EU Regulations on the Traceability and Detection of GMOs: Difficulties in Interpretation, Implementation and Compliance

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DOI: 10.1079/PAVSNNR20072077
  • 34.53 · French National Institute for Agricultural Research
  • 32.53 · French National Institute for Agricultural Research
Europe has probably the strictest GMO regulation in the world. Its objectives are to give max-imum protection of public health and the environment, while at the same time providing a science-based regulatory structure where biotechnology can flourish. In contrast to the situation in the USA, European opinion on the health and environmental biosafety of GMOs has been highly polarized, with the result that the public has expressed the desire of having an informed choice in what they are eating. Consequently, the European Union has introduced legislation on the trace-ability and detection of GMOs, including labelling of food and feed containing GMOs, or derived products thereof, above a defined threshold of fortuitous presence. This review article sum-marizes EC regulations, directives and recommendations on traceability and labelling, and dis-cusses the practical problems involved in their implementation. These include the definition of the labelling threshold and the units of measure, sampling of large cargos, mixtures of GMOs, stacked genes, unauthorized GMOs, unknown GMOs and asynchronous approval. The ways in which the EC integrated project Co-Extra is contributing to the resolution of these problems are also discussed.
EU regulations on the traceability and detection of GMOs: difficulties
in interpretation, implementation and compliance
John Davison* and Yves Bertheau
Address: Institut National de la Recherche Agronomique (INRA), F-78026, Versailles, France.
*Correspondence: John Davison. Email:
Received: 6 July 2007
Accepted: 2 October 2007
doi: 10.1079/PAVSNNR20072077
The electronic version of this article is the definitive one. It is located here:
CAB International 2007 (Online ISSN 1749-8848)
Europe has probably the strictest GMO regulation in the world. Its objectives are to give max-
imum protection of public health and the environment, while at the same time providing a science-
based regulatory structure where biotechnology can flourish. In contrast to the situation in the
USA, European opinion on the health and environmental biosafety of GMOs has been highly
polarized, with the result that the public has expressed the desire of having an informed choice in
what they are eating. Consequently, the European Union has introduced legislation on the trace-
ability and detection of GMOs, including labelling of food and feed containing GMOs, or derived
products thereof, above a defined threshold of fortuitous presence. This review article sum-
marizes EC regulations, directives and recommendations on traceability and labelling, and dis-
cusses the practical problems involved in their implementation. These include the definition of the
labelling threshold and the units of measure, sampling of large cargos, mixtures of GMOs, stacked
genes, unauthorized GMOs, unknown GMOs and asynchronous approval. The ways in which the
EC integrated project Co-Extra is contributing to the resolution of these problems are also
Keywords: Genetically engineered organisms, DNA amplification, EC regulations, GMO detection,
Traceability, Labelling, Adventitious presence, Biosafety
Review Methodology: We searched the following databases: CAB Abstracts and Medline. In addition, we used the references from
the articles obtained by this method to check for additional relevant material. For regulations and their interpretation, we extensively
accessed the web sites of EC Europa, the Belgian Biosafety Database, the Joint Research Center (JRC), the Community Reference
Laboratory (CRL), the European Network of GMO Laboratories (ENGL), the Convention on Biological Diversity, the Official Journal of
the European Union Euro-lex, the US Department of Agriculture, the Codex Alimentarius and the EC integrated project Co-Extra.
Introductory Background
Genetic engineering in plants was discovered in 1983 by
three different groups, one in Belgium, and two in the
USA. Since that time, plant biotechnology has expanded
enormously and is now the basis of a major multinational
industry. Genetically modified plants for human con-
sumption or animal feed are mainly grown in the USA and
Canada, with increasing production in Brazil, Argentina
and China. Europe cultivates only a small amount of GM
crops (mainly GM maize grown in Spain), though this is
likely to increase in the future.
The expansion of GMOs in world agriculture, as well as
its economic aspects, possible advantages and risks, has
been well documented [1–14]. First-generation GM plants
offered economic advantages to the farmer (herbicide
tolerance, insect resistance and viral resistance), as well as
probable environmental benefits (decreased herbicide and
insecticide utilization (resulting in decreased pollution))
and reduced agricultural tillage (leading to less soil ero-
sion), while facilitating a more manageable daily life for the
farmer (easier planning of pesticide applications and
reduced pesticide exposure among farm-workers). The
first-generation GM plants still comprise more than 98%
CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2007 2, No. 077
of world GMO harvests. Second-generation GM plants
are being developed that should offer advantages to the
consumer, such as enhanced food quality, and increased
vitamin and trace-metal content. Third-generation GM
plants may provide better cellulose for paper-making
and reduction of polluting lignin treatments; drought
and salt tolerance to resist global warming and to feed
growing world populations; and enhanced biofuel pro-
duction or quality. Another category, still largely in the
experimental phase, comprises GM plants able to produce
pharmaceuticals (so-called biopharming) e.g. blood and
hormone peptides, proteins, antibodies, antigens and
vaccines. It should be noted that such plants will not
usually be suitable for human consumption and that
separation of supply chains will be more necessary. In
this sense, the current work on the coexistence, separa-
tion of GM and non-GM supply chains and traceability, as
performed by the EC integrated project Co-Extra
(coexistence and traceability in the GM and non-GM
supply chains), provides a good training and validation tool
of the quality assurance systems of biotechnology com-
GMO Legislation
Worldwide regulations on GMOs can mostly be divided
into two completely different types. The first, typified by
the USA, relies on the principle of substantial equivalence
and asks the question whether, or not, GMO-derived
products are substantially the same as their generally
accepted non-GMO counterparts. If this is judged to be
so, then little further regulation, other than the food
safety requirement, is required. On the other hand, GM
products that are clearly not substantially equivalent,
such as high oleic-acid GM canola, or vitamin A-enhanced
GM rice, would require labelling. Repeated escapes of
non-approved GMOs (Bt10 corn (maize), StarLink corn,
LLRICE 601 (Liberty Link) and Chinese rice Shyaniou 63)
may result in changes in the present USA GMO regula-
tions. The second viewpoint, initiated by Europe, con-
centrates on the method of production, arguing that
GMOs are produced by a different technology or pro-
duction process, and thus require special regulation.
