BioRisk 8: 89-I | 0 (20 | 3) Apeer-reviewed open-access journal

doi: 10.3897/biorisk.8.4035 RESEARCH ARTICLE & B | O R IS k

www.pensoftonline.net/biorisk

How to track genetically modified (GM) plants in the
field? The VDI standard method of floristic mapping
of GM plants as an efficient tool

Ulrich Sukopp', UIf Schmitz?

| Federal Agency for Nature Conservation, Division II 1.3 — Monitoring, Konstantinstr. 110, 53179 Bonn,
Germany 2 Institute of Botany III, Universitatsstr. 1, 40225 Diisseldorf, Germany

Corresponding author: Ulrich Sukopp (ulrich.sukopp@bfn.de)

Academic editor: /. Settele | Received 25 September 2012 | Accepted 11 March 2013 | Published 8 August 2013

Citation: Sukopp U, Schmitz U (2013) How to track genetically modified (GM) plants in the field? The VDI standard
method of floristic mapping of GM plants as an efficient tool. BioRisk 8: 89-110. doi: 10.3897/biorisk.8.4035

Abstract

The commercial use of genetically modified (GM) organisms is regulated in the EU by law. Thus, moni-
toring the environmental effects of GM organisms after placement on the market is a mandatory task of
the respective consent holder. Since many relevant monitoring procedures lack standardisation, the Asso-
ciation of German Engineers (VDI) has commissioned expert groups with the development of guidelines
covering appropriate methodologies. As part of this project, the VDI Guideline 4330 Part 10 was set up
(Bleeker et al. 2011) describing a standardised procedure for floristic mapping of spontaneously occurring
(non-cultivated) GM crops, their wild potential crossing partners and their hybrid offspring. Areas to be
mapped are those where such plants are expected to be found, e.g. on former fields and in the vicinity of
current or former fields of GM plants. In the case of transportation, processing or use of GM plants as
animal feed, these are areas surrounding the processing, storage, handling and usage facilities, including
access routes to and from the facilities. The concept of adverse environmental effects caused by the dispers-
al and outcrossing of GM plants is briefly introduced. The necessity of floristic mapping in the context of
post-market environmental monitoring of GM plants is demonstrated taking oilseed rape as an example.
The development of the Guideline VDI 4330 Part 10 is described and its contents are summarised. An
important conclusion on the relevance and efficiency of the floristic mapping method is that strict stan-
dardisation ensures a high level of EU wide reproducibility and comparability of the results.

Keywords
Genetically modified plants, post-market environmental monitoring, VDI guidelines, standardised meth-

ods, floristic mapping, wild crossing partners, hybrid offspring, oilseed rape, Brassica napus

Copyright Ulrich Sukopp, Ulf Schmitz. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

90 Ulrich Sukopp & Ulf Schmitz / BioRisk 8: 89-110 (2013)

Introduction

The commercial use of GM organisms may have various environmental impacts at
the level of species, habitats or ecosystems (Sukopp and Sukopp 1993, Letourneau
and Burrows 2002, Velkov et al. 2005, Dolezel et al. 2007, Bartz et al. 2009, EFSA
2010). This happens for example, if GM crops spontaneously spread and persist out-
side cultivation or cross with other sexually compatible plants (Ellstrand and Hoff-
man 1990, Nijs et al. 2004, Pilson and Prendeville 2004, Gressel 2005, FitzJohn et
al. 2007, Wilkinson and Ford 2007). Therefore, the commercial use of GM organ-
isms is regulated in the EU by law in order to prevent adverse environmental effects
caused by such use. According to Directive 2001/18/EC on the deliberate release
into the environment of genetically modified organisms (EC 2001) and to Regula-
tion (EC) No 1829/2003 on genetically modified food and feed (EC 2003), moni-
toring the environmental effects of GM organisms after placement on the market is
a mandatory task of the respective consent holder. Before placing on the market any
GM organism has to pass an approval procedure led at present by the Directorate-
General for Health and Consumers of the European Commission. In the application,
monitoring procedures have to be described in a monitoring plan in detail. Once
the application is consented, the monitoring plan has to be fully implemented with
reporting obligations at regular intervals.

