Toksiske alger og algetoksiner. Mikrobiologisk klassificering & forureningsovervågning i muslingefiskeriet 2004

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1 Toksiske alger og algetoksiner Mikrobiologisk klassificering & forureningsovervågning i muslingefiskeriet 2004 Toxic algae and algal toxins microbiological classification & chemical contamination surveillance in The Danish Bivalve Mollusc Fishery 2004

2 Udarbejdet for:, Mørkhøj Bygade 19, 2860 Søborg Udarbejdet af: Thyra Bjergskov, Kevin Jørgensen, Fødevareinstituttet, DTU Bjarne Ring Thorbjørnsen,, Fødevareregion Nord, Kontrolafdeling Viborg Per Andersen, Orbicon A/S Analysearbejde blev udført af: Fødevareinstituttet, Afd. For Fødevarekemi, Mørkhøj Bygade 19, 2860 Søborg Orbicon A/S, Jens Juuls Vej, Viby J. LAB Research (Scantox), Hestehavevej 36A, Ejby, 4623 Lille Skensved Højmarklaboratoriet a. s., Adelvej 11, Højmark, 6940 Lem St., Fødevareregion Nord, Kontrolafdeling Nord, lab. Esbjerg, Høgevej 25, 6705 Esbjerg Ø AnalyCen A/S, Vesterballevej 4, 7000 Frederecia Statistik: John Pedersen, Orbicon A/S, Jens Juuls Vej 18, 8260 Viby J. Rentegning: Kirsten Nygaard, Orbicon A/S, Jens Juuls Vej 18, 8260 Viby J. Redigering: Gitte Spanggaard, Orbicon A/S, Jens Juuls Vej 18, 8260 Viby J. Dato: Oktober 2007 ISBN: ISSN: (algetoksiner i muslinger) ISSN: (fødevarerapport) 2007:17

3 INDHOLDSFORTEGNELSE SAMMENFATNING OG ANBEFALINGER... 5 ENGLISH SUMMARY... 9 FORORD OVERVÅGNINGENS FORMÅL OVERVÅGNINGSSYSTEMET Lovgivningsmæssige grundlag Opdeling i produktionsområder Overvågningsprocedure Udtag og indsendelse af prøver Åbning af produktionsområder Skærpet overvågning eller lukning Påvisning af algetoksiner i muslinger Rense- og konditionerings- og udsandingsanlæg Undersøgelse af vandprøver for forekomst af alger Prøvetagning Kvalitative undersøgelser Kvantitative undersøgelser Undersøgelse af muslinger for forekomst af algetoksiner Musetest Kemiske analyser Databehandling og lagring Grænseværdier Alger Algetoksiner Omkostninger i forbindelse med overvågningen Arbejdsgruppe vedrørende toksindannende alger OVERSIGT OVER GEOGRAFISK OG TIDSMÆSSIG UDBREDELSE AF POTENTIELT TOKSISKE ALGER OG ALGETOKSINER Restriktioner i forbindelse med fiskeriet Tidsmæssig og geografisk udbredelse af algetoksiner Limfjorden Algernes geografiske og tidsmæssige udbredelse Lukninger, skærpet overvågning og forekomst af toksiner i muslinger Administrative reguleringer Reguleringer grundet potentielt toksiske alger og/eller algetoksiner: Delkonklusion Mariager Fjord Jyllands østkyst syd for Djursland Algernes geografiske og tidsmæssige udbredelse Lukninger, skærpet overvågning og forekomst af toksiner i muslinger Administrative reguleringer Reguleringer grundet potentielt toksiske alger og/eller algetoksiner Delkonklusion Isefjord og Roskilde Fjord Algernes geografiske og tidsmæssige udbredelse Lukninger, skærpet overvågning og forekomst af toksiner i muslinger Administrative reguleringer Reguleringer grundet potentielt toksiske alger og/eller algetoksiner.. 84

4 3.6.3 Delkonklusion Vadehavet og Ho Bugt Algernes geografiske og tidsmæssige udbredelse Lukninger, skærpet overvågning og forekomst af toksiner i muslinger Administrative reguleringer Reguleringer grundet potentielt toksiske alger og/eller algetoksiner Delkonklusion VURDERING OG KONKLUSION VEDRØRENDE TOKSISKE ALGER OG ALGETOKSINER REFERENCER BILAGSOVERSIGT Bilag Medlemmer af arbejdsgruppen vedrørende toksindannende alger Bilag Kommissorium for arbejdsgruppen vedrørende toksindannende alger Bilag Registrerede koncentrationsniveauer (x 1000 celler/l) af de forskellige toksiske alger i alle produktionsområder i Bilag Ugegennemsnit af de forskellige potentielt toksiske alger (x 1000 celler/l) for hvert hovedområde Bilag Figur med angivelse af forekomst af DSP-toksiner i muslinger over grænseværdien i Bilag Kort med angivelse af konstaterede forekomster af DSP-toksiner over den gældende grænseværdi (160 µg OA/kg) i muslinger i Bilag Information vedrørende Dinophysis sacculus opblomstringen i Isefjorden Præsentation fra ICES/IOC WGHABD Bilag Den mikrobiologiske klassificering af produktionsområderne og zoneudmeldinger 204 Bilag Oversigt over produktionsområder, i hvilke der klassificeredes zone A områder i Bilag Oversigt over produktionsområder, hvori der ønskedes opklassificering til zone A, men som forblev klassificeret med zone B eller C status i Bilag Overvågning af forurenende kemiske stoffer i produktionsområderne