Extensive EU legislation, including GMO detection, trace-
ability and labelling, has been introduced to support this
viewpoint. More than 40 countries (including EU member
states (MS)) have introduced traceability and labelling
regulations [15–17]. These mainly follow either the EU
or USA regulatory patterns and may differ according to
whether labelling is mandatory or voluntary, and to
whether there is a tolerated threshold for the adventi-
tious presence of GMOs.
EU Legislation
While GM foods are more or less accepted in the USA
[18], European consumers have shown considerable
reluctance to accept them. Part of the reason is because
of a number of food safety scares (‘mad-cow disease’ and
the related Creuzfeld–Jacob syndrome, foot and mouth
disease in livestock, Listeria in refrigerated products, Sal-
monella in eggs, dioxin in chickens, milk and meat, radio-
activity from Chernobyl, and, more recently, ‘bird flu’),
has left the general public distrustful of the food safety
records of European governments and companies, and
thus easily influenced by the non-governmental anti-GMO
organizations (despite the fact that GMOs have nothing in
common with these issues). Moreover, the position of
retailers, who refuse to market GM products, has rein-
forced the consumers’ fears (although, at the same time,
the retailers cite consumer rejection to explain their ban).
The anti-science attitude of NGOs, retail companies and
many politicians, as well as its effect on European com-
petitivity, was recently severely criticized by the EU Trade
Commissioner, Peter Mandelson [19].
In order to reassure the European public on food
safety and more particularly the question of GMOs, the
European Community has developed a series of regula-
tions (Table 1) [20–33] to ensure GM safety, detection
and traceability and labelling. For general traceability of
GMO food and feed, the most important regulations
are Regulation (EC) 258/97, labelling of new foods
and new ingredients; Directive 2001/18/EEC, deliberate
release and commercialization; Regulation (EC) 178/
2002, creation of the European Food Safety Authority
(EFSA) [34] and a general obligation for traceability;
Regulation (EC) 1829/2003, commercialization of GMO
food and feed (labelling thresholds and GMO detection
methods); and Regulation (EC) 1830/2003, specific trace-
ability and labelling requirement for GMOs. Regulation
(EC) 1946/2003 is concerned with the trans-boundary
movement of living modified organisms (LMO) under
the Cartagena Protocol on Biosafety, of which the EU is a
Food safety assessment is the responsibility of EFSA and
covers food additives, animal welfare, plant health, aller-
gies, mycotoxins, biological hazards, chemical and biolo-
gical contaminants [35]. It also assesses the safety of
GMOs (seed, food, feed and derivatives). EFSA is an
independent scientific body providing advice on all aspects
of food safety, and a positive EFSA assessment is neces-
sary for authorization to place food on the European
market. Under Regulation (EC) 1829/2003, once a posi-
tive EFSA assessment has been obtained, and once vali-
dated GMO detection methods and certified reference
material (CRM) are available (both being provided by the
applicant company), the application is then sent by the EC.
On the basis of the opinion of EFSA, the EC drafts a
proposal for granting or refusing the authorization, which
it submits to the Section on GM Food and Feed of the
Standing Committee on the Food Chain and Animal
Health. If this Standing Committee accepts the proposal,
it is finally adopted by the EC. Otherwise, it is passed
on to the Council of Ministers, which has a time limit of
2 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
three months to reach a qualified majority for, or against,
the proposal. In the absence of such a decision (which
is frequently the case), the EC adopts the proposal. A
European rapid alert bulletin (RASFF) allows any stake-
holders to retrieve information from European enforce-
ment laboratories on food safety issues, including
detection of unapproved GMO [36]. The costs of com-
pliance with the EU approval procedure may be very high
[37], so that only very large international biotechnology
companies are able to afford it.
The operation of GMO food control systems (e.g.
detection, labelling and traceability methods; the subject
of this report) are not within EFSA’s remit, and remain
the responsibility of the EC, through the CRL, and the
Competent Authorities of individual MS. It should be
noted, since it is a source of frequent miscomprehension,
that EC traceability and labelling regulations are not
concerned with GMO safety, risk evaluation or risk
management, since food that does not have a positive
EFSA assessment does not reach the market. Traceability
data on food and feed, including GMOs, may serve,
however, to enable the recall of products from the
supermarkets in the case of unforeseen mishaps, such
as the accidental or deliberate contamination of food
chains. Traceability is a non-discriminatory and inex-
pensive requirement since most of the companies
already have quality assurance protocols in place and
since numerous analyses are routinely carried out for
multiple purposes, including establishing vitamin content.
Quality-assurance procedures offer several advantages to
the companies such as specific market niches, efficient
low-cost withdrawal of products and easier implementa-
tion of control procedures for future mandatory
requirements (e.g. traceability and labelling of allergens in
food and feed).
EC guidelines have recently been developed for the
coexistence of GMOs and non-GMOs in the food chain
[38, 39]. These guidelines will provide a basis for national
regulations by the relevant national Competent Autho-
rities, although several MS would prefer a European sys-
tem rather than individual national measures. To aid
national Competent Authorities, the EC has recently
created a new ‘co-existence bureau’ specific for coex-
istence issues, at JRC-IPTS, Seville, Spain.