According to Directive 2001/18/EC, post-market environmental monitoring has
to follow standard methodologies, wherever available and appropriate (see also EC
2002, EFSA 2011, Bf£N et al. 2011). Since many relevant monitoring procedures are
not standardised so far, there is a strong need to newly develop suitable standards (Ber-
horn et al. 2005). For this reason, the Association of German Engineers (VDI) as an
independent technical standardisation body has commissioned several expert groups
with the development of so called VDI Guidelines covering appropriate monitoring
methodologies (see Ziighart et al. 2013). The scientific and administrative experts dis-
cussed and decided on needs and priorities of standardised methods. The outcome
are specific regulations partly published and partly still in progress (see overview on
Guidelines VDI 4330 — 4333 in Tab. 2 and Tab. 3 in Ziighart et al. 2013). The exact
description of monitoring techniques should enable the staff to collect and analyse
data in a scientifically and statistically sound manner as required for the post-market
environmental monitoring of GM organisms (EC 2002).

Technical guidelines facilitate monitoring activities since they provide standardised
methods ensuring data quality and comparability over time and space (Graef et al. 2007).
This is particularly important if data are collected in large areas and over longer periods of
time. Post-market environmental monitoring of GM organisms is expected to last at least
10 years and may cover the entire EU territory. Therefore, comparison of data relies on
highly standardised field methods. ‘This applies also for floristic mapping data if required.

In our contribution, we explain the necessity of floristic mapping in the context
of post-market environmental monitoring of GM plants that may spread and persist
or form hybrid offspring. Further, we address the question of adverse environmental

How to track genetically modified (GM) plants in the field? The VDI standard method... i

effects caused by the dispersal and outcrossing of GM plants. Oilseed rape serves as a
prominent example of a common GM crop that can be monitored using standardised
procedures. Development and contents of the Guideline VDI 4330 Part 10 on floristic
mapping (Bleeker et al. 2011) are briefly explicated. Finally, conclusions are drawn on
the relevance and efficiency of the VDI standard method of floristic mapping.

Floristic mapping in the context of post-market environmental monitoring
of GM plants

Floristic mapping is the spatial and temporal recording and subsequent cartographic
representation of the occurrence of plants in the landscape. For this purpose, the name
of the plant taxon and the locality must be determined. Furthermore, as a minimum
requirement, the observation date and the name of the observer must be recorded. ‘The
resulting data can be stored in a data bank or can be shown on a map of the study area.

The use of GM plants can comprise cultivation including transport of seed and harvest
material to and from fields. If GM plants are used for the production of food or feed, GM
raw material as well as processed commodities containing GM material are transported
between storage yards, harbours, railway stations, reloading areas and processing plants.
During cultivation, transport, storage, handling or processing of GM plants, viable plant
material (e.g. seeds, fruits, tubers) can get into the environment e.g. through incidental
spillage. If this is leading to spontaneously growing and reproducing GM plants, further
events can be triggered: (1) ongoing spontaneous dispersal of GM plants, (2) establish-
ment of persistent populations of GM plants, (3) outcrossing of GM plants into potential
crossing partners occurring in the wild, (4) establishment and spontaneous dispersal of
hybrid offspring resulting from those crosses. Outcrossing and the production of hybrid
offspring can occur also via pollen transfer from GM crops to wild crossing partners.

Since the early 1990s, concepts have been elaborated including monitoring meth-
ods for the above mentioned environmental effects of GM plants in various European
countries (see among others Sukopp and Sukopp 1993, 1997, Traxler et al. 2001,
Ziighart and Breckling 2003, Heissenberger et al. 2003, ACRE 2004, Ziighart et al.
2005, 2008, Pascher et al. 2010, EFSA 2011, BfN et al. 2011). In Germany, the first
projects developing and testing suitable floristic field methods started at the end of the
20" century. Theenhaus et al. (2005) developed monitoring methods for a Bavarian
landscape between Neustadt an der Donau and Kelheim including floristic mapping
of Brassicaceae species as potential crossing partners of oilseed rape and sampling of
leaf material for molecular biological analysis. Menzel (2006) investigated the spread
of conventional oilseed rape and the distribution of potential crossing partners in and
around the city of Bremen. Similar surveys were carried out by Haeupler et al. (2004)
in the region around Detmold (North Rhine-Westphalia) and by Elling (2008) in the
region around Osnabriick (Lower Saxony).