5 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 5 SAMMENFATNING OG ANBEFALINGER I denne rapport over toksiske alger og algetoksiner i muslingefiskeriet i 2004 gøres status for moniteringen af alger og algetoksiner i de danske produktionsområder, hvori der høstes og opdrættes toskallede bløddyr, pighuder, sækdyr og havsnegle (muslinger m.m.). Endvidere omfatter rapporteringen også klassificeringer af produktionsområderne, det vil sige de mikrobiologiske zoneudmeldinger, samt overvågningen for kemiske forureninger. Formålet med opsamlingen af data og erfaringer er at forbedre det faglige grundlag for gennemførslen, udviklingen, administrationen og kontrollen af overvågningen af høst af muslinger m.m. Forekomst af algetoksiner Der blev registreret DSP-toksiner i koncentrationer over de af EU fastsatte grænseværdier i danske muslinger i i alt 10 produktionsområder/uger på henholdsvis den jyske østkyst, i Isefjorden, i Vadehavet og i Limfjorden. Antallet af tilfælde med DSPtoksicitet i muslinger i 2004 er således lavere end registreret i de 2 foregående år. Der blev ikke i 2004 registreret hverken PSP-, ASP- eller AZP-toksiner i koncentrationer over grænseværdierne i muslinger fra danske produktionsområder. Der blev ikke i 2004 registreret forgiftninger af konsumenter forårsaget af algetoksiner. Restriktioner for fiskeriet på grund af forekomst af algetoksiner og potentielt toksiske alger Der blev indført restriktioner (lukning og skærpet overvågning) for en række produktionsområder/uger overfor muslingefiskeriet i alle hovedområder i 2004 (se figur 3). Restriktionerne blev indført på grund af forhøjede koncentrationer af potentielt toksiske alger samt for Isefjorden, den jyske østkyst og Limfjordens vedkommende også på grund af påvisning af DSP-toksicitet. Ud af et totalt antal produktionsområder/uger på 828 forsøgt åbnet i 2004 forblev 8% (59 produktionsområder/uger) lukket på grund af forekomst af giftige alge og/eller algetoksiner i muslingerne. Forekomsten af giftige alger og/eller algetoksiner i muslingerne medførte relativt mange lukninger af fiskeriet på den jyske østkyst og i Isefjord/Roskilde Fjord, hvor henholdsvis 13% og 17% af de forsøgte åbninger forblev lukkede, mens lukningerne af produktionsområder i Limfjorden på grund af forekomst af giftige alger/algetoksiner i muslingerne kun udgjorde 4%. Forekomst af alger Den tidsmæssige forekomst af de fleste arter var normal i Dog fortsatte forekomsten af Dinophysis-arterne Dinophysis acuminata og Dinophysis acuta, som i , længere ind i efterårsperioden i Isefjorden og på den jyske østkyst end i de tidligere år.

6 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 6 Koncentrationsniveauerne var for enkelte arters vedkommende forhøjede i forhold til de tidligere års undersøgelser. Dette gjaldt bl.a. i forbindelse med opblomstringen af Pseudo-nitzschia i sommerperioden på den jyske østkyst samt for Dinophysis acuminata's og Dinophysis acuta s vedkommende i Isefjorden, på den jyske østkyst og i Vadehavet. For Dinophysis acuminata s vedkommende blev der også registreret en større tidsmæssig udbredelse og et højere koncentrationsniveau i Limfjorden. Dinophysis norvegica blev igen i 2004 registreret kortvarigt og i lave koncentrationer i alle områder, hvilket er i modsætning til perioden i begyndelsen af 1990 erne, hvor der blev registreret markante opblomstringer af denne art. Der ser således ud til, at være sket et skifte i dominansforholdene indenfor slægten Dinophysis i de danske produktionsområder, hvor de meget toksiske arter D. acuminata og D. acuta har overtaget dominansen på bekostning af D. norvegica. Dette skifte kan muligvis skyldes den generelle stigning i havtemperaturerne, da D. norvegica har sin hovedforekomst i det nordlige Atlanterhav, mens D. acuminata og D. acuta er almindeligt forekommende også i de varmere sydeuropæiske farvande. De stigende havtemperaturer kan også være årsag til, at arten Dinophysis sacculus, som er en sydeuropæisk art, nu også er blevet registreret i danske produktionsområder. Anbefalinger for overvågningen af algetoksiner og toksiske alger Som omtalt i de tidligere års rapporter vurderes det at være af stor betydning, at der fastlægges procedurer for, hvorledes der kan udtages prøver, og hvem der skal udtage prøverne i de særlige opblomstringsperioder og kritiske situationer, som ønskes nærmere undersøgt, og at der bevilliges de nødvendige økonomiske midler til dækning af udgifterne. F.eks. kunne det ønskes, at prøvetagningen blev fortsat i en række produktionsområder, selv om fiskeriet stoppes på grund af for høje koncentrationer af toksiske og potentielt toksiske alger og forekomst af algetoksiner i muslingerne. Dette for at få et bedre grundlag for at validere den igangværende moniteringsprocedure. På nuværende tidspunkt er der ikke et tilstrækkeligt datagrundlag til en fuldstændig validering af overvågningssystemet - det vil sige, til at vurdere, om der er en god eller dårlig sammenhæng mellem de registrerede koncentrationer af potentielt toksiske alger og akkumuleringen af algetoksiner i muslingerne, samt hvor hurtigt muslingerne renser sig for algetoksiner. Forholdene er således, dels fordi der ikke udtages prøver i produktionsområder, som lukkes, og dels fordi muslingefiskerne, oftest af økonomiske årsager, ikke ønsker at fiske og udtage daglige prøver til analyser, når der er indført skærpet overvågning med daglige algetoksinanalyser og algeanalyser. Dette gør det umuligt at registrere, om de forhøjede koncentrationer af toksiske og potentielt toksiske alger medfører akkumulering af algetoksiner i muslingerne. For at denne validering kan foretages, skal prøvetagningen af både alger og muslinger fortsætte i perioder med både lukning og skærpet overvågning i mindst 1-2 uger efter der er registreret forhøjede koncentrationer af alger.

