampling data collected over a longer period of time) and because there are comparatively more phytoplankton sampling sites in Zone G than in other zones. Note also that there is no distinction made in the phytoplankton monitoring results on the database between toxic and non-toxic species/strains of Pseudo-nitzschia. These data cannot therefore be used as an accurate indication of the occurrence of potentially toxic phytoplankton in New Zealand overall. However, it is pertinent in providing an indication of the occurrence of potentially toxic

31

phytoplankton with respect to the scale of the marine biotoxin monitoring programme thus far.

Species

Total No. of

samples

Percentage of samples in which

species were present (%)

Percentage at level to

trigger shellfish

testing (%)

Percentage at level to

trigger voluntary industry

closure (%)

Percentage at level to

issue public health

warning (%)

Alexandrium sp.

9124

4.67

4.11

0.53

0.03

Pseudo-nitzschia sp.

9134

65.40

6.95

2.38

n/a

Gymnodinium breve

9126

0.28

0.02

0.01

0.01

Dinophysis acuta

9116

4.31

0.31

0.33

n/a

Dinophysis acuminata

9120

4.93

0.76

0.50

n/a

Table 2.2: Cumulative results of phytoplankton monitoring for all Biotoxin

Zones by potentially toxic species using all of the available data. Data for Zone G is the period 1994-1999, and for all other zones from 1997-1999. Pseudo-nitzschia species data includes both non-toxic and potentially toxic species, as no differentiation is made in the database, or in the regulatory trigger levels. Trigger levels for Pseudo-nitzschia species assume that Pseudo-nitzschia cells comprise more than 50% of the total biomass of the sample.

It can be seen from both of the preceding tables that the percentages of samples taken that resulted in regulatory closure to harvesting or a public health warning, due either to biotoxin or phytoplankton levels, was comparatively low. However, these figures represent the cumulative dynamics of the monitoring programme over the last six years, during which time the monitoring regime has changed considerably. As will be seen in the following sections, biotoxin occurrence can also vary significantly from year to year. Care therefore needs to be taken in the use of these data in a predictive sense. However, if it can be assumed that the marine biotoxin monitoring programme has been somewhat representative of shellfish harvesting patterns over the last six years, then these data suggest that the incidence of biotoxin levels that represented a risk to shellfish consumers was relatively low.

32

2.4 RESULTS OF ANALYSIS - PSP 2.4.1 Geographic Distribution The distribution of potentially toxic Alexandrium species in New Zealand is shown in Figure 2.2. These data are drawn from the phytoplankton monitoring data on FoodNet, as described in Section 2.2.3. Species included in the analysis were those for which regulatory levels exist in the marine biotoxin monitoring programme: Alexandrium minutum, A. ostenfeldii, A. catenella, A. tamarense, and A. c.f. tamiyavanichi. Figure 2.3 shows the sites throughout New Zealand where PSP toxins above the regulatory level, and above the level of detection have been found in shellfish tested in the marine biotoxin monitoring programme (Refer to Section 2.2.7 for details of the method of analysis used). Neither of these figures provides quantitative information about the frequency of occurrence of potentially toxic phytoplankton or PSP in shellfish samples. Figure 2.2 indicates that the distribution of potentially toxic Alexandrium species is widespread around New Zealand. Their distribution does not appear to be limited by latitude, nor by biogeographic region. This is not surprising, since strains of Alexandrium species may be found in a variety of environments: for example, Alexandrium tamarense can be found in sub-Arctic and temperate areas (e.g. Taylor, 1984; Cembella et al., 1988), as well as tropical regions (Reyes-Vasquez et al., 1979). The occurrence of PSP toxins in shellfish is similarly widespread (Figure 2.3). There were variations in toxin presence between adjacent sites. While there are many sites at which PSP has not been detected in shellfish, the sites where it has occurred include both the north and south of the country, and the western and eastern coasts. The widespread distribution of PSP-producing phytoplankton suggests that there is potential for the occurrence of PSP in shellfish anywhere around the coastline should the right conditions for an increase in the population of Alexandrium species occur. Table 2.3 shows the occurrence, and levels above the regulatory limits of potentially toxic Alexandrium species by Biotoxin Zone in New Zealand as described in Section 2.2.3. (Note there are no phytoplankton monitoring sites in Zones F or K.). This table summarizes the results of analysis of two sets of data: the first (“Total Samples”) is the data set of all the phytoplankton samples recorded on the FoodNet and Marlborough Sounds Shellfish Quality Assurance Programme databases. The second set of data includes all the samples recorded on these databases within an “Identified Time Interval” (i.e. 19/12/97-25/5/99). The data relating to the occurrence of PSP-producing Alexandrium species using the total number of samples on the database is the best information that we have on an individual zone basis. The quantity of data varies considerably from zone to zone – for example, thanks to the Marlborough Sounds Shellfish Quality Assurance Programme, a full set of phytoplankton data from the beginning of 1994 through to June 1999 has been included for most sites in Zone G. However, in order to compare

33

the relative occurrence of Alexandrium between zones, the data relating to the identified time interval must be used. Figure 2.2: Distribution of potentially toxic species of Alexandrium throughout

New Zealand (to June 1999).

Potentially toxic Alexandriumspecies detected at 22 of the 26sites in the Marlborough Sounds

Potentially toxicAlexandrium species detected

No potentially toxicAlexandrium species detected

Distribution of PotentiallyToxic Alexandrium species in

New Zealand

34

Figure 2.3: Distribution of PSP at shellfish sample sites at two levels (above the

level of detection, and above the regulatory level of 80 µg PSP/100g shellfish tissue) throughout New Zealand (to June 1999).

Sites with samples above regulatory level forPSP

Sites at which PSP has been detected

Frequently sampled sites where PSP has notbeen detected

Distribution of PSP inNew Zealand

There are 18 regularly monitoredsites in the Marlborough Soundsarea. PSP above the regulatorylevel has occurred in shellfish atone site. PSP above the level ofdetection has occurred inshellfish at a further three sites.

35

From analysis of the “Identified Time Interval” data set, the zones can be ranked in descending order of frequency of occurrence as follows: A, E, H, D, B, I, J, G, C. However, it must be remembered that this analysis is based on only 17 months of recorded data, which is a comparatively short time interval.

Zone

A

B

C

D

E

G

H I

J

Percentage occurrence in

Total samples (%).

9.7

7.5

1.9

10.1

7.1

3.9

8.5

4.5

3.8

N= 432 107 583 622 240 6486 82 468 104 Percentage of Total

samples with 100-400 cells/L (%)

6.9

7.5

1.7

6.3

5.8

3.7

8.5

4.5

2.9

Percentage of Total samples with 500 to 4900

cells/L (%)

2.8

0.0

0.2

3.4

1.3

0.2

0.0

0.0

1.0

Percentage of Total samples with 5,000 cells/L or more (%)

0.0

0.0

0.0

0.5

0.0

0.0

0.0

0.0

0.0

Percentage occurrence in the identified time

interval (%)

13.4

7.9

2.2

8.9

10.6

3.1

9.2

5.3

5.2

N= 304 76 456 532 152 1941 76 304 76 Percentage of samples in

the identified time interval with100-400

cells/L (%)

9.5

7.9

2.2

5.3

8.6

3.0

9.2

5.3

3.9

Percentage of samples in the identified time

interval with 500-4900 cells/L (%)

3.9

0.0

0.0

3.4

2.0

0.1

0.0

0.0

1.3

Percentage of samples in the identified time

interval with 5,000 cells/L or more (%)

0.0

0.0

0.0

0.2

0.0

0.0

0.0

0.0

0.0

Table 2.3: Percentage occurrence of potentially toxic Alexandrium species,

and percentage occurrence above the regulatory levels (100 - 400 cells/L, 500 – 4,900 cells/L and ≥ 5,000 cells/L) in total samples and in samples from 19/12/97 to 25/5/99 (the “Identified Time Interval”) by zone.

To show the spread of the data in the “Identified Time Interval” data set, the same ranges of data (100-400 cells/L; 500-4,900 cells/L) have been represented graphically as “Box and Whisker Plots” (where five or more data points exist) or, where there are less than five data points, numbers are simply stated. This summary is presented in Figure 2.4. Zone D is the only Zone where there has been a level of potentially toxic Alexandrium species in excess of the level requiring a Public Health Warning ( ≥ 5,000 cells/L), with a maximum level of 6,500 cells/L (of A. catenella from Te Kaha on 20/12/98). The next highest level was in Zone E, where 4,700 cells/L were recorded (A. minutum

36

from Tolaga Bay on 18/1/99). In both Zones A and D, 75% of the samples that were in the range to trigger a voluntary industry closure were less than 1,500 cells/L, and 95% were less than 3,000 cells/L. Samples recorded in the range to trigger flesh testing were predominantly in the lower half of the range – in Zones A, C, D, E, G, and I, 75% of the samples were 200 cells/L or less. Zones A, E, H, and I had median values of 100 cells/L. These median values were not visible on the Box and Whisker Plot since they were the same as the 25th percentile in the distribution of cell counts in this range. Zone C and G had a median value of 200 cells/L. These values were the same as the 75th percentile.

Alexandrium spp.

Zone

A D

Ran

ge o

f dat

a ab

ove

the

Indu

stry

Vol

unta

ryC

losu

re L

evel

(500

-490

0 ce

lls/L

itre)

0

500

1000

1500

2000

2500

3000

3500Alexandrium spp.

Zone

A B C D E G H IRan

ge o

f dat

a ab

ove

the

Fles

h T

estin

gTr

igge

r L

evel

(100

-400

cel

ls/L

itre)

0

100

200

300

400

500

N=29

N=6

N=10

N=28

N=13

N=58

N=7N=12 N=18

N=16

Zone D: Above Public Health Warning Level (5000 cells/L): 6 500 Zone E: Above Industry Voluntary Closure Level (500 cells/L): 500, 800, 4 700 Zone G: Above Industry Voluntary Closure Level (500 cells/L): 600, 600 Zone J: Above Flesh Testing Trigger Level (100 cells/L): 100, 100, 100 Above Industry Voluntary Closure Level (500 cells/L): 800

Figure 2.4: Box and whisker plots showing the frequency distribution of (a)

potentially toxic Alexandrium species above the level to trigger flesh testing (100 cells/L) by zone, and (b) potentially toxic Alexandrium species above the level to trigger a voluntary closure to commercial harvesting (500 cells/L) in the two zones (A & D) where these levels occur. Data are also given for levels above the Public Health Warning limit (i.e. ≥ 5,000 cells/L).

37

Zone

Total No.

of Samples

Number of samples in which PSP

was detected

Percentage of samples

in which PSP was detected

No. of samples above

regulatory level

Percentage of samples

above regulatory

level

Maximum toxin level (µg/100g)

A 3127 157 5.02 1 0.03 83 B 1902 48 2.52 0 0.00 C 3172 6 0.19 0 0.00 D 4262 743 17.43 91 2.14 1007 E 1226 9 0.73 0 0.00 F 1897 2 0.11 0 0.00 G 6320 23 0.36 2 0.03 127 H 1127 3 0.27 0 0.00 I 2184 1 0.05 0 0.00 J 1269 10 0.79 0 0.00 K 374 0 0 0 0.00

Table 2.4: Summary by zone of the total occurrence of PSP toxins detected in

shellfish samples, and PSP toxins above the regulatory level of 80µg/100g detected in shellfish samples from 1/7/93 to 30/6/99.

A summary of PSP toxin occurrence in shellfish for all sites in all zones from July 1993 to end of June 1999 is provided in Table 2.4. It can be seen that PSP was detected in a much higher percentage of samples in Zone D than in other zones, and that only Zones A, D and G had levels of PSP above the level for closure to harvesting (i.e. 80 µg/100g). The 1,007 µg/100g maximum toxin level in Zone D (at Tokata on 21/4/97) was also considerably higher than those in Zones A or G (Zone A was only just above the level for closure). Examination of the data where there were high numbers of samples in which PSP was detected, revealed that many of the “detects" in Zone A and D related to long-running levels of PSP toxins in tuatua (specifically at Tokerau Ramp in Northland (Zone A), and Ohope Beach, Bay of Plenty (Zone D)). This potentially suggests two things: one, that the occurrence of PSP in particular areas might be related to the presence of “seed beds” of toxic Alexandrium cysts, causing repeated toxicity in that area. Secondly, that the variation in occurrence of PSP toxins is not geographical but related to the species of shellfish being sampled in each area. There have been no surveys undertaken to determine the presence of Alexandrium cysts in the sediments in either of these areas (L. Mackenzie, Cawthron Institute, pers. comm.). However, some investigation of the comparative differences in levels of PSP in different species of shellfish was possible using the available data. It should be noted that the analysis presented in Table 2.4 did not use standardized data, and that potential differences in toxin accumulation characteristics between shellfish species, the clumping of sample sites, and variations in sampling regimes, both through time and between sample sites has been ignored. In an attempt to reduce some of this bias, a similar analysis was carried out using data from a single species, from sites that had been consistently monitored over the whole time period, as described in Section 2.2.7. Because they were the most widely sampled species, Greenshell™ mussels were chosen as the sample species. The data were standardized to account for the changes in the shellfish sampling programme since the introduction of phytoplankton sampling, as described in Section 2.2.7.

38

Table 2.5 provides a summary of the PSP toxin occurrence in Greenshell™ mussels by zone from July 1993 to the end of June 1999. The scope of the data is less than that in the previous table as Greenshell™ mussels had only been consistently monitored at sites in Zones C, D, G, H, and I over that time period. No data were available from the other zones. However, these data suggest that the occurrence of PSP toxins in Greenshell™ mussels in Zone D was higher than that in the other zones.

Table 2.5: Summary by zone of the occurrence of PSP toxins in Greenshell™

mussels from consistently monitored sample sites from 1/7/93 to 30/6/99.

Given that there is potential for the accumulation of PSP to differ between species, these differences were investigated within the limits of the available data. Table 2.6 provides a summary of the occurrence of PSP in the major shellfish species sampled in the marine biotoxin monitoring programme.

Species

Total

No. with

detectable levels of

PSP

Percentage of samples

above detectable level (%)

No. of samples

above the regulatory

level

Percentage above the regulatory level (%)

GreenshellTM mussel

10587

121

1.14

15

0.14

Blue mussel 1616 6 0.37 0 0.00 Pacific oyster 3293 22 0.67 0 0.00 Dredge oyster 623 9 1.44 0 0.00 Scallop 2848 88 3.09 0 0.00 Tuatua 2189 738 33.71 78 3.56 Pipi 2023 13 0.64 0 0.00 Cockle 1696 0 0.00 0 0.00 Paua 485 2 0.41 0 0.00

Table 2.6: Summary of the occurrence of PSP toxins in the major shellfish

species sampled at all sites in the marine biotoxin monitoring programme from 1/7/93 to 30/6/99.

These data indicate that a higher percentage of tuatua samples contained a detectable level of PSP than other shellfish species sampled, and that tuatua samples had a greater percentage of PSP levels above the regulatory level of 80 µg/100g. However, these data are not sufficient to indicate species differences with respect to biotoxin accumulation and retention, since the toxin accumulation might be site specific rather than species specific.

Zone

Total No. of

Samples

Percentage of Samples with PSP above Detectable

Levels (%)

Percentage of Samples with PSP above the

Regulatory Level (%)

C 1192 0.0 0.0 D 743 5.7 1.3 G 3709 0.5 0.05 H 599 0.5 0.0 I 307 0.0 0.0

39

While the data are scarce, there are a few occasions on which several species of shellfish from the same site have been monitored concurrently and tested for PSP. The most comprehensive data are summarized in Figures 2.5 to 2.7. Note that in these figures, sample results below the level of detection are portrayed as zero.

050

100150200250300350

1/03/9

8

1/04/9

8

1/05/9

8

1/06/9

8

1/07/9

8

1/08/9

8

1/09/9

8

1/10/9

8

1/11/9

8

1/12/9

8

1/01/9

9

Time

PSP

Leve

l (µ

g/10

0g)

GreenshellmusselsTuatua

Figure 2.5: Comparison of levels of PSP in GreenshellTM mussels (Perna

canaliculus) and tuatua (Paphies subtriangulata) at Ohope Beach from March 1998 to January 1999.

0

50

100

150

200

250

7/07/9

314

/07/93

21/07

/9328

/07/93

4/08/9

311

/08/93

18/08

/9325

/08/93

1/09/9

38/0

9/93

15/09

/93

Time

PSP

Leve

l (µ

g/10

0g)

MusselTuatuaScallop

Figure 2.6: Comparison of levels of PSP in mussels (species not specified in database), tuatua (Paphies subtriangulata) and scallop (Pecten novaezelandiae) at Waihi Beach July 1993 to September 1993.

40

05

101520253035404550

28/07

/93

11/08

/93

25/08

/93

8/09/93

22/09

/93

6/10/9

3

20/10

/93

3/11/9

3

17/11

/93

1/12/9

3

Time

PSP

Leve

l (µ

g/10

0g)

MusselScallop

Figure 2.7: Comparison of levels of PSP in mussels (species not specified in

database) and scallops (Pecten novaezelandiae) in Rangaunu Bay, Northland from July 1993 to December 1993.

In addition to the data illustrated, several one-off comparisons have been made: Concurrent samples taken from Ngunguru (Northland) in October 1993 indicated that pipi (Paphies australis) from the site had a level of 34.5 µg PSP/100g tissue, while cockles (Austrovenus stutchburyi) had no detectable PSP. Blue mussels (Mytilus edulis aoteanus) from Tairua Harbour (eastern Coromandel Peninsula) on 11/07/99 had no detectable levels of PSP, whereas tuatua sampled concurrently from the same site had a level of 95 µg PSP/100g tissue (which was above the regulatory limit of 80 µg/100g. Only the data comparing the PSP levels in Greenshell™ mussels and tuatua at Ohope Beach (Figure 2.5) have sufficient samples to undertake robust quantitative analysis. A two-sample t-test, assuming unequal variance, indicated that the toxin levels in Greenshell™ mussels and tuatua from Ohope Beach were significantly different (p<0.05) over the time period of the recorded data, identifying that on average tuatua had higher levels of PSP than mussels. In the ten-month study at Ohope Beach (Figure 2.5), 35.1% of tuatua samples were above the regulatory limit for PSP, compared to only 5.9% of GreenshellTM mussel samples over the same time. This is of potential significance in terms of the risk of PSP to consumers of non-commercially gathered shellfish. Qualitative examination of the data suggests that not only do tuatua retain PSP toxins longer, but they also accumulate comparatively more toxin than mussels when a toxic event occurs in the phytoplankton. However, in terms of maximum toxin levels measured to date, GreenshellTM mussels may also accumulate high levels of PSP toxin e.g. D41-Whangaparoa, on the 22 April 1996, with a level of 556 µg PSP/100g tissue. Similarly, the data suggest that scallops may accumulate and retain PSP toxins to a

41

greater extent than mussels, but to a lesser extent than tuatua. There may also be differences in accumulation and retention of PSP toxins between pipi and cockles. Whether these differences are from environmental or physiological causes is unable to be identified from the data available in this study. However, inter-specific differences in accumulation of PSP toxins have been observed in controlled field experiments undertaken overseas (e.g. Shumway et al., 1990). The results of our analysis are not in any way definitive, and much more data collection would be required to investigate this further. However, they do suggest that differences between species in PSP toxin accumulation and retention have the potential to impact significantly on the risk of PSP to consumers, and that these differences should be taken into consideration in the design of the marine biotoxin monitoring programme. 2.4.2 Temporal Distribution Phytoplankton monitoring data on the FoodNet database had been recorded for an insufficient period for a meaningful analysis of temporal patterns. However, some analysis of seasonal patterns in potentially toxic Alexandrium species in Zone G was possible using the data supplied by the Marlborough Sounds Shellfish Quality Assurance Programme. Five years of data (1994-1999) were analysed to investigate seasonal patterns in occurrence as described in Section 2.2.3(b). The results showed that in the Marlborough Sounds in the winters of 1995-1999, 1.1% of phytoplankton samples contained potentially toxic Alexandrium species above the level to trigger flesh testing (N=1361), while in the summers (1994-1999), 7.1% of samples contained this level (N=1625). In spring and autumn, these percentages were 3.3% (N=1619) and 3.0% (N=1647) respectively. While these results are rudimentary, this suggests that there may be a higher chance of occurrence of PSP toxins in shellfish in the Marlborough Sounds in summer than in winter. The seasonality of PSP toxin occurrence in shellfish was analysed using data from sample sites that had been regularly and consistently monitored from 1/7/93 to 30/6/99 as described in Section 2.2.7. This analysis was based on the incidence of PSP toxins above the detectable level in shellfish, for each zone, because the incidence of PSP toxins above the regulatory level was low (implying a relatively low risk to consumers). The data were standardised to take account of differences in the number of sites per zone. Data from 48 sites were included in the analysis. Fourteen of these sites were in Zone G. There were no data available from Zones J or K (due to discontinuous sampling at all sites in these zones). Due to the fact that regular weekly sampling was discontinued in sites in Zone D with persistent levels of PSP toxins in tuatua, these sites were not included in this analysis. The risk of PSP to consumers is related to both the occurrence of PSP events, and the duration of retention of toxins in the shellfish. In this analysis, it is assumed that the presence of any PSP toxins in shellfish is indicative of an increased likelihood of PSP levels sufficient to be a risk to public health. As depicted in Figure 2.8, there appears to be no clear seasonal pattern relating to the potential risk of PSP to consumers of shellfish.

42

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec

Month

Cum

ulat

ive

No. o

f PSP

"D

etec

ts"

per M

onth

by

Zone

Zone A Zone B Zone C Zone D Zone E Zone F Zone GZone H Zone I

Figure 2.8: The six-year cumulative incidence of PSP toxins above the

detectable level at sites within each zone by month, recorded from 1st July 1993 to 30th June 1999. The data has been standardised to account for differences in the number of sites per zone.

Using the same set of data, the variation in PSP toxin occurrence from year to year was investigated, as described in Section 2.2.7. The temporal distribution of PSP levels above the detectable level in shellfish samples from the same consistently sampled sites is presented in Figure 2.9.

0

100

200

300

400

500

600

Mar-93

Jul-9

3

Nov-93

Mar-94

Jul-9

4

Nov-94

Mar-95

Jul-9

5

Nov-95

Mar-96

Jul-9

6

Nov-96

Mar-97

Jul-9

7

Nov-97

Mar-98

Jul-9

8

Nov-98

Mar-99

Jul-9

9

Nov-99

Time

PSP

Leve

l (µg

STX/

100g

Tis

sue)

Zone A Zone B Zone D Zone G Zone H

Figure 2.9: Distribution of detectable levels of PSP toxin in shellfish from

consistently monitored sites from July 1993 to June 1999. This figure shows that that the frequency of occurrence of detectable levels of PSP in shellfish from each zone may vary from year to year, for example, PSP toxins occurred in Zone G sites in 1993-94 and again in the summer of 1997-98. With the

43

exception of the period from July 1993 to July 1994, when there were relatively high numbers of PSP detects below the regulatory level, there appear to be no obvious year to year patterns. It should be noted however, that there is not a consistent seasonal occurrence from year to year of PSP toxins within each zone: for example, PSP toxicity occurred in Zone G in the summer of 1993-94, but was absent in the summer of 1994-95. Similarly, PSP toxicity was not recorded at the Zone D sites included in this analysis prior to the summer of 1995. There are similar variations in the incidence of PSP toxicity in Zone A. This is important in terms of risk analysis. While analysis of PSP toxin data on a purely geographical basis might suggest that Zones A and D are the areas of highest risk to shellfish consumers with respect to PSP, the temporal data suggests that extrapolation of historical data to predict future occurrence of PSP toxicity by area should be undertaken with great caution. A summary of the occurrence of El Nino/La Nina climate patterns over this time period is presented in Appendix III. There appears to be no obvious correlation between the occurrence of PSP toxins, (either across NZ as a whole, or within zones) with these broad climate patterns. More information on the population dynamics of potentially toxic Alexandrium species in each area is required before any predictability of risk would be possible. Most Alexandrium species have a life cycle that involves alternation between asexual and sexual reproduction. Anderson (1998) discusses the physiology and bloom dynamics of toxic Alexandrium species overseas. Anderson (1998) suggests that generally Alexandrium blooms have a “life-span” – a relatively short period of time in which these species are found in the water column as motile vegetative cells. At other times, Alexandrium species reside in the sediments as resting cysts (hypnozygotes). It is commonly thought that cyst “seedbeds” provide the inoculum for many Alexandrium blooms. These may not necessarily be discrete beds of cysts, but may be due to a widespread distribution of cysts in the sediment in general. The dynamics of Alexandrium species life cycles (for example, the triggers to encystment and excystment) are complex and not particularly well understood. They appear to involve the interaction of a series of factors such as temperature, nutrient availability, oxygen availability and endogenous rhythms (internal clock mechanisms) (Anderson, 1998). The bloom dynamics of Alexandrium species are the result of the interaction between the biological and behavioural characteristics of Alexandrium (e.g. life cycle, dormancy times of hypnozygotes, swimming behaviour, diel vertical migration through the water column etc.) and environmental and hydrographical factors (temperature, nutrients, salinity, currents, tides, wind etc.). Patterns of occurrence of Alexandrium may also be influenced by larger-scale factors such as El Nino/La Nina weather patterns (Erickson & Nishitahi, 1985), and 18.6 year cycles of lunar tidal modulation (White, 1987). There are insufficient data available to analyze the occurrence of Alexandrium species in significant densities in New Zealand in terms of the interaction of these micro- and meso-scale factors. While some seasonal differences in Alexandrium occurrence in Zone G may be indicated from the phytoplankton monitoring data over the last five years, there are no apparent seasonal patterns in the occurrence of PSP toxins in shellfish in New Zealand overall, and there are inter-annual differences in PSP occurrence by zone. Predictions of risk based on historical data would be inappropriate until a much greater depth of understanding

44

about the dynamics of Alexandrium blooms in New Zealand is gained through long-term studies. 2.4.3 Phytoplankton as a Predictor of PSP in Shellfish The phytoplankton monitoring data and shellfish toxin testing data on the FoodNet database were analysed to determine the probability that phytoplankton monitoring would fail to predict the occurrence of PSP above the regulatory level in shellfish. (Refer to Section 2.2.4 for methodology). Prior to the commencement of phytoplankton records on the database at the beginning of May 1997, there were residual low levels of PSP in tuatua at sites in the Bay of Plenty. Some level of activity in this area continued through to the end of the period of analysis in June 1999. During this time there were four instances at Ohope Beach (Site D37) in which the level of PSP increased from consistently below the regulatory level (around 30-40 µg/100g) to over the regulatory level of 80 µg/100g. Three of these occasions were associated with levels of potentially toxic Alexandrium species above the level to trigger industry voluntary closure to harvest (500 cells/L). In the fourth instance, early in June 1999, when there was an increase in toxin level from 48 µg/100g to 139 µg/100g, no potentially toxic Alexandrium species were detected in the phytoplankton samples concurrent with the shellfish tests, or the week before, although 200 cells/L of Alexandrium margalefii (a species considered to be non-toxic) were identified in the sample the week before the occurrence of elevated toxin levels. However, there were 100 cells/L of Alexandrium catenella in the phytoplankton sample two weeks prior to the elevated toxin levels. Assuming that the identification of Alexandrium margalefii was made correctly, and that it does not produce PSP toxins, these results suggest that either only low numbers of Alexandrium catenella are required to increase shellfish toxicity from residual levels of 48 µg/100g to 139 µg/100g, or that a low level of precision in the phytoplankton sampling or counting methods resulted in failure to detect toxic species that were present. The lack of a consistent relationship between apparent increases in PSP content in shellfish containing initial residual levels of PSP toxin and potentially toxic phytoplankton, could be due not only to changes in shellfish toxin levels over time, but to a high degree of variability between shellfish at any one time. Studies on overseas shellfish species have shown a high variation in toxin levels between individual shellfish in the same area. The degree of variation between individuals was found to increase with decreasing toxin levels (White et al., 1993). We have been unable to find any instances recorded on the FoodNet database of replicate sampling to measure the variation between individuals or pooled samples of the same species at one time. It is suggested that such information could be significant in the determination of the risk of PSP to shellfish consumers in New Zealand. It would also assist in the interpretation of monitoring data for management purposes. The results of the above analysis support the continuation of weekly shellfish testing for PSP when there are residual levels of PSP below the regulatory level present in shellfish. There were no instances of PSP toxin rising from zero levels to above the regulatory level for public warnings/closure to industry harvesting at any shellfish monitoring sites associated with phytoplankton sites during the time over which phytoplankton

45

data are recorded on the FoodNet database. This meant that we were unable to investigate the probability that phytoplankton levels would fail to indicate the presence of PSP toxin in shellfish. However, we did investigate the relationship between detectable levels of PSP in shellfish and the presence of potentially toxic Alexandrium species. In six out of eight instances where PSP toxins rose to detectable levels in shellfish, potentially toxic phytoplankton species were recorded above the level to trigger shellfish testing or voluntary closure by industry. In the two instances with no associated phytoplankton levels, the PSP levels in the shellfish were relatively low (33 µg/100g and 35 µg/100g). Further long-term data are required in order to ascertain the reliability of phytoplankton monitoring as a predictor of levels of PSP above the regulatory level in shellfish. A wide range of factors impact on the ability to predict shellfish toxicity by sampling phytoplankton. Characteristics of shellfish species with respect to their feeding, and accumulation and retention of toxins over a period of time, combined with temporal and spatial variability in phytoplankton abundance, complicate the use of weekly phytoplankton monitoring in prediction of shellfish toxicity. However, good sampling design, incorporating appropriate site selection, appropriate levels of precision in both sampling and counting of phytoplankton, combined with action levels that are appropriate for limitations of the sampling design, potentially result in a sound monitoring programme. Consideration of the phytoplankton data with respect to PSP toxicity in shellfish suggests that low numbers of potentially toxic Alexandrium species may be significant. Where low numbers are significant, precision in sampling and counting may be significant factors. Precision is the variability of repeated sample estimates of mean abundance, and is limited by the effort that can be expended to collect and analyse samples (i.e. cost). The measurement of precision provides information that is important in deciding the value to be placed on any quantitative result. In a robust marine biotoxin monitoring programme, the phytoplankton trigger levels take into consideration the precision of sampling – i.e. at a lower level of precision, the trigger levels are set more conservatively. In the case of monitoring for PSP, the levels at which Alexandrium species trigger shellfish sampling are set at the level of detection for the phytoplankton counting method used. Numbers of toxic species in a phytoplankton sample are based on counting cells settled out from one 10 mL sample (Kirsten Todd, Cawthron Institute, pers. comm.). A level of 100 cells/L (which is the concentration that triggers shellfish sampling) thus equates to a count of 1 cell in 10 mL. In counting phytoplankton, the level of precision is related to the number of individuals counted. There has been much discussion in the literature regarding the number of cells that should be counted to give a satisfactory level of precision, and a variety of approaches have been proposed. Based on an assumption of a non-aggregated distribution of individuals, it has been suggested that for counts less than 50, the limits of expectation of population means based on single estimates of abundance may be obtained from fiducial limits to the Poisson distribution. For more than 50 cells per sample, the individuals may be distributed normally (Lund et al., 1958, Venrick, 1978). This information can be used for comparing different counts. If one actual count lies within the confidence limits of

46

the other, there is no significant difference between them (Lund et al., 1958). The marine biotoxin monitoring programme for non-commercially harvested shellfish has two phytoplankton trigger levels for Alexandrium species (100 cells/Litre to trigger shellfish monitoring, and 5,000 cells/Litre to trigger a Public Warning). It is noted that, at a 95% confidence level, the respective limits of expectation indicate that counts representing these two trigger levels are significantly different under the current counting protocol. However, using the current counting protocol, determination of the limits of expectation at a 95% confidence level indicate that the two lower trigger levels in the shellfish industry programme (100 cells/Litre and 500 cells/Litre), are not significantly different (based on methods described in Lund et al., 1958; also Figure 29 - Venrick, 1978; Table 7 - Andersen, 1996). While this does not directly relate to the marine biotoxin monitoring programme for non-commercially harvested shellfish, it does raise the question of whether the precision of the counting methods has been sufficiently considered in the determination of phytoplankton monitoring methods. 2.4.4 Conclusions Despite the challenges presented by data collection not designed with long-term analysis in mind, several conclusions regarding the risk of PSP to shellfish consumers in New Zealand may be drawn from the analysis of biotoxin monitoring data undertaken in this review. These are summarised below. • Alexandrium species and PSP toxicity in shellfish are widespread around New

Zealand and have occurred in a variety of marine environments. Latitude or biogeographic region does not limit their occurrence.

• If it can be assumed that the monitoring programme is representative of shellfish

harvesting in New Zealand, the incidence of PSP toxins in shellfish at a level that represents a potential risk to consumers has been comparatively low in most areas. Levels above the regulatory level of 80 µg/100g have been detected in only Zones A, D, and G in 0.03%, 2.14%, and 0.03% of the total samples respectively.

• Analysis of the temporal distribution of PSP toxins in shellfish in New Zealand

indicates there may be some year to year variation in the number of samples with detectable levels of PSP. Overseas research suggests that long-term cycles in abundance of toxic Alexandrium species occur (e.g. 18.6 year cycles). A better understanding of the factors controlling the incidence of Alexandrium blooms in New Zealand, based on robust long-term data collection, is required to facilitate a good assessment of the risk of occurrence of PSP toxins in shellfish at levels that are harmful to consumers.

• The monitoring data collected over the last six years suggest that there were

differences in occurrence of PSP toxins between zones. In addition, temporal analysis indicates that occurrence of biotoxins varied inter-annually by zone. Overseas research suggests that Alexandrium blooms are controlled by the interaction of a complex set of variables, some of which operate over long time scales. It is therefore suggested that historical data may not provide a good

47

estimate of risk in the future with respect to differences between zones in the distribution of PSP toxins in New Zealand.

• While five years of phytoplankton monitoring data from the Marlborough Sounds

suggested that there may be some seasonal differences in the frequency of occurrence of potentially toxic Alexandrium species, there were no similar patterns obvious in the occurrence of PSP toxins in shellfish by zone across New Zealand. This suggests that until further long term phytoplankton data are available, any seasonal variations in the marine biotoxin monitoring programme for PSP toxins should arise from seasonal differences in shellfish harvesting patterns, not assumptions about seasonal variations in the occurrence of PSP.

• Limited data are available to investigate the differences in uptake and

accumulation of PSP toxins between different species of shellfish, and further data are required for rigorous analysis. However, analysis of existing data suggests that differences between species in accumulation and retention of PSP toxins have the potential to impact significantly on the risk of PSP to consumers. It needs to be ensured that these differences are taken into account in the design of the marine biotoxin monitoring programme.

• There were no data available to test the probability that phytoplankton monitoring

would fail to detect levels of PSP above the level to trigger shellfish testing. However, in 25% of instances where lower levels of PSP were detected in shellfish, there were no potentially toxic Alexandrium species recorded in the phytoplankton monitoring either concurrently or in the previous week. Phytoplankton monitoring also failed to detect any potentially toxic Alexandrium species associated with the increase of residual low levels of PSP in shellfish to above the regulatory level in 25% of the cases for which there are data. Both these instances are based on small sample sizes, and further data are required to test the robustness of the phytoplankton monitoring programme with respect to detection of PSP toxins in shellfish. However, the results of the latter analysis support the continuation of weekly shellfish testing for PSP when there are residual levels of PSP below the regulatory level present in shellfish.

48

2.5 RESULTS OF ANALYSIS – ASP 2.5.1 Geographic Distribution The distribution of Pseudo-nitzschia species throughout New Zealand is shown in Figure 2.10. These data are drawn from the phytoplankton monitoring data on FoodNet, as described in Section 2.2.3. Unfortunately, on the database there is no distinction between potentially toxic and non-toxic species, so the map represents the geographic distribution of all Pseudo-nitzschia species around the New Zealand coast, both non-toxic and potentially toxic. The distribution of potentially toxic species may not necessarily correlate with that of all species collectively. Figure 2.11 shows the sites throughout New Zealand where ASP toxins above the regulatory level, and above the level of detection, have been found in shellfish tested in the marine biotoxin monitoring programme. (Refer to Section 2.2.7 for details of the method of analysis used). Neither of these figures provides quantitative information about the frequency of occurrence of potentially toxic phytoplankton or ASP in shellfish samples. However, they do indicate that Pseudo-nitzschia species have been found at all the phytoplankton monitoring sites throughout New Zealand. The distribution of ASP toxins in shellfish is also widespread throughout New Zealand. ASP toxins have been detected in shellfish from the far north of the North Island to the south of the South Island, on both western and eastern coasts, and also in the Chatham Islands to the east. These figures suggest that latitude, or biogeographic zone, does not limit the distribution of Pseudo-nitzschia species, and that given the right environmental conditions, ASP toxicity in shellfish could potentially occur anywhere in New Zealand. Table 2.7 shows the occurrence, and levels above regulatory limits of Pseudo-nitzschia species by Biotoxin Zone in New Zealand, as described in Section 2.2.3. Note that there are no phytoplankton monitoring sites in Zones F or K. This table represents an analysis summary for two sets of data: the first (“Total Samples”) is the data set of all the phytoplankton samples recorded on the FoodNet and Marlborough Sounds Shellfish Quality Assurance Programme databases. The second set of data includes all the samples recorded on these databases within an “Identified Time Interval” (i.e. 19/12/97-25/5/99). The percentage occurrence of Pseudo-nitzschia species measured from the full data set (Total Samples) suggests that Pseudo-nitzschia species are commonly found in the phytoplankton (percentage occurrence ranges from 58% in Zone C to 80.5% of the samples in Zone H). All zones have had levels of Pseudo-nitzschia species sufficient to trigger shellfish testing for ASP, and 78% of the zones have had levels sufficient to trigger an industry voluntary closure (all zones except Zones H and J). The data relating to the occurrence of Pseudo-nitzschia species using the total number of samples on the database is the best information that we have on an individual zone basis. The quantity of data varies considerably from zone to zone – most zones include data from late 1997 to May 1999, but data for the Marlborough Sounds in

49

Pseudo-nitzschia species detectedat all 26 phytoplankton sites in theMarlborough Sounds

Pseudo-nitzschia speciesdetected

No Pseudo-nitzschiaspecies detected

Distribution of Pseudo-nitzschiaspecies in New Zealand

Figure 2.10: Distribution of Pseudo-nitzschia species at sites where

phytoplankton has been monitored throughout New Zealand (to June 1999).

50

Sites with samples above regulatorylevel for ASP

Sites at which ASP has been detected

Frequently sampled sites where ASPhas not been detected

Distribution of ASP inNew Zealand

ASP detected at onesite at the ChathamIslands

There are 18 regularly monitoredshellfish sample sites in theMarlborough Sounds area. ASPabove the regulatory level hasoccurred in shellfish at one site.ASP has been detected in all but 9other regularly monitored sites.

Figure 2.11: Distribution of ASP at shellfish sample sites at two levels (above

the level of detection and above the regulatory level of 20 µg Domoic acid/g shellfish tissue) throughout New Zealand (to June 1999).

51

Zone G are available from 1994 to 1999, from the Marlborough Sounds Shellfish Quality Assurance Programme database. However, in order to compare the relative occurrence of Pseudo-nitzschia species between zones, the data relating to the identified time interval must be used. From analysis of the “Identified Time Interval” data set, the zones can be ranked in descending order of frequency of occurrence of Pseudo-nitzschia species as follows: J, I, A, B and H, G, D, E, C. However, it must be remembered that this analysis is based on only 17 months of recorded data, which is a comparatively short time.

Zone

A

B

C

D

E

G

H I

J

Percentage occurrence in

Total samples (%). N=

73.3

438

72.9

107

58.0

584

77.2

622

75.7

243

63.1

6486

80.5

82

72.0

468

74.0

104

Percentage of Total samples with 50,000-

199,900 cells/L (%)

6.2

6.5

0.5

7.9

4.5

7.6

3.7

8.3

1.9

Percentage of Total samples with 200,000

cells/L or more (%)

1.1

5.6

0.3

5.0

0.4

2.5

0.0

3.8

0.0

Percentage occurrence in identified time interval

(%) N=

76.6

304

73.7

76

48.9

456

66.7

532

64.5

152

71.5

1941

73.7

76

78.3

304

81.6

76

Percentage of samples in the identified time

interval with 50,000-199,900 cells/L (%)

4.3

5.3

0.4

5.3

5.3

8.9

3.9

8.6

2.6

Percentage of samples in the identified time

interval with 200,000 cells/L or more (%)

3.9

3.9

0.2

2.4

0.0

2.9

0.0

5.6

0.0

Table 2.7: Percentage occurrence of Pseudo-nitzschia species, and percentage

occurrence above the regulatory levels (50,000 cells/L and ≥ 200,000 cells/L, assuming that Pseudo-nitzschia species are greater than 50% of the total phytoplankton) in the Total Samples and in the samples from 19/12/97 to 25/5/99 (the “Identified Time Interval”) by zone.

To display the spread of the data in the “Identified Time Interval” data set, the same ranges of data (50,000-199,000 cells/L and ≥ 200,000 cells/L) have been represented graphically as “Box and Whisker Plots” (where five of more data points exist) or where there are less than five data points, as numbers simply stated. This summary is presented in Figure 2.12.

52

Pseudo-nitzschia

Zone

A D G I

Ran

ge o

f dat

a ab

ove

the

Indu

stry

Vol

unta

ry

Clo

sure

Lev

el (>

200

000

cells

/Litr

e)

0

1x106

2x106

3x106

4x106

5x106

6x106

7x106Pseudo-nitzschia

Zone

A D E G I

Ran

ge o

f dat

a ab

ove

the

Fles

h T

estin

g T

rigg

er

Leve

l (50

000

to 1

99 9

00 c

ells

/Litr

e)

0

50x103

100x103

150x103

200x103

250x103

N=13 N=28 N=8 N=173 N=26

N=12 N=13 N=57 N=17

Zone B: Above Flesh Testing Trigger Level (50 000 cells/L): 95 000, 72 000, 61 000, 89 000 Above Industry Voluntary Closure Level (200 000 cells/L):

273 000, 427 000, 219 000 Zone C: Above Flesh Testing Trigger Level (50 000 cells/L): 121 000,

76 000 Above Industry Voluntary Closure Level (200 000 cells/L):

236 000 Zone H: Above Flesh Testing Trigger Level (50 000 cells/L): 89 000, 58 000, 187 000 Zone J: Above Flesh Testing Trigger Level (50 000 cells/L): 85 200, 80 000

Figure 2.12: Box and whisker plots showing the frequency distribution of (a)

Pseudo-nitzschia species above the level to trigger flesh testing (50,000 cells/L) by zone, and (b) Pseudo-nitzschia species above the level to trigger a voluntary closure to commercial harvesting (200,000 cells/L) in the four zones (A, D, G and I) where these levels occur.

Most Pseudo-nitzschia samples in the range to trigger shellfish testing (i.e. 50,000-199,000 cells/L) occurred at the lower end of the range – 75% of all samples were less

53

than 125,000 cells/L. In the upper range of samples with 200,000 cells/L or more, 75% of samples are less than 1,000,000 cells/L. The maximum density found was one sample in Zone G (Whangakoko Bay, on 29/12/97), which was in excess of 6,000,000 cells/L. Overall, these data indicate that Pseudo-nitzschia species are common throughout New Zealand waters, and can occur in very high densities. While there are molecular probes available to distinguish between potentially toxic and non-toxic species of Pseudo-nitzschia, these are not used as part of the regulatory monitoring programme. They are however used informally, particularly by the commercial shellfish industry to determine whether voluntary closures to harvesting should be implemented. Had these results been recorded on the database, there would have been information available regarding the occurrence of individual toxic species of Pseudo-nitzschia. However, studies undertaken by Rhodes et al., (1998a), on the composition of Pseudo-nitzschia blooms around New Zealand in 1996 using whole cell DNA probes and immunochemical assays indicated that species assemblages may differ regionally. The occurrence of Domoic acid in Northland scallops coincided with a bloom dominated by P. australis, and toxicity in the Bay of Plenty occurred during a bloom of P. turgidula and P. fraudulenta. A summary of ASP toxin occurrence in shellfish for all sites, in all zones, from July 1993 to June 1999 is provided in Table 2.8.

Zone

Total No.

of Samples

Number of samples in which ASP

was detected

Percentage of samples

in which ASP was detected

No. of samples

above the regulatory

level

Percentage of samples above the regulatory

level

Maximum toxin level

(µg/g)

A 2007 294 14.6 33 1.64 600 B 879 78 8.9 0 0.00 C 1698 53 3.1 0 0.00 D 2285 262 11.5 1 0.04 72 E 743 12 1.6 1 0.13 22 F 1276 11 0.9 0 0.00 G 5069 172 3.4 1 0.02 187 H 591 3 0.5 0 0.00 I 1418 30 2.1 0 0.00 J 714 16 2.2 0 0.00 K 215 2 0.9 0 0.00

Table 2.8: Summary by zone of the total occurrence of ASP toxins detected in

shellfish samples, and ASP toxins above the regulatory level of 20 µg/g detected in shellfish samples from 1/7/93 to 30/6/99.