The State Agency for Nature, Environment and Consumer Protection of North
Rhine-Westphalia is so far the only German state authority having implemented a

92 Ulrich Sukopp & Ulf Schmitz / BioRisk 8: 89-110 (2013)

regular monitoring programme regarding the potential spread of GM plants in the en-
vironment (K6nig 2009). In 2004, the observations started as part of a much broader
biodiversity monitoring programme. Sampling of leaf material of spontaneously oc-
curring oilseed rape and eight potential crossing partners is carried out in representa-
tive parts of the landscape. The molecular biological analysis proved the presence of

DNA sequences of GM oilseed rape in three samples in 2004 (Konig 2009).

GM plant dispersal and formation of hybrid offspring

‘The success of spontaneous GM plant dispersal and outcrossing events highly depends on
many different influencing factors and conditions. Among these factors are: (1) the level
of cross-pollination, (2) the degree of allogamy, (3) the level of domestication of the GM
crop, (4) the presence and frequency of sexually compatible wild plants, (5) the amount of
production of hybrid seeds, (6) the selective advantage of certain new traits of the hybrid
plants, (7) the availability of suitable sites for the establishment of hybrid plant popula-
tions (Ellstrand and Hoffman 1990, Sukopp and Sukopp 1994, Bleeker et al. 2007). The
resulting dispersal of GM plants or of their hybrid offspring can be recorded in the field
with the help of floristic mapping. The probability of the before mentioned processes
highly increases in the presence of crop-weed-complexes (Harlan 1965, 1982, Hammer
1991). In many cases, both crops and weedy relatives are the result of a very similar evo-
lutionary process. Most crop species are known to have formed at least in some parts of
the world closely related weedy forms that can easily produce offspring with the respective
crop variety. In English literature, this phenomenon is named crop ferality (Gressel 2005).
Crop-weed-complexes are described e.g. for sugar beet (Desplanque et al. 1999, Soukup
and Holec 2004, Sukopp et al. 2005), oilseed rape (Hall et al. 2000, 2005, Salisbury 2002,
FitzJohn et al. 2007) and bread wheat (OECD 1999, Zaharieva and Monneveux 2006).

Adverse environmental effects caused by GM plant dispersal and formation
of hybrid offspring

Floristic mapping in the context of post-market monitoring of the environmental effects
of GM plants and their use is triggered by (1) the hypothesis that viable GM plant mate-
rial may spread or (2) by the hypothesis that GM plants may outcross with other sexually
compatible plants. While the first hypothesis applies to all commercial uses of viable GM
plants or viable GM plant material (e.g. seeds), the second hypothesis presupposes the
presence of sexually compatible crossing partners. If there are so far no crossing partners
known in the area under survey, monitoring according to the second hypothesis is not
necessary. An example for a common crop plant of the last type in Central Europe are
potatoes. However, if crossing partners may occur in the study area, the second hypoth-
esis specifically triggers the search for such crossing partners and for potential hybrid

offspring (Sukopp and Sukopp 1993, 1994, 1997, Sukopp and Weddeling 2007, Bleeker

How to track genetically modified (GM) plants in the field? The VDI standard method... 93

et al. 2007, Schmitz et al. 2008). It should be noted, that the GM plants can be used
(e.g. cultivated or processed) either directly in the area under survey or in other more or
less remote regions. In the last case, the GM plants may be — most often — accidentally
introduced into the study area by means of transportation as it was documented for oil-
seed rape in Canada (Yoshimura et al. 2006), in Japan (Aono et al. 2006, Kawata et al.
2009, Nishizawa et al. 2009), in the USA (Schafer et al. 2011) and in Switzerland (Schoe-
nenberger and D’Andrea 2012). In Guideline VDI 4330 Part 10, lists of known crossing
partners of two common European crops are presented (Bleeker et al. 2011). These refer
to oilseed rape (Brassica napus) and to bread wheat (Triticum aestivum). Both lists contain
ephemeral, established or cultivated species found in Germany of which at least one suc-
cessful spontaneous crossing or one successful experimental crossing using hand pollina-
tion with the respective crop species is known to have occurred in or outside Germany.