7 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 7 Endvidere vil det være en stor fordel, hvis der kunne igangsættes en mere systematisk prøveudtagning på faste udvalgte positioner, så sammenhængen mellem forekomsten af potentielt toksiske alger og algetoksiner i muslinger kan analyseres på et mere sikkert grundlag. Resultater af klassificeringen og den mikrobiologiske zoneudmelding i produktionsområderne i 2004 I 2004 blev der søgt om åbning af 166 zone A-områder, heraf opfyldte 140 (84,3%) kriterierne, mens 22 (13,3%) forblev udmeldt som zone B-områder og 4 områder, svarende til 2,4% af områderne, blev udmeldt som zone C-område. Som følge af ønsker om klassificeringer af zone A-områder blev i alt 498 enkeltprøver af toskallede bløddyr analyseret for fækale coliforme, og 166 salmonella-analyser blev gennemført. I alt 140 zone A-områder blev klassificeret og udmeldt inden for i alt 31 produktionsområder, hvoraf 3 var opdræt i produktionsområder (produktionsområderne 8, 14 og 18), hvor der også blev udmeldt zone-a områder i forbindelse med fiskeri efter bundmuslinger, og 4 var opdræt i produktionsområder (produktionsområde 3, 6 (2 opdrætsanlæg) og 49), hvor der ikke blev udmeldt zone-a områder i forbindelse med fiskeri efter bundmuslinger. 94 zone A-områder blev udmeldt i 18 produktionsområder i Limfjorden. 21 zone A-områder blev udmeldt i 7 produktionsområder på Jyllands østkyst. 21 zone A-områder blev udmeldt i 5 produktionsområder i Isefjorden. 4 zone A-områder blev udmeldt i 4 produktionsområder i Vadehavet. I alt 22 undersøgte zoner, som blev analyseret, forblev klassificeret som B, heraf 16 zoner i Limfjorden, 3 zoner på Jyllands østkyst, 1 zone i Isefjorden, og 2 zone-b udmeldinger i Vadehavet. Der blev foretaget 4 zone-c udmeldinger. 3 i Limfjorden i produktionsområderne 1, 6 og 8 (opdrætsanlæg), samt en på den jyske østkyst i produktionsområde 30. Overvågningen af forurenende kemiske stoffer i produktionsområderne s overvågningssystem, som har fungeret siden 1970 erne, omfatter overvågning af blandt andet kemiske forureninger i fødevarer. Overvågningssystemet kører i 5-års perioder, således at ændringer i forureningsniveauerne kan følges. Der findes dog kun få data på forureninger i toskallede bløddyr beregnet til konsum. Indtaget af toskallede bløddyr er lavt i Danmark. Derfor bidrager toskallede bløddyr ikke væsentligt til indtaget af forurenende stoffer. Ifølge Dansk Husstandspanel (GfK, 2000) er danskernes indkøb af skaldyr (krebsdyr og toskallede bløddyr) 2,3 g/dag per person og indkøbet af muslinger, som konserves, er 0,03 g/dag per person (ifølge data indhentet i andet halvår 1999 og første halvår 2000).

8 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 8 Resultater af overvågningen for kemiske forureninger i muslinger i 2004 I 2004 blev der udtaget i alt 8 prøver af blåmuslinger og 13 prøver af østers. Prøverne er udtaget i områderne 1 og 2 i Nissum Bredning. Prøverne blev analyseret for monobutyltin, dibutyltin, tributyltin, monophenyltin, diphenyltin og triphenyltin. Prøverne blev ligeledes analyseret for bly, cadmium og kviksølv. Resultaterne af analyserne ses i bilag 11. Konklusion på analyserne Der er ingen EU grænseværdier for organotinforbindelserne, men en provisorisk TDI værdi for summen af organotinforbindelser er fastlagt til 0,25 mikrogram per kg legemsvægt. Resultaterne giver foreløbig kun anledning til, at yderligere undersøgelser bør gennemføres. Analyseresultaterne for bly, cadmium og kviksølv lå alle under grænseværdierne, med følgende resultater: 0,0811-0,428 mg Pb/kg, 0,0828-0,461 mg Cd/kg og 0, ,0219 mg Hg/kg. Anbefalinger for klassificering og for overvågningen af forurenende kemiske stoffer Det anbefales, at der udarbejdes en rapport over den mikrobiologiske klassificering gennem alle årerne med overvågning, således at den kan danne baggrund for en eventuel permanent udmelding af større zone A-områder i visse produktionsområder. Det anbefales ligeledes, at der tages stilling til, om frekvensen for prøveudtagning er acceptabel. Det anbefales endvidere, at der for kommende nye produktionsområder og nye opdrætsanlæg foretages den lovpligtige klassificering ved mikrobiologiske undersøgelser inden produktionsområderne udmeldes, og inden der gives tilladelse til start af nye opdrætsanlæg. Med hensyn til overvågningen af kemiske forureninger anbefales det, at der iværksættes en permanent overvågning i henhold til reglerne og således, at man over en 5-årig periode mindst har indsamlet analyseresultater fra alle produktionsområder. Det anbefales ligeledes, at der for kommende nye produktionsområder foretages den lovpligtige undersøgelse (kortlægning) for kemisk forurening inden produktionsområderne udmeldes, og inden der gives tilladelse til start af nye opdrætsanlæg.

