EU Harmonised Standard Operating Procedure for detection of Lipophilic toxins by Mouse Bioassay

Transcription

1 EUROPEAN UNION REFERENCE LABORATORY FOR MARINE BIOTOXINS EU Harmonised Standard Operating Procedure for detection of Lipophilic toxins by Mouse Bioassay Version 6, December 2013 Coordination: European Union Reference Laboratory for Marine Biotoxins (EURLMB) Agencia Española de Seguridad Alimentaria y Nutrición (AESAN) c/ Fonte das Abelleiras, 4 - Edificio CITEXVI Vigo, Spain [email protected]

2 CONTENTS Foreword... 3 Introduction Purpose Scope and Application Specificity Principie Reagents Apparatus and Materials Mice Analytical Procedure (Hepatopancreas Tissue) Analytical Procedure (Whole Flesh)... 9 ANNEX: Protocol for the selective extraction of Yessotoxins (YTXs) Bibliography a.14 Page 2 of 14

3 Foreword This Standard Operating Procedure has been prepared by the Working Group on lipophilic toxins via bioassay of the European network of National Reference Laboratories (NRL) for Marine Biotoxins. Members of the Working Group: France NRL, Ireland NRL, Italy NRL, Norway NRL, United Kingdom NRL, coordinated by the Community Reference Laboratory for Marine Biotoxins (CRLMB, Spain). Introduction Lipophilic marine biotoxins can be accumulated in different molluscan shellfish presenting a health risk to humans if contaminated shellfish are consumed. To protect public health, monitoring programmes for marine biotoxins have been established in many countries for detecting the presence of these compounds in shellfish tissues. The regulatory structure in the EU includes a series of regulations for the control of lipophilic toxins. Regulation (EC) No 853/2004 [1], Annex III Section VII Chapter V No. 2 (c, d, e), lays down the maximum levels for several kinds of toxins which belong to the lipophilic toxin group, including the classic diarrheic shellfish poisoning toxins (DSP): okadaic acid (OA) and dinophysistoxins (DTXs); as well as pectenotoxins (PTXs), yessotoxins (YTXs) and azaspiracids (AZAs). Regarding the analytical methods for the analysis of these compounds, Commission Regulation (EC) No 2074/2005 [2], amended by Commission Regulations (EC) No 1664/2006 and No 1244/2007, indicates the recognised testing methods for marine biotoxins for the purpose of Regulations (EC) No 853/2004 and No 854/2004. For the analysis of lipophilic toxins it is stated that biological and alternative detection methods can be used. With regard to biological methods, Regulation (EC) No 2074/2005 establishes that a series of mouse bioassay (MBA) procedures, differing in the test portion (hepatopancreas or whole body) and in the solvents used for extraction and purification, may be used; indicating that sensitivity and selectivity depend on the choice of solvents used for extraction and purification and that this should be taken into account when a decision is made on the method to be used in order to cover the full range of toxins. Legislation does not specify a detailed protocol of the mouse bioassay leading to the application of different conditions and a lack of harmonization in the EU. For this reason, a Working Group on lipophilic toxins via bioassay was established within the EU-CRL/NRLs for marine biotoxins network, with the aim of considering further refinement and standardization of the MBA protocol. The group has worked on the refinement of a Mouse Bioassay Standard Operating Procedure (MBA-SOP), which has been evaluated through the Proficiency Testing exercises organised annually since 2004 by the CRLMB, as well as with an ad hoc refinement study on the original protocol. As a result of this work, the present SOP is proposed. Page 3 of 14