These results indicate that ASP toxin has been detected in all zones. However, samples above the regulatory level have occurred in only Zones A, D, E, and G. They occurred as comparatively low percentages of the total samples tested – in Zones D, E and G there was only one sample each above the regulatory level out of all the samples tested. In Zone E, this sample was only just above the regulatory level (2 µg/g of Domoic acid above the regulatory level of 20 µg/g). However, the samples from the other zones were significantly higher – particularly from Zone A, which had a maximum Domoic acid level of 600 µg/g (a scallop sample from Doubtless Bay on

54

28/11/94). Closer examination of the data and questioning of ESR revealed that this sample was the gut portion of the scallop (P. Truman, ESR, pers. comm.) – a sample of whole scallops from the same site on the same day had a level of 136 µg/g. This variation between toxin levels in different parts of the scallop may contribute to an over estimation of the maximum value in Zone A. The maximum level of toxin in whole scallop samples (as distinct from a portion of shellfish) in Zone A was 210 µg/g from the Cavelli Islands on 2/11/93. As reported in the previous review of the marine biotoxin monitoring data (Wilson & Sim, 1996), different anatomical parts of scallops appear to retain different levels of Domoic acid. This highlights a potential problem with the consistency and accuracy of reporting of scallop data with respect to parts of the scallop analysed in specific samples in the early years of the monitoring programme. However, it appears from the limited amount of data (See Table 2.9) that the highest levels of Domoic acid are found in the gut and skirt of the scallop (which is the portion that is most commonly not eaten). Lower levels are found in the roe, and still lower levels in the muscle. These differences are taken into account in the testing for commercial harvesting, but to a lesser extent with respect to the non-commercial harvesting of scallops. Date/Site code

Domoic acid level in

sample of whole

scallops (µg/g)

Domoic acid level in

sample of scallop guts

and skirt (µg/g)

Domoic acid level in

sample of scallop roe

(µg/g)

Domoic acid level in sample

of scallop muscle and roe

(µg/g)

5/11/96/A05A 90 34 17/11/96/A05A 60 30 28/11/94/A06A 136 600 25 10 7/12/94/A06A 471 23 12 14/12/94/A06A 80 445 26 14/11/95/A08A 47 22 19/11/95/A08A 21 21 27/12/95/A08A 29 7

Table 2.9: Results of analysis for ASP in whole and portions of scallops

sampled from the same site at the same time. Table 2.9 tends to suggest that scallop muscle has the lowest levels of Domoic acid when compared to other parts of the scallop. However on one occasion (19/11/95) levels in the combined muscle and roe were identical to that found in the roe alone. Examination of the events in which Domoic acid had been detected at levels above the regulatory level revealed that most of the samples were scallops from the far north of New Zealand. We therefore undertook further analysis of the geographical distribution taking into consideration the potential differences between shellfish species. It should be noted that the analysis presented in Table 2.8 did not use standardized data, and that potential differences in toxin accumulation characteristics between shellfish, the clumping of sample sites and variations in sampling regimes (both through time and between sample sites), have been ignored. In an attempt to reduce some of this bias, a similar analysis was carried out using data from a single species only, from sites that had been consistently monitored over the whole time period, as

55

described in Section 2.2.7. Because they were the most widely sampled species, with consistent year-round sampling, GreenshellTM mussels were chosen as the sample species. The data were standardized to account for the changes in the shellfish sampling programme since the introduction of phytoplankton sampling, as described in Section 2.2.7. Table 2.10 provides a summary of ASP occurrence in GreenshellTM mussels by zone from July 1995 to the end of June 1999. The scope of the data is less than that in Table 2.8 as GreenshellTM mussels had only been consistently monitored at sites in Zones C, D, G, H and I over that time period. No data were available from the other zones. This summary suggests that the occurrence of Domoic acid in GreenshellTM mussels in Zone D was higher than in the other zones. No samples of GreenshellTM mussels above the regulatory level were found over this period.

Zone

Total No. of

Samples

Percentage of Samples with ASP above

Detectable Levels (%)

Percentage of Samples with ASP above the

Regulatory Level (%) C 816 0.3 0.0 D 179 14.0 0.0 G 2480 0.0 0.0 H 396 0.0 0.0 I 205 2.9 0.0

Table 2.10: Summary by zone of occurrence of ASP toxins in GreenshellTM

mussels from consistently monitored sample sites from 1/7/95 to 30/6/99.

Zone

Total No. of Samples

Percentage of Samples with ASP above

Detectable Levels (%)

Percentage of Samples with ASP above the

Regulatory Level (%) A 129 74.4 7.75 C 136 8.1 0.00 D 508 26.6 0.00 F 245 2.5 0.00

Table 2.11: Summary, by zone, of occurrence of ASP toxins in scallops from

sample sites consistently monitored over the same time period each year (beginning of July to end of January, excluding February to June each year) from 1/794 to 31/1/99.

Table 2.11 presents a similar summary of the occurrence of Domoic acid in scallops from sites that had been consistently monitored over the same months each year for the years 1994-1999. Since scallops are only monitored seasonally when the scallop harvesting season is open, a time period when the maximum of scallop sample sites were regularly monitored each year was identified for this analysis. This period was for seven months each year, from the 1st of July one year, through to the 31st of January the following year, beginning July 1994, and finishing January 1999. Only 8 sites from 4 zones (Zones A, C, D and F) had consistent data for this period over all years. The summary presented in Table 2.11 shows that scallops in Zone A had a higher number of samples in which Domoic acid was detected over this period. Zone A was

56

the only zone of the four that had samples with Domoic acid levels above the regulatory level of 20 µg/g. The results presented in Tables 2.10 and 2.11 suggest that the differences between zones in total occurrence of Domoic acid (Table 2.8) in shellfish may not merely be due to the varying species composition of shellfish samples taken in each zone, as results for the same shellfish species were highly variable between zones. The factors influencing blooms of potentially toxic Pseudo-nitzschia species and the production of Domoic acid appear complex and are not well understood. Overseas studies have shown that toxin production may be linked to the balance of nitrogen and silicate in the water (Bates et al., 1998). Toxin production has been observed when pulses of nitrate (for example due to run-off from rainfall after prolonged dry periods) occur when silicates are limiting growth. There has also been speculation that human activities, such as marine farming, may cause changes in the nutrient levels that promote Domoic acid production. There has recently been some lively discussion on the “Phycotoxins List” (phycotoxins@lists.listquest.com) debating this issue with respect to a long-running ASP event in Scotland. To date, no studies have been undertaken relating Domoic acid production to environmental factors in New Zealand. However, an environmental study being undertaken by shellfish farmers in the Marlborough Sounds (Zone G), Coromandel and the Mahurangi Harbour (Zone C) could provide data for the basis of such a study. Given that there is potential for the accumulation of ASP to differ between species, these differences were investigated within the limits of the available data. Table 2.12 provides a summary of the occurrence of ASP in the major shellfish species sampled in the marine biotoxin monitoring programme. It should be noted that while there may be potential for planktivorous fish to accumulate ASP toxins (e.g. Work et al. 1993), there is no record on the FoodNet database of planktivorous fish species in New Zealand having been tested for ASP.

Species

Total

No. with detectable levels of

ASP

Percentage of samples above

detectable level

No. of samples

above the regulatory

level

Percentage above the regulatory

level

GreenshellTM mussel

8451

133

1.57

5

0.06

Blue mussel 1438 29 2.02 0 0.00 Pacific oyster 2362 29 1.23 0 0.00 Dredge oyster 451 2 0.44 0 0.00 Scallop 2537 790 31.14 41 1.62 Tuatua 1211 12 0.99 0 0.00 Pipi 928 1 0.11 0 0.00 Cockle 599 9 1.50 0 0.00 Paua 236 3 1.27 0 0.00

Table 2.12: Summary of occurrence of ASP toxins in the major shellfish

species sampled in the marine biotoxin monitoring programme from 1/7/93 to 30/6/99.

57

These data indicate that a higher percentage of scallop samples contained a detectable level of ASP than other shellfish species sampled, and that scallop samples also had a greater percentage of ASP levels above the regulatory level of 20 µg/g. However, these data alone are not sufficient to indicate species differences with respect to biotoxin accumulation and retention, since the toxin accumulation might be site specific rather than species specific. While the data are scarce, several species of shellfish from the same site have been monitored concurrently and tested for ASP on a few occasions. The most comprehensive data are summarised in Figures 2.13 and 2.14. Note that in these figures sample results below the level of detection have been depicted as zero.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

7/09/9

8

14/09

/98

21/09

/98

28/09

/98

5/10/9

8

12/10

/98

19/10

/98

Time

ASP

Lev

el (µ

g/g)

Greenshell MusselScallop (Roe)

Figure 2.13: Comparison of levels of ASP in Greenshell™ mussels (Perna

canaliculus) and scallop (Pecten novaezelandiae) at Takaka River (site G07) over a six week period. (Note the scallop samples were roe only).

58

0

0.5

1

1.5

2

2.5

3

7/09/9

8

14/09

/98

21/09

/98

28/09

/98

5/10/9

8

12/10

/98

19/10

/98

26/10

/98

2/11/9

8

Time

ASP

Lev

el (

µg/g

)

Greenshell musselScallop (Roe)Cockle

Figure 2.14: Comparison of levels of ASP in Greenshell™ mussels (Perna

canaliculus), scallop (Pecten novaezelandiae), and cockles (Austrovenus stutchburyi) at Four Fathom Bay (site G87) over a two month period.

Figures 2.13 and 2.14 identify that at the same site different shellfish species can accumulate toxin very differently, with scallops tending to accumulate the highest amounts of ASP. Note that the scallop samples tested were roe only, so based on previous comparisons of ASP levels between different parts of the scallop (refer to Table 2.9), the toxin levels in the whole scallops would have been much higher. Several one-off comparison were made between Domoic acid levels in mussels and whole scallops:

Sample Site

Date

Domoic acid level in scallop sample (µg/g)

Domoic acid level in GreenshellTM mussel sample

(µg/g) Cavelli Is. (A08A) 2/11/93 210 11.4 Cavelli Is. (A08A) 15/11/93 48.6 4.0 Cavelli Is. (A08A) 29/11/93 44.6 4.8 Rangaunu Bay (A05A) 29/9/93 8.6 0 Rangaunu Bay (A05A) 5/10/93 16.2 0 Rangaunu Bay (A05A) 15/11/93 11.7 2.0

Table 2.13: Comparison of Domoic acid levels in scallop (Pecten

novaezelandiae) and mussel (Perna canaliculus) samples taken concurrently from the same sites.

The data in Table 2.13 also suggest that there may be differences in toxicity levels between species at the same site. In the three sets of samples from the Cavelli Islands, the toxin levels in all the mussel samples were below the regulatory level of 20 µg/g, while all three scallop samples had toxin levels significantly above this level.

59

Unfortunately none of these comparisons have sufficient data to allow any rigorous quantitative analysis. It should be noted that while sample results are recorded as having been taken from the same site, it is uncertain how closely the samples were actually sited. With the limited data available to date it is not possible to comment on the possible differences in toxin retention time between species. The results of this analysis are not in any way definitive, and much more data collection would be required to investigate this further. The differences may be due to physiological differences between species, or environmental differences. However, these results do suggest that differences between species in ASP toxin accumulation have the potential to impact significantly on the risk of ASP to consumers, and that these differences should be taken into consideration in the design of the marine biotoxin monitoring programme. 2.5.2 Temporal Distribution The results of phytoplankton monitoring for Pseudo-nitzschia species from three sites in the Hauraki Gulf (from January 1995 to July 1999), 25 sites in the Marlborough Sounds and Port Underwood (from January 1995 to July 1999) and one site at Collingwood (from August 1996 to July 1999) are presented in Appendix IV(B). (A map showing the sites in the Marlborough Sounds is presented in Appendix IV(A)). Sites in the Marlborough Sounds (Zone G) tended to have a greater range of cell densities of Pseudo-nitzschia sp. than those sites from the Hauraki Gulf area (Appendix IV(B)). An easy comparison can be made by comparing the number of cases at each site where Pseudo-nitzschia levels were above 200 000 cells/L. Only 2 instances above this level were recorded in the Hauraki Gulf sites, one from the Tamaki Strait on the 14 August 1995, and one at Kopake on the 21 January 1997 (see Appendix IV(B), 1).). In comparison, over the same time period (January 1995-1 July, 1999), the Marlborough Sounds sites commonly consisted of periods above this arbitrary level. At the Hauraki Gulf sites no clear seasonal trends were apparent between years. However, some seasonality was apparent in the Marlborough Sounds area (see Appendix IV(B), 2).). Both Kenepuru Entrance (G08) and Schnapper Point (G35) showed increased levels of Pseudo-nitzschia (generally >100,000 cells/L) between August and September each year. Little Nikau Bay (G44), Nikau Bay (G36), Nydia Bay (G09), and Southeast Bay (G38) all showed increased levels (>100,000 cells/L) in March/April of all years except 1999, as well as an increase in cell density (>100 000 cells/L) in August and September, similar to that mentioned for the previous site grouping. Crail Bay (G15), Laverique Bay (G37), West Beatrix Bay(G31), and Brightlands Bay(G27) did not show any consistent seasonal patterns over the period of interest. The period from October 1995 to early January 1997 was defined by consistently lower numbers of Pseudo-nitzschia sp. at all sites, when compared to the long term data series. Hallam Cove (G10), Richmond Bay (G28), Waitata Bay (G26), Ketu Bay (G41) and Cannon Bay (G16) did not show any consistent seasonal patterns over the period of interest. As described for the Crail Bay group, the period from October 1995 to early

60

January 1997 was defined by consistently lower numbers of Pseudo-nitzschia sp. at all sites, when compared to the long term data series. Forsyth Bay (G39), Anakoha Bay (G17), Puketea Bay (G18) and Oyster Bay (G13) did not show any consistent seasonal patterns over the period of interest. However, at Forsyth Bay increases in cell density were apparent seasonally from December to January in every year except 1995. Pseudo-nitzschia sp. density for these sites reached exceedingly high levels of up to 1,560,000 cells/L. East Bay (G19), Horohora Bay (G12), Whangakoko Bay (G11) and Opihi Bay (G40) appeared to have a consistently high density (> 100,000 cells/L) of Pseudo-nitzschia sp. over most of the yearly cycle. However very low densities were consistently recorded in the months of June and July of all years studied. Pseudo-nitzschia sp. density for these sites reached high levels of up to 2,430,000 cells/L. Samples from Collingwood Farms in Golden Bay (monitoring began in August 1996) did show increased density in September/October of each year sampled. Phytoplankton monitoring data on the FoodNet database has been recorded for an insufficient period for a meaningful analysis of temporal patterns in other zones. The seasonality of ASP toxin occurrence in shellfish was analysed using data from sample sites that had been regularly and consistently monitored from 1/7/95 to 30/6/99 as described in Section 2.2.7. (This excluded data from sampling of scallops, which are only monitored during the scallop harvesting season). This analysis is based on the incidence of ASP toxins above the level of detection in shellfish, for each zone, because the incidence of ASP toxins above regulatory levels was low (implying a relatively low risk to consumers). The data were standardized to take into account differences in the number of sites per zone. Data from 48 sites were included in the analysis. Fourteen of these sites were in Zone G. There were no consistently monitored sites in Zone K (Chatham Islands) over this time period. The risk of ASP to consumers is related to both the occurrence of ASP events, and the duration of retention of toxins in shellfish. As depicted in Figure 2.15, there appears to be a possible broad seasonal pattern with increased risk of ASP to consumers of shellfish from mid winter (August) to mid summer (December). This contrasts with seasonal patterns in North America, where blooms of Pseudo-nitzschia tend to occur predominantly in the late summer and autumn (Bates et al., 1998).

61

0.00

1.00

2.00

3.00

4.00

Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov DecMonth

Cum

ulat

ive

No.

of A

SP

"Det

ects

" pe

r Mon

th b

y Zo

ne

Zone A Zone B Zone C Zone D Zone E Zone F Zone GZone H Zone I Zone J

Figure 2.15: The six-year cumulative incidence of ASP toxins above the level of

detection at sites within each zone by month, recorded from 1st July 1995 to 30th June 1999. The data have been standardized to account for differences in the number of sites per zone.

05

101520253035

Apr-95

Aug-95Dec-9

5Apr-9

6Aug-96

Dec-96

Apr-97

Aug-97Dec-9

7Apr-9

8Aug-98

Dec-98

Apr-99

Time

ASP

Lev

el (µ

g/g)

Zone A Zone B Zone C Zone D Zone G Zone I Zone J

Figure 2.16: Distribution of detectable levels of ASP toxin, in shellfish from

consistently monitored sites from April 1995 to April 1999. Using the same set of data, the variation in ASP toxin occurrence from year to year was investigated, as described in Section 2.2.7. The temporal distribution of ASP levels above the detectable level in shellfish samples from the same consistently sampled sites is presented in Figure 2.16. This figure shows that the frequency of occurrence of detectable levels of ASP in shellfish from each zone may vary from year to year. However, Zone A appeared to have the most consistent seasonal pattern with detectable levels of ASP toxin between August and December of all years analysed. Overseas studies suggest that different species of Pseudo-nitzschia bloom in different environmental conditions, resulting in seasonal differences in abundance

62

of individual species (Bates et al., 1998). There have been no studies on the dynamics of Pseudo-nitzschia blooms in New Zealand. The overall occurrence of Domoic acid in shellfish across all zones also varies from year to year. A summary of the broad El Nino/La Nina climate patterns is presented in Appendix III. There appears to be no obvious correlation between the occurrence of ASP toxins (either across New Zealand as a whole, or within zones) with these broad climate patterns. For example, the most intensive periods of detectable levels of ASP have been from August to December in 1996 and 1998. In August/September 1996, La Nina conditions prevailed, turning to El Nino from October 1996 through to June 1998. From July 1998, the conditions were once again typical of La Nina. While environmental conditions no doubt do impact on the occurrence of blooms of Pseudo-nitzschia, the relationship is not so simple as to allow prediction of risk by such broad climate patterns. At this early stage, it is not possible to tell whether the pattern of increased incidence of detectable levels of ASP in the months of August to December indicates a long-term temporal trend. 2.5.3 Phytoplankton Monitoring as a Predictor of ASP in Shellfish The ability of phytoplankton counts to reliably indicate the potential presence of Domoic acid above the regulatory level could not be rigorously investigated due to the paucity of phytoplankton monitoring data – there was only one instance recorded at this level in the time period for which phytoplankton data were available. (Most high levels of ASP occur in scallops, and in general there is no phytoplankton monitoring on scallop beds). The relationships between levels of Pseudo-nitzschia species in the water, and two ranges of toxicity in shellfish (<1 µg DA/g shellfish tissue and 1-19.9 µg DA/g shellfish tissue) were therefore investigated. Because levels of toxin are greatest in the stationary phase of a Pseudo-nitzschia bloom, and shellfish toxicity may not occur until after this, Pseudo-nitzschia numbers were examined in phytoplankton for up to three weeks prior to the date of the shellfish sample. There was one instance of Domoic acid in shellfish above the regulatory level of 20 µg/g – this was a level of 187 µg/g in a sample of GreenshellTM mussels from Kenepuru Entrance (Zone G, Marlborough Sounds) on 20/12/94. None of the levels of Pseudo-nitzschia species in the phytoplankton samples either concurrent with the shellfish sample, or in any of the four preceding weeks exceeded the level to trigger shellfish testing at this site. (The maximum level recorded in this time was 27,300 cells/L). However, very high numbers of Pseudo-nitzschia species were recorded at many other sample sites in the Marlborough Sounds over this period, with no associated shellfish toxicity. It is likely that this particular phytoplankton sampling site does not provide a good indication of the toxin status of the shellfish present, as it is positioned in a current from the outer Sounds. This may increase the temporal variation of phytoplankton populations at the site. In 97.5% of the events in which trace levels of Domoic acid occurred in shellfish (N=41), Pseudo-nitzschia species were present in the phytoplankton samples either concurrently with the shellfish sample, or up to three weeks prior to the shellfish sample.

63

With levels of Domoic acid between 1.0 - 19.9 µg DA/g shellfish tissue, 100% of the events coincided with the presence of Pseudo-nitzschia species in phytoplankton samples (N=14) either concurrently with the shellfish sample or up to three weeks prior to the shellfish sample. Because the species of Pseudo-nitzschia present in the phytoplankton samples are not reported individually, and different species (and strains within species) differ in toxicity, it is not possible to meaningfully investigate the relationship between numbers of Pseudo-nitzschia cells and toxin levels in shellfish using the data available from the FoodNet database. Analysis is further complicated by the lack of independence of the variables, since levels of Pseudo-nitzschia species in excess of 50,000 cells/L may trigger shellfish sampling. This becomes significant if quantitatively analyzing events based on the incidence of any level of shellfish toxicity, resulting in the necessity to distinguish between routine shellfish samples and shellfish samples triggered by phytoplankton levels. It is pertinent to comment however, that in the data analysis it was noted that some levels of Pseudo-nitzschia species associated with trace levels of toxicity in shellfish were very low: in four instances Pseudo-nitzschia counts were less than 1,000 cells/L. However, Pseudo-nitzschia species occur commonly as a component of the phytoplankton in many areas, and thus these trace levels may be residual toxin levels arising from low numbers of toxic species. From the data available, it is not possible to conclude whether the levels of cell counts that trigger shellfish sampling in the marine biotoxin monitoring programme are appropriate for New Zealand Pseudo-nitzschia species, or whether phytoplankton monitoring is a robust proxy indicator of the presence of Domoic acid in shellfish. The data certainly suggest that phytoplankton monitoring may not always predict significant levels of Domoic acid in shellfish. However, the sample sizes are very small, and further data are required for a robust analysis. 2.5.4 The Use of Whole Cell DNA Probes for Pseudo-nitzschia Species as a

Predictor of Risk of Shellfish Toxicity At the request of the Ministry of Health, the effectiveness of the use of whole cell DNA probes as an indicator of the risk of ASP toxicity in shellfish, was investigated. Whole cell DNA probes have been developed by Cawthron Institute, as a means of easily identifying species of Pseudo-nitzschia. These probes (commonly referred to as “gene probes”) are used by the shellfish industry in the event of Pseudo-nitzschia numbers high enough to trigger a voluntary closure, to determine whether a voluntary closure should be implemented pending shellfish test results. The probes also appear to have been used in the non-commercial monitoring programme in deciding whether shellfish samples in addition to routine samples should be taken for toxin testing when Pseudo-nitzschia numbers reach trigger levels. The probes distinguish Pseudo-nitzschia australis, P. fraudulenta, P. multiseries, P. delicatissima, P. pungens, P. heimii, and P. pseudodelicatissima. There is some cross-reactivity between the probe for P. australis (a toxin-producing species), and the non-toxic P. multistriata. However, these species are easily distinguishable by shape under a light microscope

64

(P. multistriata has a distinctive sigmoid shape). P. delicatissima and P. turgidula are detected by the same probe, but these species are thought to be identical, with closely similar toxicity and morphology (L. Rhodes, Cawthron Institute, pers. comm.). The results of gene probe analyses undertaken for the shellfish industry and Ministry of Health, and as part of Dr Lesley Rhodes’ doctoral research project, were supplied to us by Cawthron Institute. These results include only the results of the gene probe tests, and for the most part, data on total Pseudo-nitzschia numbers and total phytoplankton numbers for each sample were obtained from the FoodNet database. In some cases where the phytoplankton samples were not taken as part of the regulatory monitoring programme, these data were obtained from Cawthron Institute. Upon requesting information regarding the interpretation of the results of the gene probe tests, we were initially informed that there was no formal or written protocol for this (K. Todd, Cawthron Institute, pers. comm.). (Lack of protocols is potentially an area of concern, as it can lead to inconsistency in the interpretation of test results). However, Cawthron Institute subsequently provided us with a written protocol for the interpretation of gene probe results, and the guidelines from this protocol have been used in our analysis. (These guidelines are outlined in Table 2.14). It should be noted that our analysis is based on advice from results of gene probe tests that would be given based on these stated protocols, and has not examined the actual advice given to industry or Ministry of Health at the time the tests were done.

Species of Pseudo-nitzschia

Risk Assessment Guidelines

P. australis, P. pungens and P. multiseries

Trigger flesh testing when the combined cell density is greater than 50, 000 cells/L

P. delicatissima and P. pseudodelicatissima

Trigger flesh testing at 100, 000 cells/L when they comprise > 50% of the total phytoplankton biomass Trigger flesh testing at 250, 000 cells/L when they comprise < 50% of the total phytoplankton biomass

P. fraudulenta

Trigger flesh testing at 250, 000 cells/L when they comprise > 50% of the total phytoplankton biomass Trigger flesh testing at 500, 000 cells/L when they comprise < 50% of the total phytoplankton biomass

P. heimii and P. multistriata

Trigger flesh testing at 500, 000 cells/L when they comprise > 50% of the total phytoplankton biomass Trigger flesh testing at 1, 000, 000 cells/L when they comprise < 50% of the total phytoplankton biomass

Table 2.14 Risk assessment guidelines for toxin flesh testing in shellfish, for

various species of the genus Pseudo-nitzschia. In our analysis, a total of 171 phytoplankton samples from September 1997 to June 1999 were examined to investigate the use of whole cell gene probes in differentiating potentially toxic or non-toxic species of the Pseudo-nitzschia genus as a predictor of the risk of shellfish toxicity. Density estimates of each Pseudo-nitzschia species identified by the whole cell gene probes were matched to concurrent and subsequent shellfish toxin testing results, to identify the relationships between risk assessment guideline trigger levels and subsequent detection of Domoic acid in shellfish tissue. Because the peak in Domoic acid found in shellfish tissue may not coincide with the peak Pseudo-nitzschia density

65

in phytoplankton samples (because Domoic acid may be released as the bloom crashes), shellfish samples for 2 weeks after each phytoplankton sample was collected were included in our analysis. Over all the instances in which whole cell gene probes had been applied to phytoplankton samples, there were no instances of shellfish toxin tissue testing over the regulatory level of 20 µg Domoic acid/g shellfish tissue. This is unfortunate, as in order to conclusively validate the method we need to be sure that the risk of significant shellfish toxicity would be predicted by the results of the gene probe tests and the accompanying protocols for interpretation of results. Consequently, no conclusive inference can be made about the effectiveness of whole cell gene probes in identifying species composition, and consequently risk assessment at this level. All analysis is based on extremely low trace levels of Domoic acid in shellfish. Had shellfish toxicity been measured weekly for three weeks after each gene probe test, the total shellfish sample data would have equated to 513 shellfish tests for the 171 phytoplankton samples investigated (i.e. 171 x 3, one test in the initial week of the phytoplankton sample and 2 follow up samples in subsequent weeks). Complete coverage by shellfish toxin testing was not obtained, with 119 (70%) shellfish toxin tests undertaken in the week the initial phytoplankton sample was obtained, and 101 (59%) and 106 (62%) in the second and third weeks respectively. This lack of comprehensive coverage of follow up in shellfish toxin testing reduces the robustness of assessment of the gene probe test and the current risk assessment guidelines for Pseudo-nitzschia sp. in relation to subsequent Domoic acid accumulation in shellfish tissue. The results of the gene probe tests for Pseudo-nitzschia species, and corresponding results from shellfish toxicity testing are presented in Appendix V. In this analysis 0.5 µg Domoic acid/g shellfish tissue indicates a trace level of Domoic acid only. Note also that no data are provided for P. multistriata, which is a non-toxic species. In five cases (3%), cell densities of Pseudo-nitzschia sp. above the risk assessment guidelines, as identified by whole cell probes, were related to trace levels (0.5 µg/g) of Domoic acid in shellfish toxin tissue testing (see Table 1, Appendix V). In only one case (at Blueskin Bay, 4 Feb 1998) was the level of Domoic acid significantly higher than this, but still well below the regulatory level of 20 µg Domoic acid/g shellfish tissue. In 8 cases (5%) the cell densities for Pseudo-nitzschia sp. were below the risk assessment guidelines, as identified by whole cell probes, but were still associated with trace levels of Domoic acid in shellfish toxin tissue testing (see Table 2, Appendix V). This may simply be due to the breakdown of Domoic acid from previously declining blooms, which is almost certainly the case for site A08 (Whangaroa), where trace levels of Domoic acid were apparent from August 1998 continuously through until the sample analysed there in early November. Similarly for site G19, (East Bay), where trace levels of Domoic acid were apparent from August 1998 continuously through until the sample analysed there in late September, and G01 (Collingwood), where a trace was detected a week prior to the phytoplankton levels measured on the 28th September 1998. For the other sites outlined in Table 3, there are no trace levels apparent in previous shellfish toxin tissue testing samples.

66

Interestingly the samples from A03 (Houhora) and the samples from B14 (Marsden Point) are comprised of 100% P. heimii, which may suggest extremely low levels of toxicity below the risk assessment guidelines in this species. The trace level found at Kennedy Bay (D06, 31/8/98) cannot be currently explained. From the total samples tested, there were 41 cases for which the whole cell gene probe data indicated phytoplankton densities above the risk assessment guidelines, but for which no corresponding toxicity was found in shellfish toxin tissue testing (Table 3, Appendix V). Of these samples, 24 phytoplankton samples (60%) did not have consistent shellfish toxin tissue testing for the full three-week period after the initial sample was taken. Because of this, no clear statement can be made about the relationship between cell density (as identified by the whole cell probe) and possible shellfish toxicity in these samples as identified by the risk assessment guidelines. Of the remaining 17 samples, most (70.6%) contained combined numbers of Pseudo-nitzschia australis, P. pungens, and P. multiseries sufficient to trigger shellfish toxicity testing (refer to Table 3, Appendix V). The lack of data relating to the use of whole cell gene probes in events where there have been significant levels of Domoic acid in shellfish tissue means that definitive conclusions cannot be drawn about the robustness of the method as an indicator of the risk of toxicity in shellfish. However, this is a potentially useful tool in biotoxin monitoring. It is suggested that some effort should be made to collect this information so that the method can be properly validated. The succession of Pseudo-nitzschia species within a bloom is of potential interest with respect to the use of whole cell gene probes, as an indicator of potential toxicity. There are a few instances when gene probes have been applied to a series of consecutive weekly samples, providing information about the species composition of Pseudo-nitzschia within a bloom. Unfortunately, most of these data have been collected in blooms of comparatively low densities. These data are presented in Appendix VI. From the small amount of data available, it is apparent that within a Pseudo-nitzschia species bloom, the dominant species can change over time. Thus, it cannot be assumed that a bloom that initially appears to be composed of non-toxic Pseudo-nitzschia species will continue with the same species composition. For this reason, in the absence of shellfish toxin testing, identification of the species composition of a bloom should continue at regular intervals. 2.5.5 Conclusions The following points summarise the conclusions that can be drawn from analysis of the marine biotoxin monitoring programme regarding the risk of ASP to shellfish consumers: • Pseudo-nitzschia species occur throughout New Zealand, and have been found at

all phytoplankton monitoring sites. They are common components of the phytoplankton assemblage from all zones. However, the data available do not distinguish between potentially toxic and non-toxic species of Pseudo-nitzschia.

67

• Shellfish samples containing ASP above the level of detection are also widespread throughout New Zealand, and have been found at the majority of sampling sites. The distribution of ASP toxicity does not appear to be limited by latitude or broad biogeographic patterns.

• If it can be assumed that the monitoring programme is representative of shellfish

harvesting in New Zealand, the incidence of ASP toxins at a level that represents a potential risk to consumers has been comparatively low in most areas. Such levels have been detected in only Zones A (in 1.64% of total samples), D (0.04%), E (0.13%), and G (0.02%).

• There appears to be a possible broad seasonal pattern in the occurrence of Domoic

acid in shellfish, with increased risk of ASP to consumers of shellfish from mid-winter (August) to mid-summer (December).

• The frequency of Domoic acid above the level of detection in shellfish may vary

from year to year, within zones and across all zones. Zone A appeared to have the most consistent seasonal pattern, with detectable levels of ASP toxin between August and December each year. However, there are insufficient data for predictions about future occurrence to be made.

• The distribution of Domoic acid throughout shellfish organs and tissues is not

uniform, and this impacts on the risk to shellfish consumers. Customary practices that include the consumption of scallop and paua guts (and possibly whole planktivorous fish) need to be considered in the design of the monitoring programme.

• Limited data were available to investigate the differences in uptake and

accumulation of ASP toxins between different species of shellfish, and further data are required for rigorous analysis. However, initial observations suggest that inter-specific differences between shellfish, whether arising from physiological or environmental causes, have the potential to impact significantly on the risk of ASP to consumers.

• Limited data were available to investigate the robustness of the use of whole cell

DNA probes in determining the risk of ASP toxins in shellfish, so no definitive conclusions could be drawn. It is suggested that this should be investigated further so this potentially useful method can be properly validated. The possibility that the dominant Pseudo-nitzschia species within a Pseudo-nitzschia bloom may change suggests that if reliance were to be placed on gene probes as an indicator of risk, they should be applied regularly through the course of a bloom.

68

2.6 RESULTS OF ANALYSIS – DSP 2.6.1 Geographic Distribution The distributions of the causative agents of DSP, Dinophysis species and Prorocentrum lima, in New Zealand are shown in Figures 2.17 and 2.18 respectively. These data are drawn from the phytoplankton monitoring data on FoodNet, as described in Section 2.2.3. Figure 2.17 indicates that both Dinophysis acuta and D. acuminata have been found at most sites throughout New Zealand. Prorocentrum lima appears to be less widely distributed. However, it is an epibenthic phytoplankton that is normally found associated with sand, sediments and macroalgae. Consequently, plankton samples taken from the water column are not a good measure of its abundance, since they may only be suspended in the water column after rough weather. It is thus likely that the distribution of Prorocentrum lima is wider than as recorded in the phytoplankton monitoring database. It is certainly common in the Hauraki Gulf (B. Hay, pers. obs.) and has also been recorded in the Marlborough Sounds from phytoplankton samples (Marlborough Sounds Shellfish Quality Assurance Programme database of phytoplankton monitoring results), and Cable Bay (Nelson) and Stewart Island (L. Rhodes, Cawthron Institute, pers. comm.). Figure 2.19 shows the sites throughout New Zealand where DSP toxins above the regulatory level, and above the level of detection, have been found in shellfish tested in the marine biotoxin monitoring programme (Refer to Section 2.2.7 for details of the method of analysis used). While there are many sites where DSP has not been detected, the sites where DSP has occurred are widely distributed, and are not limited by latitude. DSP has occurred at sites on both the eastern and western coasts. None of these figures provide quantitative information about the frequency of occurrence of DSP-producing phytoplankton, or DSP in shellfish samples. However, it can be seen that DSP-producing phytoplankton are found throughout New Zealand. DSP toxins have been recorded in shellfish at sites from both eastern and western coasts and do not appear to be limited by latitude. Table 2.15 shows the occurrence, and levels above regulatory limits, of potentially toxic Dinophysis acuminata by Biotoxin Zone in New Zealand, as described in Section 2.2.3. Table 2.16 shows the same data for Dinophysis acuta. (Note that there are no phytoplankton monitoring sites in Zones F or K. Note also the differences in the regulatory levels between the two species due to differences in toxicity). These tables summarise the results of analysis of two sets of data: the first (“Total Samples”) is the data set of all the phytoplankton samples recorded on the FoodNet and Marlborough Sounds Shellfish Quality Assurance Programme databases. The second set of data includes all the samples recorded on these databases within an “Identified Time Interval” (i.e. 19/12/97-25/5/99). As can be seen from Table 2.15, Dinophysis acuminata occurred in less than 10% of the total samples in all zones, except Zones E and I. Zone I had the highest percentage occurrence.

69

Dinophysis acuta detected at 15 out of 26sites in the Marlborough Sounds.Dinophysis species detected at all 26sites.

Dinophysis acuta andDinophysis acuminata detected

Dinophysis species but noDinophysis acuta

No Dinophysis species detected

Distribution of DinophysisSpecies in New Zealand

Figure 2.17: Distribution of potentially toxic species of Dinophysis throughout

New Zealand (to June 1999).

70

Prorocentrum lima has not beendetected in routine samples recorded onFoodNet at any of 26 sites in theMarlborough Sounds.

Prorocentrum limadetected at site

No Prorocentrum limadetected at site

Distribution of Prorocentrumlima.

Figure 2.18: Distribution of potentially toxic Prorocentrum lima throughout

New Zealand (to June 1999).

71

Sites with samples above regulatorylevel for DSP

Sites at which DSP has been detected

Frequently sampled sites where DSPhas not been detected

Distribution of DSP inNew Zealand

There are 18 regularly monitoredshellfish sample sites in theMarlborough Sounds area. DSPtoxicity above the regulatory levelhas been found in shellfish at 1 site.DSP toxicity in shellfish has beendetected in all but 11 other regularlymonitored sites.

Figure 2.19: Distribution of DSP at shellfish sample sites at two levels (above

the level of detection, and above the regulatory level of 20 µg DSP/100g shellfish tissue) throughout New Zealand (to June 1999).

72

Zone

A

B

C

D

E

G

H I

J

Percentage occurrence in

Total samples (%). N=

9.5

432

4.7

107

0.7

583

3.7

618

12.1

240

1.34

6486

2.4

82

18.2

468

0.0

104

Percentage of Total samples with 1,000-1,900

cells/L (%)

0.5

0.0

0.0

0.2

0.0

0.9

0.0

2.4

0.0

Percentage of Total samples with 2,000 cells/L or more (%)

0.0

0.0

0.0

0.0

0.0

0.7

0.0

0.4

0.0

Percentage occurrence in identified time interval

(%) N=

8.9

304

1.3

76

0.0

456

3.0

532

15.8 152

7.9

1941

2.6

76

16.8

304

0.0

76

Percentage of samples in identified time interval with 1,000-1,900 cells/L

(%)

0.0

0.0

0.0

0.2

0.0

1.1

0.0

1.6

0.0

Percentage of samples in identified time interval

with 2,000 cells/L or more (%)

0.0

0.0

0.0

0.0

0.0

0.5

0.0

0.0

0.0

Table 2.15: Percentage occurrence of Dinophysis acuminata, and percentage

occurrence above the regulatory levels (1,000–1,900 cells/L, and ≥ 2,000 cells/L) in the total samples, and in samples from 19/12/97-25/6/99 (the “Identified Time Interval”) by zone.

The data relating to the occurrence of Dinophysis using the total number of samples on the database are the best information that we have on an individual zone basis. The quantity of data varies considerably from zone to zone – for example, a full set of data from the beginning of 1994 through to June 1999 has been included for most sites in Zone G (from the Marlborough Sounds Shellfish Quality Assurance Programme database), but most other zones have data only from the end of 1997. However, in order to compare the relative occurrence of DSP-producing species between zones, the data relating to the “Identified Time Interval” must be used. From the analysis of the “Identified Time Interval” data set, it can be seen that Dinophysis acuminata has occurred in all zones except zones C and J. But in only three zones: D, G, and I, at levels of ≥ 1,000 cells/L. The data presented indicate that the zones can be ranked in a descending order of frequency of occurrence of D. acuminata levels as follows: I, E, A, G, D, H, and B. Prior to the 19/12/97, the only other zone that contained levels above the regulatory level was Zone A. Zone G was the only zone to obtain levels high enough for an industry voluntary closure. However, it must be remembered that this analysis is based on only 17 months of recorded data, which is a comparatively short time interval. To show the spread of the data in the “Identified Time Interval” data set, the same ranges of data (1,000-1,900 cells/L and ≥ 2,000 cells/L) have been represented

73

graphically as “Box and Whisker Plots” (where five or more data points exist). Where there are less than five data points, numbers are simply stated. This analysis is presented in Figure 2.20.

Dinophysis acuminata

ZoneG I

Ran

ge o

f dat

a ab

ove

the

Fles

h T

estin

g Tr

igge

r L

evel

(1 0

00 to

1 9

00 c

ells/

Litr

e)

0200400600800

100012001400160018002000

Dinophysis acuminata

ZoneG

Ran

ge o

f dat

a ab

ove

the

Indu

stry

Vol

unta

ry

Clo

sure

Lev

el (>

2 00

0 ce

lls/L

itre)

0

2000

4000

6000

8000

10000

N=21

N=9

N=5

Zone D: Above Flesh Testing Trigger Level (1000 cells/L): 1 500

Figure 2.20: Box and whisker plots showing the frequency distribution of (a)

Dinophysis acuminata above the level to trigger shellfish testing (1,000-1,900 cells/L), and (b) Dinophysis acuminata above the level to trigger a voluntary closure to commercial harvesting ( ≥ 2,000 cells/L) for Zone G where these levels occurred.

Zone D had only one sample above the level to trigger shellfish testing ( ≥ 1,000 cells/L). The median value of samples in the range to trigger shellfish testing in Zone G was 1,300 cells/L, with 75% of the samples below 1,400 cells/L, and 95% of the samples below 1,700 cells/L. The median of the samples in the range to trigger shellfish testing in Zone I was 1,500 cells/L, with 75% of samples less than 1,550 cells/L, and 95% of the samples below 1,580 cells/L. The median value for samples above the level to trigger an industry voluntary closure in Zone G was 3,600 cells/L, with 75% of samples less than 4,200 cells/L and 95% of samples less than 8,120 cells/L. The maximum cell count was 9,400 cells/L (from Opihi Bay on 8/3/99).

74

Zone

A

B

C

D

E

G

H

I

J

Percentage occurrence in Total samples (%).

N=

4.2

432

5.6

107

0.0

583

1.8

618

1.7

238

5.3

6486

3.7

82

13.1

466

1.0

104

Percentage of Total

samples with 500-900 cells/L (%)

0.0

0.0

0.0

0.2

0.0

0.4

0.0

1.3

0.0

Percentage of Total samples with 1,000 cells/L or more (%)

0.0

0.0

0.0

0.0

0.0

0.7

0.0

1.7

0.0

Percentage occurrence in identified time

interval (%) N=

3.3

304

5.3

76

0.0

456

2.1

532

2.6

152

1.1

1941

3.9

76

9.2

304

0.1

76

Percentage of samples in the identified time interval with 500-900

cells/L (%)

0.0

0.0

0.0

0.2

0.0

0.3

0.0

0.3

0.0

Percentage of samples in the identified time interval with 1,000 cells/L or more (%)

0.0

0.0

0.0

0.0

0.0

1.0

0.0

1.0

0.0

Table 2.16: Percentage occurrence of Dinophysis acuta, and percentage

occurrence above the regulatory levels (500-900 cells/L and ≥ 1000cells/L) in total samples and in samples from 19/12/97 to 25/6/99 (the “Identified Time Interval”) by zone.

The occurrence of Dinophysis acuta (Table 2.16) was relatively low: it occurred in less than 10% of the total samples in all zones except Zone I, where it was present in 13.1% of samples. D. acuta did not occur at all in Zone C over the time period for which we have data. Table 2.16 identified that D. acuta in excess of the levels to trigger shellfish testing ( ≥ 500 cells/L), occurred in only three zones: Zones D, G, and I. Only Zones G and I had samples with cell densities above the level to trigger voluntary industry closure ( ≥ 1,000cells/L). The “Identified Time Interval” data set showed that Zones G and I had the greatest frequency of occurrence of cell densities of D. acuta above the regulatory level of 500 cells/L. The data presented indicate that the zones can be ranked in a descending order of frequency of occurrence of D. acuta levels as follows: I, B, H, A, E, D, G, J and C. To show the spread of the data in the “Identified Time Interval” data set, the same ranges of data (500-900 cells/L and ≥ 1,000 cells/L) have been represented graphically as “Box and Whisker Plots” (where five or more data points exist). Where there are less than five data points, numbers are simply stated. This analysis is presented in Figure 2.21.

75

Dinophysis acuta

Zone

G

Ran

ge o

f dat

a ab

ove

the

Fles

h Te

stin

g Tr

igge

r L

evel

(500

to 9

00 c

ells

/Litr

e)

0

200

400

600

800

1000Dinophysis acuta

Zone

GR

ange

of d

ata

abov

e th

e In

dust

ry V

olun

tary

Clo

sure

Lev

el (›

1 00

0 ce

lls/L

itre)

0

2000

4000

6000

8000

10000

12000

N=5

N=20

Zone D: Above Flesh Testing Trigger Level (500 cells/L): 800 Zone I: Above Flesh Testing Trigger Level (500 cells/L): 500 Above Industry Voluntary Closure Level (1000 cells/L): 1 000, 1 200, 6 700 Figure 2.21: Box and whisker plots showing the frequency distribution of (a)

Dinophysis acuta above the level to trigger shellfish testing (500-900 cells/L), and (b) Dinophysis acuta above the level to trigger a voluntary closure to commercial harvesting ( ≥ 1,000 cells/L). Data from zones with less than 5 data points is also presented.

Zone D had only one sample above the level to trigger shellfish testing ( ≥ 500 cells/L). The median of the samples in the range to trigger shellfish testing in Zone G was 600 cells/L, with 75% of the samples below 700 cells/L, and 95% of the samples below 780 cells/L. Zone I had only one sample above the level to trigger shellfish testing ( ≥ 500 cells/L). The median value for samples above the level to trigger an industry voluntary closure in Zone G was 3,400 cells/L, with 75% of samples less than 4,800 cells/L and 95% of samples less than 7,340 cells/L. The maximum cell count from Zone G was 10,000 cells/L (from Wedge Point on 8/12/98). Zone I also contained 3 counts above the industry voluntary closure level, with the highest count reaching 6,700 cells/L (from Caroline Bay, on 3/5/99).

76

Zone

A

B

C

D

E

G

H I

J

Percentage occurrence in Total samples (%).