Post-market environmental monitoring aims at detecting adverse effects of GM
plants and their use on human health and the environment. Such adverse environmental
effects or damages occur when a relevant conservation resource is significantly adversely
affected (for the development of this definition see Sukopp and Sukopp 1993, 1994,
Bartz et al. 2005, 2009, Kowarik et al. 2008, Heink et al. 2012). The identification
of a significant adverse effect comprises the determination of its magnitude and of the
value of the affected conservation resource. For the implementation of this concept,
suitable criteria must be found and thresholds must be agreed on (Kowarik et al. 2008).
The magnitude of adverse environmental effects is affected by the extent of persistence
and spread of GM plants, by the frequency of outcrossing events and by the extent of
persistence and spread of the resulting hybrid offspring. All these events and processes
alone do not determine a damage to conservation resources. According to a cause-effect-
hypothesis, such events can trigger other events that may finally cause a damage e.g. if
escaped hybrid offspring can establish highly dominant populations which replace popu-
lations of a protected native plant species. In this example, a certain conservation goal is
significantly impaired (for further details of the concept see Kowarik et al. 2008). Since
outcrossing of GM plants or the spread and persistence of GM plants or hybrid offspring
can be a step towards adverse environmental effects, post-market environmental moni-
toring of GM plants has to address those processes. The monitoring results should help
confirm or reject the initial hypothesis concerning outcrossing, persistence and spread of
the GM plants and of potential hybrid offspring (BEN et al. 2011). In the context of the
environmental risk assessment of GM plants, it has to be considered that future fitness
estimations do not only depend on genotypes but rather on the interactions between
a particular plant phenotype and its specific environment. Since future environmental
changes are not fully predictable, a substantial uncertainty remains in such fitness estima-
tions. Therefore, according to the precautionary principle, the persistence of a population
of GM plant individuals can already be an issue of concern (see Kowarik et al. 2008).

Taking oilseed rape as an example, Breckling et al. (2003) and Breckling and Men-
zel (2004, 2005) discussed the need to consider potential adverse effects on conserva-
tion resources caused by the commercial use of GM oilseed rape in risk research, risk
assessment and for post-market environmental monitoring.

94 Ulrich Sukopp & Ulf Schmitz / BioRisk 8: 89-110 (2013)

Oilseed rape as an example of spread, persistence and outcrossing of a
common crop

Oilseed rape is a well-known example of a widespread crop species that generates large
numbers of volunteers on arable land, easily spreads to suitable habitats outside crop
land and hybridises with sexually compatible wild relatives. Feral (spontaneous or sub-
spontaneous) populations of oilseed rape and its hybrids can originate from three main
sources: (1) land cultivated with oilseed rape, (2) places where oilseed rape is handled,
stored or processed such as oil mills and harbours, (3) from accidental spillage during
transport along roads, railways, rivers or canals.

Oilseed rape (Brassica napus) is a close relative of a number of other crop species
(e.g. B. rapa, B. oleracea) and wild species of the Brassicaceae family (e.g. Sinapis arven-
sis, Raphanus raphanistrum) and can spontaneously produce fertile offspring with several
of those species (e.g. SchefHer and Dale 1994, Lefol et al. 1996, 1997, Salisbury 2002,
Warwick et al. 2003, Chévre et al. 2004, Knispel et al. 2008, Elling et al. 2010). A re-
cent comprehensive overview on hybridisation within Brassica and allied genera is given
by FitzJohn et al. (2007). After the year 2000, first evidence of outcrossing events under
field conditions with GM oilseed rape as the gene donor and other crops or weedy spe-
cies of arable land as receptors was published (e.g. hybridization between GM oilseed
rape and Brassica rapa or Sinapis arvensis; Halfhill et al. 2004, Daniels et al. 2005).

Conventional and GM oilseed rape can establish feral (spontaneous or subsponta-
neous) populations on various types of non-cropped land (Pessel et al. 2001, Loos et al.
2004, Yoshimura et al. 2006, Elling 2008, Elling et al. 2009, Kawata et al. 2009, Ni-
shizawa et al. 2009, Schafer et al. 2011, Schoenenberger and D’Andrea 2012). These
populations occur frequently along transportation routes (e.g. on roadsides, railway
ground, waste ground in harbours) and close to processing plants (e.g. oil mills, crush-
ing facilities). The spatial and temporal dynamics of populations and seed dispers-
al along roads were described in detail for feral oilseed rape on the M25 motorway
around London (Crawley and Brown 1995, 2004). A similar study on seed spillage
and seed dispersal of various plant species including oilseed rape was conducted along a
motorway tunnel in Berlin (von der Lippe and Kowarik 2007a, 2007b). In France and
Germany, case studies have shown that feral oilseed rape probably establishes persis-
tent populations that do not or only partly depend on seed recruitment from cultivated
populations (Pessel et al. 2001, Loos et al. 2004, Pivard et al. 2008).