9 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 9 ENGLISH SUMMARY Introduction This status report for year 2004, compile information on the Danish monitoring program on toxic and potentially toxic algae/phytoplankton and algae toxins in bivalve molluscan shellfish echinoderms, tunicates and marine gastropods (mussels etc.) in relation to the Danish mussel fishery as well as presenting the microbiological classifications and the chemical contaminants monitoring results for year The experience gained in year 2004 is documented and will be used during the annual review and revision of the monitoring program. The monitoring program covers the commercial fishery on the following bivalve molluscs: blue mussel (Mytilus edulis), common cockle (Cardium edule), surf clam (Spisula spp.), scallop (Pectinidae spp.) as well as oysters (Ostrea edulis). Legislation, Regulatory Systems and Monitoring Programmes Algae toxins and toxic and potentially toxic algae From 1984 a voluntary surveillance programme was carried out, but in 1990 this was made an intensive mandatory monitoring for toxic and potentially toxic phytoplankton and for algae toxins in relation to commercially caught mussels in Denmark. Official regulations concerning sampling and quantification of toxic phytoplankton and algae toxins in relation to the Danish mussel fisheries are given in " the order no. 202 laying down the health conditions for fishery, handling, production and placing on the market of bivalve molluscs, 15 th April 1993" and in the guidelines no of 27 th April 1993 and no of 28 th February The Danish legislation implements the EEC Council Directive of 15 th July 1991 laying down the health conditions for the production and the placing on the market of live bivalve molluscs, 91/492/EEC, with later amendments. Microbiological classification of production areas The order no. 202 and the 2 mentioned guidelines include the requirements and procedures for the classification of production areas and the order no. 699 of 6 th August 2003 includes the microbiological limits for the production areas A, B and C. Chemical contaminants Since the 1970 ties a national coordinated monitoring programme on chemical contaminants has been in force with cycles of 5 years. The content of lead, cadmium, mercury and tin in fish and fishery products has been regulated since the 1980ies with maximum limits. These limits were national legislation until 2002 when the EEC regulation on contaminants went into force. Monitoring The fishing areas are divided into production areas/grids of km 2, see the figures 1 and 2). A classified zone A area within the production areas is of the size 3 x 3 nautical mile.

10 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 10 According to the order no. 202 all production areas are closed unless samples are taken 1 week before commencement of commercial fishing is wanted in production areas and the results of the analyses show algal toxins and toxic phytoplankton concentrations below the regulatory limits, which means that production areas can be opened by The Regional Veterinary and Food Control Authority in Viborg. To keep a production area open samples of water and mussels must be taken by each fisherman and aquaculture establishment on their first day of the fishing-week. This sampling must continue every week as long as an area is wanted opened for fishery. The water samples and the sample of mussels are taken from the same nautical position and each of the samples must be marked with the geographical co-ordinates and name of the location and number of the production area, and forwarded to the appropriate laboratories. Usually only 1 or 2 of the sample sets sent in from each production area are chosen for analysis. On basis of the analytical results The Regional Veterinary and Food Control Authority in Viborg always decide upon the status of the fishery in each production area. Normally, production areas are havested each week. Intensive sampling of water and mussels If the concentrations of toxic and potentially toxic phytoplankton are elevated but not exceeding the limits, table 1 (p. 13), or if algae toxins are present under the limits, table 2 (p. 15), intensive sampling can be introduced. Furthermore, intensive sampling can be initiated if the concentrations of toxic algae are high but below the regulatory limits coinciding with periods with sunny, warm and/or calm weather, because of increased risk of local high concentrations of toxic algae and accumulation of algal toxins. Intensive sampling means that each fisherman or aquaculture establishment must take samples of each mussel lot, and no catch is permitted for the market before the result of the analysis are received by the Regional Veterinary and Food Control Authority in Viborg, and found not positive for algae toxins over the limit. The number of samples to be analyzed from each lot (1-5) is decided upon by the Regional Veterinary and Food Control Authority in Viborg. As examples restrictions are enforced if the numbers of Dinophysis acuminata or Alexandrium tamarense reach/or are above 500 cells/l and if Dinophysis acuta reach/or are above 40 cells/l. Furthermore, there have been several closings and restrictions because of blooms of Pseudo-nitzchia spp. Closing production areas If the limits for toxic and potentially toxic phytoplankton are exceeded, table 1, or if algae toxins are present over the limits, table 2, then the fishing areas concerned are closed.