At present, there is a lack of EC agreement on a
threshold for the fortuitous presence of GMOs in seeds
for planting, though such guidelines will be developed
in European countries. The current opinion among EU MS
varies from a total rejection of GMO in seeds, to the
permissive acceptance of 0.3–0.7% of GMO in seeds,
depending on the biology of the plant concerned, fol-
lowing a ‘gentlemen’s agreement’ between some MS
based on a previously published recommendation of the
Scientific Committee of Plants [40]. This lack of standar-
dization between MS, and even between competent
authorities within a MS, may clearly hamper the devel-
opment of national coexistence rules for fields, crops and
Table 1 European regulations governing the traceability and detection of GMO food and feed
Directive 90/220/EEC covers the notification for a deliberate release and of the placing on the market of GMOs. Directive
90/220/EEC was repealed by Directive 2001/18/EEC.
Directive 90/219/EEC covers the contained use of genetically modified organisms. Directive 90/219/EEC was modified
by Directive 98/81/EEC.
Directive 2001/18/EEC covers the deliberate release of GMOs in the environment (field trials and cultivation),
in the absence of specific containment measures. It also regulates commercialization (importation, processing and
transformation) of GMOs into industrial products.
Regulation (EC) 178/2002 resulted in the creation of EFSA and in a general obligation for traceability of at least one
step forwards and one step backwards in the food chain.
Regulation (EC) 1946/2003 is concerned with the trans-boundary movement, and accompanying documentation, for
LMOs destined for deliberate release, or for food and feed or for immediate processing, under the terms of the Cartagena
Protocol on Biosafety.
Regulation (EC) 1829/2003 covers mainly the commercialization of food and feed. It facilitates GMO detection by
obliging the providers of GMO plants to disclose methods for their detection (Regulation (EC) 1981/2006 provides for a
fee to be paid by the applicant to the CRL for this service). These methods are then verified and validated by the CRL/
JRC, with the support of the ENGL, before being made public. This regulation imposes labelling for authorized GMOs
above a threshold of 0.9%. Labelling is not required for conventional or organic food and feed containing the adventitious,
or technically unavoidable, presence of authorized GMOs at levels less than 0.9%. Unauthorized GMO are not permitted
entry in the EU, even at levels less that 0.9%.
Regulation (EC) 1830/2003 concerns the traceability and labelling of genetically modified organisms and the traceability
of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC. It imposes
a specific traceability requirement on GMOs, over and above that of the general traceability regulation 178/2002.
Traceability archives must be kept for five years.
Regulation (EC) 65/2004 establishes a system for the development and assignment of unique identifiers for genetically
modified organisms.
John Davison and Yves Bertheau 3
World Trade Organization GMO Dispute
Argentina, Canada and USA viewed the 1999–2003 EC de
facto moratorium on GMO authorizations as a trade
barrier that was based upon economic rather than sci-
entific reasons, and brought the case before the WTO.
The final WTO judgment (2006) was largely in favour of
the plaintiffs [41–43]. However, the European labelling
and traceability requirements were not questioned. It is
still too early to determine the effects of this judgment on
the EC attitude and upon GMO cultivation and importa-
tion into the EU.
Cartagena Protocol
The Cartagena protocol on biosafety seeks to protect
biological diversity from the potential risks (for example
by horizontal gene transfer), that could be posed by living
modified organisms (LMO) resulting from modern bio-
technology. National biosafety databases and lists of
experts must be established through the Biosafety
Clearing House.
In the Cartagena Protocol, a particularly important
section 18.2.a [44] defines the requirements and doc-
umentation for the trans-border movement of GMOs
intended for use as food and feed. It requires that ship-
ments of GMOs, whose identity is known through the
system of identity preservation [45–47] must be labelled
‘contains GMO’. In contrast, GMOs for which the identity
is not known by the system of identity preservation must
be labelled ‘may contain GMO’. This latter is a temporary
measure, until 2010, to permit member countries to put
in place the identity preservation system. Curiously, the
‘may contain’ label does not carry the obligation to list
species of GMOs other than those that constitute the
main shipment. From this it is clear that the Cartagena
Protocol article 18.2.a does not fulfil the EC import
requirements under Regulation (EC) 1829/2003. Con-
versely, most members of the Protocol refused any sys-
tem of labelling above a given threshold percentage of
adventitious GMO presence (as in place in Europe). Trace-
ability is an important part of the Cartagena protocol, as
exemplified by the application of Regulation (EC) 1946/
2003 for exports to third countries (though not for
internal movement within the EC). Naturally, however,
the EC reserves the right to apply Regulation (EC) 1829/
2003 thresholds to all shipments destined for Europe.
Some Latin American countries, such as Argentina, with
very large agricultural surfaces, and rapidly developing
identity preservation systems, are able to apply large
buffer zones, with the objective that the percentage of
adventitious presence of GMOs is extremely low (< 0.1%,
as requested by most of the customers). Such shipments
should have no problem in complying with EC regulations.
The Cartagena Protocol also includes a working party
on liability and redress, which, in the context of the
Protocol, concerns the question of the responsibility for
damage caused by the trans-boundary movement of
LMOs [48]. As witnessed by several GMO scandals
(StarLink, Bt10, LLRICE601 and Prodigene GM corn), the
possibility exists that non-GMO food, feed and seed may
become contaminated by GMO material, possibly as a
consequence of physical contamination during harvesting,
transport or processing, or by horizontal gene flow via
pollen. Such contamination may reduce the value of
conventional or organic products. Liability and redress
negotiations have proved difficult and are presently
without agreement. Legislation is currently in place
or under consideration in several countries, including
Denmark. Naturally, all liability and redress cases will rely
on analytical and documentary-based traceability meth-
ods. Co-Extra is currently studying the legal issues of
trans-borders gene flow and different liability and redress
Interpretation and Application of the GMO
The EU General Food Law Regulation 178/2002/EC
defines traceability, similarly to ISO standards, as ‘the
ability to trace and follow food, feed, food producing
animals and other substances intended to, or expected to,
be incorporated into food or feed, through all stages of
production, processing and distribution’. EC regulations
outline the general principles required for GMO
traceability, though specific details of implementation
(discussed below) may be lacking or unclear. A further
complication is that, while EC Regulations are binding on
MS, there is flexibility in the way in which EC Directives
are transposed into national law. One example is the
transposition of Directive 2001/18/EEC into French law,
which should have been transposed in 2002, but was
partly transposed (as a decree) only in 2007. The lack of
precise GMO field locations in the publicly available part
of the French national register of GMO cultures may
impede the use the of gene flow models developed by
research programmes such as Co-Extra. Similar problems
and national differences can be expected for national
coexistence laws, as observed by the different isolation
distances required by national regulations. Only a few
European countries have developed a legal frame of
coexistence (e.g. Denmark), while others (e.g. France) are
requesting EC guidance and standards.
