COMMISSION REGULATION (EU) No 15/2011  
of 10 January 2011  
amending Regulation (EC) No 2074/2005 as regards recognised testing methods for detecting 
marine biotoxins in live bivalve molluscs  
(Text with EEA relevance)  
THE EUROPEAN COMMISSION,  
Having regard to the Treaty on the Functioning of the European 
Union,  
Having regard to 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 (  1 ), and in 
particular Article 11(4) thereof,  
Having regard to Regulation (EC) No 854/2004 of the European 
Parliament and of the Council of 29 April 2004 laying down 
specific rules for the organisation of official controls on 
products of animal origin intended for human consumption (  2 ), 
and in particular Article 18(13)(a) thereof,  
Whereas:  
(1) Regulation (EC) No 854/2004 lays down specific rules 
for the organisation of official controls on products of 
animal origin and Regulation (EC) No 853/2004 lays 
down specific requirements concerning hygiene rules 
for food of animal origin. Implementing measures for 
those Regulations as regards recognised testing methods 
for marine biotoxins are set out in Commission Regu ­
lation (EC) No 2074/2005 of 5 December 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 Regu ­
lation (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 (  3 ). It is necessary to modify 
those implementing measures in the light of new 
scientific evidence.  
(2) In July 2006 the Commission requested the European 
Food Safety Authority (EFSA) to provide a scientific 
opinion to assess the current limits and methods of 
analysis with regard to human health for various 
marine biotoxins as established in the Community legis ­
lation, including new emerging toxins. The last of a series 
of opinions was published on 24 July 2009.  
(3) The mouse bioassay (MBA) and the rat bioassay (RBA) 
are the official methods for the detection of lipophilic 
biotoxins. The Panel on Contaminants in the Food Chain of EFSA noted that these bioassays have short ­
comings and are not considered an appropriate tool for 
control purposes because of the high variability in results, 
the insufficient detection capability and the limited 
specificity.  
(4) Recently developed alternatives to the biological methods 
for the determination of the marine biotoxins with lower 
limits of detection (LOD) have successfully been tested in 
prevalidation studies.  
(5) A liquid chromatography-mass spectrometry (LC-MS/MS) 
method was validated under the coordination of the 
European Union Reference Laboratory on marine 
biotoxins (EU-RL) in an inter-laboratory validation 
study carried out by the Member States. This method is 
publicly available for consultation in the web page of the 
EU-RL (http://www.aesan.msps.es/en/CRLMB/web/home. 
shtml). This validated technique of liquid chroma ­
tography (LC) mass spectrometry (MS) should be 
applied as the reference method for the detection of lipo ­
philic toxins and used as matter of routine, both for the 
purposes of official controls at any stage of the food 
chain and own-checks by food business operators.  
(6) Any other recognised method, different from the liquid 
chromatography (LC) mass spectrometry (MS), could be 
applied for the detection of lipophilic toxins provided 
that they fulfil the method performance criteria stipulated 
by the EU-RL. Such methods should be intra-laboratory 
validated and successfully tested under a recognised profi ­
ciency test scheme. If the results are challenged, the 
reference method shall be the EU-RL LC-MS/MS method.  
(7) To allow Member States to adapt their methods to the 
chemical method, the biological methods should 
continue to be used for a limited period of time. After 
this period, the biological methods should be used not as 
a matter of routine and only during the periodic moni ­
toring of production areas for detecting new or unknown 
marine toxins.  
(8) Therefore, Regulation (EC) No 2074/2005 should be 
amended accordingly.  
(9) The measures provided for in this Regulation are in 
accordance with the opinion of the Standing 
Committee on the Food Chain and Animal Health,EN 11.1.2011 Official Journal of the European Union L 6/3  
( 1 ) OJ L 139, 30.4.2004, p. 55.  
( 2 ) OJ L 139, 30.4.2004, p. 206.  
( 3 ) OJ L 338, 22.12.2005, p. 27.HAS ADOPTED THIS REGULATION:  
Article 1  
Annex III to Regulation (EC) No 2074/2005 is amended in 
accordance with the Annex to this Regulation.  Article 2  
This Regulation shall enter into force on the 20th day following 
its publication in the  Official Journal of the European Union . 
It shall apply from 1 July 2011.  
This Regulation shall be binding in its entirety and directly applicable in all Member States.  
Done at Brussels, 10 January 2011.  