N=

0.5

432

0.0

107

0.0

583

0.2

618

0.4

240

0.1

6486

0.0

82

0.4

468

0.0

104

Percentage of Total

samples with 500-900 cells/L (%)

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Percentage of Total samples with 1,000 cells/L or more (%)

0.5

0.0

0.0

0.0

0.0

0.0

0.0

0.2

0.0

Percentage occurrence in identified time

interval (%) N=

0.0

304

0.0

76

0.0

456

0.2

532

0.0

152

0.0

1941

0.0

76

0.3

304

0.0

76

Percentage of samples in the identified time interval with 500-900

cells/L (%)

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Percentage of samples in the identified time interval with 1,000 cells/L or more (%)

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.3

0.0

Table 2.17: Percentage occurrence of Prorocentrum lima, and percentage

occurrence above the regulatory levels (500-900 cells/L and ≥ 1000cells/L) in total samples, and in samples from 19/12/97 to 25/6/99 by zone.

Zone I: Above Industry Voluntary Closure Level (1000 cells/L): 1 600 The occurrence of Prorocentrum lima was exceptionally low (Table 2.17). P. lima only occurred in samples in Zones A, D, E, G, and I. The only incidence of P. lima being higher than the industry voluntary closure level of 1,000 cells/L was at Akaroa on the 8/6/98 at 1,600 cells/L.

77

Zone Total No.

of Samples

Number of samples in which DSP

was detected

Percentage of samples

in which DSP was detected

No. of samples above

regulatory level

Percentage of samples

above regulatory

level

Maximum

toxin level

(µg/100g) A 2113 32 1.5 2 0.09 33 B 1009 3 0.3 0 0.00 C 2085 13 0.6 0 0.00 D 2111 3 0.1 0 0.00 E 772 4 0.5 1 0.13 39 F 1468 2 0.1 0 0.00 G 5796 137 2.4 71 1.22 96 H 682 3 0.4 0 0.00 I 1519 27 1.8 8 0.53 86 J 817 5 0.6 0 0.00 K 200 0 0 0 0.00

Table 2.18: Summary by zone of the total occurrence of DSP toxins detected in

shellfish samples, and DSP toxins above the regulatory level of 20 µg/100g detected in shellfish samples from 1/9/94 to 30/6/99. “Total No. of Samples” includes all acetone screen tests, plus DSP ELISA tests done without a prior acetone screen test.

A summary of DSP toxin occurrence in shellfish for all sites, in all zones, from September 1994 until the end of June 1999 is presented in Table 2.18. The percentages are calculated as percentages of the total Acetone Screen tests plus the DSP ELISA’s done without a prior acetone screen, not the total DSP ELISA tests alone. This full total is used because the Acetone Screen test and the DSP ELISA together constitute the test for DSP in the Marine Biotoxin Monitoring Programme. It can be seen that the overall percentage occurrence of detectable levels of DSP was relatively low, and that there were only four zones in which levels of DSP above the regulatory level of 20 µg/100g have been detected: Zones A, E, G and I. Most of the DSP detected in Zone G was from one site, Wedge Point (G23) in the Marlborough Sounds. Blue mussels at this site contained persistent levels of DSP from November 1994-August 1995, November 1995 to July 1996, and October 1996 to March 1997. The DSP in Zone I was also predominantly from one site, Akaroa Harbour (I04), where shellfish toxicity persisted from February 1995 to October 1995. The shellfish species sampled at this site were also Blue mussels. It is interesting to note that both these long-running events occurred in relatively protected waters. Blooms of Dinophysis species in Europe have been associated with persistent stratification over summer, fed with nutrients from upwelling pulses (Reguera et al., 1993; Palma et al., 1998). We are uncertain as to the influence of these factors in the New Zealand events. It should be noted that the analysis of data presented in Table 2.18 did not use standardized data, and that differences in toxin accumulation characteristics between shellfish species, the clumping of sample sites, and variations in sampling regimes, both through time and between sample sites, have been ignored. In an attempt to reduce some of this bias, a similar analysis was carried out using data from a single species, from sites that had been consistently monitored over the whole time period,

78

as described in Section 2.2.7. Because they were the most widely sampled species, Greenshell™ mussels were chosen as the sample species. The data were standardized to cope with the changes in the shellfish sampling programme since the introduction of phytoplankton sampling, as described in Section 2.2.7. Table 2.19 provides a summary of the DSP toxin occurrence in Greenshell™ mussels, by zone, from September 1994 to the end of June 1999. The scope of the data is less than that in the previous table as Greenshell™ mussels had only been consistently monitored at sites in Zones C, D, G, H, and I over that time period. No data were available from the other zones. None of the Greenshell™ mussel samples had levels of DSP above the regulatory level. The percentage of samples with DSP above the level of detection was low. The greatest percentage occurrence was in Zone I.

Zone

Total No. of

Samples

Percentage of Samples with DSP above Detectable

Levels (%)

Percentage of Samples with DSP above the

Regulatory Level

C 816 0.5 0.0 D 179 0.0 0.0 G 2480 0.4 0.0 H 396 0.3 0.0 I 205 1.0 0.0

Table 2.19: Summary by zone of occurrence of DSP toxins in Greenshell™

mussels from consistently monitored sample sites from 1/9/94 to 30/6/99.

Given that there is potential for the accumulation of DSP to differ between species, these differences were investigated within the limits of the available data. Table 2.20 provides a summary of the occurrence of DSP in the major shellfish species sampled in the marine biotoxin monitoring programme from 1/9/94 to 30/6/99.

Species

Total (Acetone Screen tests)

No. with detectable levels of DSP by ELISA

Percentage of samples above

detectable level

No. of samples

above the regulatory

level

Percentage above the regulatory

level

GreenshellTM mussel

8670

18

0.21

1

0.01

Blue mussel 1403 127 9.05 77 5.49 Pacific oyster 2332 15 0.64 2 0.09 Dredge oyster 412 3 0.73 0 0.00 Scallop 1958 0 0.00 0 0.00 Tuatua 1220 5 0.41 0 0.00 Pipi 1202 2 0.17 0 0.00 Cockle 503 0 0.00 0 0.00 Paua 296 5 1.69 1 0.34

Table 2.20: Summary of occurrence of DSP toxins in the major shellfish

species sampled in the marine biotoxin monitoring programme from 1/9/94 to 30/6/99.

This summary indicates that a higher percentage of Blue mussel samples contained detectable levels of DSP than any other shellfish species sampled, and that Blue

79

mussels also had a greater percentage of DSP levels above the regulatory level of 20 µg/100g shellfish tissue. Greenshell™ mussels, Pacific oysters and paua (that is, whole paua including the gut) also included samples with levels of DSP above the regulatory level. The data in Table 2.20 are not sufficient to indicate species differences with respect to biotoxin accumulation and retention, since the toxin accumulation could be site specific rather than species-specific. Unfortunately, the only occasions recorded on the FoodNet database where different species have been sampled concurrently from the same site and tested for DSP have been times when no DSP toxins have been detected in any of the samples. However, a study was undertaken by Lincoln Mackenzie from the Cawthron Institute in which both GreenshellTM mussels (Perna canaliculus) and Blue mussels (Mytilus edulis aoteanus) were sampled concurrently from the same site (Wedge Point, Site G23) during a multi-species dinoflagellate bloom in the spring of 1996 (Mackenzie et al., 1998b). The predominant species in the bloom were Dinophysis acuta and Protoceratium reticulatum. Mouse bioassays using the acetone screen test indicated that both the Blue mussels and the GreenshellTM mussels contained significant toxin levels. While significant amounts of Okadaic acid were found in both the Blue mussels and plankton concentrates from water samples, only trace amounts of Okadaic acid and DTX-1 were detected in the GreenshellTM mussels. However, HPLC analysis of plankton from the GreenshellTM mussel gut, and from P. reticulatum cultures from water samples, indicated the presence of yessotoxin derivatives. This suggested that two different species of mussel feeding on the same phytoplankton assemblage accumulate different toxins. The apparent differences in DSP toxin occurrence between Wedge Point and other adjacent sampling sites in the Marlborough Sounds may be due to differences in the species of shellfish sampled, rather than toxin occurrence. The DSP ELISA Check-Kit does not detect DTX-3 or Okadaic acid diol esters. It is thus possible that the incidence of DSP is under-reported in the data. In addition, because the marine biotoxin monitoring programme does not incorporate monitoring for yessotoxin or pectenotoxin, there are no records available regarding the occurrence of these toxins on the FoodNet database. 2.6.2 Temporal Distribution The results of phytoplankton monitoring for Dinophysis species from three sites in the Hauraki Gulf (from January 1995 to July 1999), 25 sites in the Marlborough Sounds and Port Underwood (from January 1995 to July 1999), and one site at Collingwood (from August 1996 to July 1999) are presented in Appendix IV(C). (A map showing the location of sites in the Marlborough Sounds is presented in Appendix IV(A)). Sites in the Marlborough Sounds (Zone G) tended to have a greater range of cell densities of Dinophysis sp. than those sites from the Hauraki Gulf area (Appendix IV(C)). An easy comparison can be made by comparing the number of cases at each site in which Dinophysis levels were above 1,000 cells/L. Only 3 instances above this level were recorded in the Hauraki Gulf sites, two from the Tamaki Strait (on the 28 June and the 17 July 1995), and one at Kopake on the 24 August 1995 (see Appendix IV(C), 1).). In comparison, over the same time period (January 1995-1 July, 1999), the majority of the Marlborough Sounds sites also had low numbers of counts above

80

this level (see Appendix IV(C), 2).), except for the composite group of sites encompassing East Bay (G19), Horohora Bay (G12), Whangakoko Bay (G11) and Opihi Bay (G40), which had consistently high numbers of counts above this level over the total sampling period. At the Hauraki Gulf sites no clear seasonal trends were apparent between years. The Marlborough Sounds sites were also largely characterised by a lack of seasonality in increased cell density of Dinophysis sp. However, East Bay (G19), Horohora Bay (G12), Whangakoko Bay (G11) and Opihi Bay (G40) appeared to have a consistently high density (>1,000 cells/L) of Dinophysis sp. over most of the yearly cycle. Very low densities were consistently recorded in the months of June to August of all years studied. Dinophysis sp. density for these sites reached exceedingly high levels of up to 10,600 cells/L. Other sites to reach comparatively high densities of Dinophysis sp. were Hallam Cove (G10) with a maximum density of 7,200 cells/L, and Wedge Point (G23) with a maximum density of 23,400 cells/L. The 17 months of phytoplankton data recorded on the FoodNet database for other zones were insufficient for temporal analysis of seasonal patterns. The seasonality of DSP toxin occurrence in shellfish was analysed using data from sample sites that had been regularly and consistently monitored from 1/7/95 to 30/6/99 as described in Section 2.2.7. (There were no such sites in Zone K). This analysis is based on the incidence per zone of DSP toxins above the level of detection in shellfish. The data were standardized to take into account differences in the number of sites per zone. Data from 48 sites were included in the analysis. Fourteen of these sites were in Zone G. While an analysis of DSP toxins above the regulatory level would perhaps have been more pertinent in terms of measuring risk to shellfish consumers, there were insufficient instances of such toxin levels within the standardized data set.

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov DecMonth

Cum

ulat

ive

No.

of D

SP

"Det

ects

" pe

r Mon

th b

y Zo

ne

Zone A Zone B Zone C Zone D Zone E Zone F Zone GZone H Zone I Zone J

Figure 2.22: The four year cumulative incidence of DSP toxins above the level

of detection at sites within each zone by month, recorded from 1/7/95 to 30/6/99. The data has been standardized to account for differences in the number of sites per zone.

81

The risk of DSP to consumers is related to both the occurrence of DSP events, and the duration of retention of toxins in shellfish. As depicted in Figure 2.22 (see previous page), there appears to be a possible broad seasonal pattern with a slightly decreased risk of DSP to consumers of shellfish over the winter months. Using the same set of data, the variation in DSP toxin occurrence from year to year was investigated, as described in Section 2.2.7. The temporal distribution of DSP levels above the detectable level in shellfish samples from the same consistently sampled sites is presented in Figure 2.23. This figure shows that the frequency of occurrence of detectable levels of DSP in shellfish from each zone may vary from year to year. On a New Zealand-wide basis, the occurrence of toxins also varies from year to year: for example, overall there were fewer samples containing detectable levels of DSP from September 1997 to September 1998 than in previous years. A summary of the broad El Nino/La Nina climate patterns is presented in Appendix III. There appears to be no obvious relationship between the occurrence of DSP toxins (either across New Zealand as a whole, or within zones) with these broad climate patterns.

0

20

40

60

80

100

120

Sep-94Jan-95

May-95Sep-95

Jan-96May-9

6Sep-96

Jan-97May-97

Sep-97Jan-98

May-98

Sep-98Jan-99

May-99

Time

DSP

Lev

el (µ

g/10

0g)

Zone A Zone B Zone C Zone G Zone H Zone I

Figure 2.23: Distribution of detectable levels of DSP toxin in shellfish from

consistently monitored sites from September 1994 to May 1999. 2.6.3 Reliability of Acetone Screen The inability of the DSP ELISA Check Kit to detect DTX-3 and Okadaic acid diol esters has already been mentioned. However, there are also other discrepancies between the acetone screen test and the DSP ELISA Check Kit. There are 349 instances in which a DSP ELISA was undertaken even though the result of the preceding (or in some cases concurrent) acetone screen test was negative.

82

Of these, there were 50 instances in which the acetone screen test was recorded as a “Not Detect” but a DSP ELISA produced a positive result. Normally a DSP ELISA is not undertaken unless the acetone screen test is positive, so the validity of these results was queried with ESR. Checking by ESR revealed that some of these anomalies were as a result of misreporting of data (P. Truman, ESR, pers. comm.). Table 2.21 (see following page) lists the corrected results, based on investigation by Penny Truman of ESR, who checked the results with the original laboratory records. It can be seen from Table 2.21 that there are 33 instances in which the results are misreported: 32 that were reported as “Not Detected” instead of “Detected”, and one in which an ELISA test was not undertaken at all, but was reported as “Not Detected”. There are sixteen instances in which the acetone screen assay was correctly reported as negative (i.e. a “Not Detect” result), but a DSP ELISA result was positive. This represents 4.6% of the total tests in which DSP ELISA was undertaken when the screen test was negative. These positive results range from 10 to 42 µg/100g. A total of 4 results (1.1% of the total tested by DSP ELISA when the acetone screen was negative) were over the regulatory limit of 20 µg/100g, and 9 of them (2.6%) were equal to, or greater than 16 µg/100g. Investigation by ESR indicated that in two cases, (Site I04, Laboratory Numbers 951438 and 954517), the ELISA test was undertaken because the mice in the acetone screen assay were very sick at 24 hours (but still alive, so reported as “Not Detected”). The DSP ELISA found toxin levels of 22 µg/100g and 11µg/100g respectively in these samples. Of the remaining thirteen samples, one was from Site C05 (Coromandel), one from Site D03 (Port Charles), one from Site G10 (Hallam Cove), four from Site I04 (Akaroa Harbour) and eight from Site G23 (Wedge Point). ESR records show that the mice in the assays of samples from Site I04 exhibited symptoms of some toxin activity: they became very sick, but subsequently recovered (P. Truman, ESR, pers. comm.). There is currently no definitive explanation for the anomalous results in any of these thirteen samples. These results could suggest one of two things: that the acetone screen test is not 100% reliable in detecting levels of DSP above the regulatory level, as recorded by the DSP ELISA Check Kit, or that cross-reactivity in the DSP-ELISA Check Kit can cause false positives. Inconsistencies in the DSP-ELISA Check Kit have been reported elsewhere (Cembella et al., 1995a). Whatever the reasons for these discrepancies, the difference between a negative acetone screen result and a level of 42 µg/100g measured by the DSP ELISA Check Kit could potentially be significant. This requires further investigation.

83

SITECODE DATE LABNO SCREEN ELISA

(µg/100g) SPECIES

A05 02/11/94 946164 DET 10 OYST-P A05A 30/01/95 950550 DET 12 SCALR A08 06/12/94 946761 DET 11 OYST-P A08A 03/01/95 950079 DET 18 SCALR A13 01/11/94 946168 DET 11 OYST-P A15 02/11/94 946179 DET 10 OYST-P A18A 02/11/94 946181 DET 11 PIPI B11 01/02/95 950584 DET 11 SCALMR C05 24/01/95 950402 DET 13 GMUSS C05 31/01/95 950554 NOT DET 16 GMUSS C09 31/01/95 950556 DET 10 GMUSS C10 02/11/94 946173 DET 12 OYST-P C10 08/02/95 950717 DET 11 OYST-P D03 23/10/95 954719 NOT DET 11 GMUSS E13 28/11/94 946621 DET 39 PAUA-G F01 03/10/94 945685 DET 11 TUATUA F12 24/01/95 950426 DET 12 OYST-P G03 23/01/95 950390 DET 13 SCALMR G05 23/01/95 950391 DET 11 SCALMR G10 18/12/95 955636 NOT DET 12 GMUSS G23 28/11/94 946545 DET 35 BMUSS G23 05/12/94 946695 DET 33 BMUSS G23 19/12/94 946969 DET 32 BMUSS G23 09/01/95 950143 DET 50 BMUSS G23 23/01/95 950383 DET 30 BMUSS G23 30/01/95 950505 DET 44 BMUSS G23 06/02/95 950617 DET 12 BMUSS G23 13/02/95 950774 DET 34 BMUSS G23 20/02/95 950874 NOT DET 17 BMUSS G23 26/02/95 950976 DET 44 BMUSS G23 06/03/95 951104 DET 42 BMUSS G23 13/03/95 951203 DET 39 BMUSS G23 20/03/95 951308 DET 70 BMUSS G23 17/04/95 951706 NOT DET 42 BMUSS G23 24/04/95 951830 NOT DET 26 BMUSS G23 29/05/95 952338 NOT DET 15 BMUSS G23 05/06/95 952430 NOT DET 18 BMUSS G23 31/07/95 953315 NOT DET 22 BMUSS G23 01/07/96 962398 NOT DET 19 BMUSS G23 15/07/96 962603 NOT DET 10 BMUSS G23 25/11/97 972914 19 BMUSS G31 10/01/95 950176 DET 13 GMUSS H03 07/12/94 946749 DET 10 TUATUA I04 28/03/95 951438 NOT DET 22 BMUSS I04 04/04/95 951541 NOT DET 12 BMUSS I04 05/09/95 953934 DET 15 BMUSS I04 12/09/95 954062 NOT DET 17 BMUSS I04 10/10/95 954517 NOT DET 11 BMUSS I04 10/10/95 954518 NOT DET 11 BMUSS J16 01/11/94 946178 DET 11 GMUSS

Table 2.21: The corrected results for the samples recorded in FoodNet as

having negative Acetone Screen test results, but positive DSP ELISA results. (DET= Detected; NOT DET= Not detected.)

84

2.6.4 Phytoplankton Monitoring as a Predictor of DSP in Shellfish The phytoplankton monitoring data and shellfish toxin testing data on the FoodNet database and the Marlborough Sounds Quality Assurance Programme database were analysed to determine the probability that phytoplankton monitoring would fail to predict the occurrence of DSP above the regulatory level in shellfish. (Refer to Section 2.2.4 for methodology). The only shellfish samples where DSP levels exceeded the regulatory level of 20 µg/100g over a time period for which there are phytoplankton monitoring data available, were from the long-running toxicity events in blue mussels at Wedge Point (G23) in the Marlborough Sounds. Over some periods, the toxin levels varied widely from week to week (for example, range 10-93 µg/100g over 20/11/95-22/7/96) in the absence of any Dinophysis or Prorocentrum lima species recorded in the phytoplankton. It would have been useful if the variation within samples had been measured by some replicate sampling during this time, to determine the extent of the unexplained increases in DSP toxin levels. There were two instances in which DSP toxin levels rose from zero to above the regulatory level (as distinct from increasing from long-running residual levels). Neither of these instances was associated with Dinophysis sp. or Prorocentrum lima densities above the regulatory level to initiate shellfish testing. Of the three instances where detectable levels of DSP occurred (in the range 10-12 µg/100g), one was associated with Dinophysis acuta densities of 2,800 cells/L, one with 400 cells/L of Dinophysis acuminata, while for the third event, no Dinophysis species or Prorocentrum lima were recorded. It should be noted that the current sampling methods used in phytoplankton monitoring may not be appropriate for detecting densities of Prorocentrum lima that reflect the exposure of shellfish to this species. Prorocentrum lima (and other species of epibenthic phytoplankton of potential concern such as Ostreopsis and Coolia species, that may produce toxins of other kinds) live in close association with substrata such as seaweed and sediment that may be in proximity to a range of shellfish species. (Prorocentrum lima has also been found on mussel ropes – L Rhodes, Cawthron Institute, pers. comm.) As such, they are not generally distributed through the water column except after turbulent weather, but may be consumed by shellfish due to their proximity. Their absence in phytoplankton samples taken from the water column may thus not reflect the exposure of shellfish to potentially toxic species. This requires further investigation. Further data are necessary for rigorous investigation of the relationship between the numbers of potentially toxic phytoplankton and DSP toxicity in shellfish.

85

2.6.5 Conclusions The following points summarise the conclusions that can be drawn from analysis of the marine biotoxin monitoring programme regarding the risk of DSP for shellfish consumers: • Dinophysis species occur widely around the coastline of New Zealand.

Prorocentrum lima occurs less widely in phytoplankton samples taken from the water column, but has been recorded in both the North and South Islands. Information from sources other than the marine biotoxin monitoring programme database suggests that this species is also widely distributed.

• The incidence of DSP toxicity is also widely distributed, and is not limited by

latitude. Detectable levels of DSP have occurred on both eastern and western coasts.

• If it can be assumed that the marine biotoxin monitoring programme is

representative of shellfish harvesting in New Zealand, the incidence of DSP toxins at a level that represent a risk to consumers has been comparatively low in most areas.

• There may be a slightly lower risk of DSP in the winter months. • The frequency of DSP occurrence may vary from year to year across the whole of

New Zealand, and between zones within New Zealand. There is insufficient data for predictions about future occurrence to be made.

• Based on a very limited amount of data, the relationship between the presence of

species of phytoplankton known to produce DSP toxins and DSP toxicity in shellfish does not appear to be predictive. More data are required to investigate this further.

• The DSP ELISA Check-Kit does not detect DTX-3 or Okadaic acid diol esters. It

is thus possible that the incidence of DSP is under-reported in the data. In addition, because the marine biotoxin monitoring programme does not incorporate monitoring for yessotoxin or pectenotoxin, there are no records available regarding the occurrence of these toxins on the FoodNet database.

• There are some discrepancies between the performance of the acetone screen test

and the DSP ELISA Check Kit. These would become more significant if the regulatory level of DSP toxins were lowered from 20 µg/100g to 16 µg/100g. This requires further investigation.

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2.7 RESULTS OF ANALYSIS – NSP AND RESPIRATORY IRRITATION

SYNDROME 2.7.1 Introduction As discussed in the introduction, the causative agents for both NSP and Respiratory Irritation Syndrome (RIS) are Gymnodinium species and Gyrodinium galatheanum. The marine biotoxin monitoring programme does not specifically encompass RIS at present. However, because RIS results from blooms of Gymnodinium species, components of the monitoring programme do provide an indication of the risk of RIS also. For this reason, both NSP and RIS are discussed in this section. The method of testing for NSP using an ether extraction method and mouse bioassay is not specific for brevetoxins, and may also detect other lipid soluble toxins. The extent to which other lipid soluble toxins found in New Zealand phytoplankton (such as, for example, DSP toxins, gymnodimine, and “Wellington Harbour toxin”) confound the results of the bioassay for NSP is not well understood. While the mouse death times and symptoms can provide some indication of the presence of unexpected toxins, these observations provide limited information. To date, such observations have not been recorded in the database of monitoring results. Some resolution of results can be obtained by additional testing: for example, by NSP ELISA or neuroblastoma assays to confirm NSP, by DSP ELISA, and by LC-MS or HPLC. However, such methods are not used routinely as part of the monitoring programme, and where they have been undertaken as part of a research programme, the results have not been recorded on the FoodNet database. In the absence of results from additional testing, the results of phytoplankton monitoring could potentially be used to reduce uncertainty about historical NSP results. However, prior to 1997 there are few phytoplankton data from outside the Marlborough Sounds or the Hauraki Gulf. There have been no distinctions made between the various “Gymnodinium mikimotoi” species (refer to Section 1.3.4), and not all known brevetoxin producers are monitored. For these reasons, the interpretation of “NSP” results is problematical. It would have been helpful for risk analysis if any additional information collected about positive “NSP” results had been recorded on the database. In this analysis and discussion, “NSP toxins” refer to the group of toxins detected in the ether extraction mouse bioassay. Where there is evidence that indicates particular toxin events can be attributed to known toxins other than brevetoxins, this is noted. 2.7.2 Geographic Distribution The distribution of potentially toxic Gymnodinium species, Gymnodinium c.f. breve and Gymnodinium c.f. mikimotoi is shown in Figure 2.24. These data are drawn from the phytoplankton monitoring data on FoodNet, as described in Section 2.2.3. Because Gyrodinium galatheanum is less than 10 micron in size, no data have been

87

collected through the phytoplankton monitoring programme on the distribution or abundance of this species (K. Todd, Cawthron Institute, pers. comm.). This figure does not provide quantitative information about the frequency of occurrence of potentially toxic phytoplankton. However, it does indicate that the distribution of potentially toxic Gymnodinium species is widespread around the New Zealand coastline. Gymnodinium c.f. mikimotoi has been found at most phytoplankton monitoring sites in New Zealand. There are many sites where Gymnodinium c.f. breve has not been found. However, its distribution includes both the North and South Islands. Unfortunately, there are no phytoplankton data from the western coast or Chatham Islands to provide additional information in the assessment of whether or not NSP could potentially occur there.

88

Figure 2.24: Distribution of potentially toxic species of Gymnodinium throughout New Zealand.

Gymnodinium mikimotoi detected at20 out of 26 sites in the MarlboroughSounds

Gymnodinium cf breve andGymnodinium mikimotoidetected

Gymnodinium mikimotoidetected

No potentially toxicGymnodinium speciesdetected

Distribution of PotentiallyToxic Gymnodinium species

in New Zealand

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Table 2.22 outlines the occurrence, and levels above the regulatory limits of Gymnodinium c.f. breve by Biotoxin Zone in New Zealand, as described in Section 2.2.3. (Note that there are no phytoplankton monitoring sites in Zones F or K). This table summarizes the results of analysis of two sets of data: the first (“Total Samples”) is the data set of all the phytoplankton samples recorded on the FoodNet and Marlborough Sounds Shellfish Quality Assurance Programme databases. The second set of data includes all the samples recorded on these databases within an “Identified Time Interval” (i.e. 19/12/97-25/5/99).

Zone

A

B

C

D

E

G

H

I

J

Percentage occurrence in

Total samples (%). N=

0.2

436

4.7

106

0.9

583

1.8

621

1.2

242

0.0

6486

0.0

82

0.0

466

1.0

104

Percentage of Total samples with 1,000 to

4,900 cells/L (%)

0.0

1.9

0.0

0.2

0.0

0.0

0.0

0.0

0.0

Percentage of Total samples with 5,000 cells/L or more (%)

0.0

0.9

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Percentage occurrence in identified time interval

(%) N=

0.3

304

2.6

76

1.1

456

2.1

532

2.0

152

0.0

1941

0.0

76

0.0

304

0.0

76

Percentage of samples in the identified time

interval with 1,000 to 4,900 cells/L (%)

0.0

1.3

0.0

0.2

0.0

0.0

0.0

0.0

0.0

Percentage of samples in the identified time

interval with 5,000 cells/L or more (%)

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Table 2.22: Percentage occurrence of Gymnodinium c.f. breve, and percentage

occurrence above the regulatory levels (1,000 to 4,900 cells/L, and ≥ 5,000 cells/L) in total samples and in samples from 19/12/97 to 25/5/99 (the “Identified Time Interval”) by zone.

Over the entire sampling period only Zones G, H and I have not encountered Gymnodinium c.f. breve. In the “Identified Time Interval” only Zones B and D have had samples with levels above the level to trigger shellfish testing (i.e. ≥ 1,000 cells/L). These specific cases are outlined below: Zone B: Above Shellfish Testing Trigger Level (1,000 cells/L): 3 000 Zone D: Above Shellfish Testing Trigger Level (1,000 cells/L): 1 100

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Over the “Identified Time Interval” no cases above the level to trigger industry voluntary closure have been recorded for Gymnodinium c.f. breve. Table 2.23 summarizes the occurrence of Gymnodinium c.f. mikimotoi, (now identified as three separate potentially toxic species) in two density ranges: 1,000-4,900 cells/L, and >5,000 cells/L, as described in Section 2.2.3.

Zone

A

B

C

D

E

G

H I

J

Percentage occurrence in Total samples (%).

N=

6.9

436

13.9

108

22.8

583

6.1

621

6.6

242

8.0

6486

23.2

82

0.9

466

1.9

104

Percentage of Total samples with 1,000-

4,900 cells/L (%)

1.1

4.8

4.5

1.0

0.8

2.6

3.7

0.0

0.0

Percentage of Total samples with 5,000 cells/L or more (%)

0.2

0.0

0.5

0.0

0.0

0.7

7.3

0.0

0.0

Percentage occurrence in identified time

interval (%) N=

8.9

304

10.5

76

22.8

456

6.4

532

9.2

152

16.5

1941

25.0

76

1.0

304

2.6

76

Percentage of samples in the identified time interval with 1,000-

4,900 cells/L (%)

1.6

1.3

5.0

0.8

1.3

3.9

3.9

0.0

0.0

Percentage of samples in the identified time interval with 5,000 cells/L or more (%)

0.3

0.0

0.7

0.0

0.0

1.8

7.9

0.0

0.0

Table 2.23: Percentage occurrence of Gymnodinium c.f. mikimotoi and

percentage occurrence above defined density levels (1,000 to 4,900 cells/L and ≥ 5,000 cells/L) in total samples and in samples from 19/12/97 to 25/5/99 (the “Identified Time Interval”) by zone.

Gymnodinium c.f. mikimotoi appears to have a more uniform distribution around the New Zealand coastline than G. c.f. breve, being identified within all the sampled zones (Table 2.23). However over the “Identified Time Interval” G. c.f. mikimotoi has not occurred above the defined densities in either zones I or J. The zones where it has been within these ranges can be ranked in descending order of frequency of occurrence as follows: H, C, G, A, E, B, and D. Zone H appears to have a greater percentage of samples in the upper density range (>5,000) than any other zone. Note that the different species that comprise the Gymnodinium c.f. mikimotoi group may have different distributions. However, because these data have been grouped we have no way of identifying this from the available information.

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To show the spread of the data in the “Identified Time Interval” data set, the same ranges of data (1,000 to 4,900 cells/L and ≥ 5,000 cells/L) have been represented graphically as “Box and Whisker Plots” (where five or more data points exist), or where there are less than five data points, numbers are simply stated. This summary is presented in Figure 2.25.

Gymnodinium c.f. mikimotoi

ZoneA C G

Ran

ge o

f dat

a be

twee

n 1

000

to 4

900

cel

ls/L

itre.

0

1000

2000

3000

4000

5000

N=5 N=23 N=76

Gymnodinium c.f. mikimotoi

ZoneG HR

ange

of d

ata

abov

e 5

000

cells

/Litr

e.

0

50x103

100x103

150x103

200x103

250x103

N=34

N=6

Zone A: Above 5 000 cells/L: 20 000 Zone B: Above 1 000 cells/L: 2 800 Zone C: Above 5 000 cells/L: 6 200, 6 600, 6 800 Zone D: Above 1 000 cells/L: 2 600, 2 200, 1 500, 1 400 Zone E: Above 1 000 cells/L: 1 800, 1 400 Zone H: Above 1 000 cells/L: 1 600, 1 600, 4 200

Figure 2.25: Box and Whisker plots showing the frequency distribution of (a)

potentially toxic Gymnodinium c.f. mikimotoi above 1,000 cells/Litre (Zones A, C, and G), and (b) potentially toxic G. c.f. mikimotoi above 5,000 cells/Litre (Zones G and H).

Figure 2.25 clearly outlines the distribution of toxin occurrence for each zone. Four zones contained counts above 5,000 cells/L. Zone A had one sample containing 20,000 cells/L at Tapeka Point on 8/12/98. Zone C had three samples just over the 5,000 cells/L cut-off point, one at Tamaki Strait on 23/2/98 (6,200 cells/L), and two at

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Waimangu Point: 6,800 cells/L on 22/2/98 and 6,600 cells/L on 22/3/98. Zone G had 34 samples above the 5,000 cells/L cut-off point, with a median value of 9,500 cells/L. However, three extreme outliers in Zone G were obvious, two of these samples were from one episode at the Oyster Bay site: 192,000 cells/L on 20/4/98 and 140,000 cells/L on 27/4/98. The remaining outlier of 48,000 cells/L was from Wedge Point on 11/5/99. In comparison to the other zones, Zone H had a far higher median value of 54,500 cells/L. One extreme outlier was also apparent in Zone H, a density of 170,000 cells/L at Dorset Point on 16/3/98. These densities were associated with a large bloom of Gymnodinium c.f. mikimotoi in the early months of 1998 (Chang et al., 1998a). This Gymnodinium species has now been identified as a separate species, and named Gymnodinium brevisulcatum (Dr Hoe Chang, NIWA, pers. comm.). Chang et al. (1998a) described the blooms of Gymnodinium c.f. mikimotoi on the eastern coast and in Wellington Harbour in the summer of 1998. The following description is summarized from that work: From Mid-January to early February 1998, sporadic kills of fish and marine fauna were reported off the Wairarapa and Kaikoura Coasts. In early February there were also reports of human respiratory symptoms in several areas of the Wairarapa coast. Respiratory irritation was also reported in Hawkes Bay, and Cape Palliser areas in February. In Wellington Harbour, there were a small number of Gymnodinium c.f. mikimotoi in samples taken in January. These numbers increased rapidly, and peaked in about mid-March, with the highest recorded cell concentration being 33 million cells/L at Mahanga Bay (note: this sample is not recorded on the FoodNet database). People in Wellington also reported respiratory irritation, and there was extensive mortality of marine flora and fauna in the harbour. “Results from two voyages conducted off the East coast of the North Island between January and February 1998 showed that Gymnodinium c.f. mikimotoi was widespread in low numbers from Cape Brett in the north to Cape Palliser in the south of the North Island”. Environmental data collected on the same voyages “indicates that the 20o isotherm offshore extended further southwards than usual, associated with a strong southward directed current. During this period, the cooling effects of El Nino on sea surface temperatures, as experienced in the spring months around New Zealand, were reversed. The warming of nearshore waters by incursions of warm oceanic waters, and the entrainment of nutrient-rich waters by upwelling were observed at several locations along the North Island east coast. The build-up of Gymnodinium species off Wairarapa and Hawkes Bay between January and February suggests that this species might have been brought into the nutrient rich inshore waters as seed stock, by shoreward intrusions of oceanic waters. In March at Foxton on the North Island western coast, a small number of Gymnodinium c.f. mikimotoi were also recorded after fish kills were reported in the area. The distribution of sea surface temperatures in early 1998 suggest it is more likely that the Gymnodinium sp. was introduced into Wellington from the west coast than from the east coast” (Chang et al., 1998a). The sample of 20,000 cells/L at Tapeka Point in Zone A in December 1998 (mentioned above) may have resulted from similar processes. This bloom was almost immediately blown offshore again by a strong easterly wind, so no potential public health impacts resulted (T. Beauchamp, Northland Health, pers. comm.).

93

Zone

Total No. of

Samples

Number of samples in which lipid

soluble toxins (ether

extract) were

detected

Percentage of samples

in which lipid soluble toxins (ether

extract) were

detected (%)

No. of samples above

regulatory level

Percentage of samples

above regulatory level (%)

Maximum toxin level

(mouse units)

A 2112 31 1.5 9 0.43 26 B 1015 4 0.4 0 0.00 C 2101 41 2.0 3 0.14 26 D 2094 10 0.5 6 0.29 44 E 763 0 0.0 0 0.00 F 1459 0 0.0 0 0.00 G 5627 3 0.1 2 0.04 27 H 675 1 0.1 1 0.15 22 I 1471 4 0.3 0 0.00 J 811 22 2.7 4 0.49 23 K 198 1 0.5 1 0.50 21

Table 2.24: Summary by zone of the total occurrence of lipid soluble toxins

detected in shellfish samples by ether extract mouse bioassay, and lipid soluble toxins above the regulatory level of 20 mouse units detected in shellfish samples from 1/9/94 to 30/6/99.

A summary of the occurrence of lipid soluble toxins detected by ether extract mouse bioassay in shellfish for all sites, in all zones from 1/9/94 to 30/6/99 is provided in Table 2.24. Note that the analysis of data presented did not use standardized data, and that differences in toxin accumulation characteristics between shellfish species, the clumping of sample sites, and variations in sampling regimes, both through time and between sample sites have been ignored. It can be seen from Table 2.24 that “NSP” was detected in only a small percentage of all the samples tested, and that Zones C and J had the highest percentage detected at 2% and 2.7% respectively. However, Zones K, and J had the highest percentage of samples above the regulatory levels, closely followed by Zone A. Zone D had the highest recorded level of 44 mouse units (Port Fitzroy on 22/2/98) (20 mouse units is the regulatory level). Closer examination of the data, and additional data gathered by ESR, provide further information about the identity of some of the “NSP” results above the regulatory level. Of the nine instances of results above the regulatory level of 20 mouse units in Zone A, seven were from Pacific oysters from the Rangaunu Harbour between 26/9/94 and 8/2/95, and one other from the same site on 14/2/96. There are limited data from phytoplankton monitoring at this time, although some data from September 1995 appears on the database. During this time, only 200 cells/L of Gymnodinium species (species not identified) were present. However, there were several records of Gymnodinium cysts (up to 19,200 on 27/9/94). There are no phytoplankton data from this site for early 1996. The Rangaunu Harbour has experienced persistent toxicity in the acetone screen test with in general no positive results from the DSP ELISA, or ether extract mouse bioassay. Samples of oysters containing “Rangaunu Harbour toxin” have produced negative results when tested for brevetoxins by neuroblastoma assay (Dr Penny Truman, ESR, pers. comm.). David Stirling of ESR is currently elucidating the identity of the “Rangaunu Harbour toxin”. It is thought that the

94

compound dubbed the “Rangaunu Harbour toxin” would not be detected in the ether extract mouse bioassay (Dr David Stirling, ESR, pers. comm.). It is therefore possible that the positive results above the regulatory level in the ether extract mouse bioassay from Rangaunu Harbour could have been low levels of brevetoxin, but this has not been confirmed by a definitive test method. The other result above the regulatory level in the ether extract mouse bioassay from Zone A was from a sample of tuatua from Tokerau Ramp on 25/1/95. There are no phytoplankton data for this site at this time, and no additional tests have been undertaken to confirm the identity of the toxin in this sample. Of the three positive results above the regulatory level in Zone C, (GreenshellTM mussels from Sites C02 (Kopake) on 27/11/95 and C38 (Awakiriapa Bay) on 26/12/95; and Pacific oysters from Site C10 (Pakihi) on 10/5/95), only two were associated with the presence of Gymnodinium species. The sample from Site C10 (Pakihi) on 10/5/95 had associated phytoplankton counts of Gymnodinium c.f. mikimotoi of 400 cells/L on 9/5/95, but 9,200 cells/L on 15/5/95. The sample from Site C02 (Kopake) on 27/11/95 had a Gymnodinium c.f. mikimotoi density of 1,200 cells/L on the same day. Both brevetoxin and yessotoxin have recently been detected concurrently by LC-MS and neuroblastoma assay techniques in historic samples from Zone C, taken in 1995 (P. Truman, ESR, pers. comm.). It is known that yessotoxin can be detected by the ether extract mouse bioassay. The identity of the toxin present in these particular samples that were above the regulatory level remains unconfirmed, but could have been either brevetoxin, yessotoxin, or a mixture of both. The six samples above the regulatory level for NSP from Zone D were all from GreenshellTM mussels from Port Fitzroy (D01) taken between late January and early March 1998. It has now been confirmed by LC-MS that these results were due to the presence of yessotoxin. Neuroblastoma assays indicated that there was no brevetoxin in these shellfish (P. Truman, ESR, pers. comm.). The two samples above the regulatory level from Zone G were both from Blue mussel samples taken from Wedge Point (G23), on 4/11/96 and 11/11/96 respectively. The positive results in the ether extract mouse bioassay coincided with low levels of DSP (22 µg/100g and 16 µg/100g respectively). There is some uncertainty about whether Okadaic acid is detected by the ether extract mouse bioassay (P. Truman, ESR, pers. comm.). There are no phytoplankton data available for this time. In this case, the identity of the toxins detected remains uncertain – they could be either brevetoxin or Okadaic acid, or indeed, yessotoxin. The sample above the regulatory level from Zone H was a sample of GreenshellTM

mussels from Dorset Point on 31/3/98. This coincided with a very dense bloom of Gymnodinium c.f. mikimotoi, which was subsequently identified as Gymnodinium brevisulcatum. This bloom produced a toxin known as “Wellington Harbour toxin”. It appears that this compound may be extracted by the ether extraction method (although the extent to which this occurs appears variable) (P. Truman, ESR, pers. comm.). Gymnodinium brevisulcatum is also known to produce brevetoxins (A. Haywood, Cawthron Institute, pers. comm.) It is thus possible that this positive sample result in the ether extract mouse bioassay could have been due to the presence of either “Wellington Harbour toxin”, or brevetoxin.

95

The four samples from Zone J above the regulatory level for NSP were dredge oyster samples taken from Foveaux Strait in September and December 1994. There are no phytoplankton data relating to these samples. Zone J is an area in which gymnodimine, produced by Gymnodinium selliforme, has been detected in shellfish. Gymnodimine is a compound that is extracted by the acetone extraction method, and kills mice rapidly (within 20 minutes, or not at all) in the acetone screen mouse bioassay. These results were unlikely to have been produced by gymnodimine, as the mice died more slowly than this. However, in a range of samples that have been tested from Zone J by neuroblastoma assay, there has been no brevetoxin detected (P Truman, ESR, pers. comm.). The identity of the toxin in these samples thus remains unconfirmed. The sample above the regulatory level from Zone K (Chatham Islands) consisted of Blue mussels collected from site K01 on 16/1/95. There are no concurrent phytoplankton data, or data from further analysis of the toxin available. Thus, in this case also, the identity of the toxin remains unconfirmed as brevetoxin. In summary therefore, the regulatory test for NSP using the ether extraction mouse bioassay is not a specific test for brevetoxin. There is a range of other compounds found in New Zealand phytoplankton potentially detected by this method. These include yessotoxin, and possibly gymnodimine and “Wellington Harbour toxin”. Rangaunu Harbour toxin and pectenotoxin are unlikely to be detected by this method. The lack of specificity of the test method means that it is difficult to assess the incidence of brevetoxin in shellfish. Of the 25 samples that have been recorded as “NSP positive” (i.e. had levels of toxins above 20 mouse units in an ether extract mouse bioassay), six have been confirmed as resulting from yessotoxin not brevetoxin, and none of the others have had brevetoxin specifically confirmed. In three cases the presence of another toxin that could confound the results was confirmed. It thus appears that although the presence of brevetoxins in shellfish has been confirmed by investigation outside the marine biotoxin monitoring programme, the incidence of brevetoxins above the regulatory level in New Zealand since September 1994 has been extremely low, if not negligible. There are no data from the marine biotoxin monitoring programme that compare the uptake of NSP between different shellfish species. However, some differences have been observed in laboratory situations. Attempts by Fletcher et al., (1998) to induce toxicity above the regulatory level of 20 mouse units in GreenshellTM mussels by feeding them with the Florida strain of Gymnodinium breve were unsuccessful. When fed G. breve with a mixture of non-toxic species, there was some evidence of selective feeding on the non-toxic species. In contrast, Pacific oysters fed G. breve in the same quantities readily ingested the algae, and subsequent mouse bioassays indicated 24-100 mouse units of NSP in the oyster tissue (Fletcher et al., 1998). While these observations are not definitive, they do suggest that there could be significant differences in the accumulation, retention and /or detoxification processes for NSP between shellfish species.