11 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 11 Criteria used to re-open a production area The levels of toxic and potentially toxic phytoplankton and of algae toxins in the mussels must be below the limits the week before the fishery is opened and continue to be so during the following harvest period. If there has been an event with algae toxins in the mussels exceeding the limits in a production area, then the area will not be opened before two consecutive weeks, shows no sign of algae toxins. Information flow and access to information The Regional Veterinary and Food Control Authority in Viborg shall give information to the mussel fishermen about the present status of all production areas. This is done via an automatic telephone answering machine ( ) and on the website Using this information system, the status of each production area can be changed immediately, and the fishermen and aquaculture establishments can always reach the information about the status. The fishermen, the mussel industry, the Danish Directorate of Fisheries, the Fishery Inspection and the local involved Regional Veterinary and Food Control Authority, the Danish Veterinary and Food Administration and The Association of the Danish Mussel-fisheries must be notified by telephone, , fax and by the automatic telephone answering machine if algae toxin test results are exceeding the limits in an open area. Public Notifications The Regional Veterinary and Food Control Authority in Viborg must inform the general public via press release, if it is likely that there is any health risk to the consumers/private collectors of bivalve molluscs. Monitoring program Each year more than 1000 paired samples of phytoplankton algae and mussels are collected and approx samples are analysed. Analyses for DSP-toxins and PSP-toxins in the mussels are carried out by mouse bioassay. Verification of DSP-toxins and PSP-toxins as well as analyses for ASPtoxin (domoic acid) are carried out using chemical methods. The main fishing area for blue mussels (Mytilus edulis) is the Limfjord, which is divided in 22 production areas. Furthermore blue mussels are fished at the East Coast of Jutland, in the Danish part of the Wadden Sea and in the Isefjord/Roskilde Fjord, which are divided into 19, 15 and 10 production areas respectively. At the west coast of Jutland, in the Danish part of the Wadden Sea, which is divided into 16 production areas, blue mussels (Mytilus edulis), surf clam (Spisula spp.), scallop (Pectinidae spp.) and occasionally common cockles (Cardium edule) are harvested. Wild stocks of the European Oyster (Ostrea edulis) are now commercially harvested in the Limfjord, and oysters are at present commenced to be cultured in the Limfjord. The total yearly catch of blue mussels, by approximately 67 fishermen, is tons. More than 90% of the production is exported.

12 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 12 The Danish production of blue mussels is one of Europe s largest and is based on natural resources. This is unusual in European context, where tons of the yearly production of approximately 1 mill. tons are based on aquaculture. The Association of the Danish Mussel-Fisheries primarily finances the monitoring program. The expenses of the monitoring system in year 2004 amount to approx. 3% of the total value of the landing value. Results from the Monitoring program DSP-toxins were detected above the limits in 1991, 1992, 1994,1997, 1999, PSP-toxins were only once detected above the limits during the period PSP-toxins were detected by the mouse bioassay but were not verified using chemical methods. Domoic acid was detected in low concentrations below the limits in two mussel samples from the East Coast of Jutland in 1993 as well as in trace concentrations in blue mussels collected in the Limfjord in Table 1. Limits according to the legislation for the concentrations of toxic phytoplankton used in the management of the Danish mussel fisheries, year Algae species Closure or special restrictions (cells/l) DINOFLAGELLATES Dinophysis acuminata 500 Dinophysis acuta 100 Dinophysis norvegica 1,000 Dinophysis rotundata 1,000 Dinophysis spp. 1,000 Protoceratium reticulatum Only if mice react Lingulodinium polyedrum Only if mice react Alexandrium ostenfeldii 500 Alexandrium tamarense 500 Alexandrium minutum 500 Alexandrium spp. 500 Prorocentrum lima 500 Prorocentrum balticum Only if mice react Prorocentrum micans Only if mice react Prorocentrum minimum Only if mice react Protoperidinium spp. Only if mice react Karenia mikimotoi Only if mice react DIATOMS Pseudo-nitzschia seriata 200,000 Pseudo-nitzschia spp. 500,000 BLUE GREEN ALGAE Anabaena spp. Only if mice react Nodularia spumigena 100, Colonies/l Sampling of toxic algae/phytoplankton The quantitative algae-samples are collected using water-sampler. Water samples are pooled samples made up of water sampled from the surface, the middle and the bottom of the water column where fishing is taking place. A sub-sample of 200 ml is collected from the bucket and preserved using Lugol's neutral fixative. The sample is

13 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 13 marked with date, sampling position, production area number, name of locality and the identity of the collector of the sample and/or company. The qualitative algae-samples are collected using a plankton net (mesh size 20 µm), drawn vertically from the sea-bottom to the surface a couple of times. The concentrated algae are poured into a bottle and preserved using Lugol's neutral fixative. The bottle is marked with date, sampling position, production area number, name of locality and the identity of the collector of the sample. Sampling of mussels etc. Mussels etc. are harvested and sub-samples of shucked cooked mussel etc. meat are collected in plastic bags marked with date, sampling position, area number, name of locality and the identity of the collector of the sample. Laboratory procedures The samples of water and mussels are analysed by private laboratories approved by The Danish Veterinary and Food Administration. The Danish National Reference Laboratory on marine biotoxins, NRL-MB, at, Danish Veterinary and Food Administration, Institute of Food Research and Nutrition is responsible for providing the status of the analytical methods to be used in the laboratories and for ring-tests to be submitted. Phytoplankton samples are analysed at the consultancy company Orbicon A/S. Mussel samples are analysed for the presence of DSP- and PSP-toxins using mouse bioassay at the company LAB Research (Scantox). Analysis for ASP-toxin (domoic acid) as well as the verification of DSP-toxins are carried out using chemical methods (table 2), at the The Danish National Reference Laboratory on marine biotoxins, NRL-MB. The quantification of toxic algae is carried out using the Utermöhl methodology in combination with epifluorescence microscopy, Andersen & Kristensen (1995). The mussels are analysed for DSP-toxins by mouse bioassay throughout the year. The DSP-toxins acetone extraction method modified from Yasumoto's mouse bioassay, Yasumoto et al. (1978), is used for normal monitoring of blue mussels. The DSP-toxins ether extraction method modified from Yasumoto's mouse bioassay, Yasumoto et al. (1984), is used as the official verification method for algal toxins in blue mussels as well as all other bivalve molluscs, echinoderms, tunicates and marine snails. Analysis for PSP-toxins is carried out by mouse bioassay using a modification of AOAC's methodology AOAC (1990), (ph = 3) as a minimum in the months April- September. Domoic acid analyses on mussels are only carried out during blooms of Pseudonitzschia spp. and the samples are analysed by HPLC (Quilliam et al. 1995). Verification for presence of PSP-toxins is done by HPLC (Lawrence et al.2004).