For the Commission  
The President  
José Manuel BARROSOEN L 6/4 Official Journal of the European Union 11.1.2011ANNEX  
In Annex III to Regulation (EC) No 2074/2005, Chapter III is replaced by the following:  
‘CHAPTER III  
LIPOPHILIC TOXIN DETECTION METHODS  
A. Chemical methodology  
(1) The EU-RL LC-MS/MS method shall be the reference method for the detection of marine toxins as referred to in 
Chapter V(2)(c), (d) and (e) of Section VII of Annex III, to Regulation (EC) No 853/2004. This method shall 
determine at least the following compounds:  
— okadaic acid group toxins: OA, DTX1, DTX2, DTX3 including their esters,  
— pectenotoxins group toxins: PTX1 and PTX2,  
— yessotoxins group toxins: YTX, 45 OH YTX, homo YTX, and 45 OH homo YTX,  
— azaspiracids group toxins: AZA1, AZA2 and AZA3.  
(2) Total toxicity equivalence shall be calculated using toxicity equivalent factors (TEFs) as recommended by EFSA.  
(3) If new analogues of public health significance are discovered, they should be included in the analysis. Total toxicity 
equivalence shall be calculated using toxicity equivalent factors (TEFs) as recommended by EFSA.  
(4) Other methods, such as liquid chromatography (LC) mass spectrometry (MS) method, high-performance liquid 
chromatography (HPLC) with appropriate detection, immunoassays and functional assays, such as the phosphatase 
inhibition assay, can be used as alternatives or supplementary to the EU-RL LC-MS/MS method, provided that:  
(a) either alone or combined they can detect at least the analogues as identified in point A(1) of this Chapter; 
more appropriate criteria shall be defined when necessary;  
(b) they fulfil the method performance criteria stipulated by the EU-RL. Such methods should be intra-laboratory 
validated and successfully tested under a recognised proficiency test scheme. The EU-RL shall support activities 
toward inter-laboratory validation of the technique to allow for formal standardisation;  
(c) their implementation provides an equivalent level of public health protection.  
B. Biological methods  
(1) To allow Member States to adapt their methods to the LC-MS/MS method as defined in point A(1) of this Chapter, 
a series of mouse bioassay procedures, differing in the test portion (hepatopancreas or whole body) and in the 
solvents used for extraction and purification, may be still used until 31 December 2014 for detecting marine 
toxins as referred to in Chapter V(2)(c), (d) and (e) of Section VII of Annex III to Regulation (EC) No 853/2004.  
(2) Sensitivity and selectivity depend on the choice of solvents used for extraction and purification and 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.  
(3) A single mouse bioassay involving acetone extraction may be used to detect okadaic acid, dinophysistoxins, 
azaspiracids, pectenotoxins and yessotoxins. This assay may be supplemented, if necessary, with liquid/liquid 
partition steps with ethyl acetate/water or dichloromethane/water to remove potential interferences.  
(4) Three mice shall be used for each test. Where two out of three mice die within 24 hours of inoculation with an 
extract equivalent to 5 g hepatopancreas or 25 g whole body, this shall be considered a positive result for the 
presence of one or more toxins as referred to in Chapter V(2)(c), (d) and (e) of Section VII of Annex III to 
Regulation (EC) No 853/2004 at levels above those laid down.EN 11.1.2011 Official Journal of the European Union L 6/5(5) A mouse bioassay with acetone extraction followed by liquid/liquid partition with diethylether may be used to 
detect okadaic acid, dinophysistoxins, pectenotoxins and azaspiracids but it cannot be used to detect yessotoxins 
as losses of these toxins may take place during the partition step. Three mice shall be used for each test. Where 
two out of three mice die within 24 hours of inoculation with an extract equivalent to 5 g hepatopancreas or 25 g 
whole body, this shall be considered a positive result for the presence of okadaic acid, dinophysistoxins, pec- 
tenotoxins and azaspiracids at levels above those laid down in Chapter V(2)(c) and (e) of Section VII of Annex III 
to Regulation (EC) No 853/2004.  
(6) A rat bioassay may be used to detect okadaic acid, dinophysistoxins and azaspiracids. Three rats shall be used for 
each test. A diarrhetic response in any of the three rats shall be considered a positive result for the presence of 
okadaic acid, dinophysistoxins and azaspiracids at levels above those laid down in Chapter V(2)(c) and (e) of 
Section VII of Annex III to Regulation (EC) No 853/2004.  
C. After the period established in point B(1) of this Chapter, the mouse bioassay shall be used only during the periodic 
monitoring of production areas and relaying areas for detecting new or unknown marine toxins on the basis of the 
national control programmes elaborated by the Member States.’EN L 6/6 Official Journal of the European Union 11.1.2011