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2.7.3 Temporal Distribution Investigation of the temporal distribution of NSP in New Zealand is limited by the short history of phytoplankton monitoring in most areas, and the non-specific shellfish toxin testing methods. However, the temporal distribution of Gymnodinium c.f. mikimotoi in the Hauraki Gulf (3 sites), Marlborough Sounds (24 sites) and at Collingwood (Golden Bay) was analysed using phytoplankton data from 1994 to 1999. There was insufficient occurrence for similar analysis of Gymnodinium c.f. breve, and no data has been collected for Gyrodinium galatheanum. Graphs of the abundance of G. c.f. mikimotoi over time are presented in Appendix IV(D). Sites in the Marlborough Sounds have been grouped into geographical areas, and graphed accordingly. Unfortunately, these data do not distinguish between the three different species that are now known to form the “Gymnodinium c.f. mikimotoi group”. Each species may have different characteristics with respect to temporal distribution. As can be seen from the graphs in Appendix IV(D), sites in the Marlborough Sounds (Zone G) tended to have a greater range of cell densities of G. c.f. mikimotoi than those sites from the Hauraki Gulf area. An easy comparison can be made by comparing the number of cases at each site where G. c.f. mikimotoi levels were above 2,000 cells/L. Only 6 cases above this level were recorded at the Hauraki Gulf sites, one from Kopake on the 18 December 1998, and five from Tamaki Strait on the 15 and 23 May 1995, 11 July 1995, 5 November 1995 and 21 February 1995 respectively. Some sites in the Marlborough sounds also had low numbers above these levels –the Kenepuru Entrance group, Little Nikau Bay group and East Bay/Horahora group. The remaining other site groups contained large numbers above this level. Interestingly G. c.f. mikimotoi was not detected at several sites until after January 1997. At the Hauraki Gulf sites, no clear seasonal trends were apparent between years. The Marlborough Sites did show some seasonality of occurrence. The site groupings relating to Little Nikau Bay, Crail Bay, Hallam Cove and Forsyth Bay, all showed increased levels of G. c.f. mikimotoi between January and July from 1997 to 1999. Interestingly, Collingwood Farms (G01) only showed increased cell densities of G. c.f. mikimotoi from January to July in 1998, but not in 1997 or 1999. Extrapolation of these data in the prediction of the occurrence of NSP toxins in shellfish in the future should be undertaken with caution. The differences in temporal distribution between the Hauraki Gulf and Marlborough Sounds suggest that patterns of occurrence may differ significantly between different geographic locations. There are also noticeable differences in abundance of Gymnodinium c.f. mikimotoi between years. These differences do not appear to be related to the broad climate patterns caused by El Nino/La Nina (refer to Appendix III for a summary of these patterns). There is no clear relationship able to be determined between the occurrence of Gymnodinium species in the phytoplankton and the occurrence of shellfish toxicity, as detected by the ether extract mouse bioassay (refer to the discussion of shellfish toxin results above the regulatory level in the previous section). Detection of such a relationship is probably confounded by the non-specific toxin testing, and the lack of analysis of Gymnodinium c.f. mikimotoi species to a species level. The lack of reporting of the potentially brevetoxin-producing Gyrodinium galatheanum could also

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potentially impact on the ability of phytoplankton monitoring to predict NSP toxicity in shellfish. 2.7.4 Conclusions The following points summarise the conclusions that can be drawn from analysis of the monitoring programme regarding the risk of NSP for shellfish consumers: • Gymnodinium c.f. breve is widespread around New Zealand, but there are many

phytoplankton sampling sites where it has not been recorded. The distribution of Gymnodinium c.f. mikimotoi is also widespread, and it has been recorded at most phytoplankton sampling sites. Distribution of the three species that make up the G. c.f. mikimotoi group is at this stage unknown.

• There have been few occurrences of Gymnodinium c.f. breve above the level to

trigger shellfish testing, and none above the level to trigger a voluntary shellfish industry closure to harvesting or the issuing of a public health warning.

• Gymnodinium c.f. mikimotoi (or the species now known as Gymnodinium

brevisulcatum) has been associated with RIS and fish kills. “Wellington Harbour toxin” has been implicated in one of these events. In the case of several major Gymnodinium bloom events, there is evidence to suggest that the seed stock for the bloom originated offshore.

• It is thought that the Gymnodinium bloom in Wellington Harbour in the summer

of 1998 originated from the western coast of New Zealand. This suggests that the commonly held assumption that there is lower risk of toxins arising from Gymnodinium species on the western coast may be unfounded.

• The incidence of “NSP toxicity” in shellfish, as reported from the ether extract

mouse bioassay, is comparatively low, as are the maximum levels of toxicity recorded. Some of this apparent “NSP” toxicity may be due to the presence of other lipid soluble toxins.

• There are possible differences in NSP toxin uptake/detoxification processes

between shellfish species. These might impact significantly on the risk of NSP to shellfish consumers.

• Occurrence of potentially brevetoxin-producing phytoplankton may vary by year

within zones. In some areas, these phytoplankton may also vary in abundance seasonally.

• Further data are required to determine the reliability of phytoplankton monitoring

as an indicator of shellfish NSP toxicity. Currently however, not all known potentially brevetoxin-producing species are included in the phytoplankton monitoring programme.

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SECTION 3: NON-COMMERCIAL SHELLFISH GATHERING AND CONSUMPTION IN NEW ZEALAND

3.1 INTRODUCTION The risk of exposure to marine biotoxins through non-commercial shellfish harvesting not only depends upon the patterns of biotoxin occurrence as described in the last section, but also upon patterns of shellfish gathering and consumption. These patterns may be affected by geographical, social and temporal components. The following sections describe the patterns of shellfish gathering in New Zealand, and the factors that influence them. 3.2 DISTRIBUTION OF SHELLFISH IN NEW ZEALAND Underlying all non-commercial shellfish gathering patterns in New Zealand is the availability of shellfish species desired by consumers. The many non-commercially gathered shellfish in New Zealand live in a wide range of habitats, of varying accessibility to man. The habitats and ranges of the most commonly gathered species are summarized very briefly below, followed by a summary of the species available in each “Biotoxin Zone”. Except where referenced separately, this information has been collated from personal observations, from Health Protection Officers in each area, and from Morton & Miller (1973), and Tortell (1981). Because of their mode of feeding, bivalve shellfish are the species of most concern with respect to the risk of biotoxins to consumers. Bivalve species can live in a range of habitats, and are widely distributed throughout New Zealand. All of the biotoxin zones contain some bivalve species that are gathered by the public. The bivalve with one of the widest distributions in New Zealand is the green-lipped (GreenshellTM) mussel, Perna canaliculus, which is found on rocky shores from low water down. It can be found in a wide range of habitats, from on wharf piles in the protected waters of harbours, to rocks on exposed open shores, and in dense beds on the bottom in moderately protected areas. It may be collected from the shore at low water, by snorkeling in the less exposed areas, or by dredging. The blue mussel, Mytilus edulis aoteanus has a more southerly distribution, being found very commonly only as far north as Wellington, and with a more patchy distribution in the North Island. Where the blue and green-lipped mussels co-exist, blue mussels tend to live in places with less wave action. Several commonly gathered bivalves are associated with rocky shores. These include the native rock oyster, Saccostrea glomerata, and the introduced Pacific oyster, Crassostrea gigas, both of which live most frequently in harbour or estuarine areas. Native rock oysters are most common in the very north of the North Island, but may occur as far south as Taranaki on the western coast, and East Cape on the east coast. Similarly, the Pacific oyster is most common in the north of the North Island, but also occurs as far south as some parts of the Marlborough Sounds, although it is rare in the

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south of the North Island. Both species of oyster live intertidally (i.e. between the low water and high water marks), and are thus relatively easily accessible to shellfish gatherers at low tide. Tuatua (Paphies subtriangulata, and Paphies donacina) are the most common bivalves on exposed open sandy beaches, and are found in the sand at the low water mark throughout New Zealand. On extremely exposed coasts, they are replaced by the toheroa (Paphies ventricosa), which lives higher up the shore than tuatua. Both tuatua and toheroa are accessible from the shore at low water. On exposed sandy shores below low water, venerid and mactrid clams are found (e.g. Bassina yatei, Dosinia anus). These species are less easy to gather, since the conditions are generally too rough for easy dredging. Pipi (Paphies australis) and cockles (Austrovenus stutchburyi) are other New Zealand-wide inhabitants of soft shores favoured by shellfish gatherers. These species can tolerate higher levels of silt and lower salinity, and live in the more sheltered waters of harbours and estuaries. Both are accessible at low tide: pipi live in the intertidal zone in coarser sand, and cockles on the tidal flats in finer silt at neap low water. In some areas, (for example, harbours or estuaries adjacent to large cities) the gathering of pipi and cockles for consumption is restricted by the water quality of the environment in which they live (M. Hart, Healthcare Hawkes Bay, pers. comm.; P. Wood, MidCentral Health, pers. comm.; B. Munro, Tairawhiti Healthcare, pers. comm.). On soft bottoms in open water and harbours throughout New Zealand, scallops (Pecten novaezelandiae) are found. These may be collected in some places by wading out into the water at low spring tide, but are most commonly gathered by dredging or diving. Another species that is commonly dredged is the dredge oyster (also known as the flat oyster), Tiostrea chilensis. Dredge oysters occur throughout New Zealand, but are most commonly associated with the cooler waters of the South Island. They live sub-tidally in beds from the low spring water down to 500 feet deep. While bivalves represent the highest risk to consumers with respect to marine biotoxins, several other species that are frequently gathered may represent a lower potential risk. These include species that graze on benthic phytoplankton or eat macroalgae covered by benthic phytoplankton, and species that are carnivores and may accumulate biotoxins from their prey. The most commonly gathered gastropods are paua (Haliotis spp.), of which the largest is Haliotis iris. Paua live sub-tidally in rocky platform reefs in the high salinity waters of the open coasts. They are found throughout New Zealand, but are more common (and larger) toward the south. Less commonly harvested are the smaller gastropods: pupu, the mud snail (Amphibola crenata), found in protected harbours and estuaries, and the rocky reef dwellers, the cats-eye (Turbo smaragdus), the topshell (Melagraphis aethiops), Cooks turban (karakea, toitoi or ngaruru) (Cookia sulcata), the whelks (kawari) (Cominella spp.) and limpets (ngakihi) (Cellana spp.).

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The sea urchin, kina, (Evechinus chloroticus) is found throughout New Zealand in rocky reef areas. Kina feed on macroalgae. In more protected waters they are found in low tidal areas, but on open, exposed shores may be found in pools in the mid-tidal zone. They are more common sub-tidally, nestling in cracks, under ledges and in hollows, and are usually gathered by snorkeling. The most sought-after crustacean is the crayfish. The most common species is the red rock lobster (Jasus edwardsii), which is found throughout New Zealand, living sub-tidally in rocky crevices and caves on open coasts. Crayfish are gathered by diving. One potential source of biotoxins currently not considered in the marine biotoxin monitoring programme is planktivorous fish. The linking of the mortality of sea lions along the central California coast to a bloom of Pseudo-nitzschia australis (Scholin et al., 2000), indicates the potential for ASP to be passed up through the food chain. A range of planktivorous fish are found widely distributed throughout the New Zealand coastline, particularly in rocky reef areas. One of the underlying factors influencing the shellfish gathering activities in each biotoxin zone is the availability of desirable species. This is a function of the ecology of the area, which is partly determined by the topography of the coast, and the degree of wave exposure. The western coast of Zone A consists of an exposed sandy coastline (Ninety Mile Beach) between Ahipara and Scott Point. (Refer to Appendix II(A) for the location of the zones). Tuatua and toheroa are dominant species in the sand of the beach. Both species are patchy in distribution, and local knowledge is usually necessary to locate denser beds. Toheroa have been exploited to such an extent that harvesting is highly restricted, but tuatua are still present in numbers that make the species attractive to gather. Associated with the rocky outcrops (e.g. at Ahipara, The Bluff, and Scott Point) are mussels, paua, kina and crayfish, and in much lower numbers, rock oysters. While there is relatively little road access to this area, vehicles are able to drive long distances along the beach at low tide, so the whole length of the beach is accessible. The same shellfish species are gathered further south in Zone F from the long exposed sandy beaches broken occasionally by rocky reefs and headlands. The Herekino, Whangape, Hokianga, and Kaipara Harbours that lead off these long exposed beaches provide an environment for species that thrive in more sheltered and silty environments. Species gathered here include native and Pacific oysters, dredge oysters, cockles, horse mussels, and pipi. There are scallop beds in the Kaipara Harbour, and green-lipped mussels can be dredged from beds near the harbour mouth. The Manukau Harbour also has abundant shellfish, although in places the ability to harvest these is compromised by the bacteriological water quality. Due to overfishing, the Auckland Regional Council has banned the gathering of shellfish on some of the beaches between the Manukau and Kaipara Harbours, and a lower daily bag limit for cockles applies in the Auckland Regional Council area. Further south, green-lipped mussels and cockles are common in the Raglan Harbour, and pipi, cockles and mussels in the Kawhia Harbour. The Manukau and Kaipara Harbours are easily accessible from Auckland, and the coast of the Hokianga Harbour is easily accessible by car and boat. The Whangape and Herekino Harbours further north are much more remote. The most popular access to the open sandy beaches in the north

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of the zone is via Dargaville. Here, and on Muriwai Beach, and the beaches south of the Manukau Heads, cars are able to drive along the beach at low tide, so infrequent road access does not necessarily significantly limit access to the length of the beach. Southwards, the coast curves westwards into the North Taranaki Bight. As the rocky reef habitat increases, green mussels, paua and kina are more abundant, and some tuatua are found in sandy areas. The southern part of Zone F from Urenui to Cape Egmont consists of gravel, cobble and boulder beaches, providing habitat for paua, kina and green-lipped mussels. Paua, kina, green-lipped mussels and pipi are abundant in the South Taranaki Bight in the north of Zone H. Further south, the coast changes to coarse-grained sandy beaches with mactrid clams, tuatua, toheroa and pipi present. The rocky areas adjacent from Paekakariki south and then east to Cape Palliser provide habitat for paua, kina and crayfish. Cockles are found in the estuarine areas near Paremata. Zone J, the western coast of the South Island, is a long exposed rocky coastline, interspersed with mixed shingle and sand beaches. With the exception of mussels on the rocky reefs, there are few bivalves suitable for gathering – crayfish are the main non-commercially gathered species other than finfish. Similar coastline extends south to the sounds in Fiordland, where scallops, paua, kina mussels and crayfish are available. In the Sounds the cliffs drop straight into the sea, and there are very few places where the intertidal zone is other than vertical. Lack of road access in much of this area limits the gathering of shellfish. At the south of the South Island, paua and kina are present as the coast runs in an easterly direction, with toheroa and mactrid clams present in Te WaeWae Bay, and toheroa at Oreti Beach. The coastline of Stewart Island is convoluted and rocky, with the major species of interest to non-commercial harvesters being mussels, paua and dredge oysters. Paterson Inlet has a wider variety of species with the addition of kina, scallops, pipi and cockles. The Foveaux Strait is famous for its abundance of dredge oysters. These are conserved through seasonal bans on harvesting. Due to the impact of Bonamia disease on the oyster population, the seasons have been highly restricted through the 1990’s, but the beds now appear to have recovered. The longer “Bluff oyster” seasons now expected will increase the risk of exposure of consumers to biotoxins in this area. Zone I includes the eastern coast of the South Island. This coast is more sheltered from the prevailing south-westerlies than the western coast. The major species for much of this rocky coast are still mussels, paua and crayfish. The mixed sand and gravel beaches from Oamaru north to Banks Peninsula provide little opportunity for the settlement of bivalve shellfish, paua or crayfish. In contrast, these species are found in the rocky reefs around Banks Peninsula, and the harbours and estuaries contain cockles and pipi. There are significant beds of cockles at Papanui. North of Banks Peninsula is a stretch of steep coarse grained sandy beach with beds of cockles present. From Amberley Beach north, the coast is comprised of exposed rocky reefs where crayfish and paua are the major species of interest to non-commercial harvesters. North of Kaikoura, the coast is well known for its abundance of paua. The eastern coast of Zone G is predominantly rocky and gravel beaches, with paua, kina and crayfish found in reefs. Mussels are found at Port Underwood. To the west inside the Marlborough Sounds and Croiselles Harbour, the coastline is steep and

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rocky, with very few intertidal areas. The main species gathered here are mussels (Blue, and green-lipped (GreenshellTM)), and scallops. While road access is limited in much of the Marlborough Sounds, most areas are easily and safely accessible by boat. Paua and crayfish are present on the outer shores of the Sounds where the coast is more exposed to wave action. To the west, dredge oysters and scallops are the major bivalve species in Tasman and Golden Bays, and significant beds of cockles occur in Tapu Bay and Pakawau Beach. Crayfish are taken from the rocky reefs of the coast that separates the two major bays. On the south-eastern coast of the North Island, paua, kina and crayfish are the predominant species of interest to non-commercial harvesters in Zone E. Some mussels are also present, and cockle and pipi occur in the few sheltered areas. The coast is a mixture of rocky reefs, rock platforms, and steeply graded coarse sand beaches interspersed in Hawke’s Bay with rocky beaches. Much of this coastline is relatively remote, with poor road access. Similar species are found on the east-facing coast of Zone D, from Cape Runaway into the Bay of Plenty. In the sandy beaches from Opotiki to Waihi Beach, tuatua are found. Pipi, oysters, cockles and mussels are found in the harbours in this bay. On the western side of the Bay of Plenty, scallop beds lie off shore and in Tauranga Harbour, and these continue north up the Coromandel Peninsula. From Waihi northwards, the coastline is again rocky, but interspersed with harbours and estuaries where oysters, pipi and cockles are found. The rocky open shores provide good habitat for crayfish, and green-lipped mussels, paua and kina are also present. There is good road access to much of this area. The range of species potentially available for non-commercial gathering for consumption by the public is related to the range of habitats available in an area – the more homogenous the environment, the less diversity in terms of desirable species for consumption. Zone C, the Hauraki Gulf and Great Barrier Island, encompasses a very large range of habitats, and thus species for consumption by the public. These range from species found in sheltered estuarine and harbour areas, through to those in moderately protected areas, and a few areas that face open water. The zone thus contains cockles, pipi, Pacific and native rock oysters, tuatua, green-lipped mussels, scallops, kina, paua and crayfish. The shellfish gathering areas in this zone are in general highly accessible to the public – the coast is well supplied with roads and boat launching facilities. However, there are some local restrictions on the gathering of shellfish, designed both to conserve shellfish stocks, and to prevent illness in consumers. The eastern coast of Zone A and Zone B in the north-east of the North Island have a similarly wide diversity of marine habitats, providing a wide range of seafood for non-commercial harvest. As with the Bay of Plenty northwards, this coastline is protected from the prevailing south-westerly swells, but is subject to wind and waves from north-easterly directions. The coastline is irregular and convoluted. The orientation of the beaches in the area, and their fetch lengths and directions are highly variable. These substantial differences in exposure and shelter result in rapid changes in beach type over quite small distances. This variability contrasts with most of the other zones except Zone C. The result of this is that Zones A, B, and C, despite that fact that they are relatively small compared to other zones, all contain most of the

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commonly gathered species of shellfish (with the exception of course of those species that do not extend this far north, such as the Blue mussel). One species not found in this area is toheroa, which prefer sandy coasts with greater wave exposure. The coastline in both Zones A and B are relatively easily accessible. Zone B is well serviced by roads, with numerous places where it is possible to launch a boat. While some of the coastline in Zone A is relatively remote with fewer roads, most areas are easily accessible by boat. Zone K is the Chatham Islands. Chatham Island itself consists of several sandy beaches, separated by rocky coasts. Paua and crayfish are common on the rocky coasts, and scallops and tuatua occur in Hansen and Petrie Bays. The adjacent Pitt Island has only two small sandy bays with the rest of the coastline being formed by rocky reefs and eroding rock platforms. Paua and crayfish occur on this coast also. The Chatham Islands are unusual in the absence of the Green-lipped mussel, and Blue mussels are also rare. In considering the shellfish available for non-commercial harvest and their relative potentials to accumulate and retain biotoxins (as discussed in the previous chapter), it is apparent that biotoxin zones may vary considerably in biotoxin risk. One issue that has not been examined in our discussion of where different shellfish species occur, is the abundance of various species. We have merely stated that particular species occur in the habitats available in each zone. There are several reasons for this: firstly, there is very little objective data available to assist in this – few surveys of shellfish abundance have been undertaken in this detail, and most information available is at the level of casual observation. Secondly, abundance of shellfish is something that may change significantly from year to year through over-exploitation, environmental factors or disease. At a local level this is something that needs to be monitored in order to keep an appropriate level of biotoxin monitoring – there is little point in monitoring for biotoxins at a site where shellfish species are no longer available for harvest by the public. If the differences between species in accumulation and retention of biotoxins are considered along with the distribution and accessibility of the shellfish species, it is evident that there are differences between biotoxin zones. Zones A, B, C, D, F and G all contain significant proportions of coastline where bivalve shellfish are present, and that are readily accessible to the public. If it can be assumed that tuatua and scallops represent shellfish with a significantly higher level of accumulation and retention of biotoxins than some other species, then these zones would represent areas where the range of available shellfish for harvest presents the greatest risk. While some other common shellfish species are absent, the Chatham Islands (Zone K) are also home to the high risk species. The west coast portion of Zone J represents a low risk area due to the relatively low numbers of bivalve shellfish available for non-commercial harvest, and the relative inaccessibility of some areas of coast. However, the Foveaux Strait area of Zone J has a significant number of dredge oysters, toheroa and scallops, and this presents a greater risk. The harbour/estuarine areas, which contain higher numbers of bivalve shellfish than the open stony beaches, may also represent areas of relatively greater risk potential. In this respect, Zone I is similar to Zone J, except there is a greater diversity of habitats, with more extensive harbour and estuarine species. The most abundant habitats in Zone E support species with a low risk of

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biotoxin contamination, like paua and kina. However, bivalve species such as mussels also occur on the open coast, and estuaries and harbours support those bivalves normally found in such habitats. Obviously the occurrence of readily available shellfish with the potential to accumulate biotoxins is just one small factor in the assessment of the risk of TSP to consumers. However, it is significant in the design of marine biotoxin monitoring programmes. 3.3 SHELLFISH GATHERING In addition to the availability of shellfish for non-commercial harvest, the quantity of shellfish harvested and consumed, and the identity of the consumers are important in biotoxin risk analysis. There have been surprisingly few published studies undertaken on non-commercial shellfish gathering in New Zealand. Results from a study on marine recreational (i.e. non-commercial) fishing done by Sylvester et al. (1991), based on a telephone survey in 1987, have been presented in a previous review of the marine biotoxin monitoring programme (Wilson, 1996). These will not be presented in detail again here except in comparison to more recent data. A more recent National Marine Recreational Fishing Survey has been undertaken for the Ministry of Fisheries (Fisher & Bradford, 1998). This study was based on a national telephone and diary survey. The survey used a preliminary telephone survey to determine the number of households that contain marine non-commercial fishers from a random selection of households with a phone. One randomly selected fisher from each of these households was asked to keep a diary of their recreational (i.e. non-commercial) fishing trips during the year. The figures provided by the diarists were scaled to give estimates for the total marine non-commercial fishing population of New Zealand. The data collected in this survey is likely to be of better quality than that collected in the telephone survey in 1987, which relied on participants to recall their fishing activities some time after the event. In both these studies, the population surveyed included only those people that have telephone connections. It is possible that those people who do not have telephones may be more reliant on non-commercially harvested seafood as a source of food. Table 3.1 shows the number of trips by survey respondents targeting the main shellfish species in each Biotoxin Zone, except Zone K (Chatham Islands) for which there are no data. These data are reworked from Fisher and Bradford (1998) so that the fishing areas in the survey data correspond with the Biotoxin Zones used in the marine biotoxin monitoring programme. Due to the positioning of the boundaries of fishing areas used in the original analysis of data by Fisher and Bradford, Zone F data from Tirua Point southwards has been included with data from Zone H. Most other area boundaries were similar to the boundaries of the Biotoxin Zones. The “Oyster” category includes Pacific oysters, native rock oysters, and oysters of unspecified type. Some of the oysters in this category in Zone G and all in Zone I are likely to be dredge oysters. The species of mussel targeted are not detailed.

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If all the main non-finfish species that are non-commercially harvested are considered, the data suggest that the greatest level of gathering activity occurs in Zone D, with significant levels in Zones E, G and B. Comparatively low levels of activity occur in Zones A and H. If trips targeting bivalve species only are considered, Zone D again has the highest level of activity, closely followed by Zone G. Comparatively high levels of activity are also indicated in Zones B, C and F, with Zones A, E, H, I and J having comparatively lower levels. Over all areas, scallops are the most actively targeted species, followed by mussels and pipi. (Note that the ability of the general public to distinguish between pipi and tuatua may be limited, so there may be some confusion between these two classes). Comparison of the percentage of trips targeting each bivalve species within each zone (Table 3.2) indicates that scallops are the most actively targeted species in 6 of the 10 zones (Zones A, B, C, D, F and G). Mussels are the most actively targeted species in Zones E and J, and pipi in Zone H. With the exception of Dredge oysters in Zones G, I and J, oysters are the species that are least frequently targeted. In both zones A and D, tuatua and pipi are the next most frequently targeted species after scallops.

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Table 3.1

Zone Bivalve Species Total % Total Other Species Total All % AllCockle Mussel Oyster D Oyster Pipi Scallop Tuatua Bivalves Bivalves Paua Cray Kina Species Species

A 2 3 2 0 9 31 9 56 3.9 4 13 3 76 2.6B 16 30 5 0 55 59 13 178 12.5 7 126 22 333 11.5C 11 35 3 0 17 72 9 147 10.4 4 98 4 253 8.7D 13 72 7 0 85 111 74 362 25.5 26 329 16 733 25.2E 7 20 1 0 7 0 1 36 2.5 122 273 14 445 15.3F 4 34 13 0 16 110 15 192 13.5 9 14 6 221 7.6G 29 37 11 35 12 178 0 302 21.3 21 71 2 396 13.6H 6 7 0 0 9 0 0 22 1.5 21 33 7 83 2.9I 15 14 2 9 10 0 5 55 3.9 61 46 6 168 5.8J 8 39 0 14 2 10 0 73 5.1 50 68 9 200 6.9Total 111 291 44 58 222 571 126 1423 325 1071 89 2908% 7.8 20.5 3.1 4.1 15.6 40.2 8.9

Cockle Mussel Oyster D Oyster Pipi Scallop TuatuaA 3.6 5.4 3.6 0.0 16.1 55.4 16.1B 9.0 16.9 2.8 0.0 30.9 33.1 7.3C 7.5 23.8 2.0 0.0 11.6 49.0 6.1D 3.6 19.9 1.9 0.0 23.5 30.7 20.4E 19.4 55.6 2.8 0.0 19.4 0.0 2.8F 2.1 17.7 6.8 0.0 8.3 57.3 7.8G 9.7 12.4 3.7 11.7 4.0 59.5 0.0H 27.3 31.8 0.0 0.0 40.9 0.0 0.0I 27.3 25.5 3.6 16.4 18.2 0.0 9.1J 11.0 53.4 0.0 19.2 2.7 13.7 0.0

Table 3.2

Zone

Distribution of Targeted Species Within Each Zone (% of Zone Total)

Table 3.1: Number of trips targeting the main shellfish species in each Biotoxin Zone recorded by diarists in 1996. (Data from Fisher & Bradford (1998)). D Oyster = Dredge oyster; Oyster=Oysters including native rock and Pacific oysters, and unspecified species; Cray=Crayfish

Table 3.2: Distibution of targeted bivalve shellfish species within each zone as a percentage of the total target trips for each zone. (Data from Fisher & Bradford (1998)). D Oyster = Dredge oyster; Oyster=Oysters including native rock and Pacific oysters, and unspecified species; Cray = Crayfish

107

Table 3.3 shows the numbers of each species taken from each biotoxin zone by respondents in the same survey. These data reflect both gathering activity, and the availability of each species in each zone. The highest numbers of mussels, pipi, tuatua and crayfish were gathered from Zone D, while the highest number of scallops, dredge oysters and cockles were harvested from Zone G. With respect to oysters (rock and unspecified), 45% were taken from Zone F, along with 15% of the mussels. Paua and crayfish were the predominant species harvested in Zone E, which recorded the highest number of paua, and second highest number of crayfish taken. The results of the survey undertaken by Fisher & Bradford (1998) show that most people surveyed generally fish near home. Some exceptions to this are the respondents from Auckland fishing in the eastern Coromandel area (Zone D). The Marlborough Sounds (Zone G) are also a popular destination for fishers residing outside that area. Respondents from both Wellington and Christchurch made a number of trips to Pelorus and Queen Charlotte Sounds. There were only a small number of trips made by South Island respondents to the North Island. The data collected in the 1996 Marine Recreational Fishing Survey (Fisher and Bradford, 1998) described here represent the most comprehensive nation-wide data that are currently available. The results are not inconsistent with what could be predicted from the ecology of the coast, the accessibility of the coast, and the location with respect to centers of population. If one can assume that the figures from this survey provide a reasonably realistic picture of the distribution of non-commercial shellfish gathering, then with some knowledge of biotoxin distribution throughout New Zealand, and the accumulation and retention of TSP toxins in different species of shellfish, risk assessment on a zone by zone basis should be possible. Estimates of the total quantity of non-commercial harvest of species within the Ministry of Fisheries Quota Management System are provided in the Report from the Fisheries Assessment Plenary, based on telephone and diary surveys (Annala & Sullivan, 1996). This report suggests that the total non-commercial harvest of cockles per year is 62 tonne. The estimate of non-commercial harvest of paua is 205 tonne per year, with an additional illegal commercial harvest of 275 tonne. The accuracy of these data is uncertain. A study was undertaken by Kearney (1999) of the ecology and management of cockles in the Whangateau Harbour, north of Auckland. This study was conducted over a 12-month period from December 1997 to December 1998, using structured sampling regimes and interviewing all harvesters on the beach during sampling times. From the data collected in his study, Kearney estimated that the total annual harvest of cockles from Lew’s Bay, Whangateau Harbour, from December 1997 to December 1998 was 27,950 kg. The contrasts with the results of the Ministry of Fisheries 1993-94 telephone and diary survey, which estimated that the total non-commercial cockle harvest in QMA1 (Cape Reinga to Cape Runaway) was 55 tonne (Annala & Sullivan, 1996). Given that the data from Kearney’s study are based on actual observation from only one of many harvestable populations in this area, this would suggest that the Ministry of Fisheries survey grossly underestimated the non-commercial harvest of cockles in QMA1. If this underestimation extends to other shellfish species also, there are obviously implications in terms of quantification of risk in the event of the occurrence of marine biotoxins.

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No.

% of Total

Cockle No.

% of Total

Mussel No.

% of Total

Oyster No.

% of Total D Oyster No.

% of Total Pipi No.

% of Total

Scallop No.

% of Total

Tuatua No.

% of Total Paua No.

% of Total Cray No.

% of Total Kina

A 30 0.3 100 0.8 200 5.4 0 0.0 715 3.6 685 3.3 1250 10.8 30 1.0 42 1.1 42 1.2B 1770 20.2 1344 11.1 306 8.2 0 0.0 5870 29.9 1259 6.0 1259 10.9 96 3.1 505 13.3 1069 29.7C 1134 12.9 1515 12.6 32 0.9 0 0.0 1591 8.1 2003 9.5 635 5.5 22 0.7 270 7.1 221 6.1D 1133 12.9 2935 24.3 925 24.8 0 0.0 7822 39.8 2435 11.6 5890 50.9 171 5.6 1252 32.9 944 26.3E 215 2.5 568 4.7 30 0.8 0 0.0 433 2.2 0 0.0 20 0.2 1229 40.0 1045 27.4 433 12.0F 350 4.0 1885 15.6 1693 45.3 0 0.0 1767 9.0 2516 11.9 1907 16.5 71 2.3 53 1.4 326 9.1G 2236 25.5 1137 9.4 490 13.1 1221 43.3 575 2.9 12003 57.0 0 0.0 164 5.3 199 5.2 16 0.4H 253 2.9 308 2.6 0 0.0 0 0.0 412 2.1 0 0.0 0 0.0 263 8.6 113 3.0 307 8.5I 1029 11.7 540 4.5 58 1.6 843 29.9 391 2.0 0 0.0 621 5.4 584 19.0 138 3.6 98 2.7J 611 7.0 1723 14.3 1 0.0 754 26.8 87 0.4 172 0.8 0 0.0 440 14.3 190 5.0 139 3.9TOTAL 8761 12055 3735 2818 19663 21073 11582 3070 3807 3595

Oyster D Oyster Cray Kina

Zone

Quantity of Species Taken by Diarists

Pipi Scallop Tuatua PauaCockle Mussel

Table 3.3: Numbers of the main shellfish species caught by diarists in each Biotoxin Zone (Data from Fisher & Bradford, 1998).D. Oyster=Dredge oyster; Oyster=Oysters including native rock and Pacific oyster, and unspecified species;Cray=Crayfish

109 109

3.4 POPULATION STRUCTURE AND SHELLFISH CONSUMPTION The question of whether some sectors of the population are more at risk because of proportionately higher rates of consumption of non-commercially harvested shellfish, is one that needs to be considered in biotoxin risk management. Kaimoana (seafood) has traditionally been extremely important to Maori, not merely as a food source, but also as a way of upholding customary obligations within and between whanau, hapu and iwi (Te Puni Kokiri, 1993). Kaimoana is still very important to Maori today. A survey of Maori households in Te Hiku o Te Ika (the far north of the North Island) showed that 11% of the households collected seafood more than once a week, 31% collected seafood at least weekly, and 52% at least fortnightly. Only 9% did not collect seafood at least monthly (n = 499) (Hay, 1996). As noted by Wilson (1996) in the last review of the marine biotoxin monitoring programme, some species of shellfish are of particular cultural significance to iwi or hapu. This may be associated with a cultural history through which particular species are regarded in a special relationship (as in the case of Te Uri o Hau with respect to toheroa), or with the traditional use of shellfish at particular times (for example, the consumption of a particular kind of shellfish by a person who is dying). Traditionally, seafood has also been important to Pacific Island and Asian peoples, and this is potentially reflected in shellfish harvesting patterns also. The population of each regional authority area by ethnic origin is presented in Figure 3.1. It can be seen that there are significant differences in the ethnic composition of the population in different regions. There has been little data gathered on non-commercial shellfish harvesting by people of different ethnic origins in New Zealand. Wilson (1996) presented data drawn from the 1987 Marine Recreational Fishing Survey, and this is summarized in Table 3.4.

Ethnicity Gathering (%) Diving (%) Maori 14 16 Pacific Islands 2 2 Other Ethnic Groups 84 82 Non-Specified 1 0

Table 3.4: Estimated percentage of non-commercial shellfish harvesting

(either by gathering or diving) by ethnicity, per year, throughout New Zealand. (Summarized from Wilson, 1996).

110 110

Auckland(1 068 654)

Northland(137 052)

Bay of Plenty(224 361)

Gisborne(45 789)

Hawke's Bay(142 794)

Wellington(414 081)

Taranaki(106 587)

Marlborough(38 403)

Otago(185 079)Southland

(97 095)

West Coast(32 514)

Tasman(37 974)

Nelson(40 275)

Manawatu-Wanganui(228 768)

EuropeanMaoriPacific IslandAsianOtherNot Specified

Waikato(350 130)

Canterbury(468 036)

Figure 3.1: Population distribution by ethnic origin for each regional authority within New Zealand (data extracted from the 1996 census, Statistics New Zealand).

111 111

Somewhat different data have been obtained from surveys based on observations of non-commercial shellfish gathering in a qualitative survey of intertidal harvesting by amateur fishers in the Auckland Metropolitan area. This survey was conducted over the summer of 1991-92 (Drey & Hartill, 1993). In this study, it was concluded that there were distinct differences in the ethnic composition of people harvesting intertidal organisms at various beaches. Although the “relatively low number of interviews conducted at many sites render any characterization of user populations at these sites suspect” (Drey & Hartill, 1993), the data did suggest that the distribution of shellfish gatherers by ethnic origin was not representative of the composition of the population as a whole, with people of New Zealand European origin being under-represented and Maori, Asian and Pacific Island peoples generally being over-represented. This study also suggested that the most favoured species of shellfish taken from hard and soft shores differed with ethnicity. Given the potential for different species of shellfish to differ with respect to accumulation and retention of biotoxins, this may also result in differing levels of risk with respect to ethnicity. A more recent, and much more detailed study has been undertaken by Kearney (1999), in his study of the ecology and management of cockles in the Whangateau Harbour, north of Auckland (discussed previously). Table 3.5 presents data gathered from observations and interviews in his study.

Ethnic Group

Total Weight Harvested (kg)

Percentage of Total

Harvested (%)

Number of Harvesters

Percentage of Total

Harvesters (%) Maori 5853 55 893 54 NZ European 2022 19 430 26 Asian 1703 16 248 15 Pacific Island 958 9 66 4 Other 106 1 17 1 Total 10,642 100 1654 100

Table 3.5: Numbers and percentages of the total weight of cockles harvested,

and harvesting population structure by ethnic group at Lews Bay, Whangateau Harbour (n = 1654). (From Kearney, 1999).

It is interesting to note that the majority of harvesters (82%) were not resident in the surrounding district. Most had residency in Manukau City (South Auckland). These studies by Drey & Hartill (1993) and Kearney (1999), while localised and undertaken within reasonable driving distance of Auckland, suggest that based on the percentage of shellfish gatherers, the risk of TSP may not necessarily be the same for all ethnic groups. Further studies are required to ascertain the differences in risk with respect to ethnicity. It should be noted that this may vary with location also – in more remote coastal areas there may be a greater reliance by people of all ethnic origins on gathered seafood.

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3.5 TEMPORAL PATTERNS Studies by Hartill & Cryer (1999) and Kearney (1999) have shown seasonal differences in non-commercial harvesting activity. These studies have shown that for a range of intertidal shellfish, non-commercial harvesting is highest in the summer, and lowest in the winter, with harvesting activity in autumn and spring at intermediate levels and varying with the species harvested. Obviously, in addition to these patterns of activity, harvesting of some species (such as scallops and dredge oysters) may be prevented by seasonal restrictions for conservation reasons. The type of day also affects the extent of non-commercial shellfish harvest, with greatest harvesting activity occurring on public holidays or weekends attached to public holidays. Harvesting activity is significantly greater on weekends compared to weekdays (Hartill & Cryer, 1999, Kearney, 1999). Anecdotal evidence also suggests that in some areas harvesting activity may increase significantly in the days before public holidays or long weekends. Mussel spat harvesters drive along Ninety Mile Beach in the north of Northland several times each day. They report that harvesting activity, particularly by Maori, is greatest in the days before public holidays or long weekends, and suggest that this is a result of preparation for visits by whanau from outside the area (C. & R. Hensley, pers. comm.). Many Health Protection Officers have reported that there are large increases in their local populations over summer, as holiday-makers from the cities move to coastal areas over the summer holidays. Seasonal variations in shellfish gathering may thus result from both an increase in activity by local residents in summer, and through a temporary increase in population over holiday periods. 3.6 CONCLUSION An investigation of available data on non-commercial shellfish gathering suggests that based on differences in shellfish availability, shellfish gathering activity and shellfish species collected, the risk of TSP varies across the Biotoxin Zones in New Zealand. Although there are insufficient data for this to be quantified, data also suggest that this risk may vary with ethnicity. Thus differences in population structure in different areas with respect to ethnicity may also impact on the risk of TSP in each zone. Temporal patterns in shellfish gathering also impact on the risk of TSP. It should be noted that times at which shellfish gathering activity is high may coincide with an increased risk of toxic phytoplankton blooms in the warmer months (See Section 2). In general, the ability to assess the risks presented by the occurrence of marine biotoxins in shellfish would greatly benefit from the collection of high quality data through studies focussed on this issue.

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SECTION 4: ANALYSIS OF EPIDEMIOLOGICAL DATA 4.1 INTRODUCTION Environmental surveillance for public health with respect to marine biotoxins in New Zealand is primarily reliant on hazard surveillance at present – that is, monitoring for toxic phytoplankton and biotoxins in shellfish. However, outcome surveillance in the form of reporting the incidence of cases of Toxic Shellfish Poisoning (TSP), is also undertaken. The current outcome surveillance system is designed to fulfill the following objectives (Baker & McNicholas, 1995): • To identify cases of TSP so that the incidence and distribution of this illness, and

the associated biotoxin(s), can be monitored. • To identify shellfish contaminated by marine biotoxins that were not detected by

routine shellfish biotoxin monitoring, so that control measures can be taken. • To assist in characterising the illness caused by biotoxins, including the dose-

response relationship between biotoxin exposure and illness. • To assist in identifying other biotoxins that are not detected by current shellfish

biotoxin testing. • To assess the effectiveness of control measures. Cases of TSP are identified by general practitioners and hospital clinicians, and reported to local Medical Officers of Health. In some instances, (for example, where they have not consulted a medical practitioner) members of the public may report cases directly. Notified cases are investigated by public health staff, and recorded locally by them on the notifiable disease database, EpiSurv. “Confirmed” or “probable” cases are defined as being those where TSP symptoms occur within relevant time frames, and which are associated with toxicity in shellfish sufficient to account for those symptoms. Where these conditions are not met, cases may either remain in the “suspect” category, or may be discounted depending on the outcome of the investigation (Ministry of Health, 1997a). Epidemiology of TSP is severely limited by the lack of biomarkers for exposure to marine biotoxins in humans (i.e. there are no recognised human diagnostic tests to determine whether a person has been exposed to marine biotoxins). There is also a lack of knowledge about the clinical symptoms of TSP, and this results in a comparative under reporting of the incidence of TSP (Fleming et al., 1995; Fleming et al., 1998). Fleming et al., (1998) summarise the situation as follows: “The lack of progress in phycotoxin disease epidemiology is due to the lack of disease and exposure biomarkers in humans. The only way to study these diseases epidemiologically has been identification through their clinical presentation, and more recently, by applying the appropriate laboratory testing to the ingested seafood. Because diagnosis could not be made accurately for either the clinical diseases or the asymptomatic cases associated with these phycotoxin exposures, it has not been possible to investigate their true incidence in human populations. Nor has it been possible, without human biomarkers, to accurately evaluate the true clinical course, treatment and prognosis of the marine toxin diseases.”

114 114

Any analysis of the epidemiological data relating to TSP in New Zealand should consider the limitations of the knowledge framework within which the data were gathered. 4.2 METHODOLOGY AND ASSUMPTIONS IN ANALYSIS The Institute for Environmental & Scientific Research (ESR) has supplied the data used in this analysis. These data were drawn from several separate databases that record cases of TSP in New Zealand (an Excel spreadsheet of 1993 cases, a database of summarized monthly reports to the Ministry of Health (1994-July 1996), an Excel spreadsheet of cases from 1994 and January to July 1996, and the current EpiSurv database (July 1996-June 1999)). Note that the details of the cases from 1995 were only available in summary form from the summarised monthly reports to the Ministry of Health. Until 1996, assessments of the status of cases were based on the following criteria: Unlikely: The case does not meet case definition, and/or shellfish testing

(leftovers/same site) was negative for PSP or NSP. Suspected: The case meets the case definition, but no shellfish (leftovers/same

site) were available for testing. Confirmed: The case meets the case definition, and shellfish testing (leftovers/same

site) was positive for PSP, or NSP. The case definition included the presence of one or more neurological symptoms within 24 hours of eating shellfish (which includes neurosensory, neuromuscular, and neurocerebellar symptoms). Subsequent to a review of the epidemiological surveillance system for cases of TSP (Baker & McNicholas, 1995), the case definitions were re-written to include all four toxins (PSP, NSP, ASP and DSP). The current case definitions are given in Section C of the Manual for Public Health Surveillance in New Zealand (ESR Communicable Disease Centre, 1996). The revised status definitions separate the previous “Confirmed” status into “Probable” and “Confirmed”, as follows: Probable: Meets case definition for suspect case, AND detection of relevant

biotoxin at or above the regulatory limit in shellfish obtained from near or same site (not leftovers) within seven days of collection of shellfish consumed by case;

Confirmed: Meets case definition for suspect case, AND detection of biotoxin in

leftover shellfish at a level resulting in the case consuming a dose likely to cause illness: Current dose level:

ASP: 0.05 mg/kg body weight DSP: ingestion of 48 µg or 12 MU

NSP: 0.3 MU/kg body weight PSP:10 MU/kg body weight(approx. 2 µg/kg body wt.)

115 115

As procedures within the surveillance system have improved, and toxin test methods have become more specific, the quality of the epidemiological data collected has improved. Data for 1993 were provided as two sets of data – one containing all the cases reported at the time, and the other only those cases that fit the case definitions for TSP (Yvonne Galloway, ESR, pers. comm.). It has been assumed that all the cases in the latter set of data could be assessed as “suspected”. In general there was no testing of left-over meals or shellfish harvested concurrently from the same sites as the shellfish consumed, so all these cases remain in the “suspected” category. The symptoms exhibited by some cases suggest strongly that had shellfish samples been available, there is a possibility that they might have established TSP as a cause of illness. In other cases, the symptoms exhibited could have resulted from a range of causes, and it is due to the lack of confirmatory tests that they remain in the TSP “suspected” category rather than being considered “unlikely”. There is no detailed computerised database of the 43 cases reported from 1995. Copies of the original questionnaires filled out relating to each case are unable to be located by ESR. Analysis is therefore based on the Results summary for reported cases of illness following the consumption of shellfish, supplied by ESR to the Ministry of Health. These summaries do not contain data about individual cases, so no information regarding age, ethnicity, hospitalisation, weight, amount of seafood consumed etc. are available over this time period. They do, however, contain an assessment of the case status from the results of the combined questionnaire data and shellfish testing for each case. For the purposes of analysis in this review, current case definitions have been used. All cases of “confirmed” status prior to July 1996 have been revised according to the current status definitions, so that they are separated into “probable” or “confirmed” status. This is unable to be done for two “probable” cases of DSP in 1995, as the data on body weights and quantities of shellfish consumed with which to calculate likely toxin doses, are not available. These two cases thus remain as “probable”. In the course of our analysis we noted some inconsistencies in the assessments made over this time period – for example, the assessment of several cases as “unlikely” where the symptoms were recorded as fitting the case definition, but samples tested as having been free of detectable levels of PSP or NSP. However, the samples were not tested for ASP. Given that these cases resulted from the consumption of scallops, which have a tendency to accumulate and retain ASP, it is our view that these cases should have been assessed as “suspected” under the stated criteria. However, given that we had only a summary of the information available to ESR, we have not altered these assessments in our analysis of the data. Data from July 1996 to June 1999, as recorded on the questionnaire forms for each reported case, are collected in the “EpiSurv” database. We have revised the status of some cases where microbiological results have subsequently provided additional information and the case status had not been updated. Of the 17 cases recorded on EpiSurv from July 1996 to the end of June 1999, two cases entered were recorded as having no known symptoms, and a further three cases had microbiological test results that strongly suggested that the symptoms were caused by bacterial contamination of the shellfish. One further case did not fit the case definitions and there were no results

116 116

from shellfish testing, so the status of this case was revised from “probable” to “unlikely”. Epidemiological data for TSP collected from January 1993 until the end of June 1999 were analyzed with respect to: numbers of “unlikely”, “suspected”, “probable” and “confirmed” cases each year; numbers of “suspected” and “probable”, cases arising from non-commercial shellfish gathering as compared to commercial harvest; shellfish species implicated in “suspected” and “probable” cases; and the areas from which non-commercially harvested shellfish were gathered in “suspected” and “probable” cases. The incidence of “probable” cases of TSP in New Zealand were compared to the incidence of other food-borne diseases here. The distribution of gender, age and ethnicity in “suspected” and “probable” cases from 1993 and 1994, and January 1996 to June 1999 were also analyzed (these data were unavailable for the intervening period). 4.3 RESULTS Table 4.1 provides a summary of the total reported cases of TSP in New Zealand by year, and their assessment as being “unlikely”, “suspected”, “probable” or “confirmed” to have been caused by marine biotoxins in shellfish consumed.