Other problems arise between the EC and the MS on
GMO cultivation. For example, Austria, Greece, Hungary
and parts of Italy, referring to the safety clause, refuse the
cultivation of GMOs and are likely to be subjected to legal
suits by the EC. Germany recently proposed severe
restriction, virtually amounting to a ban, on MON810
corn. The European Court of Justice has recently
found the Austrian GMO ban to be illegal. In contrast, the
GMO ban in Hungary recently received support from
4 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
the EU Environment Council, in clear contradiction with
EC regulations and GMOs approvals. Finally, some new
MS are already growing illegal GMOs.
There is often a gap between regulations and their
interpretation and implementation, and GMO regulations
are no exception (see below). Numerous explanatory
texts and guidelines have been published, by the EC, and
others to aid in the understanding of EC regulations on
GMOs [49–60].
Traceability of GMOs
Traceability follows GMO products through all stages of
the production and distribution line, i.e. from farm to fork
and vice versa. Traceability starts with the company that
develops the GMO that has received EC authorization
and has provided the CRL with a quantitative identifica-
tion method. As GMOs can be approved in EU for several
purposes (e.g. for import and food transformation) it
implies that the resulting cultivars were previously
inscribed into the European register of cultivars, as for any
variety of crops. This company would then be obliged to
inform any European purchaser of the seed that it is
genetically modified. The company must also keep a reg-
ister of operators who have bought the seed. Next, the
farmer is required to inform any purchaser of the GM
nature of the crop and to maintain a register of operators
to whom the harvest was sold. National registers are also
mandatory but the level of information for stakeholders
may vary between EU countries. The process of pre-
validating and validating detection methods, as well as of
developing CRM, is a time-consuming system that drasti-
cally delays the EU approval system and thus increases the
worldwide asynchrony of approvals.
The current legislation covers all GMOs that have
received Community authorization for the placing on the
market, including food and feed, containing or consisting
of GMOs. Examples are GM seeds, and bulk quantities or
shipments of whole GM grain e.g. soybean and maize. The
regulations also cover food and feed products which are
derived from GMOs. This includes starch, highly refined
oil or flour produced from a GM maize or lecithin from
soybean or rapeseed. In some highly processed products,
the analyte (DNA or protein) may be undetectable, owing
to high purity of the product or to degradation during
processing, so that only paper trails may be used to trace
the origin of the products. This lack of analytical trace-
ability may leave open the opportunity of fraud, particu-
larly when there is a price difference between the GM and
non-GM products.
European regulations, particularly the horizontal
General Food Law (Regulation 178/02) and its hygiene
package, as well as regulations for new food and new
ingredients (Regulation 258/97) applying to GMOs,
represent the most stringent rules of traceability in the
world. A typical decision tree under EC regulations is
shown in Figure 1. Such regulations and related analytical
methods may serve as a model, be modified for other
safety purposes, such as the traceability of allergens or
toxin producing organisms [61].
The purpose of sampling is to obtain a sample repre-
sentative in characteristics and composition of the lot
from which it was taken, including unwanted ‘hidden’
contamination. Numerous sampling methods for e.g. oil-
seeds and cereals have been previously standardized
by either international organizations (e.g. ISO) or as-
sociations (e.g. AACC) or national enforcement admin-
istrations (e.g. USDA–GIPSA). Several reviews of the
sampling methods have been published [62–64]. The
GMO sampling procedure is particularly difficult and
costly when dealing with very large cargoes where
the potential GMO presence is neither homogeneous, nor
restricted to a single type. The standardization of sampling
plans has suffered from the different viewpoints of Europe
(defended by the CEN), and third countries (e.g. USA).
The Codex Alimentarius discussions are, however, still
underway and are of considerable importance to the
Cartagena protocol.
For covering the European legislation needs, the JRC
launched, several years ago, a research programme, Kelda
[65–67], and a software package, KeSTE [68], which were
taken into consideration in a European recommendation
[69]. However, this recommendation was not effectively
implemented following an attempt by MS at determining
the costs of such a sampling plan. In general, the cost
inhibits the use of adequate sampling plans, as long as
safety issues are not foreseen. Consensus sampling plans
are urgently required for GMO detection in international
trade, and the ability to use existing sampling plans and
samples (e.g. mycotoxins) would simplify and reduce costs
for the whole process.