4 1 Purpose The purpose of this Standard Operating Procedure (SOP) is to detail the conduct of the analysis in the determination of lipophilic toxins: okadaic acid and dinophysistoxins (OA-group toxins), pectenotoxins (PTX group), azaspiracids (AZA group) and yessotoxins (YTX group) in bivalve molluscs, echinoderms, marine gastropods, and tunicates. Furthermore the SOP includes in the Annex a Protocol for the selective extraction of the YTX group when their presence or co-occurrence is suspected. 2 Scope and Application The method is based on that of Yasumoto et al., 1984 [3]. This method is applicable to molluscan shellfish in the live, frozen or processed state. It can be used the whole body or hepatopancreas of molluscan shellfish as analytical sample, considering that: 2.1 For live, fresh or frozen bivalve molluscs with a large hepatopancreas, and for which the whole flesh is eaten entirely (i.e. mussels, oysters, razor clams etc), the hepatopancreas procedure should be used (Please refer to Section 8). 2.2 For live, fresh or frozen small bivalve molluscs with a small hepatopancreas (i.e. clams, cockles etc), bivalve molluscs without hepatopancreas, tunicates, echinoderms, marine gastropods, the whole flesh procedure should be used (Please refer to Section 9). 2.3 In the cases of cooked products the whole flesh procedure should be used (Please refer to Section 9). It is important to remark that this method is not intended for the analysis of these kind of products, although has been found to be suitable in certain cases. 3 Specificity The method can detect: okadaic acid and dinophysistoxins (OA-group toxins), pectenotoxins (PTX group), azaspiracids (AZA group) at the regulatory level as laid down in Regulation (EC) No 853/2004 and yessotoxins (YTX group) even below the regulatory level. When the presence of one of these groups is detected, a secondary confirmatory technique is recommended to confirm the group presented in the sample. Other acetone soluble toxins such as brevetoxins, gymnodimine, spirolides, pinnatoxin, ciguatoxin and palytoxin may be also detected. Page 4 of 14

5 4 Principle The method is based on the extraction of lipophilic compounds with acetone followed by partition between diethyl ether or dichloromethane and water. The organic solvent is evaporated and the resultant residue suspended in an aqueous solution of 1% Tween 60. One ml of this extract is injected intraperitoneally into albino mice Swiss strain weighing between 17 g and 22 g. The death of 2 out of 3 injected mice, within a 24 hours observation period, constitutes a positive result according to Commission Regulation (EC) No 2074/ Reagents 5.1 Acetone grade (99% or greater purity) 5.2 Diethyl ether (DEE) or Dichloromethane (DCM) GPR grade (99% or greater purity). It is advised that the organic solvent presaturated with water prior to analysis may be used 5.3 Deionised water. Water presaturated with DEE or DCM may be used as an option 5.4 1% aqueous Tween 60 solution Preparation of 1% Tween 60 solution (example): 1. Put ca. 10 ml of Tween 60 in a bottle with the cap loosened, place it into a water bath at approximately 40ºC-45ºC and invert frequently until it is in a liquid state 2. Using a pipette, place x ml of Tween 60 stock into a suitable volumetric flask y ml of warm distilled water is added to the flask via a measuring cylinder to give a 1% solution. Ensure the Tween 60 is fully dissolved in the water 3. The 1% Tween 60 may be stored either at room temperature or in the fridge 4. Discard after 1 month (maximum). 6 Apparatus and Materials 6.1 General. Use usual laboratory material and equipment and, in particular, the following: 6.2 Glassware 6.3 Scalpels, scissors, knifes 6.4 Analytical balance calibrated and verified to cover the daily working range 6.5 Strainers Page 5 of 14

6 6.6 Tweezers 6.7 Electric blender or Ultra-Turrax homogenizer (or equivalent) 6.8 Refrigerated centrifuge (optional) capable of 3000 rpm or 1200g 6.9 Rotary evaporator with controlled vacuum system and water bath calibrated at 40ºC-45ºC 6.10 Round bottom flasks (100 ml, 250 ml, 500 ml and 1000 ml) 6.11 Disposable 1ml sterile syringes (with 0.1 ml graduation) with gauge needle 6.12 Glass separatory funnels 6.13 Filter paper (Whatman Nº 1 or similar) 6.14 Timers 7 Mice Use male albino mice Swiss strain (CD1 is recommended) weighing between 17 g and 22 g, from a standard stock colony. All mice must be purchased from an approved supplier who operates licensed breeding premises under the legislation in force. The stock colony must be managed following the Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. The housing and care of the mice should be managed in accordance with Commission Recommendation 526/2007 of 18 June 2007 on the guidelines for the accommodation and care of animals used for experimental and other scientific purposes. 8 Analytical Procedure (Hepatopancreas Tissue) For live, fresh or frozen bivalve molluscs with a large hepatopancreas, and for which the whole flesh is eaten entirely (i.e. mussels, oysters, razor clams etc), the hepatopancreas procedure should be used. 8.1 Preparation of raw samples Thoroughly clean the outside of shellfish with fresh cold running water. Open by cutting adductor muscles. Do not use heat or anaesthetics before opening the shells, and do not cut or damage the soft tissues at this stage. Rinse inside with fresh cold running water to remove sand or other foreign material. Page 6 of 14