Year

1993

1994

1995

1996

1997

1998

1999 (6

months)

Total number of cases reported

302

82

43

21

3

1

5

Number

assessed as unlikely

163

55

13

11

2

1

0

Number

assessed as suspected

139

26

23

10

2

0

4

Number

assessed as probable

0

1

7

0

0

0

1

Number

assessed as confirmed

0

0

0

0

0

0

0

Table 4.1: Summary of the reported cases of TSP by year as assessed with

respect to the likelihood of the causative agents being PSP, ASP, NSP or DSP toxins.

117 117

Over the time-period analysed (1993 to mid 1999) no confirmed cases of TSP have been reported in New Zealand (consequently further analysis does not include this category). Of all the TSP cases identified as “probable” from 1993 to June 1999, data on hospitalisation are only available for one case. This case was not hospitalised. The incidences of “probable” cases of TSP are compared with the incidence of two other illnesses associated with the consumption of shellfish in Table 4.2. Campylobacter is the most common food-borne illness, predominantly associated with poor food handling. Vibrio parahaemolyticus is a halophilic bacterium that is naturally present in marine waters and silts, and that can become concentrated in shellfish due to their filter-feeding activity. Because illness from Vibrio parahaemolyticus is not notifiable, it is possible that these numbers are understated (hence not being stated as an official rate per 100,000 in Table 4.2). It can be seen that the incidence of TSP is very much lower than that of Campylobacter, and very slightly lower than that of Vibrio parahaemolyticus.

TSP Campylobacter Vibrio parahaemolyticusYear Number of

cases Rate per 100,000

Number of cases

Rate per 100,000

Number of cases

1994 1 0.03 7,714 213.2 Not available 1995 7 0.19 7,442 205.7 2 1996 0 0 7,628 210.8 3 1997 0 0 8,848 244.5 3 1998 0 0 11,578 320.0 6 Table 4.2: Comparison of the numbers of cases and rates per 100,000 people

in New Zealand between “probable” cases of TSP, and illnesses caused by Campylobacter and Vibrio parahaemolyticus. (Source: Epidemiology Group, ESR).

Of the “suspected” and “probable” cases of TSP in 1993-94, and from January 1996 to June 1999, 48.3% were male, and 50.6% were female (N=178). The gender of 1.1% of the cases was not recorded. Data were not available for 1995 cases. Of the total “suspected” and “probable” cases from January 1993 to June 1999 (N=213), 30.0% were from commercially harvested shellfish, and 67.7% from non-commercially harvested shellfish. In 2.3% of the cases, the shellfish source was not recorded on the database. Most of the cases in 1993 occurred prior to the instigation of a comprehensive marine biotoxin monitoring programme. Of the total cases in 1993 (N=139), 21.6% were from commercially harvested shellfish, and 74.8% from non-commercially harvested shellfish. The source of 3.6% of the shellfish was not recorded. Of the “suspected” and “probable” cases from the beginning of 1994 to June 1999 (i.e. excluding the cases from 1993), 45.9% were from commercially harvested shellfish, and 54.1% from non-commercially harvested shellfish (N=74). However, eight of the nine “probable” cases (88.9%) arose from non-commercially harvested seafood, and 11.1% from commercially harvested seafood. The case arising from commercially harvested shellfish meets the case definition for NSP, and arose from the consumption of mussels, harvested from Big Glory Bay in Zone J. Of the cases arising from non-

118 118

commercially harvested shellfish, one was a case of PSP arising from tuatua gathered at Ohope Beach (Zone D), and 7 cases of DSP were from mussels gathered at Akaroa (Zone I). The age distribution of the “suspected” and “probable” cases of TSP in 1993-94 and from January 1996 to June 1999 is shown in Table 4.3 with reference to the New Zealand population as a whole. Data were not available for 1995 cases.

Age (years)

Number

Percentage of Total Cases

(%)

Age Structure of New Zealand Population

(%) <10 2 1.1 15.7 10-19 9 5.1 14.7 20-29 26 14.6 14.2 30-39 47 26.4 15.9 40-49 46 25.8 13.7 50-59 25 14.0 10.7 60-69 12 6.7 7.3 70-79 6 3.4 5.1 80+ 1 0.6 2.7 Not recorded 4 2.3

Table 4.3: Age distribution of the “suspected” and “probable” cases of TSP in

1993-94, and from January 1996 to June 1999, compared to the age structure of the total New Zealand population (based on 1996 Census data, Statistics NZ).

Table 4.4 shows an analysis of the “suspected” and “probable” cases of TSP in 1993-94 and from January 1996 to June 1999 by ethnic origin, with reference to the ethnic composition of the New Zealand population as a whole. Data were not available for cases from 1995.

Ethnic Origin

Percentage of Cases from Non-

Commercially Harvested Seafood (%)

Percentage of Cases from Commercially Harvested Seafood

(%)

Percentage of Total New Zealand

Population (%)

NZ European 78.0 85.5 71.7 Maori 14.6 9.1 14.5 Pacific Island 4.1 1.8 4.8 Other 2.4 0.0 9.0 Not recorded 0.9 3.6 Total No. of cases (N)

123

55

Table 4.4: Analysis of “suspected” and “probable” cases of TSP from non-

commercially and commercially harvested seafood in 1993-94 and from January 1996 to June 1999 by ethnic origin, compared to the ethnic composition of the NZ population. (NZ population figures from 1996 census data, Statistics NZ).

For all the “suspected” and “probable” cases of TSP from 1993-94 and 1996-June 1999, the source of seafood (i.e. non-commercially harvested or commercially

119 119

harvested) for each ethnic group was analysed. These results are presented in Table 4.5. Data were not available for cases from 1995.

Ethnic Origin

Number of Cases

Percentage from Non-Commercially Harvested Seafood

(%)

Percentage from Commercially

Harvested Seafood (%)

NZ European 143 67.1 32.9 Maori 23 78.3 21.7 Pacific Island 6 83.3 16.7 Other 3 100.0 0.0 Not Recorded 3 33.3 66.7

Table 4.5: Analysis of source of seafood by ethnic group for “suspected” and

“probable” cases from 1993-1994 and 1996-June 1999. The cases arising from non-commercial gathering of shellfish have been analyzed with respect to the areas from which shellfish were taken. For this purpose, the Biotoxin Zones A-K have been used. The results of this analysis are presented in Table 4.6. A map illustrating the geographical areas covered by each zone and a summary of these data are presented in Figure 4.1.

Number of Cases in each Zone

Zone 1993

1994

1995

1996

1997

1998

1999 (6 months)

Total No. of Cases

Total No. of Cases excluding

1993 A 7 3 0 1 0 0 0 11 4 B 36 1 0 0 0 0 0 37 1 C 17 0 3 0 0 0 0 20 3 D 13 2 4 0 1 0 1 21 8 E 13 0 0 0 0 0 0 13 0 F 5 4 1 1 0 0 0 11 6 G 0 0 4 1 0 0 4 9 9 H 1 0 0 0 0 0 0 1 0 I 3 0 6 0 0 0 0 9 6 J 0 0 3 0 0 0 0 3 3 K 0 0 0 0 0 0 0 0 0

Not known

9

0

0

0

0

0

0

9

0

Table 4.6: Geographical distribution of sites from which shellfish were

gathered in “suspected” and “probable” cases of TSP arising from non-commercially harvested shellfish January 1993-June 1999. (Zones are illustrated in Figure 4.1).

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Zone A: Tauroa Point to Cape BrettCases: 11 (4)

Zone B: Cape Brett to CapeRodney. Cases: 37 (1)

Zone C: Cape Rodney to CapeColville. Cases: 20 (3)

Zone D: Cape Colville to CapeRunaway. Cases: 21 (8)

Zone E: Cape Runawayto Cape Palliser. Cases:

13 (0)

Zone F: Tauroa Point to CapeEgmont. Cases: 11 (6)

Zone G: Cape Farewellto Cape Campbell.Cases: 9 (9)

G

H

Zone H: Cape Egmont toCape Palliser.Cases: 1 (0)

Zone I: CapeCampbell to Bluff.

Cases: 9 (6)

Zone K: ChathamIslands. Cases: 0 (0)

Zone J: Cape Farewellto Bluff. Cases: 3 (3)

Figure 4.1: Distribution of “suspected” and “probable” cases of TSP arising

from non-commercially harvested shellfish. Number of cases = Total number of cases from January 1993-June 1999; (Number in brackets = Number of cases January 1994-June 1999).

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An analysis of the shellfish species consumed in the “suspected” and “probable” cases of TSP arising from non-commercially and commercially harvested shellfish is presented in Figure 4.2. Where several shellfish species were consumed and it is not possible to either identify the individual species, or identify separately which of several species is implicated in the TSP, the shellfish has been classified as “Combination”.

05

10152025303540

Musse

l

Scallo

p

Combin

ation

Tuatua Pipi

Oyster

Paua

Crayfis

hKina

Cockle Crab

Shellfish Species

Num

ber o

f Cas

es

Commercially harvested speciesRecreationally harvested species

Figure 4.2: Graph showing the number of “suspected” and “probable” cases

of TSP from January 1993 to June 1999 arising from the consumption of different seafood species. Figures for cases arising from non-commercially harvested seafood and commercially harvested seafood are shown separately.

ESR does not collect data regarding the incidence of respiratory irritation syndrome caused by brevetoxins or other lipid soluble toxins. There have been three incidents of respiratory irritation syndrome in New Zealand. One occurred in Orewa, north of Auckland in the summer of 1993 (Bates et al. 1993), one on the east coast of the North Island from Hawkes Bay to Cape Palliser in the summer of 1998, and the other in the Wellington Harbour area, also in the summer of 1998 (Chang et al., 1998a). In the two former events, several hundred people living on the coast or visiting the beach were affected. Approximately 80 people reported symptoms associated with the Wellington Harbour event. Symptoms included sore throat, dry coughing, and nose and eye irritation. People swimming in the Wairarapa area also reported skin irritation. It has been reported that asthmatic people may be more seriously affected than others (Catherine Hayes, Health Protection Officer, Wairarapa Health district, Hutt Valley Health, pers. comm.).

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4.4 DISCUSSION Because of the large number of variables impacting on the reported incidence of TSP in New Zealand, interpretation of the epidemiological data needs to be undertaken with care and caution. Interpretation of the data needs to take into consideration the following factors: • The amount of good data quality data is limited: Although 457 cases potentially

related to TSP have been reported between January 1993 and June 1999, only 9 of these (1.97%) have been corroborated as probably being some form of TSP (i.e. established as “probable” or “confirmed” status). There have been increases over time in the range and specificity of tests undertaken for toxins in leftover/same site samples of shellfish. In 1993, tests were not done on leftover/same site samples, so no cases could be either downgraded to “unlikely”, or up-graded to “confirmed”. Similarly, the testing of leftover/same site samples for only one or two toxin groups in the early years means that cases that may have resulted from, say ASP or DSP (which were commonly excluded from testing at that time), have not been established as “probable’ or “confirmed” cases. In addition, where tests from leftover or same site samples have been taken, and microbiological tests have been undertaken, the results have frequently not been entered into the database. A distortion of the proportion of cases in the “suspected” category is the result of these inconsistencies, and this must be considered when trying to draw conclusions from year-to-year comparisons. The lack of leftover samples, failure to collect “same site” samples in a timely manner, and incomplete data from 1995 (resulting in the inability to calculate the dose received in two cases with “probable” status), have also resulted in failure to corroborate “suspected” cases as “probable” or “confirmed”.

• It is not known what percentage of TSP cases actually get reported to the medical

authorities. It is likely that people suffering from neurological symptoms after consuming shellfish would seek the advice of their doctor, since these symptoms are somewhat unusual and likely to worry the sufferer. The level of familiarity of doctors with the symptoms of TSP, the proportion of cases that are recognised and reported as such, and the incidence of asymptomatic cases, are not known.

• It is likely that the incidence of DSP is under-reported in these data. The

symptoms of DSP are similar to those of many illnesses of microbiological sources related to shellfish consumption. In addition, cases arising from pectenotoxin, or from a suite of DSP toxins in which DTX-3 is predominant, will not be identified as TSP under the present surveillance system. Similarly, the surveillance system does not include yessotoxin.

• Data on the incidence of NSP prior to the introduction of testing using the acetone

extraction method as a screen test, followed by a bioassay using an ether extraction method (refer to section 1.2) may not accurately reflect the incidence of NSP. This may impact on the validity of the confirmation of an NSP case prior to September 1994, (currently assessed as “probable”).

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• Due to the absence of biomarkers, there are no recorded data on the effects of long-term exposure to low levels of any of the biotoxins.

The epidemiological data suggest that there is a general trend toward a reduction in the number of cases of TSP being reported (Table 4.1). There are several factors that could potentially contribute to this trend: a) the overall decrease in marine biotoxin events since 1993; b) an increasingly effective marine biotoxin monitoring programme, ensuring that fewer people are exposed to marine biotoxins; and c) a decreasing awareness amongst medical practitioners of the symptoms of TSP. It is suggested that medical practitioners be reminded of the symptoms of TSP, and the requirement to report suspected cases. One general practitioner questioned about TSP by the authors commented that “the general public are kept better informed about TSP than are medical practitioners”. While the gender distribution of TSP cases is similar to that of the general population in New Zealand, the age structure appears somewhat different (Table 4.3). There appears to be a lower than expected number of cases of people less than 20 and 80+ years old, and a higher than expected number of cases in the 30-59 age group. While this could potentially be due to an increased susceptibility of the 30-59 age group to TSP, it may also be a result of a higher level of consumption of shellfish. Similarly, the lower level in the younger and older age groups could result from lower levels of shellfish consumption. Within the limits of the data collected, the distribution of cases of TSP from non-commercially harvested seafood with respect to ethnic origin is similar to that of the total population in New Zealand (Table 4.3). With respect to commercially harvested shellfish, the higher than expected percentage of NZ European cases, and slightly lower percentage of Maori and Pacific Island cases may be a reflection of how shellfish is obtained by these groups. In cases arising from both commercially and non-commercially harvested shellfish, people of ethnic origin other than NZ European, Maori or Pacific Island have lower reported incidence of TSP than would be predicted from the population structure. The results are also surprising given the data on ethnicity of shellfish gatherers presented in Section 3, which suggests that Maori and Asian populations potentially form a much more significant percentage of non-commercial harvesters of shellfish (for example, 54% and 15% respectively in one study in the Whangateau Harbour). The distribution of cases of TSP does not reflect this activity. One reason for this could be differences in medical presentation and reporting rates between people of different ethnic origins. Except in cases where ethnic origin has not been recorded, a greater percentage of cases in all ethnic groups arose from non-commercially harvested seafood (Table 4.5). NZ Europeans were the group with the lowest percentage of cases from non-commercially harvested seafood. This may be a reflection of a lower level of non-commercial harvesting activity by this group. With respect to a comparison between the incidence of cases of TSP arising from commercial and non-commercial harvesting, the high percentage of cases related to non-commercial harvesting in 1993 is a reflection of the geographical location of shellfish contaminated with biotoxins, and the location of major commercial

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harvesting areas elsewhere. The event also occurred over a summer holiday season when non-commercial harvesting was particularly high. The comparison between the incidence of cases of TSP from commercial and non-commercial harvesting since the institution of the marine biotoxin monitoring programme is more pertinent to the comparative effectiveness of commercial and non-commercial programmes. The percentage of total cases from non-commercial harvesting is only slightly higher than that from commercially harvested shellfish (54.1% compared to 45.9%). However, if only the “probable” cases of TSP are considered, the result is somewhat different: eight out of nine “probable” cases (88.9%) arose from the consumption of non-commercially harvested shellfish. This suggests that the non-commercial marine biotoxin monitoring programme is less effective at protecting public health than the commercial marine biotoxin monitoring programme. This is not surprising, given the differences between the programmes with respect to the level of control over harvesting in the event of biotoxin occurrence.

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SECTION 5: RISK ASSESSMENT 5.1 INTRODUCTION A commonly used public health risk analysis model consists of three phases: risk assessment, risk management, and risk communication (USEPA, 1993). This section of the report provides a risk assessment with respect to Toxic Shellfish Poisoning (TSP) arising from consumption of non-commercially harvested shellfish in New Zealand. “Risk assessment defines the potential safety and health effects from exposure of individuals and populations to hazardous materials and situations” (USEPA, 1993). The USEPA risk analysis model outlines four interrelated steps to risk assessment (USEPA, 1993). These are: • Hazard identification: identification of the environmental agent of concern, its

adverse effects, target populations and conditions of exposure. • Dose-response assessment: determination of the degree of the effects at different

doses. • Exposure assessment: estimation of the magnitude, duration and frequency of

human exposure to pollutants of concern, and the number of people exposed via different pathways.

• Risk characterisation: combination of the information obtained from the hazard

identification, dose-response assessment, and exposure assessment to estimate the risk associated with each exposure scenario considered, and to present information on uncertainties in the analysis.

The following sections contain a risk assessment for each of PSP, ASP, DSP, NSP and respiratory irritation syndrome. 5.2 PARALYTIC SHELLFISH POISONING 5.2.1 Hazard Identification The toxins that cause Paralytic Shellfish Poisoning (PSP) comprise a suite of naturally occurring neurotoxic tetrahydropurine derivatives, which act as sodium channel blocking agents. Cembella et al., (1995b) summarised the toxin syndromes of PSP: “Paralytic Shellfish Poisoning is a neurotoxic syndrome resulting primarily from the blockage of neuronal and muscular Na+ channels. Binding to the Na+ channel prevents propagation of the action potential that is essential to the conduction of nerve impulse and muscle contraction. In vertebrates, the peripheral nervous system is particularly affected; typical symptoms of poisoning include tingling and numbness of the extremities, progressing to muscular incoordination, respiratory distress, and muscular paralysis leading to death by asphyxiation in extreme cases”. Initial symptoms occur within 30 minutes of ingestion, and in extreme cases, respiratory paralysis may occur within 2-24 hours after ingestion (Hallegraeff, 1995). The

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fatality rate may exceed 10%, particularly when medical attention is not available (Wilson, 1996). There are no known antidotes to the toxin. Treatment includes pumping out the patient’s stomach, and the application of artificial respiration. In cases that are not fatal, there are no lasting effects (Hallegraeff, 1995). Exposure to the toxin does not confer immunity. In New Zealand, several species of the dinoflagellate Alexandrium produce biotoxins known to cause PSP. They include Alexandrium angustitabulatum, A. minutum, A. ostenfeldii, and an unidentified species isolated from Marsden Point in Northland. Alexandrium tamarense is known to produce PSP toxins overseas, but as yet the New Zealand strains have not been tested or implicated in the occurrence of PSP toxins in shellfish. Given possible variations in toxin production between different strains of the same species, and that phytoplankton monitoring has not always identified the origin of detectable levels of PSP in shellfish, it is possible that other species of Alexandrium in New Zealand waters will also produce PSP toxins. Different strains of Alexandrium have different toxin profiles (Cembella et al., 1987). Since different toxin derivatives have different toxicity, the toxin profile impacts on the overall toxicity of the strain. Because of the tendency of shellfish to accumulate PSP toxins from toxic phytoplankton, shellfish consumers are exposed to PSP toxins when they consume shellfish that have been harvested from areas where PSP-producing phytoplankton are present. Shellfish consumers are thus the target population for PSP. Shellfish consumers are reliant on a marine biotoxin monitoring programme to determine whether shellfish are contaminated with PSP toxins – it is not possible to ascertain this by the appearance of the shellfish, or the water from where they are harvested. The comparatively low densities of toxic Alexandrium species required to produce significant PSP levels in shellfish are not obvious as a “bloom” in the water. The preparation of the shellfish may impact on the risk of PSP to the consumer – for example, whole scallops are more toxic than scallop roe. However, exclusion of high risk portions of the shellfish is the only step that can be taken in the preparation of shellfish for consumption to reduce the risk of PSP. Cooking does not destroy PSP toxins. The risk of PSP varies not only with the degree of exposure of the shellfish to PSP-producing phytoplankton and the toxin profile of the phytoplankton, but also with the species of shellfish. Analysis of coincident monitoring data suggests that the presence of PSP-producing Alexandrium species in shellfish growing water results in significantly higher levels of toxicity in tuatua than in GreenshellTM mussels. Qualitative analysis of a very limited amount of data on toxicity levels in other species suggests that scallops do not become as toxic as tuatua, but accumulate higher PSP toxin levels than GreenshellTM mussels, pipi and cockles. These differences may be related to differences in ingestion of toxin, or in detoxification processes within the shellfish. These results are not in any way definitive, and more data collection is required to investigate this further.

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5.2.2 Dose-Response Assessment A summary of international data on PSP dose-response relationships for adults was provided in the previous review of the marine biotoxin monitoring programme (Wilson, 1996). Wilson (1996) concluded that “It is estimated that 200 to 500 µg of saxitoxin will produce at least mild symptoms, 500 to 2,000 µg is likely to cause moderate to severe symptoms, and consumption of over 2,000 µg is likely to produce serious and possibly lethal consequences. However, the variability in PSP toxicity is quite marked”. There has been one probable case of PSP in New Zealand. The patient had consumed an estimated 50 g of tuatua taken from Ohope Beach in January 1999. Tuatua taken from the same site two days later were tested and found to contain 230 µg STX eq./100g. If it can be assumed that the PSP level in the shellfish had not decreased significantly over the two day interval, this suggests an estimated dose of 115 µg of saxitoxin was consumed by the 60 kg patient. Symptoms experienced included: nausea, vomiting, diarrhoea, numbness and tingling sensation in face and hands, unstable walking, blurred vision, difficulty swallowing, slurred speech, weakness and drowsiness, a rash on the skin, headaches, back pain and aching joints. 5.2.3 Exposure Assessment Given the data available, it is extremely difficult to provide a meaningful exposure assessment that is in any way quantitative. Analysis of marine biotoxin monitoring data in Section 2 identified that PSP toxin levels in shellfish varied within zones from year to year, and that overall occurrence of PSP in shellfish also varied annually across New Zealand as a whole. As a result of this, and in consideration of the complex set of variables that may impact on PSP toxin occurrence, it was concluded that predictions of risk based on historical data would be inappropriate until a much greater depth of understanding about the dynamics of Alexandrium blooms in New Zealand has been gained through long-term studies. Exposure assessment related to the consumption of non-commercially gathered shellfish is further complicated by potential differences in toxin ingestion and detoxification between different shellfish species, variations in the distribution of shellfish species, and lack of robust data regarding the location and quantity of shellfish gathered and consumed by non-commercial harvesters. However, the following observations regarding the risk of exposure to PSP can be made: • The frequency of occurrence of PSP levels in shellfish above the level that

represents a risk to consumers has been relatively low (one or two samples over 6 years) or zero in most areas since July 1993. An exception to this is in the Bay of Plenty, where there has been a long-running occurrence of PSP, with levels both above and below the regulatory level in many of the months between March 1995 and the present. It is not possible to quantify the overall frequency of occurrence of PSP toxin levels that represent a potential risk to consumers, due to temporal and spatial stratification in the sampling regime.

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• With exception to the Bay of Plenty, the maximum toxin levels in most areas have been low (82 µg/100g and 127 µg/100g compared to a regulatory level of 80 µg/100g ). The maximum toxin level in the Bay of Plenty (Zone D) was 1007 µg/100g, which is high enough to cause moderate to severe symptoms in human consumers. In the Bay of Plenty, the site with the greatest number of samples with PSP toxin levels above the regulatory level between 1/7/93 and 30/6/99, (Site D37, Ohope Beach), contained PSP toxin levels in samples over 18 weeks that were sufficient to produce mild symptoms in consumers (PSP >115 µg/100g and <500 µg/100g). PSP levels sufficient to produce moderate to severe symptoms (>500 µg/100g and <2,000 µg/100g) occurred on 1 week during this period at this site. This is potentially an understatement of risk, since sampling was discontinued when toxin levels were high. At one of the most consistently monitored sites in this area, D41 (Whangaparoa), PSP toxin levels sufficient to produce mild symptoms occurred on 4 weeks, and levels sufficient to produce moderate to severe symptoms on 1 week. At Tokata (D38), a sample site that has not been consistently monitored, toxin levels sufficient to produce mild symptoms were recorded on 2 weeks, and sufficient to produce moderate to severe symptoms on 3 weeks. Again, this is probably understated, as sampling was discontinued at times when toxin levels were high. PSP toxin levels in shellfish at other sites in the Bay of Plenty that were inconsistently monitored had between 1 week and 12 weeks on which toxin levels were sufficient to produce mild symptoms in shellfish consumers. The numbers of people that would have been potentially exposed to PSP toxins in this area in the absence of a marine biotoxin monitoring programme are uncertain, due to inadequate data on non-commercial harvesting of shellfish. However, the diary survey of Fisher & Bradford (1998) suggested that Zone D is an area where a comparatively high level of non-commercial shellfish gathering activity has occurred (25% of the total New Zealand fishing trips targeting bivalve species).

• Available data suggest that the risk of exposure to PSP toxins varies with the

species of shellfish, with tuatua being a shellfish that represents a high risk. Further work is required to clarify these differences.

• Phytoplankton monitoring indicates that potentially toxic Alexandrium species are

widely distributed in New Zealand. The mechanisms related to the disappearance of previously persistent PSP toxicity in shellfish at Tokerau Beach, and the persistence of PSP toxicity in the Bay of Plenty are not understood. Until they are, it cannot be assumed that PSP will persist in the Bay of Plenty, or not occur elsewhere.

• There are no obvious seasonal patterns in the occurrence of PSP toxicity in

shellfish. However, research suggests that for a range of inter-tidal shellfish, non-commercial harvesting is highest in the summer, and lowest in the winter (Kearney, 1999; Hartill & Cryer, 1999).

5.2.4 Risk Characterisation Meaningful quantitative assessment of the risks associated with PSP through the consumption of non-commercially harvested shellfish is not possible due to the lack of robust information about consumption of non-commercially harvested shellfish.

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Data collected from the marine biotoxin monitoring programme, combined with research results overseas, are insufficient to make robust predictions about the frequency of occurrence, or location of PSP toxins in New Zealand in the future. However, several scenarios are presented in order to examine the kinds of human impacts that PSP could have in New Zealand. (Even these scenarios are difficult to construct, due to the discontinuity of sampling in Zone D at times when PSP toxins were present). Data from the diary survey of recreational fishing in New Zealand by Fisher & Bradford (1998) indicated that diarists recorded taking 4,341 tuatua from Zone D in the 12 months of the survey. Fisher and Bradford (1998) apply a scaling factor of 139.9 to calculate the total non-commercial harvest figures from the figures recorded by the diarists. Using this scaling factor, and if an average meal size of tuatua is 100g, equating to 12 tuatua, the average number of meals per week of non-commercially harvested tuatua from Zone D would be 973. In a year like July 1995-June 1996, there were approximately 2 weeks when PSP levels in shellfish in the Bay of Plenty would produce moderate to severe symptoms in consumers, and 2 weeks when consumption of shellfish would result in mild PSP symptoms. (These figures are estimated over all the tuatua sites where PSP toxin levels occurred, taking into account discontinuities in the sampling). This suggests that in the absence of a marine biotoxin monitoring programme, and assuming that there is no communication of risk that prevents consumers eating shellfish in the event of PSP occurrence, 1,946 people could suffer moderate to severe PSP symptoms, and 1,946 people mild PSP symptoms each year. These figures are calculated from estimated consumption of tuatua alone (apparently the species that represents the greatest risk, and harvested from the area in Zone D where the toxin occurred), and do not include exposure to toxins through consumption of other species of shellfish. In reality, communication of the risk through media reports means that the actual number of people likely to become ill would be somewhat lower. In contrast, in a scenario similar to the July 1994-June 1995 year, there would be no cases of PSP in Zone D, and none from elsewhere. Data from Hay (1996) found that 31% of Maori households in the far north of New Zealand collected seafood for consumption at least once a week, and 52% at least fortnightly. Information from a variety of Maori informants suggests that approximately 30% of this seafood is shellfish. This means that in the Far North, approximately 10% of the Maori households collect shellfish at least weekly, and about 16% at least fortnightly. If similar toxin levels as occurred in Zone D in 1995-96 were to occur in the far north, then a significant percentage of the Maori population would be at risk from PSP. At this point it is worth considering these scenarios somewhat objectively. The wide disparity between the results of the Fisher & Bradford (1998) diary survey, and the results of the study by Kearney (1999) with respect to non-commercial harvest of cockles, brings into question the reliability of the harvest data used in these scenarios. This highlights the need for more comprehensive and robust data in order to estimate the risk of PSP and other marine biotoxins accumulated by shellfish in New Zealand.

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5.3 AMNESIC SHELLFISH POISONING 5.3.1 Hazard Identification Amnesic Shellfish Poisoning is caused by Domoic acid (Wright et al., 1989). Domoic acid is an excitatory amino acid derivative acting as a glutamate agonist on the Kainate receptors of the central nervous system (Cembella et al., 1995b). “This secondary amino acid is considered to be a more potent neuroexcitor than Kainic acid, which when systemically injected into specific parts of the brain is known to have degenerative effects. Domoic acid is considered to be the primary toxin involved in ASP, although isomeric forms (e.g. iso-domoic acid) of lesser potency also occur naturally”(Cembella et al., 1995b). Pennate diatoms within the genus Pseudo-nitzschia produce Domoic acid implicated in ASP. Domoic acid has been confirmed in some but not all New Zealand isolates of Pseudo-nitzschia australis, P. pungens, P. turgidula, (Rhodes et al., 1996), P. delicatissima and P. pseudodelicatissima (Rhodes et al., 1998b). Two other species are a frequent component of Pseudo-nitzschia blooms: P. heimii and P. multistriata (L. Rhodes, Cawthron Institute, pers. comm.), but to date neither have been found to be toxic. A mild case of ASP causes nausea, vomiting, diarrhoea, and abdominal cramps, within 3-5 hours. In severe cases, patients suffer a decreased reaction to deep pain, dizziness, hallucinations, confusion, short term memory loss, and seizures (Hallegraeff, 1995). Most severe cases have been found to show continued selective memory loss, particularly short-term memory (Todd, 1993). Because of the tendency of shellfish to accumulate ASP toxins from toxic phytoplankton, shellfish consumers are exposed to ASP toxins when they consume shellfish that have been harvested from areas where ASP-producing phytoplankton are present. Shellfish consumers are thus the target population for ASP. The age of the consumer may affect the severity of the symptoms of ASP experienced. For example, there appears to be a close association between memory loss and age: those people under 40 years old are more likely to have diarrhoea, and those over 50 to have memory loss (Todd, 1993). Other symptoms are not related to age. However, people in poor health are more likely to be more severely affected (Todd, 1993). Shellfish consumers are reliant on a marine biotoxin monitoring programme to determine whether shellfish are contaminated with ASP – it is not possible to ascertain this by the appearance of the shellfish or the water from where they are harvested. Risk may be reduced somewhat in the preparation of the shellfish for eating: toxin levels may vary with the parts of the shellfish consumed (for example, scallop guts tend to contain higher levels of ASP toxins than the roe or muscle). Exclusion of the high risk portions of shellfish can reduce the risk of ASP. It has been suggested that cooking, and subsequent discarding of the cooking water, might reduce the risk of ASP since the toxins are water soluble (Wilson, 1996). Experiments with Dungeness crabs showed that cooking appears to decrease the level of Domoic acid in the viscera while translocating small amounts into the meat (Loscutoff, 1992). However, how this applies to shellfish is unknown.

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The risk of ASP may also vary with shellfish species consumed. Limited data from concurrent sampling of different shellfish species from the same sites suggest that scallops accumulate and retain higher levels of Domoic acid than mussels or cockles. More data are required to investigate this further. 5.3.2 Dose-Response Assessment A summary of international data on ASP dose-response relationships for adults was provided in the previous review of the marine biotoxin monitoring programme (Wilson, 1996). Wilson concluded: The minimal dose for acute symptoms has been estimated as being in the 5-10 mg range, with moderate to severe symptoms, including memory loss, associated with doses in the 60-290 mg range. There have been no confirmed cases of ASP in New Zealand. 5.3.3 Exposure Assessment The following observations can be made regarding the risk of exposure to ASP: • The frequency of occurrence of ASP levels in shellfish above the level that

represents a risk to consumers has been relatively low, with zero or only one sample over 6 years in most zones. An exception to this very low frequency is in Zone A, where 33 samples above the regulatory level have occurred since July 1993.

• The frequency of occurrence of Domoic acid, above the level of detection in

shellfish, may vary from year to year, within zones, and across all zones. Both Pseudo-nitzschia species, and the occurrence of low levels of Domoic acid in shellfish, are widely distributed throughout New Zealand. There are insufficient data for robust predictions about future occurrence to be made.

• In Zone A, all Domoic acid levels above the regulatory level have occurred in

scallop samples, with the exception of three GreenshellTM mussel samples. In Zone A, 7.75% of scallop samples taken between 1/7/94 and 30/6/99 contained levels of ASP above the regulatory level (Note that scallop sampling is seasonal, and includes the time of year when ASP is most frequent). Qualitative analysis suggests that there may be significant differences in accumulation and retention of Domoic acid between different shellfish species, and that scallops may be a species that represents a comparatively high risk to consumers. 74.4% of Zone A scallop samples taken in the harvesting season between 1/7/94 and 30/6/99 contained detectable levels of Domoic acid, as did 26.6% of scallop samples in Zone D. While there are currently no known chronic effects caused by long-term ingestion of low levels of Domoic acid, these frequencies would be a cause for concern should this be the case.

• Inconsistent sampling confounds quantification of the magnitude of toxin levels.

Observations suggest that the toxin levels vary in different parts of scallops, with the highest levels found in the gut and skirt, and lower levels in the roe and muscle. The maximum level of Domoic acid found in whole scallops was 210 µg/g, while a level of 600 µg/g was found in the gut and skirt in another sample.

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The maximum level in GreenshellTM mussels was 187 µg/g. Assuming that mild symptoms of ASP arise from a dose that is 50 µg/g – 599 µg/g (i.e. 5-59.9 mg/100g), then consumers of non-commercially harvested shellfish in Zones D & G would have been exposed to ASP for one week at one site over 6 years. Domoic acid level in scallops (either whole, or muscle and roe only) from Zone A were sufficient to produce mild ASP symptoms in consumers of 100g of shellfish for 2 weeks in July 1993-June 1994, 3 weeks in 1994-95, and 1 week in 1996-97. None of the levels in mussels in Zone A were sufficient to produce even mild symptoms assuming consumption of only 100g.

• There appears to be a possible broad seasonal pattern in the occurrence of Domoic

acid in shellfish, with increased risk of exposure to ASP for consumers of shellfish from August to December. Research suggests that for a range of inter-tidal shellfish, non-commercial harvesting is highest in the summer, lowest in the winter, and at intermediate levels in spring and autumn (Kearney, 1999; Hartill & Cryer, 1999). However, there are no data available regarding seasonality of non-commercial harvest of sub-tidal shellfish such as scallops (although the time period when scallops may be legally harvested runs from July through to February).

5.3.4 Risk Characterisation Quantitative estimation of the risk of ASP in New Zealand can only be undertaken if large assumptions about non-commercial harvesting patterns and consumption are made. If scenarios are created using the occurrence of ASP in scallop beds in Zone A as a model, assumptions need to be made about the level of non-commercial harvesting from each bed. A range of scallop beds in Zone A appears to have exhibited ASP toxicity above regulatory levels in different years: the Whangaroa/Cavelli Islands area in 1993 and 1995, Doubtless Bay in 1994, and Rangaunu Bay/Houhora Bay in 1996-97. From the diary survey of Fisher & Bradford (1998), the total non-commercial harvest of scallops in Zone A can be estimated. An assumption is made that the harvest is divided equally across the beds in all areas, and that the non-commercial harvest of scallops from Spirits Bay (in the far north) is negligible because it is less accessible to small boats, and further from population bases. It is assumed that the average scallop meal from non-commercially harvested scallops would consist of 12 scallops, equating to 200g in weight. Under these assumptions, there would be 83 meals of scallops harvested from each bed each week of the scallop harvest season. In a year similar to July 1996-June 1997, there would be 9 weeks over which Domoic acid levels were sufficient to produce mild symptoms of ASP, provided that only the muscle and roe of the scallops were consumed. In the absence of any publicity about illness, this would mean that 747 people could potentially be exposed to ASP. However, given that the occurrence of Domoic acid is likely to be in consecutive weeks, and some publicity of illness is likely, then the number of mild cases of ASP is likely to be somewhere between 747 and 83. Conversely, in a year similar to 1997-98, there would be no cases of ASP from this area.

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The widely recognised cultural importance of seafood for Maori, and some survey data (e.g. Kearney, 1999) suggest that Maori may be disproportionately affected by marine biotoxins. As discussed previously, results from a survey by Hay (1996) and subsequent discussion with Maori informants suggest that 10% of Maori households eat non-commercially harvested shellfish each week, and approximately 16% at least once a fortnight. Data from the diary survey of Fisher & Bradford (1998) indicates that in Zone A, 55% of non-commercial fishing trips targeting bivalve shellfish target scallops. If it could be assumed that harvest quantities are proportional to target trips (a very large assumption) then it follows that 5.5% of the Maori community consume scallops each week in Zone A and approximately 8.8% at least fortnightly. This suggests that a significant proportion of the Maori population would be exposed to mild ASP in a year similar to 1996-97. The scenarios presented have utilised Zone A as a model, because this is the area where Domoic acid has most frequently occurred to date. However, given the wide distribution of both low levels of Domoic acid in shellfish and Pseudo-nitzschia species, ASP events could potentially occur in other areas also. If for example, similar toxin levels as occurred in Zone A in 1996-97 were to occur in the eastern Coromandel area (Zone D), in one of three major scallop harvesting areas (Mercury Islands, Slipper Island, or Whangamata to Waihi), the potential cases of ASP would be higher. Using harvest data from the diary survey of Fisher & Bradford in the Eastern Coromandel, it is estimated that there are 687 200g meals of scallops consumed every week over the scallop season. If these are distributed equally over the scallop beds, and a toxin scenario similar to that in Zone A in 1996-97 were to occur at one location, then in the absence of a marine biotoxin monitoring programme, there would potentially be 2,061 mild cases of ASP in the year. (This assumes that there is no publicity to prevent continuing exposure). Should a similar incident occur in the Nelson area, then harvest figures suggest that the number of cases could be up to five times higher. The risk of exposure, and the severity of the impact, are increased if the shellfish guts (particularly scallop guts) are consumed. There are no robust data on the numbers of people who consume whole scallops, as distinct from muscle and roe. Forty-four percent of cases on the epidemiological databases who had eaten scallops had consumed the whole shellfish. However, this proportion is not representative of scallop consumers in general, since those who eat whole scallops are more at risk of TSP, and thus more likely to be represented on the epidemiological database. It does however, indicate that whole scallops are consumed by some members of the community. It should be noted that discarding parts of the shellfish is contrary to Maori traditional practices related to moumou kai. Moumou kai requires that people gather just what they need, and that they eat all that is gathered without waste (Wyllie, 1995). However, in scenarios like those presented from Zone A, the consumption of whole scallops would not increase the numbers of moderate to severe ASP cases. However, if only the gut and skirt were consumed, then it is likely that a greater number of severe cases of ASP would occur under this scenario. Although the scenarios presented have resulted in only mild cases of ASP, the dose-response figures are not adjusted for body weight. It has also been assumed that only 200g of shellfish are consumed. Variations in body weight, and amount of shellfish consumed will also cause variation in the severity of ASP symptoms.

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5.4 DIARRHETIC SHELLFISH POISONING As discussed in Section 1.3.3, there are several different toxins in the “DSP group”. However, because they are frequently found together, and are all extracted by the acetone extraction method, they tend to be grouped together. All these toxins, whether diarrheagenic or not, are included in the risk assessment in this section. 5.4.1 Hazard Identification The toxins in the “DSP group” can be sub-divided into several groups: Okadaic acid and the closely related dinophysistoxins: the pectenotoxins, which are polyether lactones consisting of three compounds with known structure (PTX 1-3) and at least two additional compounds with presumed slightly modified skeletons; and thirdly, yessotoxin (YTX), with two sulphate esters, which resemble the brevetoxins (Aune & Yndestad, 1993). The DSP toxins Okadaic acid and dinophysistoxins DTX1 and DTX3, produce diarrheagenic effects. (DTX-3 hydrolyses back to the more toxic DTX-1 in the body). These substances are potent inhibitors of at least two sub-classes of protein phosphatases (PP1 and PP2A), and this mode of action may be linked to diarrhoea, degenerative changes in the absorptive epithelium of the small intestine, and to tumour production. Symptoms of human intoxication occur within 30 minutes to a few hours (seldom more than 12 hours) and include diarrhoea, nausea, vomiting, and abdominal pain (Hallegraeff, 1995). Although no human mortalities have been reported, the affliction can be highly debilitating for several days. The longer term potential effects of OA and DTX1 are of more concern. Okadaic acid and DTX1 have tumour-promoting activity through their inhibition of the activity of protein phosphatases 1 and 2 (Bialojan & Takai, 1988). In laboratory tests, Okadaic acid has also been shown to exhibit some mutagenic properties and immunotoxic effects (Aune & Yndestad, 1993; Fessard, 1998). Laboratory trials have also suggested that DTX1 has enterotoxic effects (Terao et al., 1990). There is some discussion about whether OA and DTX1 have long term tumour promoting and mutagenic effects if only low levels of toxin are ingested. This remains controversial. Most, but not all, research on this has been done using high toxin levels and administration by intraperitoneal injection. Fessard (1998) summarised the research on the potential carcinogenic effects of OA, and concluded that “It can be suggested that OA can be considered as a genotoxic compound forming (directly or indirectly) DNA adducts and that its promotion capacity is in fact due to the Tumour Necrosis Factor ∝ (TNF- ∝µ ) induction. In that case, we must be aware that the risks incurred for human health, mainly during chronic intoxications, are underestimated. Therefore, the toxin levels allowed for consumption should be reconsidered”. A Working Group on Toxicology of Yessotoxins, Pectenotoxins and the Okadaic Group Toxins has been set up by the European Community Reference Laboratory on Marine Biotoxins. At a meeting of this Group in February 1999, it was concluded that “According to the data on tumour promotion activity of Okadaic acid, there is no evidence that there may be a risk of a long term hazard for shellfish consumers under the current regulations regarding diarrheagenic substances. There

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is no good evidence of the genotoxic potential of OA. Only the diarrheagenic activity of OA should be a matter of concern to establish regulatory limits for these toxins”. It was suggested that the level of 16 µg/100g should offer sufficient protection to consumers (note that the regulatory level in New Zealand is currently 20 µg/100g). Few studies have yet been undertaken on the impact of yessotoxin. Pathological effects on the heart and liver have been shown after intraperitoneal injection of mice. Experimental studies based on oral ingestion have suggested that high levels of yessotoxin produce pathogenic effects in the gastrointestinal tract. More recent studies suggest that yessotoxin may have little impact when ingested orally at the levels likely to be encountered in shellfish (Ogino et al., 1997). Until recently, research on yessotoxin was hindered by a lack of knowledge of causative agents limiting the toxin available to work with. However, the identification of Protoceratium reticulatum as a producer of yessotoxin (Satake et al., 1997) will facilitate research in this area. In the mean time, the limited knowledge of the toxicity of yessotoxin means that it remains a public health risk because of potential long-term effects. Research into the toxic effects of pectenotoxin is severely limited by the inability to culture Dinophysis species (the known producers of pectenotoxins) in the laboratory. Until recently, there was no evidence of human illness caused by pectenotoxins. However, pectenotoxin has been implicated in a TSP incident that occurred in New South Wales, Australia, in which a number of people became ill, with symptoms that included vomiting, and diarrhoea (P. Truman, ESR, pers. comm.). Mackenzie et al. (1999) suggest that the pectenotoxins PTX-2 and PTX-2 Seco acid may be the predominant DSP-toxins in New Zealand, and may be of greater concern to human health than Okadaic acid. This conclusion was based on the following (Mackenzie et al., 1999):

• PTX-2 induces diarrhetic symptoms and severe mucosal injuries in the small intestine of mice by intraperitoneal injection and oral administration (Ishige et al., 1988; Terao et al., 1986)

• PTX-2 causes liver necrosis in mice by oral administration (Terao et al., 1986)

• PTX-2 has potent and selective cyto-toxicity against human lung, colon and

breast cancer cell lines (Jung et al., 1995).