Assay individual
No labelling
GMO quantification
Food or feed sample
Adventitious presence
Deliberate presence
Figure 1 A decision tree for labelling GMO food and feed
John Davison and Yves Bertheau 5
Detection of GMOs
Detection of GMOs may be performed by a variety of
methods. Bioassays may be used, for example with
application of herbicide on plantlets. Their ease of use,
minimal personnel training and low cost, makes bioassays
useful (e.g. it is used by the Association of Certified Seed
Certifying Agencies for seed certification). Near Infra Red
(NIR) has been tested, within Co-Extra, on soybean and
could distinguish GM and non-GM in a high percentage of
cases [70]. NIR does not directly detect GM analytes
(DNA or protein), but rather compositional and struc-
tural changes. It is of interest since it is non-invasive and
thus non-destructive, although it still requires consider-
able development. Protein-based immunological methods
may be used to determine the presence of a transgenic
protein in food and feed samples [71–74]. However,
protein-based methods do not permit the low levels of
detection required under EC regulations (i.e. with LOD
better than one seed among 1000), except when used in
combination with multiple control plans by attributes
(also called sub-sampling). They also perform poorly
with processed products. However, even for raw mate-
rial, such as kernel, most of the problems with protein-
based detection arise from the varying protein content
(e.g. between transformation events, cultivars and also
due to environmental influence). They are thus most
useful for preliminary on-site screening of growing crops
and harvested samples when only a limited number of
traits are concerned (e.g. Roundup Ready
soybean in
USA and Argentinean Identity Preservation systems). A
recent report [75] describes the use of fluorescent beads
coupled to specific antibodies, for high throughput, high
sensitivity detection of GM proteins. A special case of a
non-immunological traceability method is the expression
of the glucuronidase gene in Hawaiian papaya, which
enables the rapid detection of fresh transgenic papaya
(fruit, leaves or roots).
At present, all CRL-validated GMO detection methods
are based on DNA analysis using quantitative polymerase
chain reaction (qPCR) [61] (next section), which permits
the required sensitivity and reproducibility of quantitative
Real-time qPCR
All GMO detection methods, considered by ENGL as
complying with EC regulations and with its performance
criteria, use the real-time qPCR. These methods and their
validation procedures have been discussed in detail [76–
86]. All validated methods are listed on the CRL web site
[87, 88], and mostly use TaqMan
chemistry from
Applied Biosystems.
The EC project Co-Extra [89] investigates alternative
chemistries and apparatus and emphasizes time and cost
reduction so as not to unduly increase the total cost of
the food and feed products being analysed. While multi-
plex qPCR has the potential of better economy by
simultaneous processing of multiple primers, it is difficult
to optimize, given the overlap in the spectra of the dif-
ferent fluorophores that are used to label the TaqMan
or MGB probes. One alternative multiplex method
separates PCR fragments according to size, by capillary
electrophoreses, and identifies them by using differently
coloured fluorescent labels [90]. Multiplex qualitative
PCR, which is more easily developed, might be used with
multiple control plans by attributes for lowering the costs,
though these provide statistical probabilities, rather than
precise quantitative data. Moreover, multiplex qualitative
PCR reactions are useful for initial screening of samples,
before the identification and quantification of the GMOs
The modular approach, recently retained at the
European level, suggests ways of independently develop-
ing and validating analytical modules and calculating
the measurement uncertainty of the several combined
modules such as DNA extraction, reference genes
and GMO PCR tests and calibration [91–93]. Standardi-
zation of different simplex detection methods, and
their use in duplex or multiplex reactions, is necessary
to improve time and cost efficiency for their use,
and accreditation in analytical laboratories. Guidelines
for multiplex qPCR development and optimization
are thus actively being developed Co-Extra, ENGL and
In addition to the use of adequate sampling plans
to minimize sampling uncertainty (discussed above),
measurement uncertainty because of a variety of factors
(technical manipulator variability, local environmental
and accommodation conditions, equipment variability
and calibrations methods) may lead to variations
within, and between, laboratories participating in valida-
tions. Statistical methods allow correction for the opti-
mization of the calibration curve and the weight of
outlying points, leading to improved trueness and preci-
sion [94]. The measurement of various dilutions of the
sample also reduces measurement uncertainty. Reliable
inter-laboratory results are dependent on methods
comparisons, evaluation validation and harmonization.
Uncertainty in the measured value may lead to costly
disputes, particularly when very large lots sizes, such as
cargoes, are involved. For that purpose, a Codex Ali-
mentarius consensus document should propose guidelines
for solving disputes. The concepts of limit of detection
(LOD) and limit of quantification (LOQ) are important. In
practice, the absolute limit of detection (LODA) in a
qPCR reaction is about five copies, while the absolute
limit of quantification (LOQA) is approximately 100
copies. Given the lack of availability of CRM below 0.1%,
the relative LOD (LODr) and LOQ (LOQr) are deter-
mined only down to this value, which is not sufficient
compared with what is requested as seed threshold
(0.1%) by some MS.
6 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
The GMO detection research in Co-Extra investigates
new methods, generally qualitative, such as microarrays
with hybridization, and SNPlex designed for multiple
target detection. A robust statistics-based approach of
multiple control plans by attributes [95, 96] would allow
the use of these methods to be used with the free Co-
Extra software OPCASA [97] for improving accuracy and
time/cost-effectiveness. The inter-laboratory validation
process of such methods is currently under study.
Methods of Detection and their Validation
To obtain the EU approval for a GM plant, it is necessary
to establish and validate a detection specific protocol.
Under regulation (EC) 1829/2003, the method of detec-
tion must be supplied by the applicant and validated by the
CRL–JRC with the support of ENGL [98], according to a
collaborative trial (ISO 5725) using the modular approach,
before being made public in the JRC database [99]. This
regulation introduced a great procedural change since
prior to its introduction, the methods of GMO detection
were developed and validated from research pro-
grammes, or by enforcement laboratories, without the
collaboration of the applicant. The new procedure also
made mandatory the payment by the notifying companies
of the pre-validation and validation costs. CRL and ENGL
provide a detailed list of minimal requirements for GMO
detection, to the applicants [80]. The applicant must then
comply by submitting a detailed protocol which is then
analysed by the CRL. The detection methods are made
public, on the CRL web site, only after CRL validation.
The CRL-JRC web site also gives reference to the current
status of all dossiers, as well as the opinions of the EFSA
panel on GMOs and decisions of the EC.