7 Remove meat from shell by separating adductor muscles and tissue connecting at hinge. Obtain carefully approx. 25 g of hepatopancreas, trying to remove all the mantle portions bonded to it. Transfer the hepatopancreas to a strainer and drain for 5 minutes. NOTE It is advised that the weight proportion of hepatopancreas:whole flesh (HP:WF) is recorded. 8.2 Extraction procedure The amount of acetone used should be equivalent to the ratio of 5:1 (acetone:hepatopancreas) for a single extraction. Homogenize 20 g ± 0.2 g of hepatopancreas tissue in 100 ml of acetone in a blender or Ultra-Turrax for 2 minutes Filter (gravity/vacuum) through paper or centrifuge under refrigeration at approx rpm (or rate equivalent to 1200 g) for 10 minutes and transfer the filtrate/supernatant to a clean evaporation flask Remove the organic solvent by rotary evaporation until cessation of dripping into the receiving flask and/or bubbling in the evaporation flask (temperature of the water bath must be maintained at 42ºC ± 2 C). To prevent excessive foaming, the vacuum may need to be released occasionally during evaporation. The flask must be checked regularly to determine the acetone evaporation end point. It is suggested that after vacuum is released, empty the receiving flask and then evaporate for a further 5 minutes Add 10 ml of deionised water to the evaporation flask, followed by 50 ml of DEE or DCM and swirl the flask. Transfer the sample extract into a separatory funnel Shake by gentle rocking motion 8-10 times. Avoid vigorous shaking. Collect the aqueous layer and any emulsion layer into the evaporation flask. Gently swirl the DEE or DCM phase to draw any remaining aqueous phase or emulsion to the funnel tap, drain and combine with the collected aqueous layer. Collect the organic layer into a clean glass container. Transfer the aqueous phase to the separatory funnel Add 50 ml of DEE or DCM to the aqueous layer, swirl the flask and transfer the aqueous phase to the separatory funnel and repeat step to combine the organic phases Repeat step Discard the aqueous layer to waste Combine the three organic extracts in the second separatory funnel. Add 20 ml of deionised water. Mix by gentle rocking motion 8-10 times. Avoid vigorous shaking. Hold funnel horizontally to maximise the water/organic interface. Allow the 2 phases to separate completely, discard the aqueous phase to waste Repeat step Transfer the organic phase to an evaporation flask. Evaporate the organic fraction to dryness using a rotary evaporator (temperature of the water bath must be 42ºC ± 2 C). Evaporation should be continued to dryness or a point where an oily residue remains. It is suggested that after vacuum is released, the receiving flask is emptied and the sample is then evaporated for a further 5 minutes Page 7 of 14

8 Make up the residue to 4 ml of 1% Tween 60. NOTE 1 In the cases where it is impractical to inject the Tween 60 1% extract due to viscosity, additional volumes of Tween 60 may be added to such a stage where the extract is injectable, (max. total volume must not exceed 6 ml). Note that this is only an exceptional case when is not possible to repeat the extraction procedure and that the injection volume at step 8.3 must be always 1 ml/mouse. NOTE 2 If it is necessary, the resuspended extract may be further homogenised via Ultra-Turrax (recommended 8mm shaft) prior to inoculation. NOTE 3 If the sample extract cannot be injected on the same day, it should not be homogenised, and should be stored overnight in a fridge or in a freezer for a max of 2 nights. Prior to injection the extract must be fully thawed and homogenised via Ultra-Turrax (recommended 8mm shaft) or vortex. 8.3 Mouse bioassay Inoculate 1 ml of the solution obtained in step (equivalent to 5 g of hepatopancreas) to each of three mice weighing 19 g - 21 g and observe for 24 hours. In case that mice out of this range are used (17-18 g or 22 g), the injection volume should be adjusted accordingly. 8.4 Interpretation of the results The death of two out of three mice within 24 hours should be interpreted as a positive result where the presence of one or more of the toxins of the following groups: OA-group toxins, PTX group and AZA group at levels above those established in the Regulation (EC) No 853/2004 or YTX group at levels even below those established in the Regulation (EC) No 853/2004. The presence of YTXs or the co-occurrence of YTXs with the other toxins of the group can be suspected if mice die with PSP-like symptoms (convulsion and jumping). It this case, the application of the Protocol described in Annex is advisable to separately detect YTX group [4]. Nevertheless, if 2 or more mice are dead within 15 min, the assay should be considered inconclusive and must be followed up by further testing according to Commission Regulation (EC) No 2074/2005. Once the toxicity criteria of Commission Regulation (EC) No 2074/2005 is met (2/3 mice dead) and in order to avoid animal suffering, and to address Council Directive 86/609/EEC of 24 November 1986, the third mouse may be culled optionally within 24 hours. Page 8 of 14