Pessel et al. (2001) identified relict plants of a no longer marketed oilseed rape
variety which had persisted on road verges for at least 8 years according to farmer sur-
veys in the region of Loir-et-Cher in France. In the same region, Pivard et al. (2008)
conducted a large-scale study on factors explaining the occurrence of feral oilseed rape
populations on field margins and road verges. Two dominant factors were identified:
(1) annual seed dispersal from surrounding fields cropped with oilseed rape, (2) per-
sistence of feral populations, mainly through persistent seed banks. ‘Therefore, the seed
bank contribution to the dynamics of feral oilseed populations needs to be considered
more seriously. Persistent seed banks combined with selfrecruitment indicate a high

How to track genetically modified (GM) plants in the field? The VDI standard method... 95

potential for the persistence also of feral GM oilseed rape outside fields. Loos et al.
(2004) described feral populations of oilseed rape on waste land in the Ruhrgebiet
in Germany that are presumably persistent (naturalisation in anthropogenic habitats
outside arable land). Elling (2008) showed that 79,5 % of the 78 examined feral popu-
lations of Brassica napus in the region of Osnabriick, Germany, persisted for more
than one year on the same locality. In Austria, Pascher et al. (2010) demonstrated
significant genetic differences between commercial varieties and feral populations of
oilseed rape. The authors concluded that feral oilseed rape is able to maintain persistent
populations that are already to a certain extent genetically separated from the crop
varieties. At present, evidence from several European studies strongly supports the
assumption that both conventional and GM feral oilseed rape persists in many places
outside agricultural land (Squire et al. 2011).

Other studies have reported feral GM oilseed rape plants or populations along
transport routes and in harbour facilities in Canada (Garnier & Lecomte 2006,
Yoshimura et al. 2006, Knispel et al. 2008, Knispel and McLachlan 2009), Japan (Saji
et al. 2005, Aono et al. 2006, Kawata et al. 2009, Nishizawa et al. 2009, 2010) and
most recently also in the USA along roadsides outside the oilseed rape cultivation area
(Schafer et al. 2011). Potential selective advantages of feral GM herbicide resistant oil-
seed rape can occur along transport routes (e.g. roadsides, railway ground) and in har-
bours particularly where complementary herbicides are applied. Schafer et al. (2011)
described spontaneous GM oilseed rape populations at roadsides that had recently
been mowed or treated with herbicides. The presence of stacked herbicide resistance
genes from different cultivated GM oilseed rape varieties was observed in feral oilseed
rape populations outside fields in western Canada giving evidence for gene flow among
those feral populations (Knispel et al. 2008).

While observing the spread of oilseed rape along railways in Switzerland, Schoe-
nenberger and D’Andrea (2012) proved the presence of GM herbicide resistant oilseed
rape on railway ground in Basel and Lugano. ‘The findings indicate that certain GM
oilseed rape varieties may be capable of establishing persistent populations outside
agricultural land, e.g. on herbicide-treated railway tracks, on handling areas and in
surrounding disturbed habitats. The question arises if this leads to a transfer of the
introduced herbicide resistance genes to wild species that are sexually compatible.

Development of Guideline VDI 4330 Part 10 on floristic mapping

In Central Europe, floristic mapping for scientific purposes looks back on a long histo-
ry. First prints of local flora books appeared at the end of the 16" century and reached
a modern scientific standard already in the 19" century (e.g. Ascherson 1864). For
Germany, a milestone at the end of the 20" century was the publication of the results
of two floristic mapping projects covering West and East Germany in two separate vol-
umes (Haeupler and Schénfelder 1988, Benkert et al. 1996). Rather comprehensive
guidelines concerning the organisation and methods of floristic mapping in Germany