14 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 14 Verification for presence of DSP-toxins, including okadaic acid (OA), Dinophysistoxin 1 (DTX-1), Dinophysis-toxin 2 (DTX-2), and DSP-toxin esters is done using a LC/MS/MS method developed at the The Danish National Reference Laboratory on marine biotoxins NRL-MB (Jørgensen and Jensen 2004). A summary of the monitoring for algal toxins is summarised in table 2. The sampling periods can be extended if e.g. results from the phytoplankton analysis prove it necessary. Table 2. Algae toxin DSP Algae toxins and EU regulatory limits as well as the minimum period of monitoring for algae toxins in relation to the Danish mussel-fisheries. Regulatory Limits Period Method Must not be positive =2 out of 3 mice die within 24 hours < 160 µg okadaic acid /kg whole body All year round Mouse bioassay, Yasumoto et al. (1978 and 1984) Official method: ether extraction (used to verify positive results from the acetone extracts as well as for analysis of other bivalve molluscs) Routine method: acetone extraction (used for Mytilus edulis under normal surveillance) PSP ASP Must not be positive = 1 or more mice die within 1 hour < 800 µg saxitoxin/ kg whole body < 20 mg domoic acid /kg whole body Minimum April-September When blooms of Pseudo-nitzschia spp. occur Verification by LC/MS/MS (Jørgensen and Jensen 2004) Mouse bioassay: AOAC, Paralytic shellfish poison. Biological method. Final action. Official methods of analysis, 15th edition, Association of Official Analytical Chemists, Washington DC. Verification by HPLC (Lawrence et al. 2004) HPLC (Quilliam et al. 1995) Control samples The establishments, producers or packers of fresh or processed mussels must, according to the legislation, take control samples of each days production or catch, and store them as references, if later examination of the mussels from special catches or batches is needed. Control samples of mussels, which are sold live for direct consumption must be kept 1 month frozen, and control samples from mussels, which are sold frozen or canned must be kept for 1 year or for the shelf life of the product. The above-mentioned establishments must ensure that all products comply with the criteria given in the legislation through own checks. Annual reports The results of the monitoring program are compiled in annual status reports in Danish with a summary in English. The monitoring program and the annual report is discussed and reviewed once a year by a working group consisting of The Danish National Reference Laboratory on Marine Biotoxins, The Danish Veterinary and Food Administration, experts on phyto-

15 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 15 plankton, experts on the chemical methods and the mouse bioassays on algae toxins and representatives from The Association of the Danish Mussel-fisheries. Monitoring results 2004 In year 2004 the fishermen and aquaculture establishments collected 1422 paired samples of phytoplankton and mussels and all 1422 paired samples were analysed. Occurrence of algae toxins DSP-toxins (okadaic acid and esters of okadaic acid) were registered in commercially harvested bivalve molluscs in concentrations exceeding the limits in 10 production areas/weeks in At the East Coast of Jutland, the observation of DSP-toxins took place during a period where the fishery was restricted and/or closed due to reaction in the mouse bioassay (abnormal behaviour death). The observations of DSP-toxins coincided with high concentrations of Dinophysis acuminata and Dinophysis acuta. In the Isefjord DSP-toxins were observed in the absence of or in situations with low concentrations of Dinophysis acuminata and Dinophysis acuta. Like in 2003, it seems likely that the DSP toxicity was caused by very low concentrations of Dinophysis acuta, which remained in the fjord during the winter of 2003 and until the beginning of It shall be noted that no consignments containing algal toxins above the regulatory limit were placed on the market, and no consumers were ill caused by DSP-toxins in Danish mussels. Restrictions imposed on the mussel fishery due to occurrence of algae toxins and/or toxic phytoplankton Restrictions (precautionary closure of production areas and/or intensified monitoring) on the mussel fishery was imposed in the Limfjord, on the east coast of Jutland as well as in the Isefjord/Roskilde Fjord and in the Wadden Sea in 59 production areas and weeks, equal to 8% of the total number of 828 areas/weeks which were tried to be opened, due to concentrations of toxic phytoplankton exceeding the limits, as well as the occurrence of algal toxins. Occurrence of toxic/potential toxic phytoplankton The seasonal as well as the geographical occurrence was normal for most species in A few species were observed in high concentrations compared to observations from the previous years, such as species from the diatom genus Pseudonitzschia which was registered in elevated concentrations in the Limfjord, at the east coast of Jutland and in the Isefjord. The Dinophysis species D. acuminata and D. acuta continued to bloom also in 2004 where as the closely related species D. norvegica kept on declining in Danish waters. The shift in abundance between the different species from the genus Dinophysis might be a result of the on-going warming of the Danish coastal waters, favouring the more tolerant warm water species D. acuminata and D. acuta on the behalf of the cold water species D. norvegica. Also the observation of a bloom of the southern Dinophysis species D. sacculus indicates the importance of the warming of the Danish coastal waters to the shift in species distribution.