• PTX-2 is implicated in cases of gastro-intestinal illness in Australia. Yessotoxin and PTX-2 and its derivatives are detected by the current mouse bioassay screening procedures but are not detected by confirmatory ELISA (DSP Check-Kit) or protein phosphatase inhibition assays. Okadaic acid and dinophysistoxins are produced by dinoflagellates in the genera Dinophysis and Prorocentrum. Species that have been found to be toxic in New Zealand include Dinophysis acuta and Prorocentrum lima (Rhodes & Syhre, 1995). Low levels of DSP toxins in shellfish have also been associated with Dinophysis acuminata in the water (L. Mackenzie, Cawthron Institute, pers. comm.). Yessotoxin is produced by Protoceratium reticulatum (Satake et al., 1997, Mackenzie et al.

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1998b). In New Zealand, Dinophysis acuta has been found to produce the pectenotoxins PTX-2 and Pectenotoxin-2 Seco acid (Daiguji et al., 1998). Because of the tendency of shellfish to accumulate DSP toxins from toxic phytoplankton, shellfish consumers are exposed to DSP toxins when they consume shellfish that have been harvested from areas where DSP-producing phytoplankton are present. Shellfish consumers are thus the target population for DSP from Okadaic acid, dinophysistoxins, yessotoxin and pectenotoxin. Shellfish consumers are reliant on the marine biotoxin monitoring programme to determine whether shellfish are contaminated with DSP toxins – it is not possible to ascertain this by the appearance of the shellfish, or the water from where they are harvested. It is possible that the portions of the shellfish eaten may vary in toxin content. The risk of DSP (including poisoning from Okadaic acid, dinophysistoxin, yessotoxin and pectenotoxin) may vary not only with the degree of exposure of the shellfish to the toxic phytoplankton, and the toxin profile of the phytoplankton, but also with the species of shellfish consumed (Mackenzie et al., 1998b; Rhodes et al., 2000). More research is required to investigate this further. 5.4.2 Dose-Response Assessment Overseas experience suggests that diarrheagenic effects from Okadaic acid and DTX toxins start at ingested amounts of 40 µg and 30 µg respectively (Aune & Yndestad, 1993). Cases from consumption of New Zealand shellfish suggest that 20-30 µg of DSP toxins may produce symptoms (Wilson, 1996). However, it is possible that some of these symptoms may have resulted from ingestion of coincident pectenotoxin that would not have been detected by the DSP ELISA. There is no information on the long term mutagenic and immunotoxic effects of Okadaic acid or dinophysistoxins ingested orally by humans. Similarly, there is no dose response information for the oral ingestion of yessotoxin by humans, with regard to either short-term or long-term effects. Illness has been reported in New South Wales, Australia, following the consumption of shellfish contaminated with pectenotoxin. Information from this event suggests that 30-60 µg of pectenotoxin was ingested by the people affected (Mike Quilliam, National Research Council of Canada, pers. comm.). However, the dose response information arising from this incident has not been published, and there is no information regarding long-term impacts or the impact of long-term ingestion at low levels. 5.4.3 Exposure Assessment The following observations regarding the risk of exposure to DSP toxins can be made: • The Dinophysis species producing Okadaic acid, dinophysistoxins, and

pectenotoxin are widely distributed throughout New Zealand, and have been found at most sample points. There are inadequate data to determine the distribution of phytoplankton producing the other “DSP” toxins: Prorocentrum lima, and Protoceratium reticulatum.

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• “Classic DSP” toxins (i.e. Okadaic acid and dinophysistoxins) above the regulatory level of 20 µg/100g shellfish tissue have occurred relatively rarely in New Zealand since monitoring for DSP began. They occurred in only 4 zones (Zones A, E, G, and I) from 1/4/94 to 30/6/99. The highest frequency of occurrence was in Zone G, where 71 samples (1.36% of total samples) above the regulatory level were recorded. Most of the DSP detected in Zone G was from one site, Wedge Point (G23) in the Marlborough Sounds. Blue mussels at this site contained persistent levels of DSP from November 1994-August 1995, November 1995 to July 1996, and October to March 1997. The DSP in Zone I (the area with the next highest frequency of occurrence of DSP toxins in shellfish) was also predominantly from one site, Akaroa Harbour (I14), where shellfish toxicity persisted from February 1995 to October 1995. The shellfish species sampled at this site were also blue mussels. There are insufficient data to comment on the distribution and frequency of occurrence of yessotoxin and pectenotoxin in shellfish in New Zealand.

• There is some indication that there may be differences in accumulation and

retention of DSP toxins between different species of shellfish, but further investigation is required to determine these differences. Blue mussels are a species of shellfish that are relatively infrequently sampled in the marine biotoxin monitoring programme, but exhibited a significantly higher frequency of occurrence of DSP toxins than other species.

• The maximum DSP toxin levels (as determined by the DSP ELISA) in Zones A,

E, G and I were 33, 39, 96 and 86 µg/100g respectively. At Wedge Point (G23), where the highest frequency of samples containing DSP levels above the regulatory level occurred, the mean of these values was 46.8 µg/100g, and the median 44 µg/100g. If it is assumed, as suggested by Wilson (1996), that ingested levels of Okadaic acid/dinophysistoxins as low as 20 µg/100g can produce DSP symptoms, then levels sufficient to produce at least mild symptoms of DSP occurred in 82 weeks in the time period between 1/9/94 and 30/6/99.

• There are insufficient data to comment on the levels of pectenotoxin or yessotoxin

in shellfish in New Zealand with respect to risk to shellfish consumers. • The frequency of occurrence of Okadaic acid and dinophysistoxins has varied

significantly between years since shellfish monitoring began. There are insufficient data to be able to predict future occurrence.

• There is a possibility that there is a slightly lower risk of DSP in the winter

months, but further data are required to clarify this. If this were the case, the times of higher risk would coincide with the times of higher non-commercial shellfish harvest activity (Kearney, 1999; Hartill & Cryer, 1999).

5.4.4 Risk Characterisation Lack of robust data makes estimation of the risks associated with DSP very difficult. There are no data on the occurrence of pectenotoxin and yessotoxin on which to base meaningful analysis. Neither is definitive information available about effective doses,

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nor chronic effects of ingestion of these toxins over a longer period of time. These uncertainties effectively increase the potential risks associated with the occurrence of pectenotoxin and yessotoxin in New Zealand. There is a dearth of robust data on which to base sensible quantitative estimation of risks associated with “classic DSP”. While data for non-commercial harvesting of shellfish are available from the Fisher & Bradford (1998) diary survey for Queen Charlotte Sound (the sound within which lies Wedge Point), there are no non-commercial harvesting data available for Akaroa Harbour alone. This limits the construction of scenarios based on previous events. However, given that Wedge Point is the site where DSP toxins have been found most frequently, it is worthwhile to consider estimations of risk in various scenarios from that site. In these scenarios, it is assumed that an effective dose may be as low as 20 µg/100g (Wilson, 1996). It is also assumed that the level of toxin at Wedge Point is indicative of toxin levels in shellfish throughout Queen Charlotte Sound (there are insufficient data from other sample points in the Sound to test this assumption and none from where the same species of shellfish have been sampled). From 1/9/94 to 30/6/95, samples of mussels contained a minimum of 20 µg/100g of DSP toxins in 26 weeks. In subsequent years (from 1/7 to 30/6 the following year) the frequency of DSP toxins at this level were as follows: 30 weeks in 1995-96, 12 weeks in 1996-97, 1 week in 1997-98; and 1 week in 1998-99. Data from the Fisher & Bradford (1996) diary survey suggests that the non-commercial harvest of mussels from Queen Charlotte Sound is approximately equivalent to 60 100g meals of mussels per week (assuming 10 mussels equals 100g meat weight). Based on a year like 1995-96, in the absence of a marine biotoxin monitoring programme, there could be 1,800 cases of DSP arising from consumption of mussels alone from Queen Charlotte Sound. Other species of shellfish are also harvested from the area, including cockles, crayfish, paua, pipi and scallops, so the number of cases could be higher depending upon the propensity of these other species to accumulate and retain DSP toxins. In contrast, in a year like 1997-98, there would only be 60 cases of DSP arising from consumption of mussels from the same area. However, reservations about the reliability of shellfish harvest data expressed in previous sections potentially apply to this analysis also. As is the case with toxins discussed in previous sections, the occurrence of DSP toxins in shellfish is likely to impact disproportionately on Maori, and to a lesser extent on Pacific and Asian peoples in some areas (Kearney, 1999). Based on data from Maori households in the Far North (Hay 1996), up to 16% of Maori would be exposed to DSP if there were no publicity of the risk of DSP within a fortnight of the initiation of an event. In particular, the potential impacts of long-term consumption of low levels of DSP toxins are also likely to impact disproportionately on Maori, since they are more regular consumers of shellfish.

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5.5 NEUROTOXIC SHELLFISH POISONING 5.5.1 Hazard Identification Neurotoxic shellfish poisoning, (NSP), is caused by lipophilic polycyclic ether compounds, known as brevetoxins. There are many brevetoxin derivatives. All these derivatives exert their toxic effect by specific binding to site-5 of voltage sensitive Na+ channels, leading to channel activation at normal resting potential (i.e., they act as sodium channel activators) (Cembella et al., 1995b). The symptoms of NSP occur within 3-5 hours. Symptoms of a mild case include: chills, headache, diarrhoea, muscle weakness and joint pain, nausea and vomiting. Severe symptoms include paraesthesia, altered perception of hot and cold, difficulty in breathing, double vision, trouble in walking and swallowing (Hallegraeff, 1995). Death may occur as a result of respiratory arrest. Species of phytoplankton known to produce NSP toxins in New Zealand include Gymnodinium c.f. breve, Gymnodinium c.f. mikimotoi, (which is now known to include three separate species), Gyrodinium galatheanum and a species of Heterosigma (Mackenzie et al., 1995a, Haywood, 1998). The NSP toxins found isolated from New Zealand shellfish include previously known brevetoxins, and new analogues (Ishida et al., 1994; Ishida et al., 1995; Morohashi et al., 1995; Murata et al., 1998). At this stage the specific toxicity of the new analogues is unknown. Because of the tendency of shellfish to accumulate NSP toxins from toxic phytoplankton, shellfish consumers are exposed to NSP toxins when they consume shellfish that have been harvested from areas where NSP-producing phytoplankton are present. Shellfish consumers are thus the target population for NSP. As with other marine biotoxins, shellfish consumers are reliant on a marine biotoxin monitoring programme to determine whether shellfish are contaminated with NSP toxins – it is not possible to ascertain this from the appearance of the water or the shellfish themselves. The risk of NSP may vary with the species of shellfish consumed. For example, based on a very limited amount of data, it appears that Pacific oysters might accumulate brevetoxins from Gymnodinium breve more readily than GreenshellTM mussels. These results are not definitive, and more rigorous research is required to investigate these differences further. 5.5.2 Dose-Response Assessment There are several limitations to dose-response estimation for NSP toxins in New Zealand. Some of the brevetoxin derivatives observed in New Zealand shellfish in the 1993 event had not previously been observed elsewhere. There have been no confirmed cases of NSP in New Zealand. There is thus no dose-response relationship for some of the brevetoxins found in New Zealand.

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Wilson (1996) extrapolated overseas data to obtain some tentative dose-response relationships that were adjusted for the acetone extraction mouse bioassay results that were used in New Zealand prior to the introduction of the acetone screen test followed by the ether extraction mouse bioassay for NSP. It is apparent from dose-response data published by Hemmert (1975) that, not surprisingly, the impact of a dose of NSP toxin depends on the body weight of the consumer. His data, drawn from cases from Florida red tide events, showed that in adults no symptoms were evident at a dose of 0.3-3.6 mouse units/kg of body weight. Mild symptoms, including distal paraesthesia, occurred at 5.1-6.8 mouse units/kg body weight, and extreme symptoms, including difficulty walking, occurred at 5.1-6.8 mouse units/kg body weight. The dose for children was much lower, with extreme symptoms occurring at 3.1-4.4mouse units/kg of body weight. These data, plus other international data, were summarised by Wilson (1996) in terms of the concentration of NSP toxin in the shellfish consumed (so that it can be related to the mouse bioassay results). This combination of data showed that doses (in adult consumers) as low as 54 mouse units/100g may produce tingling in the mouth, 115 mouse units/100g may produce mild symptoms. Conversely, 295 mouse units/100g may produce no symptoms. Distal paraesthesia, and in some cases, extreme symptoms such as difficulty in walking, may occur at 340-350 mouse units/100g. Doses as low as 94 mouse units/100g may result in extreme cases in children. It is not known how well these data fit the brevetoxins found in New Zealand shellfish. 5.5.3 Exposure Assessment The following observations can be made regarding the risk of exposure to NSP toxins through consuming non-commercially harvested shellfish in New Zealand: • The distribution of potentially toxic Gymnodinium species is widespread

throughout New Zealand, and they have been detected at most sample sites. The distribution of NSP toxicity in shellfish is not well understood due to the non-specific test methods employed in the marine biotoxin monitoring programme, the results of which may be confounded by other lipid soluble toxins. However, the presence of brevetoxins in shellfish has been confirmed at Rangaunu Harbour, Coromandel and the Bay of Plenty (Ishida et al., 1994; Ishida et al., 1995; Morohashi et al., 1995; Murata et al., 1998), in oysters, mussels and cockles.

• Since the introduction of the ether extraction mouse bioassay in September 1994,

“NSP” levels above the regulatory level of 20 mouse units/100g have occurred relatively rarely. This toxin level has not occurred at all in Zones B, D, E, F, or I. Recorded instances in Zones G and H are unlikely to be due to brevetoxin (most likely to be Okadaic acid/yessotoxin, and “Wellington Harbour” toxin, respectively). They occurred at a frequency of less than 0.5% of samples in each of the other zones between 1/9/94 and 30/6/99. Temporal and geographic stratification of the sampling regime means that this level is indicative only. However, it is apparent that the incidence of possible NSP has been very low.

• The maximum “NSP” toxin levels in Zones A, C, G, H, J and K were all less than

30 mouse units/100g. (There were no “NSP” toxins above the regulatory level in

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samples from the other zones). These levels are unlikely to produce significant illness in adult human consumers.

• There is little doubt that NSP occurred in shellfish in New Zealand in 1993.

However, there are insufficient data to determine the frequency of occurrence, magnitude, or duration of the event, since the monitoring results are confounded by the acetone extraction method used at the time, and the lack of differentiation between NSP and other lipid soluble toxins (such as gymnodimine, DSP toxins, yessotoxin, pectenotoxin etc.). While the frequency and magnitude of presumed NSP toxins in shellfish in New Zealand since September 1994 have been very low, it is pertinent to note that significant blooms of Gymnodinium species producing other lipid soluble compounds have occurred during this time. Should such a bloom of a brevetoxin-producing species occur, then there would be a significant risk of exposure to NSP through the consumption of shellfish.

• It is likely that there are differences in accumulation and retention of NSP toxins

between different shellfish species, and that this impacts on the risk of exposure to consumers. However, research is required to investigate this rigorously.

• To date, NSP (i.e. the occurrence of brevetoxins in shellfish) has only been

confirmed from the Bay of Plenty northwards, on the eastern coast. However, there are insufficient data to predict a greater level of risk in some areas than others.

5.5.4 Risk Characterisation As with toxin groups discussed previously, the characterisation of risks associated with NSP in New Zealand is hampered by lack of robust data. Based on available information, in a future scenario similar to any of the years from 1994-1999, it is unlikely that there would be any significant illness in adults due to NSP (assuming a meal size of 100-200g, with no adjustment for variations due to body weight). There are insufficient dose-response data available to determine whether illness would occur in children at the maximum toxin levels that occurred in these years. It has been assumed that the toxicity of the brevetoxin derivatives found in New Zealand is similar to those in Gymnodinium breve in Florida. However, it cannot be assumed that there is no risk of NSP in the future. The occurrence of significant Gymnodinium blooms containing other lipid soluble bioactive compounds (possibly seeded from oceanic populations), and the occurrence of some NSP toxicity in shellfish in 1993 (albeit unquantifiable), suggest that there is potential for NSP to occur in shellfish here. There are insufficient data to quantify the impact of such an event in terms of the likely number of cases. However, data from Kearney (1999) suggests that Maori might be significantly disproportionately at risk, and to a lesser extent, Pacific and Asian peoples also. Data from Hay (1996) discussed elsewhere suggests that in the far north, up to 16% of the Maori population would be exposed to NSP toxins during such an event if there were no publicity of the risk within a fortnight of the initiation of the event.

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While not NSP, it is also pertinent to comment on the potential risk associated with “Wellington Harbour toxin”. This toxin was detected in shellfish, and killed mice by IP injection (Hoe Chang, NIWA, pers. comm.). However, there were no reported cases of TSP resulting from this event. The oral toxicity of the compound is as yet unknown. 5.6 RESPIRATORY IRRITATION SYNDROME 5.6.1 Hazard Identification Respiratory Irritation Syndrome (RIS) is caused by aerosolised marine biotoxins arising from blooms of toxic phytoplankton in the sea. Toxins known to cause RIS include brevetoxins (for example, from Gymnodinium breve) (Pierce, 1986), and the as yet unidentified toxin that was isolated from the newly identified Gymnodinium brevisulcatum in Wellington Harbour during the summer of 1998 (Hoe Chang, NIWA, pers. comm.). In the laboratory, purified brevetoxins block neuromuscular transmission, and cause a severe bronchoconstriction in animal models. The mode of action of the “Wellington Harbour toxin” is unknown. During blooms of the toxic phytoplankton, the toxins become aerosolised in the process of the bursting of bubbles caused by wind-generated whitecaps and breaking waves. The presence of toxic algal cells is not necessary for the occurrence of toxins in sea spray – with brevetoxins, sea spray containing fragments of Gymnodinium breve cells, and cell-free extracts have both been found toxic. This suggests that toxicity in aerosols can persist for some time after the algal cells have disappeared from the water column, so phytoplankton monitoring is unlikely to provide a good estimate of the risk of persistence of a RIS event. The toxin profiles of the aerosolised toxins may not be in the same proportions as the toxins in the phytoplankton. Pierce (1986) suggested that factors such as solubility, volatility, surface sorption characteristics, and the presence of other naturally occurring organic surface active substances would have an important effect on the extent to which each toxin is aerosolised, potentially altering toxin aerosol composition even within the same red tide bloom event. Selective accumulation in aerosols of four most potent brevetoxins to levels 20 to 25 times that in the algal cells has been shown for aerosols from G. breve (Baden et al., 2000). Brevetoxin is rapidly absorbed into the body by inhalation. It is highly soluble in cellular lipid, and crosses cell membranes with high efficiency (Baden et al., 2000). It is likely that some of the effects of aerosolised brevetoxins are systemic, arising from brevetoxins absorbed into the body across membrane surfaces. There is no information on the mode of absorption of the “Wellington Harbour toxin”. Most of the airborne toxin effect is experienced near the surf zone. The respiratory irritation is observed most intensively within a few kilometres of the beach, indicating a rapid settling or dispersion of the aerosolised toxins (Pierce, 1986). Symptoms of RIS caused by brevetoxins include sore throats, eye and nose irritation, involuntary dry coughing and sneezing, watery eyes, copious rhinnorhea, and

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difficulty breathing. Aerosolised “Wellington Harbour toxin” produced similar symptoms, but also caused headaches and skin rash (reported by marine hatchery workers at NIWA, Wellington, J. Illingworth, pers. comm.). People with pre-existing lung disease appear to have more lower airway symptoms from aerosolised brevetoxins than people with no prior history of reactive lung disease (Kirkpatrick et al., 2000), and elderly people are more susceptible than young people (Dr S. Shumway, Southampton College, pers. comm.). Asthmatics are affected by contact with both brevetoxin and “Wellington Harbour toxin” aerosols. Studies of RIS caused by brevetoxin overseas found that exposure induced asthma attacks in 80% of the asthmatics studied (Baden et al., 2000). Those people living in coastal areas, or visiting the coast, are affected. There may also be some occupational exposure by people working on the coast – for example, by lifeguards, hatchery workers, shellfish farmers and fishermen. Wearing cotton face masks, or moving into air-conditioned space indoors immediately reduces, and subsequently eliminates, respiratory irritation. Residue remaining on skin and mucous membranes can re-intoxicate by rubbing or touching previously affected areas (Baden et al., 2000). Anecdotal accounts of repeated exposure to brevetoxin aerosols from Florida red tides suggest sensitisation, but this has not been verified or quantified (Baden et al., 2000). The long-term impacts of repeated exposure to aerosolised brevetoxin and “Wellington Harbour toxin” are unknown. 5.6.2 Dose-Response Assessment There is no dose-response information available for human exposure to aerosolised brevetoxins or aerosolised “Wellington Harbour toxin”. A study that includes an investigation of the dose-response to aerosolised brevetoxins in humans is currently being undertaken by Baden and co-workers. The results of this study are not yet available. However, studies of aerosolised brevetoxins using sheep have shown that exposure to femtomolar concentrations produce bronchoconstriction. Environmental inhalation of aerosolised brevetoxins produced a massive mortality of manatees in 1996 (158 deaths) (Baden et al., 2000). 5.6.3 Exposure Assessment There have been several respiratory irritation events recorded in New Zealand since the beginning of 1993. The first event, at Orewa in the summer of 1993, is presumed to have been caused by brevetoxins, but no identification of aerosol toxins was undertaken. However, shellfish in some areas in the Hauraki Gulf did accumulate brevetoxins over this time, as confirmed by toxin analysis in shellfish tissue (Ishida et al., 1994; Ishida et al., 1995; Morohashi et al., 1995; Murata et al., 1998). The second series of events occurred in the summer of early 1998, on the coasts of Wairarapa and Hawkes Bay, and in Wellington Harbour. The nature of the aerosolised toxins that were causative agents in this series of events is unknown (the “Wellington Harbour toxin”), as is the quantity of toxin in the aerosol. There is no formal record of the duration of any of these events, nor of the numbers of people affected. Anecdotal evidence suggests that several hundred people were affected in each event, over a period of several weeks.

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If it can be assumed that the limited data that has been gathered can be used to predict future exposure to RIS in New Zealand, then the following might be expected: • Gymnodinium sp. blooms with the potential to cause RIS may occur every three

years. In the years when these blooms occur, widespread and/or several events might occur. These blooms may last for up to three months, and may change location on the coast during this time. The conditions that cause exposure to RIS are likely to occur at some time during this period and may continue for up to several weeks.

• RIS is more likely to occur in the months December-March. Since this is the

period of high recreational use of the coastline, exposure to RIS is not just limited to the population normally resident in coastal areas, but may be several orders of magnitude higher.

• The location of RIS events appears more likely on the eastern coast of the North

Island, but events on the western coast, or further south may also be possible. • The most intense exposure to RIS occurs at the coast, but may be experienced up

to a few kilometres inland from the shore, depending on the quantity of toxin in the aerosol and the strength and direction of the wind.

The numbers of people likely to be exposed to RIS are difficult to predict, since they depend on the specific location of Gymnodinium blooms. It is not possible to predict such specific locations from the data available. Obviously, if the blooms occur in areas where the adjacent coast is highly populated by residents and visitors, the numbers of people exposed are likely to be higher than in areas that are largely uninhabited or sparsely populated. 5.6.4 Risk Characterisation Given the uncertainties in the analysis, it is difficult to present an overall estimation of the risk associated with RIS in New Zealand. The variations in occurrence of Gymnodinium sp. blooms with the potential to cause RIS are likely to occur over a much longer period than the period for which we have data available. The events that have been recorded so far have occurred in somewhat different hydrographic and environmental conditions, and at different locations. In addition, there is no information available about the toxicity or mode of action of the “Wellington Harbour toxin”, nor any data on human dose-response or long-term impacts of either the “Wellington Harbour toxin” or aerosolised brevetoxins. Predictions of risk therefore need to be made with great caution. There are several scenarios that can be considered in estimating the risks associated with RIS in New Zealand. One scenario is years like 1994-1997 and 1999, in which no cases of RIS occurred. Based on the data that are currently available, it is likely that some years in the future will match this scenario.

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The potential risks associated with blooms of Gymnodinium producing RIS toxins vary according to the size, density and location of the blooms, and the wind conditions at the time. The numbers of people affected could vary from thousands (for example in the event of a dense bloom associated with a strong on-shore breeze off Auckland’s North Shore or Northland holiday resorts) to few. Amongst those people exposed, asthma sufferers are likely to be most seriously affected, with the possibility that 80% may suffer asthma attacks upon contact with the toxins. In New Zealand, 20% of children under the age of 15, and 10% of adults, suffer from asthma (statistics from the Asthma Society Inc. Auckland). This suggests that approximately 9.8% of people exposed to RIS could potentially suffer asthma attacks as a result (based on approximately 23% of the population being under the age of 15). This needs to be considered in the management of risk associated with RIS. Other considerations in the estimation of risk of RIS in New Zealand are the factors that are unknown with respect to the “Wellington Harbour toxin”. These include the unknown mode of action of the toxin, the lack of information about effects of prolonged exposure, or likely chronic effects.

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SECTION 6: SUMMARY BY AREA Following is a brief summary of the factors impacting on the risk of TSP, and local issues in each Biotoxin Zone. The location of each zone is presented on a map in Appendix I(D). 6.1 ZONE A Zone A extends from Tauroa Point on the north-west coast of Northland around to Cape Brett on the eastern coast. The western coast of Zone A consists of an exposed sandy coastline (Ninety Mile Beach) between Ahipara and Scott Point. Tuatua and toheroa are dominant species in the sand of the beach. Both species are patchy in distribution, and local knowledge is usually necessary to locate denser beds. Associated with the rocky outcrops (e.g. at Ahipara, The Bluff, and Scott Point) are mussels, paua, kina and crayfish, and in much lower numbers, rock oysters. While there is relatively little road access to this area, vehicles are able to drive long distances along the beach at low tide, so the whole length of the beach is accessible. Locals report a consistently high level of shellfish harvesting off this beach: “Hundreds of shellfish come off this beach every day, all year round”. There are high levels of harvesting before public holidays, as people prepare for visits from whanau from outside the area (C & R Hensley, mussel spat collectors, Ninety Mile Beach, pers .comm.). The eastern coast of Zone A has a wide diversity of marine habitats, providing a wide range of seafood for non-commercial harvest. The coastline is irregular and convoluted. The orientation of the beaches in the area, and their fetch lengths and directions are highly variable. These substantial differences in exposure and shelter result in rapid changes in beach type over quite small distances. This coast in Zone A contains most of the commonly gathered species of shellfish (with the exception of course, of those species that do not extend this far north, such as the blue mussel). One species not found in this area is toheroa, which prefer sandy coasts with greater wave exposure. A survey by Fisher & Bradford (1998) indicated that scallops are the most actively targeted species for non-commercial harvest in this area, followed by crayfish, tuatua and pipi. Paua, mussels, cockles, oysters and kina are also targeted (Table 3.1, Section 3). The same survey showed that the trips targeting bivalve shellfish species from this zone comprised only 3.9% of the total trips in New Zealand. If trips targeting paua, kina and crayfish are included, the trips from Zone A only represent 2.6% of the total trips in New Zealand. These low figures may be in part due to the comparatively small size of this zone compared to most other zones. It is also possible that significant harvesting activity has not been reported in the survey, which was undertaken for the Ministry of Fisheries – for example, the harvesting of toheroa, which is prohibited by regulation except for customary purposes. A survey of consumption of non-commercially harvested seafood in the north of this zone by Hay (1996) showed that 11% of Maori households collected seafood more than once a week, 31% at least weekly, and 52% at least monthly. If 30% of that seafood were shellfish species (as suggested from anecdotal evidence), Maori consume significant quantities of non-commercially harvested shellfish in this region.

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The coastline is relatively easily accessible. While some of the eastern coastline in Zone A is relatively remote with fewer roads, most areas are easily accessible by boat. The population of Northland is not high, but it is within proximity of the dense population in the Auckland area. Maori are a significant proportion of the population. There are large increases in population over the summer holiday period. Low levels of PSP have been found in shellfish from both the western and eastern coasts of Zone A. Most of these occurred in 1993 and early 1994, with some activity late in 1995 (October- December) on the eastern coast at Houhora Bay and the Cavelli Islands, and in January 1996 at Waipapakauri (on the western coast). Most of the instances of PSP arose in samples of tuatua taken from Tokerau Beach between July 1993 and October 1996. Tuatua exhibited residual low levels of PSP throughout this time. Only one sample above the regulatory level was detected in Zone A between 1/7/93 and 30/6/99 – this occurred at Houhora Harbour in November 1996. This level was only 83 µg/100g (the regulatory level is 80 µg/100g). Since 1996, the PSP activity in Zone A has been very low, with only two samples with very low levels detected in 1997, and none in 1998 or 1999. The current monitoring regime has been designed on the premise that there is likely to be little toxin activity on the western coast of New Zealand. This is not supported by the data collected across New Zealand. (Author note: Subsequent to the preparation of this report a large bloom of Gymnodinium catenatum has occurred on the western coast of Zone A). Shellfish from Zone A contained ASP levels above the regulatory level (20 µg/g) in 33 samples taken between 1/7/93 and 30/6/99. All except three of these samples were scallops. Detectable levels were found in 294 samples (14.6%) over the same time period. The highest level found in a whole shellfish was 210 µg/g in scallops from the Cavelli Islands on 2/11/93. DSP above the regulatory level of 20 µg/100g was detected in two shellfish samples taken from Zone A between 1/9/94 and 30/6/99. These were samples of Pacific oysters taken in consecutive weeks in July 1995 from Rangaunu Harbour, and had DSP levels of 33 µg/100g and 26 µg/100g respectively. Detectable levels of DSP were found in 32 shellfish samples (1.5% of total samples taken) over the same time period. These included a sample from Waipapakauri on the western coast of the zone. An unidentified toxin, called “Rangaunu Harbour toxin” has caused persistent toxicity in shellfish in the Rangaunu Harbour. The identity and oral toxicity of this toxin is unknown. However there is no evidence of any human illness having resulted from the consumption of shellfish from the Rangaunu Harbour. There are 9 instances of shellfish samples from the period 1/9/94 to 30/6/99 that have contained toxins above the regulatory level of 20 mouse units when tested with the ether extract mouse bioassay for NSP. Seven of these were from shellfish taken from the Rangaunu Harbour, some of them at a time when significant numbers of Gymnodinium cysts were identified from phytoplankton samples. It is thus possible that these toxin levels were due to brevetoxin. There have been no probable or confirmed cases of TSP from Zone A, and four suspected cases since the beginning of 1994.

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6.2 ZONE B Zone B extends from Cape Brett to Cape Rodney. Zone B has wide diversity of marine habitats, providing a wide range of seafood for non-commercial harvest, including most of the commonly gathered species of shellfish. One species not found in this area is toheroa, which prefer sandy coasts with greater wave exposure. A survey by Fisher & Bradford (1988) suggested that crayfish, scallops, pipi and mussels were the most actively targeted species for non-commercial harvesting in this area, followed by kina and cockles. Tuatua, paua and oysters are also targeted (Table 3.1, Section 3). The same survey also indicated that trips in this zone targeting bivalve species represent 12.5% of all trips in New Zealand. If trips targeting paua, kina and crayfish are included, trips in this zone represent 11.5% of all trips in New Zealand. If it can be assumed that the results of this survey are representative of non-commercial harvesting activity, this suggests that Zone B has a comparatively significant level of non-commercial harvesting for shellfish. Zone B is well serviced by roads, with numerous places where it is possible to launch a boat. It has a comparatively low population, with Whangarei being the major population centre. However, the zone is in close proximity to the dense population in the Auckland area, and there are large increases in the population in the zone over the holiday periods in the summer months. There have been no instances of PSP above the regulatory level in shellfish between 1/7/93 and 30/6/99. The 48 instances of low levels of PSP occurred predominantly at in tuatua at Oakura and Waipu in 1993-95. Only one sample was above the regulatory level in 1996 (again, in tuatua at Oakura) and one in 1998 in pipi from Waipu. These samples reflect the tendency of tuatua to hold low residual levels of PSP for long periods of time. No shellfish from Zone B contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. Detectable levels were found in 78 samples (8.9% of total samples taken) over the same time period. All these were samples of scallops from the Bream Bay area between 1994 and 1997. DSP above the regulatory level of 20 µg/100g was not detected in any shellfish samples taken from Zone B between 1/9/94 and 30/6/99. Detectable levels of DSP were found in 3 shellfish samples (0.3% of total samples taken) over the same time period. There have been no shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99. However, four samples over this time had detectable levels of toxin using this assay. The identity of this toxin has not been confirmed as brevetoxin. There have been no probable or confirmed cases of TSP in Zone B, and one suspected case since the beginning of 1994 to June 1999.

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6.3 ZONE C Zone C includes the Hauraki Gulf from Cape Rodney to Cape Colville at the north of the Coromandel Peninsula, including Great Barrier Island. Zone C encompasses a very large range of habitats, and thus species for consumption by the public. These range from species found in sheltered estuarine and harbour areas, through to those in moderately protected areas, and a few areas that face open water. The zone thus contains cockles, pipi, Pacific and native rock oysters, tuatua, green-lipped mussels, scallops, kina, paua and crayfish. The shellfish gathering areas in this zone are in general highly accessible to the public – the coast is well supplied with roads and boat launching facilities. However, there are some local restrictions on the gathering of shellfish, designed both to conserve shellfish stocks, and to prevent illness in consumers. A survey by Fisher & Bradford (1998) indicated that crayfish and scallops were by far the most frequently targeted species in this area, followed by mussels, pipi, cockles and tuatua. Lower numbers of trips targeted paua, kina and oysters (Table 3.1, Section 3). The same survey showed that trips targeting bivalve shellfish in this zone represent10.4% of total non-commercial harvesting trips in New Zealand. The Auckland region in Zone C is a highly populated area, with significant ethnic diversity, including comparatively high numbers of Maori, Pacific Island people, and people of Asian origin. The more remote areas in this zone, such as Coromandel Peninsula, are still within easy access of Auckland, and this is reflected in the increase in population over holiday periods. Zone C has exhibited an extremely low level of PSP activity, with no samples found above the regulatory level of 80 µg/100g, and only six samples with detectable levels (5 of them in November-December 1993). No shellfish from Zone C contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. Detectable levels (but extremely low levels) were found in 53 samples (3.1% of total samples taken) over the same time period. DSP above the regulatory level of 20 µg/100g was not detected in any shellfish samples taken from Zone C between 1/9/94 and 30/6/99. Detectable levels of DSP were found in 13 shellfish samples (0.6% of total samples taken) over the same time period. There have been three shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99. Two of these were associated with the presence of Gymnodinium c.f. mikimotoi in the phytoplankton. However, the identity of this toxin has not been confirmed as brevetoxin. There have been no probable or confirmed cases of TSP in Zone C, and 3 suspected cases from the beginning of 1994 to June 1999.

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6.4 ZONE D Zone D extends from Cape Colville to Cape Runaway. In the sandy beaches from Opotiki to Waihi Beach, tuatua are found. Pipi, oysters, cockles and mussels are found in the harbours in this bay. On the western side of the Bay of Plenty, scallop beds lie off shore and in Tauranga Harbour, and these continue north up the Coromandel Peninsula. From Waihi northwards, the coastline is rocky, but interspersed with harbours and estuaries where oysters, pipi and cockles are found. The rocky open shores provide good habitat for crayfish, and green-lipped mussels, paua and kina are also present. There is good road access to much of this area. A survey by Fisher & Bradford (1998) indicated that crayfish and scallops were the most frequently targeted species by non-commercial harvesters. Significant numbers of pipi, tuatua and mussels were also targeted, followed by paua, kina, and cockles (Table 3.1, Section 3). The same survey also indicated that 25.5% of all non-commercially harvested shellfish (bivalves) come from Zone D. This was the highest percentage for any zone. Maori form a significant proportion of the population, and there is a significant component of non-commercial harvesting by Maori, as well as gathering by the general public. Early in the biotoxin programme, concern was expressed by Maori in the western Bay of Plenty that the sampling programme did not specifically monitor their gathering sites. There has been a problem with persistent PSP toxicity in the Bay of Plenty, over a time period spanning 1993-1999. This has occurred in the open beach sites, predominantly in tuatua, which have a tendency to retain PSP toxins. Over this time, 91 samples above the regulatory level of 80 µg PSP/100g shellfish tissue, have been recorded. The maximum level was 1007 µg/100g. For the period 1993-June 1999, this area exhibited the highest frequency of PSP toxin activity of any area in New Zealand, and also the highest toxin levels. These levels represent a potential risk to consumers. While some of the persistent toxicity may be as a result of long-term sequestering in tuatua tissue, there may also be seed beds of cysts of Alexandrium species, causing repeated blooms in the area. It is not possible to determine this from available information. One shellfish sample from Zone D contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. This was a sample of scallop meat and roe taken in December 1994 from Rangiwaea. Detectable levels (but extremely low levels) were found in 262 samples (11.5% of total samples taken) over the same time period. DSP above the regulatory level of 20 µg/100g was not detected in any shellfish samples taken from Zone D between 1/9/94 and 30/6/99. Detectable levels of DSP were found in 3 shellfish samples (0.1% of total samples taken) over the same time period.

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There have been six shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone. However, the identity of this toxin has been confirmed as yessotoxin, not brevetoxin. There has been one probable case of TSP in Zone D, and 7 suspected cases between the beginning of 1994 and June 1999. The probable case of PSP arose from the consumption of tuatua gathered from Ohope Beach. It is highly likely that one of the suspected cases was also PSP, since a phytoplankton sample gathered from the nearest sample site the following day, contained 10,300 cells/L of Alexandrium catenella. A suggestion has been made that public warnings with respect to marine biotoxins in shellfish should be species specific, to allow some continued harvesting of species that have not accumulated biotoxins (E. Ashcroft, Pacific Health, pers. comm.). This would involve monitoring several species of shellfish at each site, and would thus be more expensive. It would have advantages in allowing some food source to remain available for harvest, and in the maintenance of credibility that is lost when the public harvest seafood in the face of a warning, but do not get sick. In addition to other factors, the effectiveness of public warnings in this case would depend on the ability of the public to absorb and interpret a more complicated warning message, and to competently distinguish between different species of shellfish. 6.5 ZONE E Zone E extends from Cape Runaway to Cape Palliser. Paua, kina and crayfish are the predominant species of interest to non-commercial harvesters in Zone E. Some mussels are also present, and cockle and pipi occur in the few sheltered areas. The coast is a mixture of rocky reefs, rock platforms, and steeply graded coarse sand beaches interspersed in Hawke’s Bay with rocky beaches. Much of this coastline is relatively remote, with poor road access. A survey by Fisher & Bradford (1998) showed that most non-commercial harvesting in this area is targeted at crayfish and paua. Of the bivalve species, mussels are the most frequently targeted, with much lower numbers of trips targeting cockles and pipi (Table 3.1, Section 3). The same survey found that this zone only represents 2.5% of the total trips targeting bivalve shellfish. However, if paua, crayfish and kina are included in the shellfish species, this zone represents 15.3% of total trips targeting shellfish – an indication of the significance of these latter species in the non-commercial harvesting in the region. The northern part of the zone is comparatively sparsely populated, with the main population centre being Gisborne. Napier and Hastings form the major population centres further south. Regional data indicate that the area has the highest proportion of Maori, relative to other regions. There have been no shellfish samples with levels of PSP above the regulatory level of 80 µg/100g in this zone, and only 9 samples (0.73%) above the detectable level. This suggests that the PSP activity in the area has been low. Samples with detectable levels comprised a variety of shellfish, in several different years at different sites.

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However, it is noticeable that all 9 samples occurred in the months November to February. One shellfish sample from Zone E contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. This was a sample of GreenshellTM mussels from Te Araroa Beach in October 1995 (22 µg/g). Detectable levels (but extremely low levels) were found in 12 samples (1.6% of total samples taken) over the same time period. DSP above the regulatory level of 20 µg/100g was detected in one shellfish sample taken from Zone E between 1/9/94 and 30/6/99. This was from paua gut from a sample taken from Riversdale Beach in November 1994 (39 µg/100g). Detectable levels of DSP were found in 4 shellfish samples (0.5% of total samples taken) over the same time period. All these results were from tests on paua gut. There have been no shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone, nor any detectable levels of toxin identified using this assay. There were no suspected, probable or confirmed cases of TSP in Zone E between the beginning of 1994 and June 1999. 6.6 ZONE F Zone F extends from Tauroa Point to Cape Egmont on the west coast of the North Island. Tuatua and toheroa are present on the long exposed sandy beaches broken occasionally by rocky reefs and headlands. The predominant species in the rocky areas include mussels, kina, paua and crayfish. The Herekino, Whangape, Hokianga, and Kaipara Harbours that lead off these long exposed beaches provide an environment for species that thrive in more sheltered and silty environments. Species gathered here include native and Pacific oysters, dredge oysters, cockles, horse mussels, and pipi. There are scallop beds in the Manukau and Kaipara Harbours, and green-lipped mussels can be dredged from beds near the Kaipara Harbour mouth. The Manukau Harbour also has abundant shellfish, although in places the ability to harvest these is compromised by the bacteriological water quality. Due to overfishing, the Auckland Regional Council has banned the gathering of shellfish on some of the beaches between the Manukau and Kaipara Harbours, and a lower daily bag limit for cockles applies in the Auckland Regional Council area. Further south, green-lipped mussels and cockles are common in the Raglan Harbour, and pipi, cockles and mussels in the Kawhia Harbour. The Manukau and Kaipara Harbours are easily accessible from Auckland, and the coast of the Hokianga Harbour is easily accessible by car and boat. The Whangape and Herekino Harbours further north are much more remote. The most popular access to the open sandy beaches in the north of the zone is via Dargaville. Here, and on Muriwai Beach, and the beaches south of the Manukau Heads, cars are able to drive along the beach at low tide, so infrequent road access does not necessarily significantly limit access to the length of the beach.

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Southwards, the coast curves westwards into the North Taranaki Bight. As the rocky reef habitat increases, green mussels, paua and kina are more abundant, and some tuatua are found in sandy areas. The southern part of Zone F from Urenui to Cape Egmont consists of gravel, cobble and boulder beaches, providing habitat for paua, kina and green-lipped mussels. A survey by Fisher & Bradford (1998) indicated that scallops are the most frequently targeted shellfish species in this zone, followed by mussels, with lower numbers of trips targeting pipi, tuatua, oysters and cockles. Non-bivalve species targeted include crayfish, followed by paua and kina (Table 3.1, Section 3). The same survey found that trips targeting bivalve shellfish species in this zone represent 13.5% of the total non-commercial harvesting trips in New Zealand. This suggests that non-commercial harvesters collect significant quantities of shellfish off this coast. There were no shellfish samples above the detectable level for PSP in this zone between 1/7/93 and 30/6/99 and only 2 samples (0.11% of total samples taken) above the detectable level for PSP. If we were to rely on the predictive nature of historic data, this would suggest that Zone F is a low risk area for PSP. However, as this report is being prepared, there is a very large bloom of Gymnodinium catenatum off this coast, resulting in high levels of PSP in shellfish. This is a good example of how unreliable short-term historic data can be in predicting the future risk of TSP. No shellfish from Zone F contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. Detectable levels (but extremely low levels) were found in 11 samples (0.9% of total samples taken) over the same time period. DSP above the regulatory level of 20 µg/100g was not detected in any shellfish samples taken from Zone F between 1/9/94 and 30/6/99. Detectable levels of DSP were found in 2 shellfish samples (0.1% of total samples taken) over the same time period. There have been no shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone, nor any detectable levels of toxin identified using this assay. There have been no probable or confirmed cases in Zone F, and six suspected cases between the beginning of 1994 and June 1999. 6.7 ZONE G Zone G includes the north of the South Island, from Farewell Spit in the west, to Cape Campbell in the east. The eastern coast of Zone G is predominantly rocky and gravel beaches, with paua, kina and crayfish found in reefs. Mussels are found at Port Underwood. Inside the Marlborough Sounds and Croiselles Harbour, the coastline is steep and rocky, with very few intertidal areas. The main species gathered here are mussels (blue and green-lipped), and scallops. There are significant numbers of cockles gathered from

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Pelorus Sound and Croiselles Harbour. While road access is limited in much of the Marlborough Sounds, most areas are easily and safely accessible by boat. Paua and crayfish are present on the outer shores of the Sounds where the coast is more exposed to wave action. There is a high level of non-commercial fishing activity in Tory Channel, where mussels, kina, crayfish and paua are collected. The Marlborough Sounds are easily accessible from the more densely populated area of Wellington, and there are significant increases in population during holiday periods. Maori account for approximately 10% of the population in the Sounds. To the west, dredge oysters and scallops are the major bivalve species in Tasman and Golden Bays, and significant beds of cockles occur in Tapu Bay and Pakawau Beach. Crayfish are taken from the rocky reefs of the coast that separates the two major bays. A survey by Fisher & Bradford (1998) indicated that the most frequently targeted species in this zone are scallops, followed by crayfish and dredge oysters, mussels and cockles. Other species targeted include pipi, and paua, with lower numbers of kina targeted. The same survey showed that trips targeting bivalve shellfish species from this zone comprise 21.3% of the total New Zealand trips. This was the second highest percentage, indicating that this zone is an area of significant non-commercial harvest of bivalves. Between 1/7/93 and 30/6/99, levels of PSP above the regulatory level of 80 µg/100g have only been found in two shellfish samples in Zone G: these were consecutive samples of GreenshellTM mussels from Anakoha Bay in January 1994. PSP has been detected in a very low percentage of samples (0.36%), mostly from Oyster Bay in 1993-94 and Anakoha Bay in January-February 1994 and November-December 1997. Two samples with detectable levels of PSP also occurred in Melville Cove in February-March 1998. There have been no detectable levels of PSP in any sites in Tasman or Golden Bays. Only one shellfish sample from Zone G contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. This was from Kenepuru Entrance in December 1994, in a sample of GreenshellTM mussels. The sample contained a level of 187 µg/g Domoic acid. Detectable levels (but extremely low levels) were found in 172 samples (3.4% of total samples taken) over the same time period. DSP above the regulatory level of 20 µg/100g was detected in 71 shellfish samples taken from Zone G between 1/9/94 and 30/6/99. Detectable levels of DSP were found in 137 shellfish samples (2.4% of total samples taken) over the same time period. Most of these samples were from Wedge Point (G23). Blue mussels from this site contained persistent levels of DSP from November 1994-August 1995, November 1995 to July 1996, and October 1996 to March 1997. It is possible that the hydrology of the Wedge Point area, combined with the diurnal phytoplankton migration favours the establishment of resident populations of Dinophysis. However, it is noted that there is a significant year to year variation in the occurrence of DSP in mussels at Wedge Point.