A difficulty with the detection methods provided by
applicants is that several companies can provide different
detection methods for a same transformation event, when
used, for example, as single GMO by the one company,
and as stacked gene by under licence to another company.
The same applies for reference genes (discussed below),
which might differ between, and even within, companies.
For instance, five different reference genes are used for
corn, even though ENGL and CRL showed a preference
for the Adh reference gene developed by INRA [100].
Recommendations of preferred detection methods are
thus expected to standardize routine analyses and
decrease the costs of adaptation and accreditation.
Another weakness of the current European regulations
is the lack of consideration of screening methods and of
negative controls of donor organisms (e.g. CaMV for
P35S), which could result in time/cost gains. In the Co-
Extra project, these have been independently developed
and should be validated.
Each GMO detection protocol also requires the inclu-
sion of CRM provided by the JRC IRRM via their on-line
catalogue [101]. A CRM contains a mixture of GMO and
non-GMO ground seeds, in various certified proportions,
and is necessary for the quantification of the amount of
GMO in the experimental samples, in terms of the mass/
mass percentage (however, for difficulties associated with
lack of commutability, see next section).
There has been much discussion on the possible
application of alternative reference material such as
plasmid-reference materials, to replace CRM. Plasmids
have the advantages of low cost, stability and continuity.
Researchers in Japan, Korea, Taiwan and Europe [102–
109] have elaborated plasmid-based methods, with a
conversion factor to compare with mass/mass-based
CRM. In the EC, plasmid-based reference materials are
only at the experimental stage, though low-cost plasmid
reference materials are now being commercialized, while
DNA certification of mass/mass CRM is underway under
the auspices of the IRMM. It is probable that plasmid
reference material will be preferred in the future, since
the availability of plant-derived CRM standards is limited
in time (e.g. Bt176 is almost no longer sold, while StarLink
has been withdrawn and is no longer available to make
Interpretation of EU Threshold Levels For the
Adventitious Presence of GMOs
Regulation (EC) 1829/2003 calls for the labelling of food
and feed products that intentionally contain authorized
GMO, or have an adventitious presence of authorized
GMOs, above a threshold of 0.9%. The term ‘adventitious
presence’ is not defined in Regulation (EC) 1829/2003
but refers to the accidental and technically unavoidable
presence of GMOs in otherwise non-GMO food and
feed [110, 111]. Every time that a non-labelled product
contains authorized GMOs, but at a level below 0.9%,
evidence must be provided that such adventitious pre-
sence could not have been avoided by routine food
and feed chain procedures (Figure 1). Above the 0.9%
threshold the product must be labelled. In contrast,
unapproved GMOs are not permitted at any level.
Furthermore, the regulation does not specify the units
of measure to be used for calculating the value of 0.9%,
and this has given rise to serious, and still unresolved,
problems of interpretation, as addressed in ENGL meet-
ings and documents. Seed producers work in terms of
number of seeds, while farmers, transporters, processors
and regulators work in terms of mass/mass ratios of GMO
to non-GMO. In contrast, the scientific measurements in
enforcement labs are, de facto, made in DNA/DNA ratios
which are then converted to mass/mass using CRM (see
While the DNA-based measurements can be accurately
determined, for a variety of different biological reasons
the conversion from DNA ratios to mass ratios has
numerous difficulties of interpretation. The EC recom-
mendation 2004/787/2000 proposes that GM copy
John Davison and Yves Bertheau 7
numbers should be expressed as the percentage, in rela-
tion to taxon-specific gene target DNA copy numbers,
calculated in terms of haploid genomes. However, this
recommendation gives rise to new difficulties that may
have non-negligible effects on the estimation of the per-
centage of GMO in a given sample. These problems have
been discussed in depth [112–115] and are based on the
non-evident relationship between DNA content and
plant mass. Firstly, there may be a non-uniform nuclear
DNA content between different cultivars of the same GM
insert. Secondly, the same plant line grown under different
environmental and agricultural conditions may have dif-
ferent DNA/mass ratios. Thirdly, different parts of a plant
may have different ploidy (e.g. the endosperm of maize is
triploid). Finally, transgenic plants that are homozygous
diploids or hemizygous diploids have DNA percentages of
100 and 50% respectively, as expressed in haploid genome
equivalents. Recommendations to improve the coherence
of the legislation with real-life practical methods of GMO
detection have recently been proposed at the ENGL level.
The DNA certification, through inter-laboratory testing
of the current CRM by ENGL laboratories and IRMM,
should also provide experimental data and insights on
these issues.
Mixtures of GMOs
Regulation (EC) 1829/2003 exempts, from labelling, food
and feed products that contain the adventitious presence
of authorized GMOs below a threshold of 0.9%. When
the sample contains a mixture of GMOs, each ingredient
(analytically translated as content per taxon) may not
exceed 0.9%, irrespective of its proportion in the final
product. A mixture of GMOs is difficult, and costly, to
detect unless there is some indication of which GMOs
may be present in the mixture. It is also difficult to dif-
ferentiate, using the current qPCR methods, a sample
containing a mixture of GMOs from one containing the
same genetic constructs present as stacked genes (next
It should be noted that because of the methods of
calculation of GMO content, numerous stakeholders
request regulations giving more tolerance on the for-
tuitous presence of trace botanical impurities (an issue
currently surveyed within Co-Extra). For example, there
may be adventitious traces of approved GMO soybean in
non-GMO corn feed, which in principle does not contain
soybean. If the fortuitous soybean is 100% GMO, then the
level of that constituent is 100%, which obviously exceeds
the 0.9% (per ingredient) limit, and thus must be labelled.