16 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 16 Recommendations for the monitoring of toxic algae and algae toxins As in the previous reports it is recommended that procedures is set up for sampling of phytoplankton and mussels etc. during periods, where the limits for phytoplankton and algae toxins are exceeded and where closures and restrictions ordering intensive daily sampling in production areas are enforced or when unexplained toxicity in the mussels etc. is registered by the mouse bioassay, possibly due to unknown toxins. For that purpose the necessary funds should be allocated. During closures no samples are taken, and when restrictions ordering intensive daily sampling in production areas are enforced, the fishermen often for economical reasons do not want to fish. The purpose is to collect the necessary data from these special periods to be used in the evaluation of the monitoring programme, to follow the possible build up of algae toxins and to assess how long time the mussels need before they have depurated themselves from algae toxins. To support the validation of the present monitoring programme it would be valuable to establish systematic sampling of both toxic phytoplankton and molluscan shellfish at a few fixed stations to make research into the relationship between the occurrence of toxic phytoplankton and algae toxins in the mussels possible. Microbiological classification of production areas and monitoring of chemical contaminants Since 1984 Denmark has had a mandatory control system on the microbiological status of the live bivalve molluscs intended for direct consumption and originating from the commercial fishery of bivalves, from the aquaculture of oysters, from the purification and from the expedition centres. This control is also carried out on imported live bivalve molluscs for direct consumption. Microbiological classification of production areas All the production areas are classified as B areas. A zone A area is according to the legislation of the size 3 x 3 nautical mile and it is always opened within a production B area, which is opened according to the monitoring procedures for algae toxins and toxic algae. Approximately 4 10 production areas have 1 or more classified zone A areas open every week. Opening of zone A areas within the production areas If a harvester or a mussel fisherman wants to open a zone A area within a production B area, he must take 3 samples of live bivalve molluscs within the specific area, and get them analysed approx. 10 days before commencement of commercial fishing is wanted. The samples of the live bivalve molluscs must be marked with the nautical position by giving the geographical co-ordinates together with other necessary information for identification, and the samples must be forward to a laboratory approved by the competent authority. Copies of the markings on the samples are routinely forwarded by The Regional Veterinary and Food Control Authority in Viborg to local fishery inspectorats, other Veterinary and Food Control Authorities, The Association of the Danish

17 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 17 Musselfisheries as well as industries not organized within The Association of the Danish Musselfisheries. If the results of the analyses show no presence of salmonella and show that the results of the presumptive E. coli / faecal coliforms analysis are found under the legislative limits, (no analytical tolerance is allowed), then a zone A area of that production areas can be opened by The Regional Veterinary and Food Control Authority in Viborg. The precise calculation of the zone A area to be opened is based on the geographical co-ordinates given by the mussel fisherman or harvester in the logbooks or on the registration documents. This zone A area has the status for 1 month. To keep a zone A part of a production area open for a longer period, 3 samples of live bivalves must be taken once every month. This sampling must continue every month as long as a zone A area is wanted opened for fishery for direct consumption. If the analytical results on a zone A part of a production area is not received by The Regional Veterinary and Food Control Authority in Viborg, then the zone A status is changed to B. Legislative Limits for the microbiological classification Zone A classification limit: Less than 300 faecal coliforms or less than 230 E. coli per 100 g of molluscs flesh and intravalvular liquid and Salmonella must not be present in 25 g of flesh. B classification limit: results must not exceed faecal coliforms or E. coli per 100 g of flesh in 90% of the samples. C classification limit: results must not exceed faecal coliforms per 100 g of flesh. All the 3 limits must be based on a five tube, three-dilution MPN-test. Information flow and access to information The opening of zone A areas is primarily financed by individual mussel and oyster fishermen or by the owners of aquaculture establishments. The Regional Veterinary and Food Control Authority, Viborg, always decides upon the microbiological status of each production area, and can change any status from A to B or even to C, if laboratory reports show results requiring this. The Regional Veterinary and Food Control Authority in Viborg shall give information to all the mussel fishermen and the establishments processing mussels, if the classification of a production area changes to zone C status. Closing zone A parts of production areas If the limits for presumptive E. coli / faecal coliforms or Salmonella are exceeded then the zone A parts of production areas return to either the B classification or to C classification status.

18 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 18 Sampling of mussels etc. Blue mussels, oysters and other bivalve molluscs etc. are harvested and raw samples with shells, for oysters 3 x 100g (approx. weight without shells), for the other species min. 2.7 kg per sample, are sampled and marked with date, sampling position, area number, name of locality and the identity of the collector of the sample. A minimum of 3 samples must be taken from each area to be opened as zone A. Laboratory procedures The samples of mussels are analysed by a private accreted laboratory approved by The Danish Veterinary and Food Administration and by some of the Regional Veterinary and Food Control Authority Laboratories. Faecal coliform bacteria, (presumptive E. coli), in live bivalve molluscs, method: Detection and enumeration of thermo tolerant coliform bacteria (presumptive E. coli) by MPN method according to Nordic Committee on Food Analysis method no. 96, 2. ed., 1994 and Bacteriological examination of fresh and frozen seafood, using: -5 tubes per each of 3 dilutions -double strength broth in the 1 st dilution -10 ml of the 1 st dilution -sample size 20 g bivalve molluscs ml dilution media for each. Salmonella, method: Salmonella. Detection in foods according to Nordic Committee on Food Analysis 71, 5. Ed., *Nordic Committee on Food Analysis. Control / counter samples The establishments, producers or packers of fresh or processed bivalve molluscs must according to the legislation take control samples of each days production or catch, and store them frozen as references, if supplementary analysis of the bivalve molluscs from specific catches or batches is needed. Control samples of mussels, which are sold for direct consumption, must be kept 1 month frozen, and control samples from mussels, which are sold frozen or canned must be kept for 1 year or for the shelf life of the product. The above-mentioned establishments must ensure that all products comply with the criteria given in the legislation through own checks. Monitoring results for 2004 for the classification of zone A areas In year 2004 a total of 166 parts of production areas were analysed because mussel fishermen / expedition centres wanted parts of production areas classified as zone A areas. Out of the 166 parts a total of 140 (84,3%) parts were classified as zone A areas, while 22 (13,3%) parts remained zone B classification and 4 parts of a production areas were classified as zone C (2,4%). In year 2004 a total of 498 single samples of mussels were analysed for the level of E. coli / faecal coliforms, and 166 analyses for Salmonella were carried out. The 140 zones A areas were classified in a total of 31 production areas 7 of which were mussel cultures.