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Several samples with detectable levels of DSP were located in Tasman Bay (The Glen and Port Motueka). There were no detectable levels found in Golden Bay. There have been two shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone. However, there is a possibility that these results may be due to Okadaic acid (or possibly, yessotoxin), not brevetoxin. The presence of brevetoxin in shellfish samples has not been confirmed in this zone. Between the beginning of 1994 and June 1999, there were seven probable cases of TSP in Zone G, and two suspected cases. All the probable cases were from non-commercially harvested shellfish. These are likely to have been caused by DSP toxins or pectenotoxin. 6.8 ZONE H Zone H is situated at the south-west of the North Island, and extends from Cape Egmont in the west, around to Cape Palliser at the south of the North Island. Paua, kina, green-lipped mussels and pipi are abundant in the South Taranaki Bight in the north of Zone H. Further south, the coast changes to coarse-grained sandy beaches with mactrid clams, tuatua, toheroa and pipi present. The rocky areas adjacent from Paekakariki south and then eastwards to Cape Palliser provide habitat for paua, kina and crayfish. Cockles are found in the estuarine areas near Paremata. A survey by Fisher & Bradford (1998) indicated that the most frequently targeted species by non-commercial harvesters in this area is crayfish, followed by paua, with lower numbers of trips targeting pipi, mussels, cockles and kina (Table 3.1, Section 3). The same survey indicated that the trips targeting bivalve shellfish in this zone comprise only 1.5% of the total trips in New Zealand. If trips to target paua, kina and crayfish are also included with the bivalve species, the trips targeting all these species in Zone H only comprise 2.9% of the New Zealand total. If one can assume that the results of this survey are representative of non-commercial harvesting activity for these species, this suggests that Zone H is an area where non-commercial harvest of shellfish is less significant than most other zones in New Zealand. There were no shellfish samples detected with levels of PSP above the regulatory level (80 µg/100g) between 1/7/93 and 30/6/99. PSP has been detected in three shellfish samples: two at Ohawe Beach (68 µg/100g on 8/11/95 and 30 µg/100 on 25/5/98), and one at Dorset Point (36 µg/100g, 7/9/98). No shellfish samples from Zone H contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. Detectable levels (but extremely low levels) were found in 3 samples (0.5% of total samples taken) over the same time period. DSP above the regulatory level of 20 µg/100g was not detected in any shellfish samples taken from Zone H between 1/9/94 and 30/6/99. Detectable levels of DSP

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were found in 3 shellfish samples (0.4% of total samples taken) over the same time period. There has been one shellfish sample above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone. However, there is a possibility that this might have been due to Wellington Harbour toxin, not brevetoxin, as it occurred at Dorset Point during a dense bloom of Gymnodinim c.f. mikimotoi (in this case, Gymnodinium brevisulcatum) that is known to have been producing “Wellington Harbour toxin”. There were no suspected, probable or confirmed cases of TSP in Zone H between the beginning of 1994 and June 1999. 6.9 ZONE I Zone I extends down the eastern coast of the South Island, from Cape Campbell to Bluff. The major species for much of this rocky coast are mussels, paua and crayfish. Tuatua are also found in some sandy beach areas (for example, Moeraki, Kaitiki Beach, and Warrington), and dredge oysters are found at Blueskin Bay. The mixed sand and gravel beaches from Oamaru north to Banks Peninsula provide little opportunity for the settlement of bivalve shellfish, paua or crayfish. In contrast, these species are found in the rocky reefs around Banks Peninsula, and the harbours and estuaries contain cockles and pipi. There are significant beds of cockles at Papanui. North of Banks Peninsula is a stretch of steep coarse-grained sandy beaches with beds of cockles present. From Amberley Beach north, the coast is comprised of exposed rocky reefs where crayfish and paua are the major non-commercially harvested species. North of Kaikoura, the coast is well known for its abundance of paua. A survey by Fisher & Bradford (1998) indicated that the species most frequently targeted for non-commercial harvest on this coast are paua and crayfish, with lower numbers of trips targeting cockles, mussels and pipi. Other species collected include dredge oysters, kina and tuatua (Tale 3.1, Section 3). The same survey indicated that the trips targeting bivalve shellfish species in this zone comprised only 3.9% of the total trips in New Zealand. If trips targeting paua, kina, and crayfish are included with the trips targeting bivalve species, the total trips from Zone I are only 5.8% of the trips in New Zealand. If the results of this survey can be assumed to be representative of non-commercial harvesting for shellfish in New Zealand, these results suggest that Zone I has a comparatively low level of non-commercial shellfish gathering. There were no shellfish samples detected with levels of PSP above the regulatory level (80 µg/100g) between 1/7/93 and 30/6/99. PSP has been detected in one shellfish sample (0.05% of total samples) over this time period: at Bull Creek in September 1995 (32 µg/100g). (Note that subsequently the Gymnodinium catenatum bloom in 2000 has resulted in high levels of PSP in shellfish in this area).

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No shellfish samples from Zone I contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. Detectable levels (but extremely low levels) were found in 30 samples (2.1% of total samples taken) over the same time period. DSP above the regulatory level of 20 µg/100g was detected in 8 shellfish samples taken from Zone I between 1/9/94 and 30/6/99. Detectable levels of DSP were found in 27 shellfish samples (2.4% of total samples taken) over the same time period. Most of these samples were from Akaroa Harbour (I04). Blue mussels from this site contained persistent levels of DSP from February 1995 to October 1995. There have been no shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone. There have been no probable or confirmed cases of TSP in Zone I, and 6 suspected cases between the beginning of 1994 and June 1999. 6.10 ZONE J Zone J encompasses all the west coast of the South Island, from Cape Farewell in the north, down to Bluff in the south, and includes Stewart Island. The western coast of the South Island is a long exposed rocky coastline, interspersed with mixed shingle and sand beaches. With the exception of mussels on the rocky reefs, there are few bivalves suitable for gathering – crayfish are the main non-commercially gathered species other than finfish. White-baiting is an important recreational and commercial fishery. Similar coastline extends south to the sounds in Fiordland, where scallops, paua, kina mussels and crayfish are available. In the Sounds the cliffs drop straight into the sea, and there are very few places where the intertidal zone is other than vertical. Lack of road access in much of this area limits the gathering of shellfish. At the south of the South Island, paua and kina are present as the coast runs in an easterly direction, with toheroa and mactrid clams present in Te WaeWae Bay, and toheroa at Oreti Beach. The coastline of Stewart Island is convoluted and rocky, with the major species of interest to non-commercial harvesters being mussels, paua and dredge oysters. Paterson Inlet has a wider variety of species with the addition of kina, scallops, pipi and cockles. The Foveaux Strait is famous for its abundance of dredge oysters. These are conserved through seasonal bans on harvesting. Due to the impact of Bonamia disease on the oyster population, the seasons have been highly restricted through the 1990’s, but the beds now appear to have recovered. The longer “Bluff oyster” seasons now expected will increase the risk of exposure of consumers to biotoxins in this area. A survey by Fisher & Bradford (1998) indicated that the species most frequently targeted for non-commercial harvest are crayfish, paua and mussels, with lower numbers of trips targeting dredge oysters, scallops, cockles, kina and pipi (Table 3.1, Section 3). The same survey showed that the trips targeting bivalve species in this zone comprised 5.1% of the total trips in New Zealand. If paua, kina and crayfish are included among the species targeted, the trips from this zone comprise 6.9% of the

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total. Most of this activity is concentrated in the southern part of the zone, as large stretches of the western coastline (in the north and south) are relatively inaccessible. There were no shellfish samples detected with levels of PSP above the regulatory level (80 µg/100g) between 1/7/93 and 30/6/99. PSP has been detected in 10 shellfish samples (0.79% of total samples) over this time period. One was from blue mussels sampled from Mussel Rocks (on the West Coast) in May 1997 (32 µg/100g), and the others from Foveaux Strait (8 in February/March 1994, 1 in February 1996), all dredge oysters. No shellfish samples from Zone J contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. Detectable levels (but extremely low levels) were found in 16 samples (2.2% of total samples taken) over the same time period. These included two samples containing traces of ASP from Mussel Rocks on the West Coast, as well as samples from the southern sites in the zone.

DSP above the regulatory level of 20 µg/100g was not detected in any shellfish samples taken from Zone J between 1/9/94 and 30/6/99. Detectable levels of DSP were found in 5 shellfish samples (0.6% of total samples taken) over the same time period.

There have been four shellfish samples above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone (equating to 0.49% of the total samples taken over this period). These samples were Dredge oysters taken from Foveaux Strait in September and December 1994. The identity of the toxin in these samples was not confirmed as brevetoxin, but is unlikely to have been gymnodimine. There has been one probable case of TSP in Zone J, and three suspected cases between the beginning of 1994 and June 1999. The probable TSP case in 1994 arose from the consumption of commercially harvested mussels from Big Glory Bay. It should be noted that the NSP assay at this time utilised an acetone extraction method, which also extracts gymnodimine. Initial rat-feeding trials, and the lack of any epidemiological evidence of illness arising from the consumption of shellfish containing gymnodimine, suggest that this compound is not orally toxic. The current marine biotoxin monitoring programme does not include gymnodimine. Note that low levels of PSP and ASP have been detected at the one sample site on the western coast of the South Island, indicating that these toxins may occur in this environment. It is questionable as to whether this one site adequately protects consumers of non-commercially harvested shellfish along the whole length of the coast. 6.11 ZONE K Zone K encompasses the Chatham Islands, which lie to the east of the main islands of New Zealand. Chatham Island itself consists of several sandy beaches, separated by rocky coasts. Paua and crayfish are common on the rocky coasts, and scallops and

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tuatua occur in Hansen and Petrie Bays. The adjacent Pitt Island has only two small sandy bays, with the rest of the coastline being formed by rocky reefs and eroding rock platforms. Paua and crayfish occur on this coast also. The Chatham Islands are unusual in the absence of the GreenshellTM mussel, and Blue mussels are also rare. Zone K is relatively small compared to other Biotoxin Zones. Based on 1996 Census figures, the total population of the Chatham Islands is 729, with a relatively high proportion (57%) of the population being Maori (Data from Statistics New Zealand). There is little information available about the non-commercial harvest of seafood in the Chatham Islands. However scallops and tuatua, species that appear to readily accumulate and retain PSP and ASP toxins, are available for harvest. Anecdotal evidence suggests that crayfish and paua are important species harvested non-commercially. A commercial scallop fishery exists in the area, and marine biotoxin monitoring, (weekly scallop samples), is undertaken by the scallop fishery during the scallop season. Weekly monitoring for marine biotoxins in non-commercially harvested shellfish was discontinued in Zone K in November 1996, and the seasonal scallop monitoring by the scallop industry currently represents the only monitoring for marine biotoxins in the zone. Due to the low level of monitoring, there are only limited data regarding the occurrence of marine biotoxins in this zone. However, the available data indicate that there were no shellfish samples with levels of PSP above the regulatory level (80 µg/100g), or above the level of detection, between 1/7/93 and 30/6/99. No shellfish samples from Zone K contained ASP levels above the regulatory level (20 µg/g) in shellfish samples taken between 1/7/93 and 30/6/99. Detectable levels (but extremely low levels) were found in 2 samples of scallop roe (0.9% of total samples taken) over the same time period.

No DSP toxins above the regulatory level of 20 µg/100g, or above the level of detection, were found in any of the shellfish samples taken from Zone K between 1/9/94 and 30/6/99.

There has been one shellfish sample above the regulatory level of 20 mouse units using the ether extract mouse bioassay over the period 1/9/94 to 30/6/99 in this zone (equating to 0.5% of the total samples taken over this period). This occurred in a sample of Blue mussels taken from Site K01 in January 1995. The identity of the toxin in these samples was not confirmed as brevetoxin.

There were no reported cases of TSP from Zone K in the period from January 1993 to June 1999. The lack of phytoplankton data from this area, and the inconsistent sampling since 1996, means that the prediction of the risk of marine biotoxin occurrence based on available data should be undertaken with great caution. There is insufficient evidence to conclude that marine biotoxins are less likely to occur in the Chatham Islands than in other areas of New Zealand. Based on data from other areas (Hay, 1996; Kearney, 1999), the high proportion of Maori in the Chatham Islands population suggests that, if shellfish are available for harvest, there would be a comparatively high proportion

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of the population at risk from TSP in the event of the occurrence of marine biotoxins. Further data, including patterns of the occurrence of potentially toxic phytoplankton, and non-commercial shellfish harvesting patterns, are required before a robust assessment of the risk of marine biotoxins in this zone can be undertaken.

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SECTION 7: BIOTOXIN MANAGEMENT IN NEW ZEALAND AND OVERSEAS

7.1 EDUCATION AND COMMUNICATION STRATEGIES Two components of biotoxin management in New Zealand have been discussed in previous sections of this report: the monitoring programme that encompasses hazard surveillance, and the outcome surveillance by reporting of cases of TSP. Educational strategies form another component of the management of biotoxins in New Zealand. These strategies are designed to perform a variety of functions: • Provide an immediate warning to the public not to consume shellfish from an

affected area when a biotoxin event occurs. • Ensure that the public has sufficient background knowledge to be able to interpret

the public warnings and take appropriate action. • Encourage people to use safe practices in the consumption of shellfish to reduce

the risk of TSP (for example, not to eat the gut of scallops or paua). • Ensure that cases of TSP are reported rapidly to local health authorities. Communication strategies include a range of different actions, some of which may be modified to suit local situations. During a biotoxin event, local health protection officers advise the public not to consume shellfish through a variety of media: press releases to news media, including local newspapers, and local radio stations (including iwi radio stations); signs posted at boat ramps and access points to shellfish gathering areas; and in some cases, signs in local stores, and messages on marine radio. A range of different languages may be used in these communications. In the event of very high or persistent toxicity, national media statements may also be made. The information to be included in the media statements in the event of a biotoxin event is specified in the Marine Biotoxin Management Plan. In the non-commercial scallop season, this also recommends the inclusion of a message that people should never eat the skirt and gut from scallops, even when taken from areas that are not subject to warnings about biotoxins. Health protection officers in many cases also directly contact local marae and iwi organisations, and local boating clubs. They may also alert local medical practitioners. It is a requirement of the National Marine Biotoxin Management Plan that Medical Officers of Health should regularly ensure that general practitioners in their areas are aware of the need to notify suspected cases so that these cases can be followed up (Section 5.1) (N.Z. Marine Biotoxin Management Board, 1995). General practitioners may also be reminded about TSP through reports of events in the NZ Public Health Report, or through newsletters from local public health service providers. However, due to the lack of major biotoxin events in most areas in the last few years, it appears from discussion with local Health Protection Officers that there has been little communication of this sort recently. (Author note: This may have changed due to the recent bloom of Gymnodinium catenatum). Educational strategies have also encompassed a variety of methods. When the 1992-93 biotoxin event occurred in New Zealand, most of the New Zealand public had never heard of marine biotoxins or TSP. The publicity through the media (television, radio and newspapers) of this event increased the awareness of the public of these

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issues. Until the restructuring of the management of marine biotoxins in New Zealand in 1997, the Communications Committee of the Marine Biotoxin Management Board was responsible for ensuring that mechanisms were in place to keep the public informed about marine biotoxins. As part of the management of biotoxins, several formal initiatives were made to educate the public about the risks of marine biotoxins. These included: • The preparation in 1994 of a leaflet “Beginners Guide to Marine Biotoxins”,

containing basic information about marine biotoxins, toxic phytoplankton, closure and opening of areas to shellfish harvesting, and the management of marine biotoxins in New Zealand. The prime target group for this leaflet was reporters, so that the information publicised through the media was accurate and informative.

• Data from the 1993 biotoxin event suggested that Maori and Pacific Island people

were possibly more likely to have reported illness after eating shellfish (19% and 3.4% respectively). The Public Health Commission identified the need to inform whanau, hapu and iwi about the potential risks of TSP for Maori by developing the Mataitai Health Education Resource Kit. This resource kit includes posters, pamphlets, and a video, about marine biotoxins, and is presented in both Maori and English. In late 1994, twelve consultation hui were held throughout the country to promote the Mataitai resource. They spanned whanau, hapu and iwi groupings in locations that were inland, estuarine and coastal. These hui recognised the preference for kanohi ki te kanohi (face to face) interaction, and were designed as “training for trainers” hui to introduce the resource to iwi. This enabled further dissemination of the resource to those who could not attend the hui. The facilitator was Tutekawa Wyllie, and technical support was provided by Public Health Commission analysts and local health protection officers (Wyllie, 1995).

Discussion in the hui covered key themes including tikanga and traditional conservation practices, and the relationship between whanau, hapu, iwi and the Crown acting through local and central governments. A number of recommendations arose from the hui, and were presented in a report by Wyllie (1995). This report also includes valuable information about traditional practices that impact on effective management of the risk of TSP for Maori (for example, practices relating to moumou kai). The Public Health Commission distributed the Mataitai Health Resource Education Kits to marae, iwi organisations and general practitioners. The pamphlets and booklets available as part of this resource were reprinted in 1996, and are still available from Public Health Services.

• At a local level (for example, in Northland) pamphlets were distributed at boat ramps advising people about the risks of marine biotoxins, and advising them not to eat the guts of scallop or paua.

There have been no studies undertaken in New Zealand to evaluate the effectiveness of the current education and communication strategies with respect to TSP. It is suggested that this would be a valuable exercise, both in terms of ensuring the cost-

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effectiveness of the management of risk of biotoxins, and in interpretation of epidemiological data that have been collected. Various factors may impact on the effectiveness of these strategies. These include factors related to whether people receive the communications, and whether they are acted upon: • Language – it is noted that the languages used on signage may not adequately

target those people at highest risk – Maori, Pacific Island, and Asian people. Especially in areas where there are high numbers of new Asian immigrants with limited English skills, the use of appropriate language on signage would improve communication of risk.

• In some isolated areas it is difficult to post adequate signage (for example, Marlborough Sounds, where access to many isolated bays is possible by boat, and people may be away from public boat ramps/piers for many days).

• Method of communication must be culturally appropriate – At the Mataitai hui in 1994 it was suggested the news media were not an effective method of communication of public warnings to Maori. They identified a need for the HPO’s to work in partnership with kaumatua in each area to ensure that the message is received appropriately: “the message is more effective coming from iwi Maori instead of Health Protection Officers” (Wyllie, 1995). This recommendation has been acted upon in some areas, but there is no evidence of any investigation of the most effective means of communicating with other “high risk” groups.

• There may be cultural pressure not to act on warnings – for example, several such issues were identified as a result of the Mataitai hui. Concern was expressed that the discarding of parts of shellfish is contrary to Maori traditional practices related to moumou kai. In addition, it was suggested that “a significant factor to Maori non-compliance concerned those iwi who lived at the moana and retained kaitiaki responsibility whereby the impact of closures affected their mana, tikanga and their identity” (Wyllie, 1995). While there has been some effort to identify such issues with respect to Maori, there has been little effort to investigate issues relating to other ethnic groups identified as being at a high risk from TSP. Such issues may impact on whether public warnings are heeded by these groups.

• There is little feedback to reinforce compliance with public warnings. In general, anecdotal evidence suggests that those members of the public who have not heeded warnings have not become ill (often due to the fact that the regulatory toxin levels are set with a safety margin), and this may result in a level of complacency in complying with warnings.

It is noted that there appears to be no strategy targeting people at the most risk with respect to respiratory irritation syndrome (e.g. people with asthma). It is suggested that protocols for communicating this risk to the public in the event of a bloom producing toxins that cause RIS, could usefully be added into the section on “Marine Biotoxin Control” in the Ministry of Health “Food Administration Manual” (Ministry of Health, 1997a).

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7.2 OVERSEAS MARINE BIOTOXIN MONITORING PROGRAMMES AND TECHNOLOGY DEVELOPMENTS

In the course of this review, the Ministry of Health indicated that they were interested in examining overseas marine biotoxin monitoring programmes, to determine whether there were any management options that could be of benefit in New Zealand. The following section briefly discusses marine biotoxin monitoring programmes overseas and associated developments in biotoxin technology. During this project, health authorities overseas were questioned by Dorothy-Jean McCoubrey (MAF Verification Authority) about the marine biotoxin monitoring programmes for non-commercially harvested shellfish in their countries. Countries where inquiries were made included Australia, Canada, Spain, Denmark, Ireland, United Kingdom, Philippines and USA. The results are summarised below. Many countries do not have a separate monitoring programme for non-commercially harvested shellfish outside the monitoring programme for the shellfish industry. This may be because either there is not a strong tradition of non-commercial harvesting (e.g. Denmark) or because non-commercial harvest of shellfish is forbidden, to protect over-exploited shellfish beds (e.g. Galicia, Spain). In Canada, most of the non-commercial harvesting areas are covered by the sampling programme that covers commercial harvesting, with some additional sampling, particularly from within National Parks. In the USA, the monitoring criteria in the National Shellfish Sanitation Programme only covers commercial shellfish that are transferred interstate, and each harvesting state develops its own programme for non-commercially harvested shellfish. In some states, such as Maine, the two programmes are integrated. Recreational harvesting in Texas and Florida is based on monitoring phytoplankton counts for Gymnodinium breve. University researchers undertake monitoring in Texas, and areas are closed when the cell counts are high. Most countries advertise public warnings through the news media, but advertising the status of shellfish gathering areas on a web page on the Internet is common. Another common method of disseminating such information appears to be through a telephone hotline where callers can dial in for recorded information. In the USA, there is a much higher level of community involvement in monitoring for biotoxins than in New Zealand. In California, Maine and Massachusetts, “volunteer observer networks” have been set up to monitor for potentially toxic phytoplankton. These are run in conjunction with volunteer groups that monitor water quality in general. We were also provided with an example of community involvement in education, in which a teacher from Maine who had her students involved in the phytoplankton monitoring programme had some students develop a fact sheet about biotoxins that went to all the doctors in their immediate region. The comment was “It was very low budget, cute, and probably got the attention of the docs. Local is always better in the US” (Paul Anderson, University of Maine, U.S.A, pers. comm.). As knowledge of the causative agents of TSP increase, there appears to be an increasing trend toward the inclusion of phytoplankton monitoring in marine biotoxin monitoring programmes. Phytoplankton is cost-effective, can provide early warning of shellfish intoxication, and the results are available promptly (Todd, 1999).

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Monitoring for toxic phytoplankton species requires equipment (i.e. plankton samplers, microscopes) and the ability to recognize potentially toxic species. The level of technical expertise required is related to the number of different potentially toxic species, and the type of species – as discussed earlier, toxic and non-toxic species may not be readily distinguishable. In some countries there is a shortage of people with the necessary skills to analyze plankton samples. In Korea, this is being overcome by the use of remote television monitoring network: microscopic images from local laboratories are transmitted to a central institute in Pusan. An expert in a central institute can identify the algae from the transmitted microscopic image, thus reducing the need for large numbers of highly trained staff (Kim, 1999). A different kind of approach to phytoplankton monitoring is being taken in some states in the USA. As mentioned, in California, Maine and Massachusetts, “volunteer observer networks” have been set up to monitor for potentially toxic phytoplankton. They have been trained in sampling techniques and phytoplankton identification, and are equipped with nets and field microscopes (Anon, 1998; Hall, 1999). Monitoring is qualitative rather than quantitative, providing a rough estimate of numbers only. Samples may be preserved and sent to a laboratory for identification if required. While these plankton observations do not replace toxicity testing, they make the monitoring programme more cost effective by focussing toxicity testing on times, locations and toxins of greatest concern. The net sampling method used in the programme is not suitable for sampling Gymnodinium species, since they are too fragile and disintegrate when sampled through a net. The system would therefore have to be modified for use in areas such as New Zealand, where NSP is a potential problem. Whether or not such a system would be feasible in areas where greater numbers of species of potentially toxic phytoplankton need to be identified is also a relevant consideration in regard to the use of this system elsewhere. More automated methods of analyzing phytoplankton samples are also being investigated - for example, the use of flow cytometry (Hofstraat et al., 1994), visual identification using neural network techniques (Culverhouse, 1995), and as mentioned earlier, the use of gene probes (Rhodes et al., 1997). However, at this stage analysis of phytoplankton samples for potentially toxic species is generally undertaken with the use of a light microscope. In New Zealand, molecular probes are being used successfully to distinguish between potentially toxic and non-toxic species of Pseudo-nitzschia, and probes are becoming available for Alexandrium species. Overseas, an instrument for autonomous collection and real-time detection of harmful algae using species-specific molecular probes is being developed (Chris Scholin, HABTech Workshop presentation, Nelson, February 2000). This instrument is being designed to collect discrete water samples autonomously, concentrate particles contained within those samples onto filter discs, and automate application of species-specific DNA probes to identify and quantify particular organisms so captured. In addition to archiving discrete samples, the instrument is also capable of transmitting results of the probe assays in real-time to a remote location for data processing and interpretation. While this is useful as a measure of risk, this technique does not indicate toxin levels, since strains within species may differ in toxin production. However, DNA probes are currently proving useful to the New Zealand shellfish industry when trying to decide whether to implement a voluntary closure to harvesting based on phytoplankton sample results, and as knowledge of toxin production in phytoplankton increases, so too will the effectiveness of this technology.

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The use of remote sensing techniques in monitoring to provide early warning of algal blooms is also being investigated overseas (Satsuki et al., 1989; Millie et al., 1992; Belliss, 1993; Wiebe, 1995; Tester et al., 1998). Given the low cell densities of some toxic phytoplankton that result in shellfish toxicity (e.g. Alexandrium sp.), these techniques are unlikely to be useful as early warning indicators in New Zealand. The most widely used toxin test method in shellfish samples is still the mouse bioassay, although HPLC is used by five of the seven countries/regions that test for ASP (Andersen, 1996). Except for ASP, there is a large variation between countries in the critical toxin concentration limits. Concern about this is frequently expressed in the literature. This is especially an issue in Europe, where the borders between EC member states are disappearing. In order to deal with this issue, the EC has nominated National Reference Laboratories on marine biotoxin analysis, and a Community Reference Laboratory with the aim of establishing an information exchange network about analytical methodology and to coordinate the standardization of these criteria (Fernandez et al., 1996). New test methods for biotoxins found in New Zealand are being developed both in New Zealand and overseas, to replace mouse bioassays. These include methods based on chemical analysis (for example HPLC, LC-MS), and in vitro assay methods that may be broadly categorized into two general sub-types: functional assays, or structural assays (Cembella et al., 1995a). Cembella et al. (1995a) summarise these as follows: “Functional assays are based upon biochemical action of the toxin (e.g. binding to the ion channels of neuroreceptors), and hence quantitation will tend to correlate well with the specific toxicity of the analyte. In the case of matrices which contain several toxic components with a similar mode of biological activity, but which vary in specific potency, such assays should yield an accurate estimate of net toxicity. In contrast, structural assays are dependent upon the conformation interaction of the analyte (toxin) with the assay recognition factor (e.g. epitopic binding sites in immunoassays). Thus cross-reactivity in such structural immunoassays is limited to components with compatible epitopic sites and often does not reflect relative biological activity or specific toxicity. This lack of broad-spectrum cross-reactivity for toxic, naturally occurring analogs is a major drawback to the use of quantitative immunoassays for screening phycotoxins in naturally contaminated samples.” Functional assays for marine biotoxins include cell culture (cytotoxicity) bioassays (e.g. neuroblastoma assays), and enzymatic tests (e.g. protein phosphatase inhibition assays). Structural assays include immunoassays of various kinds (e.g. ELISA techniques). Each method has advantages and disadvantages. Chemical analysis methods are accurate in their determination of what toxins are present, but may lack sensitivity. They require expensive analytical instruments, and often involve complex sample extraction procedures. Thus they can only be performed in centralised laboratories. Analytical instrumental detection methods are based on sequential processing of samples, so it is more difficult to process large numbers of samples (Cembella et al.,

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1995b). In New Zealand, LC-MS methods are being developed for detection of yessotoxin and pectenotoxin, but accessibility to equipment may be a problem if large numbers of samples need to be processed. (Author note: Recent developments initiated by the shellfish industry have resolved this issue). On the other hand, the processing of large numbers of samples may reduce the cost, since the set-up times per sample would be reduced. Neuroblastoma assays, based on the activity in sodium channels, are being developed in New Zealand to test for brevetoxins (NSP) and saxitoxins (PSP). These techniques are relatively sensitive, and are specific to toxin activity. Neuroblastoma assays could also be combined with biosensor technology to produce a test that can be undertaken outside a laboratory by relatively unskilled people. The protein phosphatase inhibition assays have been developed to detect DSP toxin analogues, Okadaic acid and DTX1 in both shellfish samples and phytoplankton. The protein phosphatase type-2 inhibition assay is more sensitive than the DSP-ELISA Check Kit (P. Truman, ESR, pers. comm.), and may be useful if the regulatory limits for DSP were to be lowered in New Zealand. Functional assays also include neuroreceptor binding assays, such as the saxitoxin radio receptor binding assay. This is basically a competitive displacement assay in which radiolabelled and unlabelled STX and/or its derivatives compete for a given number of available receptor sites in a preparation of rat-brain synaptosomes. The disadvantage of these types of assays is the use of vertebrate animals. Work is progressing on the use of cloned receptors in these assays. Immunoassays are structural assays. ELISA techniques and sandwich hybridization assays currently present a promising option for “dock-side” techniques. ELISA technology is well established, and widely used in other applications. However, these techniques do have disadvantages, including the susceptibility for generating false positives due to the presence of non-toxic (or less toxic) congeners, or failure to detect all toxigenic components when a complex suite of toxin analogues is present. One of the difficulties in developing immunoassays is the difficulty in obtaining a supply of toxins to work with. Currently this is particularly pertinent to pectenotoxins, since the phytoplankton that produce these toxins cannot be cultured in the laboratory sufficiently well to obtain quantities of toxin. There are currently very limited options for mitigation or control of toxic algal events. Because of the economic impact of harmful algal blooms, research into the control of algal blooms is being undertaken overseas. There are several potential methods of controlling blooms being investigated. These include the use of bacteria and viruses to kill phytoplankton populations (Nagasaki et al., 1995, Imai et al., 1995, Yoshinaga et al., 1995a & 1995b, Gastrich et al., 1998, Nagai & Imai, 1998, Yoshinaga et al., 1998), and the use of clay flocculents to disperse blooms (Kim, 1998; Bae et al., 1999; Sun et al., 1999; Sengco et al., 1999; Sengco et al., 2000). The environmental impacts of releasing algicidal bacteria or viruses into algal blooms have not been determined, and at this stage would seem to be a high-risk option for control. Clay flocculents are being used successfully in Korea to control blooms, and this may present a viable option for the control of large blooms in the future. However, this technique may be more applicable to blooms of a non-toxic nature (such as those that impact on

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aquaculture), and would need to be approached with some caution. An attempt was made to control a Gymnodinium breve red tide in Florida in the 1950’s with the application of copper, which lysed the algal cells. This was unsuccessful in terms of toxin reduction, since the lysed cells released the toxin into the water, which resulted in increased and persistent toxicity in the water (Steidinger, 1983). It is possible that the application of flocculents might have a similar effect on Gymnodinium species, or indeed other species here. The issues of the environmental impact of applying large quantities of clay into our coastal waters would also have to be investigated, and weighed against the impact of toxic phytoplankton blooms. Although some research is being undertaken to investigate the environmental conditions under which toxic algal blooms occur in New Zealand (e.g. Sharples et al., 1998), these processes are still not well understood. Data from an environmental monitoring programme established by the mussel farming industry in the Marlborough Sounds, and more recently by the oyster farming industry at Coromandel and Mahurangi Harbour, could provide valuable information if linked with data from the marine biotoxin monitoring programme. Currently there is little action able to be taken to prevent the occurrence of toxic phytoplankton in New Zealand. However, the threat of introduction of new species of toxic algae to New Zealand by ballast water is being addressed in a Government strategy under which new international regulations request ships to exchange ballast water in mid-ocean away from coastal influences. There are technical difficulties associated with exchanging ballast water on large ships, and research to determine alternative practicable measures to reduce the risk from ballast water is being undertaken (Ministry of Fisheries, 1998). 7.3 NEW ZEALAND BIOTOXIN MANAGEMENT IN A GLOBAL

CONTEXT In a review of New Zealand’s non-commercial marine biotoxin monitoring programme, it is pertinent to consider some factors from a wider context than those covered by the risk analysis model, which has a very sharp focus. Some of these factors are summarised below: • There is an apparent increase and/or increasing awareness, of marine biotoxins

globally. This may be linked to global warming, the distribution of toxic algal species via ballast water in ships, and/or increasing concern and understanding about health issues globally.

• People are becoming more concerned about environmental quality. There is an

awareness of the potential for human activities to impact on the occurrence of marine biotoxins, and a concomitant lack of understanding about the processes that cause toxic phytoplankton blooms.

• Social values are changing in the western world, including New Zealand, with

respect to animal rights. It is suggested that the mouse bioassay will not continue to be acceptable as a toxin detection technique.

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• New toxin detection techniques are being developed, presenting opportunities for improvement in monitoring techniques.

• On a world-wide scale, New Zealand is unusual in that a wide range of marine

biotoxins have been detected here. This adds a level of complexity to the process of monitoring for marine biotoxins that is not found elsewhere.

Along with the specific assessment of risk, these factors provide part of a broad framework within which options for management of the risk of TSP and respiratory irritation syndrome are developed.

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SECTION 8: DISCUSSION AND CONCLUSION This section provides a summary of the conclusions drawn from analysis presented in previous sections, and outlines potential options for cost-effective management of the risk presented by marine biotoxins in New Zealand. More detailed discussion of options is provided in Part 2 of this report under separate cover (Hay et al., 2000). Following the analysis and collation of information presented in previous chapters, some broad observations relating to the risk of TSP and RIS can be made: • In broad overview, the situation in New Zealand with respect to marine biotoxins

is characterised by:

• Wide distribution of potentially toxin-producing phytoplankton throughout New Zealand.

• Periods of low frequency of biotoxin occurrence followed by periods of

higher frequencies, generally in relatively localised areas. Biotoxins may then persist in a localised area for a period of time, sometimes in one shellfish species/at one location.

• Possible seasonal patterns in the occurrence of some biotoxins in shellfish

(for example, ASP), and not in others (e.g. PSP).

• In periods of low biotoxin activity, some toxins are present very rarely (e.g. NSP toxins) and others are common at low levels in some areas (e.g. Domoic acid).

• Possible differences in the accumulation and retention of biotoxins by

different New Zealand shellfish species. These differences are potentially significant in terms of the risk of TSP to consumers.

• Currently there is a poor understanding, both here and internationally, of the

factors influencing the occurrence of toxic phytoplankton blooms. There is insufficient information to be able to predict the future occurrence of marine biotoxins in New Zealand with confidence.

• There is a possibility that the impact of human activity may increase the risk of

marine biotoxins – for example, through global warming, addition of nutrients to the marine environment, and transportation of toxic species from place to place. This would suggest that there might be an increasing risk of TSP and RIS in the future.

• The oral toxicity of some of the marine biotoxins found in New Zealand,

including the effect of long-term ingestion of low levels of toxin, are still unknown. The current outcome surveillance may not detect these impacts. This uncertainty needs to be taken into account in the management of risk of biotoxins.

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• The estimation of the risk of TSP and RIS in New Zealand is limited by a significant lack of robust data. This includes data regarding interspecific and intraspecific differences in toxin accumulation and retention by shellfish for each toxin type, and long-term data regarding geographic and temporal patterns in biotoxin occurrence. There are very significant discrepancies between the few studies that have been undertaken on non-commercial shellfish harvesting, and this suggests that further studies are required in this field.

• The potential risks presented by marine biotoxins in New Zealand are not

distributed evenly across the population. There is a disproportionate potential impact on sectors of the population that consume more non-commercially harvested shellfish (for example, Maori, and possibly Pacific and Asian peoples), on older people or people in poor health, and on asthmatics. Any change to the management of risk of marine biotoxins needs to consider any strategic health objectives or goals relevant to the sectors impacted. The objectives contained in the current strategy for public health specifically mention the improvement, promotion and protection of the health of Maori and Pacific Island peoples, and the reduction of disability and death rates from asthma in children.

• The options for the public to minimise or avoid the risk of TSP in the absence of a

marine biotoxin monitoring programme are limited. Risk can be reduced by avoidance of consumption of high-risk portions of shellfish, such as scallop and paua guts. However, in the absence of a marine biotoxin monitoring programme, the only way of avoiding the risk of TSP is by not consuming non-commercially harvested shellfish. Some sectors, such as Maori, to whom seafood is of cultural and economic importance, are likely to find this option unacceptable (Wyllie, 1995).

• The options for effective surveillance for TSP and RIS are limited. Environmental

surveillance for public health involves three types of complementary surveillance: hazard, exposure and outcome surveillance. Monitoring for toxic phytoplankton and biotoxins in shellfish constitutes hazard surveillance, and this is the method of surveillance that is strongly relied upon both in New Zealand and internationally. Other forms of surveillance are limited by lack of knowledge: there is a lack of biomarkers to detect the exposure of humans to marine biotoxins, and this means that exposure surveillance is not possible. Outcome surveillance is also hindered by lack of knowledge about the clinical symptoms of TSP, resulting in an under-reporting of cases (Fleming et al., 1995; Fleming et al., 1998). At present, chronic effects caused by long term consumption of low levels of toxins are highly unlikely to be recognised as arising from marine biotoxins at all. Until these issues are resolved, reliance on hazard surveillance in the management of marine biotoxins with respect to public health remains the most effective option.

Within the strategic framework for public health (Section 1.4), and the global context discussed in Section 7, a range of broad issues relating to the management of marine biotoxins in New Zealand require consideration. These encompass both strategic and technical issues. In the course of this review it has become apparent that there is a dearth of information on which to base robust risk analysis, and that the mere passage of time

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on the present course of action will not significantly alter this situation. Uncertainty increases the overall level of risk. The marine biotoxin monitoring programme is currently focused only on utilising the data gathered for immediate needs (i.e. is it currently safe to harvest shellfish from this area?). It is suggested that a more proactive, strategic approach to the collection and use of data would result in substantial improvements in cost-effectiveness in the future. This would involve:

a) Improved management of the data collected in the marine biotoxin monitoring programme. This would ensure that all relevant information is recorded consistently and in a manner that is accessible for informed analysis, preferably on a central database. In addition to many problems associated with the recording of historical data, current deficiencies include: differences between laboratories in the way in which low levels of toxins are quantified on the database, and lack of facility (e.g. fields in the database) to record all relevant data. Relevant data include information about changes to protocols (e.g. changes in test methods, changes in phytoplankton species names etc.), environmental data, results of determination of phytoplankton species by gene probe, results of any additional testing undertaken in addition to regulatory requirements (e.g. LC-MS, neuroblastoma or ELISA assays for NSP) and information about mouse deaths in mouse bioassays. An annual audit of the epidemiological data would be beneficial to ensure the quality of the data entered, and to identify cases where results from testing have not been entered, or case status updated.

b) A sampling strategy to ensure that robust, long-term data are available to

detect patterns in the geographic and temporal distribution of biotoxins. Monitoring data need to be gathered consistently and regularly from sufficient representative sites within each zone. The initiation of additional monitoring is suggested in some areas, for example, the collection of regular phytoplankton data from suitable sites on the western coast (such as sites within harbours). The Chatham Islands (Zone K) also represents an area from which very little data have been collected. The collection of phytoplankton data from this area, possibly through the use of volunteers, would be beneficial in future risk assessment.

c) A co-ordinated management strategy that ensures sampling and

analytical techniques used in the monitoring programme are scientifically validated, and that changes in the programme are consistent with the results of risk analysis and long-term strategies to increase cost-effectiveness. Co-ordination is required to ensure that the marine biotoxin monitoring programme does not progressively change in a “piecemeal” fashion.

As part of a strategy to improve the long-term cost-effectiveness of the marine biotoxin monitoring programme for non-commercially harvested shellfish, an increased emphasis on advocacy and facilitation of relevant research is suggested.

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This includes research to:

• Determine inter-specific differences between shellfish with respect to biotoxin uptake, accumulation and detoxification processes.

• Determine the oral toxicity, including the impacts of long-term ingestion of low levels of toxin, for those compounds currently known to be toxic to mice by IP injection, but for which oral toxicity is unknown.

• Relate environmental parameters and processes to the occurrence of toxic phytoplankton, and toxin levels within species.

• Validate new toxin testing methods for biotoxins found in New Zealand (such as the PSP MISTTM Alert Kit).

• Rigorously determine non-commercial shellfish harvest patterns. • Evaluate the effectiveness of education and communication strategies with

respect to the management of the risk of marine biotoxins in New Zealand. The Biotoxin Research Strategy provides a forum for facilitating relevant research. An increased emphasis on promotion of research relevant to the risk of marine biotoxins from non-commercially harvested seafood need not result in additional cost to the Ministry of Health. One of the cross-cutting themes identified as underpinning all public health goals, objectives and targets is “Building strategic alliances” (Ministry of Health, 1997b; see Section 1.4 of this report). While participation in the Biotoxin Technical Committee is an example of such an alliance, increased incorporation of this theme in the strategy for management of marine biotoxins could have some benefit. For example, there are environmental data being collected by a range of other organisations (such as shellfish industry associations, Regional Councils etc.) for other uses. Similarly, a range of organisations such as the Ministry of Fisheries, Regional Councils, Maori, and community groups, are interested in non-commercial harvest of shellfish. Strategic alliances with organisations with research interests in common could assist in facilitation of these research outcomes. Maintenance of cost-effectiveness in the marine biotoxin monitoring programme is obviously very important. It is pertinent to consider whether, in the light of a re-evaluation of risk arising from analysis of historical data, costs could be reduced by broad reduction in temporal or spatial components of the monitoring programme. Several factors are relevant to consideration of this issue: • There are sufficient data to suggest that in the absence of a non-commercial

marine biotoxin monitoring programme, a significant number of people could become ill as a result of TSP in some years. Some of these people would be severely ill, with the risk of death. For example, one scenario suggests that 50% of the PSP cases (amounting to nearly 2000 people) would have moderate to severe symptoms. The long-term effects of ingestion of “DSP” toxins, including Okadaic acid, dinophysistoxins, yessotoxin and pectenotoxin, are unknown, but potentially present a risk. The incidences of these two latter toxins are unknown because they are not currently included in the monitoring programme. The levels of Domoic acid detected in shellfish to date would only produce relatively mild symptoms of ASP in adults, (although one scenario suggested that up to 750 people could be affected). The potential impact of NSP remains somewhat unknown due to a lack of robust data on shellfish toxicity levels from the 1993 event, but has been very low in subsequent years.

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• In reviewing the non-commercial marine biotoxin monitoring programme, the

relevance of historical data in predicting future occurrence of marine biotoxins needs to be considered. There are now 5-6 years data from monitoring the occurrence of biotoxins in shellfish. Temporal and geographic stratification of sampling, and inconsistent monitoring data (for example, discontinuities and changing sample sites) mean that the actual historical occurrence of biotoxins in shellfish throughout New Zealand cannot be rigorously quantified. International data suggest that there may be long-term cycles (e.g. 18-19 years) in the occurrence of biotoxins (White, 1987). The time over which marine biotoxins have been monitored in New Zealand is thus relatively short. Internationally, there are many instances where significant levels of toxins have “unexpectedly” appeared. These factors, plus the widespread distribution of potentially toxic phytoplankton, suggest that the use of present historical data in the prediction of future risk of marine biotoxins should be undertaken with extreme caution.

• The risk of exposure of the New Zealand public to TSP from the consumption of

non-commercially harvested shellfish is dependent, not just on the occurrence of biotoxins in the marine environment, but also on the patterns of non-commercial shellfish harvesting and potential differences in accumulation and retention of biotoxins by different species of shellfish. Definitive data are lacking in both these areas.