9 9 Analytical Procedure (Whole Flesh) For live, fresh or frozen small bivalve molluscs with a small hepatopancreas (i.e. clams, cockles etc), bivalve molluscs without hepatopancreas, tunicates, echinoderms, marine gastropods, the whole flesh procedure should be used. In the cases of cooked products the whole flesh procedure should be used. 9.1 Raw samples (in shell) Thoroughly clean the outside of shellfish with fresh cold running water. Open by cutting adductor muscles. Do not use heat or anaesthetics before opening the shells, and do not cut or damage the soft tissues at this stage. Rinse inside with fresh cold running water to remove sand or other foreign material. Remove tissue from shell by separating adductor muscles and tissue connecting at hinge. Transfer tissues to a strainer and drain for 5 minutes. Homogenize in a blender or Ultra-Turrax ca. 150 g of shellfish meat. 9.2 Processed samples If the sample is frozen allow to thaw to room temperature. Rinse with fresh water. Transfer tissues to a strainer and drain a few minutes. Homogenize in a blender or Ultra-Turrax ca. 150 g of shellfish meat. 9.3 Canned samples Samples in brine or water. Place entire contents of can in a strainer. Allow the sample to drain, discard the liquid and rinse with distilled water. Drain for two minutes. Samples in oil and oily sauces. Discard any non-shellfish ingredients e.g. spice. Homogenize all the contents of the can in a blender or Ultra-Turrax. 9.4 Extraction procedure The amount of acetone used should be equivalent to the ratio of 3:1 (acetone: shellfish meat) for the first extraction and 2:1 (acetone: shellfish meat) for the second Weigh 100 g ± 1 g of the homogenate, using a top pan balance Measure 300 ml acetone in a measuring cylinder, add to the sample and homogenize in a blender or Ultra-Turrax for 2 minutes Filter the extract (gravity/vacuum) through filter paper or centrifuge under refrigeration at 3000 rpm (or rate equivalent to 1200 g) for 10 minutes and transfer filtrate/supernatant to a clean evaporation flask Page 9 of 14