However, labelling would reduce its market value. The
feed companies may be tempted to add quite large
quantities of non-GM soybean (in this example) to be sure
that the GMO soybean content in will be less than 0.9% in
the final product. Such a practice is costly, though not
illegal. This problem is likely to get worse since the
number of commercially grown GMO taxa, and thus the
possibility of cross-contamination, is increasing.
Stacked GMOs
Stacked GMOs contain two, or more, transgenic con-
structs specifying two, or more, traits (e.g. herbicide
tolerance and insect resistance) and are usually obtained
by genetic crosses between the parent plants to give
hybrids [116, 117]. Double and triple stacked GMO plants
are currently grown in several countries, and eight-
stacked GMOs are under development. In Europe,
stacked GMOs are not authorized for cultivation, but
nonetheless, feed produced from stacked GMOs can be
imported. An EFSA guidance document giving the
requirements for safety approval, under Regulation (EC)
1929/2003, of GMO plants containing stacked transfor-
mation events has recently been published [118].
Using currently available PCR-based detection meth-
ods, mixtures GMOs and stacked genes containing the
same events are difficult to differentiate in a cost-effective
manner. For this reason, the CRL pragmatically accepts
methods able to separately detect each transformation
event. One analytical approach for detecting stacked
genes performs the PCR reaction on individual kernels,
though this is very costly [119]. Several methods using
PCR associated with statistical analyses using multiple
control plans by attributes are currently being tested
in the Co-Extra project. For qualitative PCR, multiple
control plans by attributes allow rapid determination of
the probability of presence of stacked genes. Similarly,
the additional information acquired by the use of qPCR
provides strong statistical arguments for suspecting the
presence for stacked genes. New mathematical models
are currently being tested. In all cases, decision-support
systems would largely help the decision-taker in such a
‘probabilistic environment’.
Unauthorized GMOs
As stated above, Regulation (EC) 1829/2003 focuses on
the ability to uniquely identify authorized GMOs. In
practice, the detection method is usually performed using
an event-specific PCR assay, which detects the unique site
of insertion of the transgene, and must be provided by
the applicant, and verified, and validated by the CRL
and ENGL. From this it follows that this information is
available only for authorized GMOs, and that only
authorized GMOs can be identified by CRL validated
methods. Regulation (EC) 1829/2003 does not, and can-
not, apply to GMOs not authorized in the EU, since there
is no legal basis for requiring this information from the
biotechnology company, who may have no intention of
commercializing these GMOs in Europe, and who may
consider information on such constructs to be part of
8 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
their intellectual property portfolio. In the cases of con-
tamination by Bt10 (Syngenta) [120] and LLRICE601
(Bayer Crop Science) [121] in shipments destined for
Europe, the CRL was able to rapidly put in place the
appropriate tests because of the cooperation of the
companies concerned. Unauthorized GMOs are not
permitted at any level in Europe, though APHIS/USDA
[122], in close collaboration with the FDA and EPA, adopt
a more tolerant attitude, provided that the GMO poses
no evident risk to health or environment, and it carries
transgenes similar to those already approved. Given the
very large number of experimental GMOs in the pipeline,
it is difficult to predict whether the problem of con-
tamination and detection of unauthorized GMOs can be
expected to become more difficult in the future, or
whether increased awareness and compliance with EC
regulations will reduce such costly incidents. A recent
study by the EC DGAgri [123] simulated various scenarios
that might arise from the low-level presence of unau-
thorized GMOs in feed destined for Europe. The most
severe result could be a shortage of feed coming from the
USA and South America, with consequent price increase
in animal products in the EU.
Unknown GMOs
Unauthorized GMOs may be either known or unknown,
depending on whether an adequate dossier containing the
complete description is available. Known, but unauthor-
ized, GMOs may be detected by using the same methods
as for authorized GMOs (previous section). Unknown
GMOs, by definition, are unauthorized. However, also by
definition, they cannot be detected by current PCR-based
techniques since the target sequences are unknown. Some
methods, under study in the Co-Extra project (matrix
approach, using e.g. DNA chip hybridization or multiplex
PCR and CGE, qualitative and quantitative differential
PCR, fingerprinting, DNA chip hybridization), attempt to
determine the probability of presence of unknown GMOs
by supposing the presence of commonly used genetic
constructs, or the residual sequences from the plasmid
vector. However, it is theoretically possible to construct a
transgenic plant that contains no previously used trans-
genic sequence (though such a scenario seems unlikely
due to the developmental work required), and such a
plant would be undetectable by present methods. A the-
oretical bioinformatics approach to this problem has
recently been published [124].
Asynchronous Approval of GMOs
A major current problem, which complicates GMO trace-
ability and detection efforts, is asynchronous author-
ization whereby a given GMO may be authorized (legal) in
one country but not authorized (illegal) in another. It thus
follows that even a very slight contamination by a GMO
authorized in one country may make the shipment illegal
in the other country, where it would be rejected at the
port of entry by the Competent Authorities. The issue of
asynchronous authorizations, and its related problems,
can only be resolved at the international level and the
USA is currently proposing discussions under the frame-
work of the Codex Alimentarius.
Future Developments
Thanks to the recent European regulation (EC) 1829/
2003, requiring the provision of a GMO detection
method by the applicant, the European research labora-
tories were able to focus their attention on cost- and
time-effectiveness, and on more prospective work such
as the detection of unknown and/or stacked GMOs, and
the development of new detection methods. The good
interaction between the research laboratories, national
enforcement laboratories, ENGL and the EC allowed
improvements in the EU regulations. The growing devel-
opment of qualitative methods such as multiplex PCR,
microarrays and SNPlex will probably induce some
changes in the necessary expertise of the analytical
laboratories charged with GMO detection. More efficient
documentary traceability will clearly decrease the work-
load of control laboratories and reduce the stakeholders’
costs. The future direction of the GMO detection
laboratories will probably be towards the use of different
detection methods, chemistries and apparatus, with an
increasing part of controls on traceability documents by
the national Competent Authorities.
