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11.1.2011 Official
Journal
of
the
European
Union
L
6/3
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,
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,
Having regard to the Treaty on the Functioning of the European
the insufficient detection capability and the limited
Union,
specificity.
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 (
(4) Recently
developed
alternatives
to
the
biological
methods

1 ), and in
particular Article 11(4) thereof,
for the determination of the marine biotoxins with lower
limits of detection (LOD) have successfully been tested in
prevalidation studies.
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 (
(5) A
liquid
chromatography-mass
spectrometry
(LC-MS/MS)

2 ),
and in particular Article 18(13)(a) thereof,
method was validated under the coordination of the
European Union Reference Laboratory on marine
biotoxins (EU-RL) in an inter-laboratory validation
Whereas:
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.
(1) Regulation
(EC)
No
854/2004
lays
down
specific
rules
shtml). This validated technique of liquid chroma
for the organisation of official controls on products of
tography (LC) mass spectrometry (MS) should be
animal origin and Regulation (EC) No 853/2004 lays
applied as the reference method for the detection of lipo
down specific requirements concerning hygiene rules
philic toxins and used as matter of routine, both for the
for food of animal origin. Implementing measures for
purposes of official controls at any stage of the food
those Regulations as regards recognised testing methods
chain and own-checks by food business operators.
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
(6) Any
other
recognised
method,
different
from
the
liquid
Parliament and of the Council and for the organisation
chromatography (LC) mass spectrometry (MS), could be
of official controls under Regulation (EC) No 854/2004
applied for the detection of lipophilic toxins provided
of the European Parliament and of the Council and Regu
that they fulfil the method performance criteria stipulated
lation (EC) No 882/2004 of the European Parliament and
by the EU-RL. Such methods should be intra-laboratory
of the Council, derogating from Regulation (EC) No
validated and successfully tested under a recognised profi
852/2004 of the European Parliament and of the
ciency test scheme. If the results are challenged, the
Council and amending Regulations (EC) No 853/2004
reference method shall be the EU-RL LC-MS/MS method.
and (EC) No 854/2004 ( 3 ). It is necessary to modify
those implementing measures in the light of new
scientific evidence.
(7) To
allow
Member
States
to
adapt
their
methods
to
the
chemical method, the biological methods should
(2) In
July
2006
the
Commission
requested
the
European
continue to be used for a limited period of time. After
Food Safety Authority (EFSA) to provide a scientific
this period, the biological methods should be used not as
opinion to assess the current limits and methods of
a matter of routine and only during the periodic moni
analysis with regard to human health for various
toring of production areas for detecting new or unknown
marine biotoxins as established in the Community legis
marine toxins.
lation, including new emerging toxins. The last of a series
of opinions was published on 24 July 2009.
(8) Therefore,
Regulation
(EC)
No
2074/2005
should
be
(3) The
mouse
bioassay
(MBA)
and
the
rat
bioassay
(RBA)
amended accordingly.
are the official methods for the detection of lipophilic
biotoxins. The Panel on Contaminants in the Food
(9) The
measures
provided
for
in
this
Regulation
are
in
( 1 ) OJ L 139, 30.4.2004, p. 55.
( 2 ) OJ L 139, 30.4.2004, p. 206.
accordance with the opinion of the Standing
( 3 ) OJ L 338, 22.12.2005, p. 27.
Committee on the Food Chain and Animal Health,

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L 6/4
EN
Official Journal of the European Union
11.1.2011
HAS ADOPTED THIS REGULATION:
Article 2
Article 1
This Regulation shall enter into force on the 20th day following
its publication in the Official Journal of the European Union.
Annex III to Regulation (EC) No 2074/2005 is amended in
accordance with the Annex to this Regulation.
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 BARROSO

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EN
11.1.2011 Official
Journal
of
the
European
Union
L
6/5
ANNEX
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.

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L 6/6
EN
Official Journal of the European Union
11.1.2011
(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.’

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