96 Ulrich Sukopp & Ulf Schmitz / BioRisk 8: 89-110 (2013)

were laid down by the Central Office for Floristic Mapping in Germany in the early
1990s (Bergmeier 1992). The results of floristic mapping are often used in landscape
planning and nature conservation, e.g. to set up targeted conservation programmes for
particular species or to compile Red Lists of rare and endangered plants (cf. Schulte
and Voggenreiter 2000, Sukopp 2001, Haeupler 2005, Sukopp and Sukopp 2006, Su-
kopp et al. 2006). For example, the data collected during the recent floristic mapping
of the city of Hamburg were published together with the latest Red List of vascular
plants of the city (Poppendieck et al. 2010). A comprehensive overview of approxi-
mately 250 flora books and plant distribution atlases, that were published between
1945 and 2010 covering either the whole country or certain regions of Germany,
was compiled by Horn et al. (2006, 2012). Some outstanding examples of recent
regional floristic mapping projects are the surveys of the Regnitz river basin (Gatterer
and Nezadal 2003), of North Rhine-Westphalia (Haeupler et al. 2003), of the city of
Hamburg (Poppendieck et al. 2010) and of the city of Berlin (Seitz et al. 2012). In
these projects, mapping was carried out with a systematic approach on the basis of a
regular grid and in a highly professional manner.

In close cooperation with the VDI, a group of seven voluntary experts worked
on the Guideline VDI 4330 Part 10 between 2008 and 2011. The work aimed at
providing standard methods that support a scientifically sound and efficient post-
market environmental monitoring of GM plants and their use. The work was em-
bedded in the conceptual framework specified in the Guideline VDI 4330 Part 1
on basic principles and strategies for the environmental monitoring of GM plants
(Ziighart et al. 2013). The participating experts brought their personal competent
view on floristic mapping to the formulation of the new guideline. Furthermore, the
committee could build on the previously released VDI Guideline on the assessment
of the diversity of ferns and flowering plants by means of vegetation records (Beis-
mann et al. 2008). First, the expert group developed an internal preliminary draft
that was then published (Bleeker et al. 2010) and subjected to a public approval
procedure (see also Ziighart et al. 2013). All received objections were discussed and
dealt with by the experts. Finally, the revised Guideline VDI 4330 Part 10 was pub-
lished (Bleeker et al. 2011).

Contents of the Guideline VDI 4330 Part 10 on floristic mapping

The Guideline VDI 4330 Part 10 is entitled “Floristic mapping of genetically modi-
fied plants (GM plants), their crossing partners and their hybrid offspring” (Bleeker et
al. 2011). The described floristic mapping methods are based on well-established and
widely applied procedures, but needed to be adapted to the specific requirements of
post-market environmental monitoring of GM plants and their commercial use. Plants
to be recorded are spontaneously occurring (non-cultivated) GM crops, their wild po-
tential crossing partners and hybrid offspring resulting from crosses between GM crops
and these partners. Planted or intentionally sown GM crops are not mapped.

How to track genetically modified (GM) plants in the field? The VDI standard method... 97

The floristic mapping according to Guideline VDI 4330 Part 10 covers only vas-

cular plants. All plants recorded in the area under survey are identified to the rank of

species and in the case of aggregates, to the rank of microspecies. Infraspecific taxa
(subspecies, varieties, etc.) can be differentiated if this information is required to ana-

lyse and interpret the mapping data. Identification is made using standard floras or
alternatively in the case of critical taxa with the aid of specialist literature. The nomen-

clature is based on up-to-date reference publications.

Guideline VDI 4330 Part 10 describes standardised methods for all steps of the

floristic mapping procedure. The ten most important issues are (Bleeker et al. 2011):

(1)
(2)
(3)

(4)

(5)
(6)

Equipment and material needed for the field work is listed.

Necessary steps to prepare the field work are explained.

The selection of individual mapping dates and the scheduling of the entire mapping
programme in the course of several years are explicated. The plants to be mapped
should be recorded at a time when they can be most easily identified. To determine
the original state (reference state) before GM plants were introduced into the envi-
ronment, mapping must begin the year before the first use of GM plants. Mapping
is then carried out annually for the entire period of GM plant use. If the use of GM
plants is interrupted or terminated, mapping must continue annually for a period
of at least eleven years after the last year of GM plant use. Mapping can be termi-
nated earlier, if no spontaneous occurrences of the plants to be mapped are found
in four consecutive years following the last year of GM plant use.