Traceability and detection of GMOs represent a growing
public request in the EU and other countries, including the
USA. In Europe, the application of PCR detection meth-
ods to whole supply chains, with very different matrices,
initially produced difficulties due to lack of prior experi-
mentation and lack of feedback. However, in recent years,
regulation was improved through strong interaction
between research labs and the Competent Authorities,
particularly through ENGL. Most methods of GMO
detection involve qPCR and protocols must be provided
to the CRL by the applicant. These methods are then
validated by the CRL with the support of ENGL, and only
then made public. Some problems still remain however,
which cause difficulties in the application and imple-
mentation of EC and regulations. As in other areas of
detection (mycotoxins, allergens, pathogens and quar-
antine), the best sampling method for large cargoes, with a
heterogeneous distribution of GMOs, remains an un-
resolved and expensive problem. Difficulties remain in the
conversion of qPCR DNA ratios to the mass/mass ratios
John Davison and Yves Bertheau 9
needed by seed producers, farmers, processors and reg-
ulatory authorities, and are still under discussion. There
are problems in differentiating simple mixtures of GMO
material from those containing stacked transgenes. Simi-
larly, unknown transgenic plants cannot be detected due
to the absence of a known PCR target sequence. Within
the EC Co-Extra programme, a variety of new improve-
ments have been introduced with the development of the
modular approach, the matrix approach and quantitative
differential PCR, which are applicable to other detection
areas. Current experimental focus is on transposition of
alternative chemistries and apparatus, new detection
methods and strategies, to reduce cost and duration and
increase accuracy and effectiveness. The EC Co-Extra
project devotes considerable effort to the dissemination
of results, to the involvement of stakeholders, and to legal
aspects of GMO traceability, such as liability and redress.
Co-Extra is also currently addressing documentary trace-
ability and decision trees and support systems, as well as
the information flow management.
Part of this work was funded by the EC FP6 research
project Co-Extra (contract 007 158) and by the EC FP6
research project PETER (contract number 031 717).
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14 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
    • "This invitation resulted in the adoption of Regulation 1829/2003 on the authorization procedure for genetically modified food and feed, and Regulation 1830/2003 concerning the traceability and labelling of GMOs and the traceability of food and feed products produced from GMOs. Even more importantly, Regulation 1946/2003 was explicitly designed to implement the provisions of the Cartagena Protocol on preventing biotechnological risks (Davison and Bertheau 2007 "
    [Show abstract] [Hide abstract] ABSTRACT: The literature on international regulatory regimes has highlighted how rival standards can create different points of convergence. Scholarly attention has also focused on how the European Union (EU) and the United States (USA) attempt to 'export' their environmental standards internationally. Here, we explore the effectiveness of these attempts by means of third states' decisions to ratify the Carta-gena Protocol on Biosafety to the Convention on Biological Diversity, a multilateral environmental agreement regulating genetically modified organisms that is promoted by the EU but opposed by the USA. Our findings confirm that both rivals are able to influence the ratification decision of states, but they also suggest that these effects may have different origins. Countries relying more heavily on US markets for food exports tend to be less likely to ratify the Cartagena Protocol, while countries that have applied for EU membership are more likely to ratify the protocol.
    Full-text · Article · May 2015
    • "The proposed regulation amends Directive 2001/18/EC to allow Member States to restrict or prohibit the cultivation of GMOs in their territory. Source: adapted from Vicario, 2010, Davison and Bertheau, 2007 and Europa, 2008b. As of January 18, 2011, the EU Register of GM Food and Feed lists 38 GM authorized products for food and/or feed use: 6 cotton varietals, 22 maize, 1 bacterial biomass, 1 yeast biomass, 3 varieties of rapeseed, 1 potato, 3 soybean types and 1 sugar beet (EC, n.d.b). "
    [Show abstract] [Hide abstract] ABSTRACT: Economies of scale are an alternative source of growth particularly at a time when countries are suffering from global economic malaise. The proposed EU-India free trade agreement holds substantial promise as this will create a combined market of over one and a half billion and generate economies of scale from intra-industry trade, which are likely to be concentrated in manufactured products such as chemicals, machinery and transport equipment. Bold action is needed on the part of politicians in both the EU and India to successfully negotiate the agreement given that this will enable both countries to reap the efficiency gains of global economies of scale, provide a significant competitive advantage over other major economies and deliver the necessary spur to shake both the EU and India out of their current economic stagnation.
    Full-text · Article · Mar 2014
    • "Labeling requires the detection and quantification of the GE food/feed or derived product in the tested food/feed or seeds or any other product when applicable. The scientific literature compiled about traceability largely deals with the following issues: (a) sampling procedures – there are no universally acknowledged sampling procedures (Davison & Bertheau, 2007); this has been the object of a EU funded research programme (Paoletti et al., 2006); "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract The technology to produce genetically engineered (GE) plants is celebrating its 30th anniversary and one of the major achievements has been the development of GE crops. The safety of GE crops is crucial for their adoption and has been the object of intense research work often ignored in the public debate. We have reviewed the scientific literature on GE crop safety during the last 10 years, built a classified and manageable list of scientific papers, and analyzed the distribution and composition of the published literature. We selected original research papers, reviews, relevant opinions and reports addressing all the major issues that emerged in the debate on GE crops, trying to catch the scientific consensus that has matured since GE plants became widely cultivated worldwide. The scientific research conducted so far has not detected any significant hazards directly connected with the use of GE crops; however, the debate is still intense. An improvement in the efficacy of scientific communication could have a significant impact on the future of agricultural GE. Our collection of scientific records is available to researchers, communicators and teachers at all levels to help create an informed, balanced public perception on the important issue of GE use in agriculture.
    Full-text · Article · Sep 2013
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