10 9.4.4 Extract residual meat with 200 ml of acetone and homogenize in a blender or Ultra-Turrax for 2 minutes Filter the extract (gravity/vacuum) through filter paper or centrifuge under refrigeration at 3000 rpm (or rate equivalent to 1200 g) for 10 minutes. Collect and combine the 2 extracts in the same evaporation flask Evaporate the acetone using a rotary evaporator set at 42 C ± 2 C. To prevent excessive foaming, the vacuum may need to be released occasionally during evaporation. Evaporation of the solvent is judged to be complete when bubbling in the distillation flask stops and no further liquid can be seen passing to the collection flask. The flask must be checked regularly to determine acetone evaporation end point. It is suggested that after vacuum is released, empty the receiving flask and then evaporate for a further 5 minutes Adjust the volume of the aqueous extract to 100 ml with distilled water by transferring to a measuring cylinder, ensuring all the extract is transferred completely. Transfer it into a separatory funnel. Measure 100 ml of DEE or DCM for the liquid/liquid partition in a clean measuring cylinder and use a small amount of this organic solvent to rinse the evaporation flask and the measuring cylinder which contained the aqueous phase and transfer the rinsing to the separatory funnel. Add the remaining organic solvent Gently shake the flask to ensure the contents are mixed. Shake by gentle rocking motion 8-10 times. Avoid vigorous shaking. Allow for DEE or DCM and aqueous phase separation. Collect the aqueous layer and any emulsion layer into the evaporation flask. Gently swirl the DEE or DCM phase to draw any remaining aqueous phase or emulsion to the funnel tap, drain and combine with the collected aqueous layer. Collect the organic layer into a clean glass container. Transfer the aqueous phase to the separatory funnel Add 100 ml of DEE or DCM to the aqueous layer and repeat step to combine the organic phases Repeat step Throw the aqueous layer away Combine the three organic fractions into a separatory funnel. Add 20 ml of deionised water and gently mix the contents. Mix by gentle rocking motion 8-10 times. Avoid vigorous shaking. Hold funnel horizontally to maximise the water/organic interface. Allow to separate completely and throw the aqueous layer away Repeat step Transfer the organic phase to an evaporation flask. Evaporate the organic fraction to dryness using a rotary evaporator (temperature of the water bath must be 42ºC ± 2 C). Evaporation should be continued to dryness or a point where an oily residue remains. It is suggested that after vacuum is released, the receiving flask is emptied and the sample is then evaporated for a further 5 minutes Make up to 4 ml of 1 % Tween 60. NOTE 1 In the cases where it is impractical to inject the Tween 60 1% extract due to viscosity, additional volumes of Tween 60 may be added to such a stage where the extract is injectable, (max. total volume must not exceed 6 ml). Note that this is only Page 10 of 14

11 an exceptional case when is not possible to repeat the extraction procedure and that the injection volume at step 9.5 must be always 1 ml/mouse. NOTE 2 If it is necessary, the resuspended extract may be further homogenised via Ultra-Turrax (recommended 8mm shaft) prior to inoculation. NOTE 3 If the sample extract cannot be injected on the same day, it should not be homogenised, and should be stored overnight in a fridge or in a freezer for a max of 2 nights. Prior to injection the extract must be fully thawed and homogenised via Ultra-Turrax (recommended 8mm shaft) or vortex. 9.5 Mouse bioassay Inoculate 1 ml of the solution obtained in step (equivalent to 25 g of shellfish meat) to each of three mice, weighing 19 g - 21 g and observe for 24 hours. In case that mice out of this range are used (17-18 g or 22 g), the injection volume should be adjusted accordingly. 9.6 Interpretation of the results The death of two out of three mice within 24 hours should be interpreted as a positive result where the presence of one or more of the toxins of the following groups: OA-group toxins, PTX group and AZA group at levels above those established in the Regulation (EC) No 853/2004 or YTX group at levels even below those established in the Regulation (EC) No 853/2004. The presence of YTXs or the co-occurrence of YTXs with the other toxins of the group can be suspected if mice die with PSP-like symptoms (convulsion and jumping). It this case, the application of the Protocol described in Annex is advisable to separately detect YTX group [4]. Nevertheless, if 2 or more mice are dead within 15 min, the assay should be considered inconclusive and must be followed up by further testing according to Commission Regulation (EC) No 2074/2005. Once the toxicity criteria of Commission Regulation (EC) No 2074/2005 is met (2/3 mice dead) and in order to avoid animal suffering, and to address Council Directive 86/609/EEC of 24 November 1986 the third mouse may be culled optionally within 24 hours. Page 11 of 14

12 ANNEX: Protocol for the selective extraction of Yessotoxins (YTXs) Scope and application The protocol will be applicable when the presence or co-occurrence of YTX group toxins is suspected in the samples. Principle The method is based on the extraction of the lipophilic compounds in the sample with acetone. Then, in order to separately extract YTX group from other lipophilic toxins (OA-toxins group, PTX group and AZA group), a dichloromethane/60% methanol partition is carried out. For both extracts, solvent is evaporated and the resultant residue suspended in an aqueous solution of 1% Tween 60. One ml of each extract is injected intraperitoneally into albino mice Swiss strain weighing between 17 g and 22 g. For the dichloromethane extract (investigation of OA-toxins group, PTX group and AZA group), the death of 2 out of 3 injected mice, within a 24 hours observation period, constitutes a positive result according to Commission Regulation (EC) No 2074/2005. In the case of the methanolic extract (investigation of YTX group), the death of 2 out of 3 injected mice, within a 6 hours observation period, constitutes a positive result for the presence of YTXs. Specificity The method can detect, separately, okadaic acid and dinophysistoxins (OA-group toxins), pectenotoxins (PTX group), azaspiracids (AZA group) at the regulatory level as laid down in Regulation (EC) No 853/2004 and yessotoxins (YTX group) at the regulatory level laid down in Regulation (EC) No 853/2004. Reagents See section 5. Methanol (MeOH), GPR grade (99% or greater purity) 60% Methanol. Methanol/water 60:40, v/v Apparatus and materials See section 6. Mice See section 7. Page 12 of 14