If the short-comings outlined in the last two paragraphs could be ignored, and it could be assumed that the occurrence of marine biotoxins will not increase (as a result of human activities, changes to the environment such as global warming, or the spread of toxic phytoplankton species through the ballast water of ships), then one could possibly conclude that cost savings through a reduction in monitoring could be achieved without a resulting increase in the risk of TSP to the public. However, it is suggested that these short-comings are too comprehensive to be totally ignored. Consequently, any cost-saving reductions in marine biotoxin monitoring (either in the temporal or spatial components of the monitoring regime, or reduction in effectiveness of biotoxin detection) must be considered in light of the question: Would an increased level of risk to the public resulting from marine biotoxins be acceptable under the current strategy for public health? This issue is significantly complicated by the lack of robust data allowing the impact of changes in the management of the risk of biotoxins to be accurately quantified. In consideration of the current public health strategy (refer to Section 1.4), it appears that an increased level of risk would be inconsistent with the strategy unless the money saved by the change could be more effectively used elsewhere. Within the scope of this review, and the data available, we are unable to assess this. Education of the public about the risks associated with marine biotoxins, and communication of risk in the event of marine biotoxin occurrence, are important aspects of the way in which the risk of marine biotoxins are managed. The issues associated with effective communication regarding marine biotoxins are not simple. As the public become more aware of the marine biotoxin monitoring programme through communication of public warnings via the news media, there is potentially an increased expectation that the marine biotoxin monitoring programme will provide

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protection from the risk of marine biotoxins. On the other hand, the effectiveness of the monitoring programme is diminished when the public consumes shellfish in defiance of a public warning, but individuals do not get sick. As one Health Protection Officer commented recently: “The public test shellfish when we warn them, and if they get sick they tell nobody. If they do not get sick, they tell everybody” (T. Beauchamp, Northland Health, oral presentation at the Marine Biotoxin Science Workshop, November 2000). It is apparent that communication of the risk of marine biotoxins to the public is not always effective. However, the extent to which this is the case is unknown, as are the impacts of this: People who have consumed shellfish in the face of a public warning are unlikely to report consequent illness unless it is very serious, and the health impacts of consistent consumption of shellfish containing low levels of biotoxins are unknown. In areas where consumption of non-commercially harvested shellfish is of particular cultural or economic importance to a comparatively high proportion of the local population, and that are subject to long closures due to biotoxin persistence in one shellfish species only, the introduction of species-specific closures could be considered. This would require knowledge of the dynamics of accumulation and detoxification of biotoxins in each shellfish species. Some additional testing to clear a species for harvest would also be required. The education and communication requirements with respect to species specific public warnings would be greater than in the case of implementation of public warnings that apply to all shellfish types. Protection from the risk of biotoxins would rely not only on the public understanding the implications of a public warning sufficiently well to take heed, but also on the ability to discriminate between different shellfish species when they are collecting them. More complex public warnings may be harder for the public to recall in detail. In this case, the use of a telephone “hotline”, containing regularly updated recorded messages of the biotoxin status of each area, could be beneficial. In addition to the benefits to the public in terms of increased access to shellfish, species specific public warnings at the end of biotoxin events would assist in maintaining the credibility of the marine biotoxin monitoring programme, by restricting the application of public warnings to only those shellfish species that actually contain marine biotoxins. In discussing potential broad changes to the marine biotoxin monitoring programme, it is noted that there is currently no specific surveillance or management of risk with respect to RIS. One option would be to utilise the current phytoplankton monitoring programme to detect the risk of RIS based on high numbers of relevant Gymnodinium species, and to issue public warnings based on this data. In effect, this is what currently happens, and only formalisation of the protocols is required. Provision of information to medical practitioners about the risks of RIS to asthmatics is also suggested. There is currently a high level of co-operation and co-ordination between the Ministry of Health and MAF and the shellfish industry with respect to monitoring for biotoxins. Any changes proposed to the marine biotoxin monitoring programme for non-commercially harvested shellfish need to take into consideration any impacts on the marine biotoxin monitoring programme for commercially harvested shellfish also. The impacts of any changes proposed in the commercial monitoring programme on the non-commercial marine biotoxin monitoring programme also need to be considered. Currently the Ministry of Health pays the shellfish industry for data

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received from their monitoring programme. Both industry and the Ministry of Health programmes use the same test methods. This has advantages in terms of cost, since it means economies of scale are able to be achieved by the testing laboratories. The volume of monitoring, (in terms of sample numbers), undertaken in the commercial programme is approximately twice that of the public health programme (Janet Young, Ministry of Health, pers. comm.). This means that the shellfish industry is in a more dominant economic position in driving any changes with respect to testing methods. In addition to concerns about the health of shellfish consumers, the design of the marine biotoxin monitoring programme for commercially harvested shellfish is influenced by several other issues. These include: • The attitude and regulations regarding various new biotoxins in overseas markets;

and • The methods overseas markets use to test for biotoxins in incoming products. Currently the shellfish industry is planning to initiate testing for DSP group toxins (Okadaic acid, DTX-1, DTX-2, DTX-3, Okadaic diol esters, pectenotoxin and yessotoxin), using LC-MS. The shellfish industry is also aware of the unacceptability of continuing to use mouse bioassays in toxin testing. The moves initiated by the shellfish industry provide the Ministry of Health with the opportunity to consider alternative toxin test methods in an environment where either economies of scale, or competition for business between competing testing laboratories, have the potential to provide downward pressure on cost. Technical developments in the field of testing for biotoxins provide opportunities for more definitive information about the identity of biotoxins in shellfish samples than the mouse bioassay currently used. Testing that focuses more specifically on identifying particular toxins, or, in the case of functional assays, specific modes of toxic activity, has advantages in the reduction of “false positive” toxicity results that may occur in mouse bioassays. However, a move to testing for specific biotoxins or types of toxin activity would remove the hazard surveillance for new biotoxins currently provided by the mouse bioassays. In this situation, robust outcome surveillance would be of increased importance. Some new biotoxin assay methods may offer cost advantages over the mouse bioassays currently used. Specific options with respect to the potential use of new test methods are discussed in Part 2 of this report under separate cover. Phytoplankton monitoring plays an important role in the monitoring of marine biotoxins in New Zealand. It can provide early warning of shellfish toxicity, and additional information that is important in the management of the risk of biotoxins. Our analysis suggests that further data are required to confirm the robustness of the current phytoplankton monitoring programme in predicting shellfish toxicity. This applies to both monitoring using counts of potentially toxic phytoplankton species, and the use of whole cell DNA probes for identification of potentially toxic Pseudo-nitzschia species. However, review of the phytoplankton monitoring data highlighted several technical issues. With respect to some phytoplankton species, the impact of the low level of precision in the phytoplankton methods currently used, and whether any additional assurance gained by improving this precision justifies additional cost, needs to be considered. In addition, the impact of succession of Pseudo-nitzschia species within a Pseudo-nitzschia bloom on the protocols for use of gene probes

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within the monitoring programme, requires further investigation. As mentioned earlier, validation of the protocols for use of whole cell DNA probes in the monitoring programme should be undertaken before they are used in decisions not to undertake shellfish toxicity testing. In addition to the technical issues raised by our review of the robustness of the phytoplankton monitoring programme, one other important issue was highlighted. This concerns the quality of critiquing of sampling protocols and analytical techniques prior to their introduction to the monitoring programme. While the quality assurance programmes of the organisations delivering sampling or analytical services to the programme ensure a consistent quality, they do not necessarily address the issue of whether the protocols used are designed to deliver the required result. Many of the technical issues that require consideration in biotoxin monitoring are complex and specialised, and the Marine Biotoxin Technical Committee regularly seeks expert advice. However, it is suggested that the Marine Biotoxin Technical Committee could benefit from consistently seeking independent advice from appropriately qualified technical specialists to peer review technical proposals when considering major changes to the marine biotoxin monitoring programme. In conclusion, the analysis of biotoxin data undertaken as part of this review identified some broad patterns relating to the occurrence of biotoxins in New Zealand. Also identified were a number of very key areas where lack of information prevents a robust assessment of the risks to public health presented by biotoxins. Robust quantification of changes in risk resulting from changes to the monitoring programme is thus not possible. We therefore cannot recommend any broad cost-saving changes in the frequency or distribution of monitoring for marine biotoxins, especially as the sectors of the public most probably disproportionately at risk include those for which there are specific strategic objectives and goals to improve public health. An alternative is to address the current barriers to risk analysis, and ensure that the data being collected in the monitoring programme is used more effectively to obtain the information required for robust risk analysis in the future. The options suggested in this review address both specific technical issues that have been highlighted in the review process, and broader issues that will improve the cost-effectiveness of the programme in the longer term. Particularly in the latter case, strategic relationships with outside organisations will be important in determining synergies with respect to both effectiveness and cost.

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193

APPENDICES

194

APPENDIX I(A): PHYTOPLANKTON TRIGGER LEVELS Phytoplankton Species Toxin Level in

composite sample to

trigger flesh sampling (Cells/L)

Industry voluntary

closure pending flesh testing results

(Cells/L)

Issue public health

warning (Cells/L)

Alexandrium minutum PSP 100 500 5,000 Alexandrium ostenfeldii PSP 100 500 5,000 Alexandrium catenella PSP 100 500 5,000 Alexandrium tamarense PSP 100 500 5,000 Alexandrium angustitabulatum

PSP 100 500 5,000

Pseudo-nitzschia species (>50% total biomass)

ASP 50,000 200,000 N/A

Pseudo-nitzschia species (<50% total biomass)

ASP 100,000 500,000 N/A

Gymnodinium c.f. breve NSP 1,000 5,000 5,000 Dinophysis acuta DSP 500 1,000 N/A Dinophysis acuminata DSP 1,000 2,000 N/A Prorocentrum lima DSP 500 1,000 N/A

195

APPENDIX I(B) THE COMMERCIAL AND NON-COMMERCIAL MONITORING PROGRAMME SAMPLING REGIMES

1) Non-Commercial Phytoplankton Monitoring Programme

Location

Site No.

Frequency

Marsden Point P021 Weekly Bay of Islands (Tapeka Point)

P020 Weekly

Whangaroa P059 Weekly when industry not sampling Whatuwhiwhi P023 Weekly Houhora Bay P023 Weekly Rangaunu Harbour P024 Weekly when industry not sampling Waimangu Point P028 Weekly when industry not sampling Tiritiri/Whangaparaoa P019 Weekly Port Fitzroy P027 Weekly when industry not sampling Waiheke P032 Weekly when industry not sampling Kennedy Bay P017 Weekly Tairua P018 Weekly Steels Reef P001 Weekly 1 September to 15 February

Fortnightly 1 July to 31 August Matakana Bank P002 Weekly Motiti Island P003 Weekly Ohope Beach P005 Weekly Te Kaha P004 Weekly Tolaga Bay Wharf P008 Weekly Hicks Bay Wharf P010 Weekly Hawkes Bay P007 Weekly Wellington Harbour entrance P009 Weekly Collingwood Farms P034 Weekly when industry not sampling The Glen – Tasman Bay Weekly when industry not sampling Port Motueka – Tasman Bay P033 Weekly when industry not sampling Takaka River/Tata Island P006 Weekly when industry not sampling Wedge Point P026 Weekly Tory Channel P025 Weekly Kaikoura P014 Fortnightly: 1 November-31 March Akaroa (The Kaik) P011 Weekly Taylors Mistake P012 Weekly Caroline Bay/Timaru P013 Weekly Blueskin Bight/Heyward Point

P015 Weekly

Riverton/Colac Bay P016 Weekly

196

2) Non-Commercial Shellfish Sampling Programme Location Site

No. Species/Frequency Toxins

Langs Beach B25C Oyster- Monthly All Waipu B10 Tuatua/Pipi- Monthly All One Tree Point B15B Pipi-Monthly when industry not

sampling All

Ngunguru/Tutukaka B05 Oyster- Monthly 1 April- 1 Sept. otherwise fortnightly

All

Oakura B01 Tuatua/Pipi/Oyster Monthly 1 April-1 Sept. otherwise weekly

All

Te Haumi A18 Pipi-Monthly when industry not sampling

All

Waiaua Bay A09 Tuatua –Monthly PSP, ASP Whangaroa A08A Oyster – Monthly when industry

not sampling All

Tokerau Beach A21 Tuatua – Monthly Tuatua – Weekly

All PSP, ASP

Houhora A02 Oyster/mussel – monthly when industry not sampling

All

Parengarenga A01 Oyster – Monthly when industry not sampling

All

Waipapakauri A27 Tuatua –Monthly 1 May-1 Sept, otherwise fortnightly

All

Hokianga F22 Oyster – Monthly All Glinks Gully F01 Tuatua – Monthly All Manukau Harbour F15/

F11 Scallop – Fortnightly 7 months Mussel – Fortnightly 5 months

All All

Kaipara Harbour F08A/ F08C

Scallop – Fortnightly 7 months Oyster– Fortnightly 5 months

All All

Waimangu Point C09 Mussel – Monthly when industry not sampling

All

Kawau Island C36 Scallop – Monthly for 7 months ASP Port Fitzroy (Nagle Cove) D02A Scallop – Monthly for 7 months ASP Port Fitzroy D01 Mussel – Monthly for 3 months All Awakiriapa (Waiheke) C38 Mussel – Monthly for 3 months

when industry not sampling All

Kennedy Bay D06 Mussel – Monthly All Tairua D12 Mussel – Monthly All Mercury Island D05 Scallop – Weekly Dec-Feb ASP Slipper Island D13 Scallop – Weekly Dec-Feb

Scallop – Fortnightly Jul-Aug ASP ASP

Raglan F16 Mussel – Fortnightly All Waihi D17 Tuatua – Weekly

Tuatua - Monthly PSP All

Pukehina D28 Tuatua – Weekly Tuatua - Monthly

PSP All

197

Non-Commercial Shellfish Sampling Programme continued… Location Site

No. Species/Frequency Toxins

“A” Buoy D19 Mussel – Monthly All Kauri Point D21 Scallop – Fortnightly 1 July-31

August Scallop – Weekly 1 Sept-15 Feb

ASP, PSP ASP, PSP

Rangiwaea D31 Scallop – Fortnightly 1 July-31 August Scallop – Weekly 1 Sept-15 Feb

ASP, PSP ASP, PSP

Motiti Island D29 Scallop – Fortnightly 1 July-31 August Scallop – Weekly 1 Sept-15 Feb

ASP, PSP ASP, PSP

Whangaparaoa D41 Mussel – Weekly Mussel – Monthly

PSP All

Tokata D38 Mussel – Weekly Mussel – Monthly

PSP All

Ohope D37 Tuatua – Weekly Tuatua – Monthly Mussel – Monthly

PSP All All

Te Araroa E15 Mussel – Monthly ASP, PSP Tolaga Bay E01 Mussel – Monthly All Pania Reef (Napier) E07 Mussel – Monthly All Ohawe H01 Mussel – Fortnightly All Oakura F20 Mussel – Fortnightly All Himitangi Beach Tuatua – Fortnightly All Riversdale E13 Paua gut – Weekly All Dorset Point H07 Mussel – Monthly All Collingwood Farms G01 Mussel – Monthly when

industry not sampling All

The Glen- Tasman Bay G03 Dredge oyster/Scallop – Monthly when industry not sampling

All

Port Motueka G05 Dredge oyster/Scallop – Monthly when industry not sampling

All

Takaka River/Tata Island G07 Dredge oyster/Scallop – Monthly when industry not sampling

All

Wedge Point G23 Mussel – Monthly Mussel – Weekly

All DSP

Tory Channel G22 Mussel – Monthly All Oaro I01 Mussel – Monthly 1 Apr-31 Oct All Akaroa (The Kaik) I04 Mussel – Weekly

Mussel – Monthly DSP All

Taylors Mistake I21 Mussel – Monthly All Dashing Rocks I09 Mussel – Monthly All

198

Non-Commercial Shellfish Sampling Programme continued… Location Site

No. Species/Frequency Toxins

Mussel Rocks J03 Mussel - Fortnightly All Mopoutahi Pt (Aramoana) I14 Mussel – Monthly

Mussel – Weekly at high risk times (plus I11, I16 & I17 at high risk times)

All All

Riverton/Colac Bay J20 Mussel – Monthly All 3). Commercial Phytoplankton and Shellfish Monitoring Programme Location Site

No. Phytoplankton Frequency

Frequency Toxins

North Island

Parengarenga, Kauanga

A01 Weekly Fortnightly Monthly

NSP/DSP ASP,PSP

Houhora A02 Weekly Summer - Weekly Winter - Monthly Summer –Fortnightly Winter - Monthly

ASP,PSP NSP/DSP

Houhora Bay A03 Weekly Summer – Fortnightly Summer – Monthly Summer – Fortnightly Winter - Monthly

ASP PSP NSP/DSP ASP,PSP,NSP/DSP

Rangaunu Hbr A05 Weekly at lease site

Weekly Monthly

NSP/DSP ASP,PSP

Whangaroa Hbr A08 Weekly Summer – Fortnightly Summer – Monthly Winter – Fortnightly Winter - Monthly

ASP,NSP/DSP PSP ASP PSP, NSP/DSP

Kerikeri/Te Puna Inlet

A14 Weekly Fortnightly Monthly

NSP/DSP ASP,PSP

Paroa Bay, Orongo Bay, Waikare Inlet

A15 Weekly at Tapeka Point

Fortnightly Monthly

NSP/DSP ASP,PSP

Parua Bay, Snake Bank, Mair Bank

B07, B13, B14, B15

Weekly Summer – Fortnightly Summer – Monthly Winter - Monthly

NSP/DSP ASP,PSP ASP,PSP,NSP/DSP

199

Commercial Phytoplankton and Shellfish Monitoring Programme continued… Location Site

No. Phytoplankton Frequency

Frequency Toxins

Kaipara Pahi Inlet

F03 None Fortnightly ASP,PSP,NSP/DSP

Mahurangi C33 None Fortnightly Monthly

NSP/DSP ASP,PSP

Kaipara Hbr Mouth

F25 None Fortnightly, if F08A indicates toxicity

ASP,PSP,NSP/DSP

Great Barrier Island

D01, D01A

Weekly Fortnightly Monthly

ASP,NSP/DSP PSP

Waiheke Island, Kauri Bay and Waimangu Point

C38, C10, C09

Weekly Fortnightly Monthly

NSP/DSP ASP,PSP

Whitianga D07 None Monthly ASP,PSP,NSP/DSP Port Charles D03 Weekly Weekly ASP,PSP,NSP/DSP Kennedy Bay D06 Weekly Monthly ASP,PSP,NSP/DSP Coromandel C02,

C05, C29

Weekly Summer - Weekly Summer – Fortnightly Winter - Fortnightly

NSP/DSP ASP,PSP ASP,PSP,NSP/DSP

Ohiwa D33 Weekly Summer – Fortnightly

PSP

Scallop Areas None Weekly Fortnightly

ASP PSP,NSP/DSP

South Island

Nydia Bay G9 Weekly Weekly

Monthly NSP/DSP ASP,PSP

Waitaria Bay G14 Weekly Weekly Monthly

NSP/DSP ASP,PSP

Crail Bay G15 Weekly Weekly Monthly

NSP/DSP ASP,PSP

Pukatea Bay G18 Weekly Weekly Monthly

NSP/DSP ASP,PSP

Brightlands G27 Weekly Weekly Monthly

NSP/DSP ASP,PSP

West Beatrix G31 Weekly Weekly Monthly

NSP/DSP ASP,PSP

Laverique Bay G37 Weekly Weekly Monthly

NSP/DSP ASP,PSP

200

Commercial Phytoplankton and Shellfish Monitoring Programme continued… Location Site

No. Phytoplankton Frequency

Frequency Toxins

Hallam Cove G10 Weekly Weekly Fortnightly Monthly

NSP/DSP ASP PSP

Whangakoko G11 Weekly Weekly Fortnightly Monthly

NSP/DSP ASP PSP

Horahora G12 Weekly Weekly Fortnightly Monthly

NSP/DSP ASP PSP

Cannon Hill G16 Weekly Weekly Fortnightly Monthly

NSP/DSP ASP PSP

Richmond Bay G28 Weekly Weekly Fortnightly Monthly

NSP/DSP ASP PSP

Oyster Bay, Croisilles

G13 Weekly Weekly Monthly

PSP,NSP/DSP ASP

Anakoha G17 Weekly Weekly Fortnightly

PSP,NSP/DSP ASP

Kenepuru Entrance

G8 None Weekly ASP,PSP,NSP/DSP

East Bay G19 Weekly Weekly ASP,PSP,NSP/DSP Opihi Bay, Waitata, Schnapper Point, Nikau Bay, South East Bay, Little Nikau Bay, Forsyth Bay

Weekly None

Port Gore

G50 Weekly-when harvesting

Weekly-when harvesting

ASP,PSP,NSP/DSP

Port Hardy

G85 Occasionally Weekly-when harvesting

ASP,PSP,NSP/DSP

Clifford and Cloudy Bays

G30 None Weekly-when harvesting

ASP,PSP,NSP/DSP

Pakawau &Collingwood [one site covers both]

G1 Weekly Fortnightly Monthly

NSP/DSP ASP,PSP

Tapu Bay G6 Weekly Monthly ASP,PSP,NSP/DSP Wainui Farms None Weekly-when

harvesting ASP,PSP,NSP/DSP

201

Commercial Phytoplankton and Shellfish Monitoring Programme continued… Location Site

No. Phytoplankton Frequency

Frequency Toxins

Dredge Oyster Fisheries [seasonal, March to 31 August]

None Weekly ASP,PSP,NSP/DSP

Scallop [seasonal] Tasman/Golden Bays / Marlborough Sounds

None Weekly ASP,PSP,NSP/DSP

Papanui Inlet Blueskin Bay

I14 Info from recreational weekly site

Weekly ASP,PSP,NSP/DSP

Continental Shelf I20 None Weekly-when harvesting

ASP,PSP,NSP/DSP

Big Glory J13 None Weekly ASP,PSP,NSP/DSP Foveaux Strait Dredge Oysters

J6 Weekly-Seasonal ASP,PSP,NSP/DSP

202

APPENDIX I(C) FLOW DIAGRAM ILLUSTRATING SHELLFISH TISSUE TESTING FOR NSP AND DSP

203

APPENDIX I (D) MAP SHOWING LOCATION OF BIOTOXIN ZONES

Zone D: Cape Colville to CapeRunaway.

Zone A: Tauroa Point to Cape BrettZone B: Cape Brett to Cape

Rodney

Zone C: Cape Rodney to CapeColville.

Zone E: Cape Runawayto Cape Palliser.

Zone F: Tauroa Point to CapeEgmont

Zone G: Cape Farewell toCape Campbell.

G

Zone H: Cape Egmont toCape Palliser.

Zone I: Cape Campbell to Bluff.

Zone K: ChathamIslands.

Zone J: Cape Farewell toBluff.

204

APPENDIX II PHYTOPLANKON SITES INCLUDED IN ANALYSIS OVER

THE “IDENTIFIED TIME INTERVAL”

Zone A: P060 Keri Keri Inlet P059 Patricks Point P020 Tapeka Point P023 Whatuwhiwhi

Zone B: P021 Marsden Point

Zone C: P029 C2

P030 C5 P031 Te Kapa P032 Tamaki Strait P028 Waimangu Point P019 Whangaparoa

Zone D: P017 Kennedy Bay

P002 Matakana Bank P003 Motiti Island P005 Ohope P027 Port Fiztroy P004 Te Kaha P018 Tairua

Zone E: P007 Napier

P008 Tolaga Bay

Zone G: G08 Kenepuru Entrance P035 Schnapper Point P036 Waitaria Bay P037 Little Nikau Bay P038 Nikau Bay P039 Nydia Bay P040 South East Bay P041 Crail Bay P042 Laverique Bay P044 Brightlands Bay P043 West Beatrix Bay P045 Hallam Cove P046 Richmond Bay P047 Waitata Bay G041 Ketu Bay P048 Cannon Bay P049 Forsyth Bay P050 Anakoha Bay P051 Puketea Bay P052 Oyster Bay

205

Phytoplankton sites included in analysis over the “Identified Time Interval” continued…

P053 East Bay P054 Horahora Bay P055 Whangakoko Bay P056 Opihi Bay P026 Wedge Point P034 Collingwood Farms

Zone H: P009 Fort Dorset

Zone I: P011 Akaroa

P013 Caroline Bay P015 Blueskin Bay P012 Taylors Mistake

Zone J: P016 Riverton

206

APPENDIX III TEMPORAL PERIODICITY OF EL NINO/LA NINA WEATHER CONDITIONS

Year Jan-Mar Apr-Jun Jul-Sep Oct-Dec 1992 El Nino El Nino El Nino El Nino 1993 El Nino El Nino El Nino El Nino 1994 El Nino El Nino El Nino El Nino 1995 El Nino El Nino La Nina La Nina 1996 La Nina La Nina La Nina El Nino 1997 El Nino El Nino El Nino El Nino 1998 El Nino El Nino La Nina La Nina 1999 La Nina La Nina La Nina La Nina

Summarised from a website of the NOAA Climate Prediction Centre-http://www.cpc.ncep.noaa.govt/products/analysis_monitoring/ensostuff/ensoyears.html

207

APPENDIX IV(A) MAP SHOWING THE LOCATION OF SAMPLING SITES IN THE MARLBOROUGH SOUNDS (ZONE G)

G08-Kenepuru Entrance G35-Schnapper Point G14-Waitara Bay G44-Little Nikau Bay G36-Nikau Bay G09-Nydia Bay G38-South East Bay G15-Crail Bay G37-Laverique Bay G31-West Beatrix Bay G27-Brightlands G10-Hallam Bay G28-Richmond Bay G26-Waitata Bay G41-Ketu Bay G16-Cannon Bay

208

Continuation of sample sites… G39-Forsyth Bay G17-Anakoha Bay G18-Pukatea Bay G13-Oyster Bay G19-East Bay G12-Horohora Bay G11-Whangakoko Bay G40-Opihi Bay G23-Wedge Point G01-Collingwood Farm

209

APPENDIX IV(B) SITE COMPARISONS OF PSEUDO-NITZSCHIA OCCURRENCE IN THE HAURAKI GULF AND MARLBOROUGH

SOUNDS/COLLINGWOOD

1). Hauraki Gulf (Zone C, 1995-1999)

Pseudo-nitzschia

Sampling DateJan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

100000

200000

300000

400000

500000800000

1000000

1200000

Kopake (Coromandel)MAF Farms (Coromandel)Tamaki Strait

The scale of the Y-axis is the same as that used for the Marlborough sites. 2). Marlborough Sounds/Collingwood (Zone G, 1994-1999) All Marlborough sites are grouped on the following 7 graphs, with the same Y-axis scale to show the relative abundance of Pseudo-nitzschia between sites. In some cases Pseudo-nitzschia numbers were greater than the given scale-bar.

Pseudo-nitzschia

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

100000

200000

300000

400000

500000800000

1000000

1200000

Kenepuru Entrance (G08)Schnapper Point (G35)Waitaria Bay (G14)

A

210

Marlborough Sounds/Collingwood (Zone G, 1994-1999) continued…

Pseudo-nitzschia

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-990

100000

200000

300000

400000

500000800000

1000000

1200000Little Nikau Bay (G44)Nikau Bay (G36)Nydia Bay (G09)South East Bay (G38)

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

100000

200000

300000

400000

500000800000

1000000

1200000Crail Bay (G15)Laverique Bay (G37)West Beatrix Bay (G31)Brightlands Bay (27)

Points omitted-Laverique Bay-678 000 West Beatrix Bay-574 000

B

C

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

0

100000

200000

300000

400000

500000800000

1000000

1200000Hallam Cove (G10)Richmond Bay (G28)Waitata Bay (G26)Ketu Bay (G41)Cannon Bay (G16)

Point omitted-Hallam Cove 719 000D

211

Marlborough Sounds/Collingwood (Zone G, 1994-1999) continued…

Pseudo-nitzschia

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-990

100000

200000

300000

400000

500000800000

1000000

1200000Forsyth Bay (G39)Anakoha Bay (G17)Puketea Bay (G18)Oyster Bay (G13)

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

100000

200000

300000

400000

500000800000

1000000

1200000

East Bay (G19)Horohora Bay (G12)Whangakoko Bay (G11)Opihi Bay (G40)

E

F

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

0

100000

200000

300000

400000

500000800000

1000000

1200000

Collingwood Farms (G01)Wedge Point (G23)

G

Note: Collingwood Farms (G01) site was not monitored until late August 1996. Wedge Point (G23) site data missing from 29-Oct 1996 to 30-Apr 1997.

212

APPENDIX IV(C) SITE COMPARISONS OF DINOPHYSIS OCCURRENCE

IN THE HAURAKI GULF AND MARLBOROUGH SOUNDS/COLLINGWOOD

1). Hauraki Gulf (Zone C, 1995-1999)

Dinophysis

Sampling DateJan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Kopake (Coromandel)MAF Farm (Coromandel)Tamaki Strait

2). Marlborough Sounds/Collingwood (Zone G, 1994-1999) All Marlborough sites are grouped on the following 7 graphs, with the same Y-axis scale to show the relative abundance of Dinophysis between sites. In some cases Dinophysis numbers were greater than the given scale-bar.

Dinophysis sp.

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

Cel

l Den

sity

(L-1

)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000Kenepuru Entrance (G08)Schnapper Point (G35)Waitaria Bay (G14)

A

213

Marlborough Sounds/Collingwood (Zone G, 1994-1999) continued…

Dinophysis sp.

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-990

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Little Nikau Bay (G44)Nikau Bay (G36)Nydia Bay (G09)South East Bay (G38)

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Crail Bay (G15)Laverique Bay (G37)West Beatrix Bay (G31)Brightlands Bay (G27)

B

C

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Hallam Cove (G10)Richmond Bay (G28)Waitata Bay (G26)Ketu Bay (G41)Cannon Bay (G16)

D

214

Marlborough Sounds/Collingwood (Zone G, 1994-1999) continued…

Dinophysis sp.

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-990

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Forsyth Bay (G39)Anakoha Bay (G17)Puketea Bay (G18)Oyster Bay (G13)

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000East Bay (G19)Horohora Bay (G12)Whangakoko Bay (G11)Opihi Bay (G40)

E

F

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Collingwood Farms (G01)Wedge Point (G23)

G

Note: Collingwood Farms (G01) site was not monitored until late August 1996. Wedge Point (G23) site data missing from 29-Oct 1996 to 30-Apr 1997.

215

APPENDIX IV(D) SITE COMPARISONS OF GYMNODINIUM c.f. MIKIMOTOI OCCURRENCE IN THE HAURAKI GULF AND

MARLBOROUGH SOUNDS/COLLINGWOOD 1). Hauraki Gulf (Zone C, 1995-1999)

Gymnodinium c.f. mikimotoi

Sampling DateJan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0

2000

4000

6000

8000

10000

Kopake (Coromandel)MAF Farms (Coromandel)Tamaki Strait

2). Marlborough Sounds/Collingwood (Zone G, 1994-1999) All Marlborough sites are grouped on the following 7 graphs, with the same Y-axis scale to show the relative abundance of G. c.f. mikimotoi between sites. In some cases G. c.f. mikimotoi numbers were greater than the given scale-bar.

Gymnodinium c.f. mikimotoi

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

Cel

l Den

sity/

L

0500

10001500200025003000350040004500500055006000

Kenepuru Entrance (G08)Schnapper Point (G35)Waitaria Bay (G14)

A

216

Marlborough Sounds/Collingwood (Zone G, 1994-1999) continued…

Gymnodinium c.f. mikimotoi

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-990

50010001500200025003000350040004500500055006000

Little Nikau Bay (G44)Nikau Bay (G36)Nydia Bay (G09)South East Bay (G38)

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0500

10001500200025003000350040004500500055006000

Crail Bay (G15)Laverique Bay (G37)West Beatrix Bay (G31)Brightlands Bay (G27)

B

C

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

0500

10001500200025003000350040004500500055006000

Hallam Cove (G10)Richmond Bay (G28)Waitata Bay (G26)Ketu Bay (G41)Cannon Bay (G16)

D

217

Marlborough Sounds/Collingwood (Zone G, 1994-1999) continued…

Sampling DateJul-94

Sep-94Nov-94

Jan-95Mar-9

5May-95

Jul-95Sep-95

Nov-95Jan-96

Mar-96

May-96Jul-96

Sep-96Nov-96

Jan-97Mar-9

7May-97

Jul-97Sep-97

Nov-97Jan-98

Mar-98

May-98Jul-98

Sep-98Nov-98

Jan-99Mar-9

9May-99

0500

10001500200025003000350040004500500055006000

Collingwood Farms (G01)Wedge Point (G23)

Gymnodinium c.f. mikimotoi

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-990

50010001500200025003000350040004500500055006000

Forsyth Bay (G39)Anakoha Bay (G17)Puketea Bay (G18)Oyster Bay (G13)

Jul-94Sep-94

Nov-94Jan-95

Mar-95

May-95Jul-95

Sep-95Nov-95

Jan-96Mar-9

6May-96

Jul-96Sep-96

Nov-96Jan-97

Mar-97

May-97Jul-97

Sep-97Nov-97

Jan-98Mar-9

8May-98

Jul-98Sep-98

Nov-98Jan-99

Mar-99

May-99

Cel

l Den

sity/

L

0500

10001500200025003000350040004500500055006000

East Bay (G19)Horohora Bay (G12)Whangakoko Bay (G11)Opihi Bay (G40)

E

F

G

Note: Collingwood Farms (G01) site was not monitored until late August 1996. 1996. Wedge Point (G23) site data missing from 29-Oct 1996 to 30-Apr 1997.

218

APPENDIX V RESULTS FROM WHOLE CELL DNA PROBES FOR PSEUDO-NITZSCHIA SPECIES

Shellfish Toxin Results (weeks after initial

phytoplankton test)

Relative density of each Pseudo-nitzschia species from whole cell gene probes

Site No

Site Name

Date Total Biomass

Pseudo-nitzschia Biomass

Wk 1 Wk 2 Wk 3 P. australis & P. pungens & P. multiseries Cells/L

P. pseudodeli-catissima & P. delicatissima Cells/L (% of Total Biomass)

P. fradulenta Cells/L (% of Total Biomass)

P. heimii Cells/L (% of Total Biomass)

A08 Patricks Point

19-Oct-98 640,200 224,000 0.50 1.00 0.50 112000 56000 (8.7)

56000 (8.7)

0

G07 Takaka 8-Dec-97 929,975 871,650 0.50 0.50 n.t. 43583 0 174330 (18.7)

653738 (70.3)

G28 Richmond 15-Dec-97 330,485 155,490 0.50 0.00 0.00 77745 0 0 77745 (23.5)

I13 Blueskin Bay

23-Dec-97 631545 417,480 0.50 0.00 0.00 208740 0 208740 (33.1)

0

I13 Blueskin Bay

4-Feb-98 221,000 219,000 12.00 0.00 0.00 0 219000 (99.1)

0 0

Table 1. Cases of cellular density above the risk assessment guidelines for Pseudo-nitzschia sp. (relative densities obtained from

whole cell gene probes) associated with positive shellfish testing results (Domoic Acid, µg/g shellfish tissue). Cell densities above the risk assessment guidelines are given in italics. n.t. = No shellfish toxin testing for that particular week.

219

Shellfish Toxin Results (weeks after initial phytoplankton

test)

Relative density of each Pseudo-nitzschia species from whole cell gene probes

Site No

Site Name Date Total Biomass

Pseudo-nitzschia Biomass

Wk 1 Wk 2 Wk 3 P. australis & P. pungens & P. multiseries Cells/L

P. pseudodeli-catissima & P. delicatissima Cells/L (% of Total Biomass)

P. fradulenta Cells/L (% of Total Biomass)

P. heimii Cells/L (% of Total Biomass)

A03 Houhora 25-Nov-97 74,890 60,705 0.50 0.00 0.00 0 0 0 60705 (81.1)

A08 Whangaroa 10-Nov-98 495,800 377,000 0.50 0.00 0.00 37700 18850 (3.8)

124410 (25.1)

188500 (38.0)

B14 Marsden Point

25-Nov-97 2,287,340 225,780 0.50 0.50 0.50 0 0 0 225780 (9.9)

B14 Marsden Point

9-Dec-97 1,832,670 680,535 0.50 0.50 0.50 0 0 0 680535 (37.1)

B14 Marsden Point

15-Dec-97 426,555 12,000 0.50 0.50 0.00 0 0 0 12000 (2.8)

D06 Kennedy Bay 31-Aug-98 20,000 10,000 0.50 n.t. n.t. 10000 0 0 0 G01 Collingwood 28-Sep-98 93,600 6,400 0.50 0.00 n.t. 5824 64

(0.1) 64

(0.1) 0

G19 G19 30-Sep-98 71,000 53,000 2.50 0.50 n.t. 44520 3710 (5.2)

4240 (6.0)

0

Table 2. Cases of cellular density below the risk assessment guidelines for Pseudo-nitzschia sp. (relative densities obtained from

whole cell gene probes) associated with positive shellfish testing results (Domoic Acid, µg/g tissue). n.t. = No shellfish toxin testing for that particular week.

220

Table 3. Cases of cellular density above the risk assessment guidelines for Pseudo-nitzschia sp. (relative densities obtained from whole cell gene probes) associated with no incidence of domoic acid in shellfish testing results (Domoic acid, µg/g tissue). Cell densities above the risk assessment guidelines are given in italics. n.t. = No shellfish toxin testing for that particular week. ** = cell density would be lower than this density as only an approximate % was given.

Shellfish Toxin Results

(weeks after initial phytoplankton test)

Relative density of each Pseudo-nitzschia species from whole cell gene probes

Site No Site Name Date Total Bio

Pseudo-nitzschia Biomass

Wk 1 Wk 2 Wk 3 P. australis & P. pungens & P. multiseries

Cells/L (% of

Total Biomass)

P. pseudodeli-catissima & P. delicatissima

Cells/L (% of

Total Biomass)

P. fradulenta Cells/L (% of Total Biomass)

P. heimii Cells/L (% of Total Biomass)

A03 Houhora 12-Nov-98 938,400 935,000 0.00 n.t. 0.00 28050 9350 (1.0)

9350 (1.0)

906950 (96.6)

A03 Houhora 28-Apr-99 425,800 382,000 0.00 0.00 n.t. 3820 370540 (87.0)

3820 (0.9)

3820 (0.9)

B14 Marsden Point 9-Feb-98 176,400 52,000 0.00 n.t. n.t. 52000 0 0 0 B14 Marsden Point 13-May-98 293,400 95,000 0.00 n.t. 0.00 76000 0 19000

(6.5) 0

B14 Marsden Point 21-Oct-98 474,000 89,000 n.t. 0.00 n.t. 55180 0 0 32930 6.9

C02 Coromandel 13-Apr-98 386,800 121,000 n.t. 0.00 n.t. 60500 0 0 0 D06 Kennedy Bay 18-May-98 154,300 130,000 0.00 0.00 0.00 104000 0 26000

(16.9) 0

D12 Tairua 7-Nov-98 789,400 507,000 0.00 n.t. n.t. 5070 5070 (0.6)

456300 (57.8)

40560 (5.1)

D19 Matakana 11-Nov-97 195,400 166,600 0.00 0.00 n.t. 133280 0 33320 (17.1)

0

D19 Matakana 8-Feb-98 618,000 564,000 n.t. n.t. 0.00 0 564000 (91.3)

0 0

D19 Matakana 13-Apr-98 224,600 176,000 n.t. 0.00 n.t. 88000 88000 (39.2)

0 0

E15 Hicks Bay 13-Dec-98 2,608,000 2,419,000 n.t. 0.00 n.t. 846650 725700 (27.8)

120950 (4.6)

725700 (27.8)

221

Table 3 continued Shellfish Toxin Results

(weeks after initial phytoplankton test)

Relative density of each Pseudo-nitzschia species from whole cell gene probes

Site No Site Name Date Total Bio

Pseudo-nitzschia Biomass

Wk 1 Wk 2 Wk 3 P. australis & P. pungens & P. multiseries

Cells/L (% of Total Biomass)

P. pseudodeli-catissima & P. delicatissima Cells/L (% of

Total Biomass)

P. fradulenta

Cells/L (% of Total Biomass)

P. heimii

Cells/L (% of Total Biomass)

G01 Collingwood 19-Jan-98 11,442,000 1,066,000 0.00 n.t. n.t. 106600** 0 959400 (8.4)

10660 (0.1)

G10 G10 10-Nov-97 172,400 101,800 n.t. 0.00 0.00 97728 0 1018 (0.6)

0

G10 G10 24-Nov-97 542,535 399,325 0.00 0.00 0.00 199663 0 199663 (36.8)

0

G13 G13 10-Jun-98 203,800 153,000 0.00 0.00 0.00 18360 130050 (63.8)

7650 (3.8)

0

G16 G16 10-Nov-97 118,200 86,400 0.00 0.00 0.00 82944 0 864 (0.7)

0

G17 G17 5-Jan-98 1,663,265 1,499,520 0.00 0.00 0.00 14996 0 149952 (9.0)

1349568 (81.1)

G28 G28 10-Nov-97 103,200 83,400 0.00 0.00 0.00 80064 0 834 (0.8)

0

G28 Richmond 17-Nov-97 176,000 89,800 0.00 0.00 0.00 89800 0 0 0 G28 Richmond 28-Dec-97 328,555 224,715 0.00 0.00 0.00 112358 0 112358

(34.2) 0

G28 Richmond 5-Jan-98 957,770 473,925 0.00 0.00 0.00 0 473925 (49.5)

0 0

G28 Richmond 23-Feb-98 330,800 96,000 n.t. 0.00 n.t. 96000 0 0 0 G28 Richmond 28-Dec-98 518,000 198,000 0.00 0.00 0.00 75240 0 61380

(11.8) 61380 (11.8)

G28 Richmond 4-Jan-99 180,800 86,000 0.00 0.00 0.00 77400 0 4300 (2.4)

4300 (2.4)

G31 G31 14-Sep-98 266,400 60,000 0.00 0.00 0.00 60000 0 0 0 G37 G37 13-Apr-98 579,000 147,000 0.00 0.00 0.00 139650 0 1470

(0.3) 1470 (0.3)

G39 G39 10-Nov-97 129,100 73,125 n.t. n.t. n.t. 70200 0 732 (0.6)

0

222

Table 3 continued Shellfish Toxin

Results (weeks after initial phytoplankton

test)

Relative density of each Pseudo-nitzschia species from whole cell gene probes

Site No

Site Name Date Total Bio

Pseudo-nitzschia Biomass

Wk 1 Wk 2 Wk 3 P. australis & P. pungens & P. multiseries

Cells/L (% of Total Biomass)

P. pseudodeli-catissima & P. delicatissima Cells/L (% of

Total Biomass)

P. fradulenta

Cells/L (% of Total Biomass)

P. heimii

Cells/L (% of Total Biomass)

G39 G39 5-Jan-98 1,381,325 1,167,240 n.t. n.t. n.t. 23345 0 11672.4 (0.8)

1108878 (80.3)

G39 G39 28-Dec-98 449,800 282,000 0.00 0.00 n.t. 56400 11280 (2.5)

169200 (37.6)

45120 (10.0)

G40 G40 10-Feb-98 681,800 493,000 0.00 0.00 0.00 295800 0 197200 (28.9)

0

G44 G44 13-Apr-98 287,800 207,000 n.t. n.t. n.t. 103500 82800 (28.8)

10350 (3.6)

0

G50 G50 10-Feb-98 2,030,800 573,000 0.00 0.00 n.t. 28650 544350 (26.8)

0

I01 Kaikoura 10-Aug-98 306,900 178,000 n.t. n.t. n.t. 5340 178000 (58.0)

1780 (0.6)

0

I04 Akaroa 5-Jan-98 363,365 309,915 0.00 0.00 0.00 309915 0 0 0 I04 Akaroa 9-Nov-98 2,116,200 1,900,000 0.00 0.00 0.00 0 1254000

(59.3) 627000 (29.6)

0

I04 Akaroa 29-Dec-98 132,700 132,000 0.00 n.t. n.t. 13200 112200 (84.6)

6600 (5.0)

0

I04 Akaroa 1-Jun-99 1,212,600 217,000 0.00 n.t. 0.00 65100 0 32550 (2.7)

130200 (10.7)

I21 Taylor's Mistake

16-Mar-98 626,000 313,000 n.t. n.t. n.t. 65730 93900 (15.0)

0 156500 (25.0)

I21 Taylor's Mistake

16-Aug-98 965,000 809,000 0.00 0.00 0.00 24270 776640 (80.5)

40450 (4.2)

0

I21 Taylor's Mistake

29-Nov-98 1,249,000 177,000 0.00 0.00 n.t. 169920 0 1770 (0.1)

1770 (0.1)

223

APPENDIX VI PSEUDO-NITZSCHIA SPECIES COMPOSITION AT THE SAME SITE OVER CONSECUTIVE WEEKS, DETERMINED BY WHOLE

CELL GENE PROBES

Marsden Point

Sampling Date19-Oct-98 26-Oct-98 2-Nov-98

Cel

l Den

sity/

L

0

10000

20000

30000

40000

50000

60000400000

500000

P. australisP. pungensP. multiseriesP. pseudodelicatissimaP. delicatissimaP. fraudulentaP. heimiiMatakana

Sampling Date

10-Nov-97 17-Nov-97 24-Nov-98

Cel

l Den

sity/

L

0

50000

100000

150000

200000

250000

224

Pseudo-nitzschia species composition at the same site over consecutive weeks, determined by whole cell gene probe, continued…

Richmond

Sampling Date10-Aug-98

17-Aug-98

24-Aug-98

31-Aug-98

7-Sep-98

14-Sep-98

21-Sep-98

28-Sep-98

Cel

l Den

sity/

L

0

10000

20000

30000

40000

Richmond

23-Mar-9

8

31-Mar-9

8

6-Apr-98

13-Apr-98

Cel

l Den

sity/

L

0

5000

10000

15000

20000

25000

P. australisP. pungensP. multiseriesP. pseudodelicatissimaP. delicatissimaP. fraudulentaP. heimii

Richmond

16-Dec-98

21-Dec-98

28-Dec-98

4-Jan-99

11-Jan-99

0

20000

40000

60000

80000

100000

Beatrix Bay

Sampling Date31-M

ar-98

6-Apr-98

13-Apr-98

20-Apr-98

0

10000

20000

30000

40000

50000

60000