19 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side zone A areas were classified in 18 of the 22 production areas in the Limfjord. 21 zone A areas were classified in 7 of the 18 production areas at the East Cost of Jutland. 21 zone A areas were classified in 5 of the 10 production areas in the Isefjord. 4 zone A areas were classified in 4 of the 18 production areas in the Wadden Sea. All the areas, which were classified as zone A within a production area, are given in annex 8. In annex 9 are shown, the 22 cases where the areas wanted for classification as zone A, remained as zone B classification. The annex show 8 cases in the Limfjord, 3 cases at the East Coast of Jutland, 1 case in the Isefjord and 2 cases in the Wadden Sea. In annex 9 are shown the 4 cases where areas wanted for classification as zone-a, was classified as a zone C. Recommendations The microbiological results over the years, show that enlargement of the size of the zone A areas is possible without compromising the food safety objectives. Certain production areas have shown results, which indicates that permanent A classification can be established. It is therefore suggested to carry out the necessary data analysis to establish such permanent A classified production areas and also analyse the data concerning permanent B classification. Monitoring of chemical contaminants in production areas In chapter V, point 4 of Council Directive 91/492/EEC, it is stated that live bivalve molluscs must not contain toxic or objectionable compounds occurring naturally or added to the environment such as those listed in the annex to directive 79/923/EEC in such quantities that the calculated dietary intake exceeds the permissible daily intake (PDI), or that the taste of the molluscs may be impaired. In chapter V, section II, point 3, part B, of Council Directive 91/493/EEC, laying down the health conditions for the production and the placing on the market of fishery products, is stated that without prejudice to the Community rules concerning water protection and management, and in particular those concerning pollution of the aquatic environment, fishery products must not contain in their edible parts contaminants present in the aquatic environment such as heavy metals and organochlorinated substances at such a level that the calculated dietary intake exceeds the acceptable daily or weekly intake for humans. In the Council Directive 79/923/EEC on the quality requirements for the shellfish waters, requirements are laid down for the testing of contaminants and radio nuclides and listed in its annex. The Ministry of Environment is monitoring the status for the chemical contaminants in the aquatic environment. The monitoring covers heavy metals such as led, mercury,

20 Toksiske alger, algetoksiner og mikrobiologiske zoner i muslingefiskeriet 2004 Side 20 cadmium, tin (TBT), arsenic, zinc, PAH (Polycyclic Aromatic Hydrocarbons) and a number of other metals together with substances such as organochlorines etc. Monitoring results and recommandations for chemical contamination in 2004 I 2004 a total of 8 samples of Danish blue mussels and 13 samples of Danish oysters were collected to be analysed for monobutyl pewter, dibutyl pewter, tributyl pewter, monophenyl pewter, diphenyl pewter og triphenyl pewter. The samples were also analysed for led, cadmium and mercury. The results of the analyses are given in Annex 11. No EU limits are given for monobutyl pewter, dibutyl pewter, tributyl pewter, monophenyl pewter, diphenyl pewter and triphenyl pewter, but a TDI limit on the sum of pewter components mentioned above is set at 0,25 microgram per kg body weight. The results are not estimated to be unsafe for human consumption, but more samples will be analysed. The analytical results on led, cadmium and mercury were all below the EU limits. The results were found to be the following: 0,0811-0,428 mg Pb/kg, 0,0828-0,461 mg Cd/kg og 0, ,0219 mg Hg/kg. TECHNICAL AND SCIENTIFIC REPORTS: The following technical and scientific reports are based upon results from the Danish monitoring system: 1990 Bjergskov, T., J. Larsen, Ø. Moestrup, H. M. Sørensen & P. Krogh (1990). Toxic and potential toxic algae in Danish waters: Management, taxonomy, ecology, toxicology. Published by The Fish Inspection Service. (in Danish/English) Hald, B., T. Bjergskov & H. Emsholm (1991). Monitoring and analytical programmes on phycotoxins in Denmark. Proceedings of symposium on marine biotoxins. (Ed. J. Marc Fremy 1991) p Andersen, P.; H. Emsholm & S. D. Jacobsen (1992). Status on the monitoring of toxic algae and algal toxins in mussels in Danish fjords and coastal areas Report to The Ministry of Fisheries. (in Danish) Andersen, P. & H. S. Kristensen (1993). Rapid and precise identification of the dinoflagellates using epifluorescence microscopy. Proceedings from the 6th International Conference on Toxic Phytoplankton, Nantes - FRANCE. p Lavoisier, London.

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