13 Sample preparation See sections 9.1, 9.2 and 9.3. Sample extraction Proceed as described from section to Liquid-liquid partitioning Transfer the extract obtained in step into a separatory funnel with 30 ml dichloromethane (DCM) and 60 ml MeOH: water 60%. NOTE It is important to have solvents presaturated into each other to avoid emulsions. Gently shake the flask to ensure the contents are mixed. Avoid vigorous shaking. Allow phases separation. Collect the lower dichloromethane layer and any emulsion layer into the evaporation flask. Keep the aqueous MeOH phase. Extract DCM phase twice with 60 ml MeOH: water 60%. Combine the three aqueous MeOH extracts. Investigation of OA-toxins group, PTX group and AZA group (DCM extract) Evaporate the DCM fraction to dryness. Make up to 4 ml with 1% Tween 60. Inoculate three mice, weighing 17 g- 22 g with 1 ml of the solution (equivalent to 25 g of shellfish meat) and observe for 24 hours. The death of two out of three mice within 24 hours should be interpreted as the presence of one or more toxins of OA-toxins group, PTX group and AZA group at levels above those established in the Regulation (EC) No 853/2004. Investigation of YTX group (aqueous MeOH extract) Make up aqueous MeOH extract to 200 ml with MeOH. Take 16 ml of this solution and evaporate to dryness. Make up to 4 ml with 1% Tween 60. Inoculate three mice, weighing 17 g- 22 g with 1 ml of the solution (equivalent to 2 g of shellfish meat) and observe for 6 hours. The death of two out of three mice within 6 hours should be interpreted as the presence of YTX group toxins at levels above those established in the Regulation (EC) No 853/2004. Some water-soluble compounds may be extracted by aqueous MeOH, resulting in false positive (i.e. PSP toxins or even PLTXs). Therefore, if 2 or more mice are dead within 40 min, the assay should be considered inconclusive and must be followed up by further testing according to Commission Regulation (EC) No 2074/2005. REMARK: The application of this Annex: Protocol for the selective extraction of Yessotoxins (YTXs), does not correspond to the new regulatory limit of yessotoxins (Commission Regulation (EU) No 786/2013). False positives could be obtained due to the presence of YTXs below the regulatory limit. Page 13 of 14

14 Bibliography [1] Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin, Off. J. Eur. Communities, L139, , [2] Commission Regulation (EC) No 2074/2005 laying down implementing measures for certain products under Regulation (EC) No 853/2004 of the European Parliament and of the Council and for the organisation of official controls under Regulation (EC) No 854/2004 of the European Parliament and of the Council and Regulation (EC) No 882/2004 of the European Parliament and of the Council, derogating from Regulation (EC) No 852/2004 of the European Parliament and of the Council and amending Regulations (EC) No 853/2004 and (EC) No 854/2004, Off. J. Eur. Communities, L338, 27-59, [3] Yasumoto, T., M. Murata, Y. Oshima, G.K. Matsumoto, and J. Clardy Diarrhetic shellfish poisoning, p In Ragelis (ed.), Seafood Toxins. ACS Symposium Series 262. American Chemical Society, Washington, DC. [4] Fernández, M.L., A. Míguez, E. Cacho, A. Martínez, J. Diogene, and T. Yasumoto Bioensayos con mamíferos y ensayos bioquímicos y celulares para la detección de ficotoxinas, p In Sar, E.A, M.E Ferrario, and B.Reguera (eds.), Floraciones Algales Nocivas en el Cono Sur Americano. Instituto Español de Oceanografía, Pontevedra, Spain. Page 14 of 14