Rules are given how to delimit the study area. In the case of crop cultivation, the
study area includes fields where GM crops were previously grown and areas of
land and paths surrounding current or former fields of GM plants. In the case
of transportation, processing or use of GM plants as food or animal feed, areas
surrounding the GM plant processing, storage, handling and usage facilities in-
cluding access routes to and from these facilities must be surveyed. ‘The general
spatial design of the mapping consists of at least ten concentric zones at intervals
of 0 m to 100 m (zone 1), 100 m to 200 m (zone 2), 300 m to 400 m (zone 3),
etc. around current or former fields of GM plants or around processing, storage,
handling and usage facilities of GM plants (see Figure 1). At the time of the
survey, the zones are systematically examined for the presence of the plants to be
mapped, starting at the innermost zone. Main access routes (roads, railway lines
and waterways) and drivable paths including their verges are examined as well
within the 2000 m wide study area around places where GM plants are culti-
vated, processed, stored or handled (see Figure 1).

Field recording starts with entering general data, such as identification number,
date and name of field observer, in the data sheet.

The location and exact outline of the area covered by the current or former GM
plant field or accordingly by the processing, storage, handling and usage facility
of GM plants is verified in the field and the boundaries marked on the prepared

map are corrected if necessary.

98 Ulrich Sukopp & Ulf Schmitz / BioRisk 8: 89-110 (2013)

4330 Part 10, Bleeker et al. 2011). The following elements are marked on a topographic map: | the site

of an oil mill (blue area) 2 ten concentric zones at intervals of 0 m to 100 m (zone 1), 100 m to 200 m
(zone 2), 300 m to 400 m (zone 3), etc. around the site (thin green lines) 3 boundary of the mapped area
at a distance of 2000 m around the site (thick green line) 4 main access routes to and from the site within
a range of 2000 m around the site (thick red lines) 5 waterway for shipments to and from the site within a
range of 2000 m around the site, and up to a minimum distance of 3000 m from the site in the direction

of flow, including regularly flooded backwaters and meanders (pink area).

(7) For the floristic mapping, the study area is systematically examined for the presence
of the plants to be mapped. Any specimen found is identified as exactly as possible by
examining its morphological characters. Usually, it is impossible to distinguish be-
tween GM and non-GM plants in the field on the basis of their apparent characters.
A reliable distinction can only be made by molecular biological analysis of plant sam-
ples in the laboratory. For this reason, plant samples are collected during mapping

in accordance with Guideline VDI 4330 Part 5 “Guidelines for the collection and
preparation of plant samples for molecular biological analysis” (Belter et al. 2010).

How to track genetically modified (GM) plants in the field? The VDI standard method... 99

(8) ‘The localities of the plants to be mapped are marked on the prepared maps in the
field using point, line and area symbols. ‘The localities are numbered on the map,
and the numbers are also entered on the field data sheet. A locality is defined as
the smallest distinguishable area that provides a uniform habitat for individuals
of the mapped taxon without large gaps (generally < 20 m for a scale of 1: 5000).
The plants of one locality must be in a comparable developmental stage. If the
individuals of a plant taxon at one site largely differ in their developmental stage,
it is required to distinguish the different stages and to quantify the respective
numbers of individuals at each developmental stage (see next paragraph). A local-
ity may contain several sampling points.

(9) For each occurrence of plants of one taxon at one locality, the total number of
individuals estimated by means of a logarithmic scale (I: 1 individual, Hl: 2 to
10 individuals, III: 11 to 100 individuals, IV: 101 to 1000 individuals, etc.) is
entered on the field data sheet.

(10) The biotope type and — if possible — the vegetation type of the individual localities
are entered on the field data sheet. This information is based on a named refer-
ence work. Further information about the locality may also be included, e. g. the
use of herbicides or the presence of patches of bare soil.

The field data are transferred from the field map to a final map in appropriate scale.
GM plants and their hybrid offspring must first be verified by molecular biological
analysis. When creating a digital map, all information about the position of occur-
rences can be processed with GIS software. Tables are prepared containing the names
of the recorded taxa arranged by locality number, the corresponding number of indi-
viduals, the area size or the length of the locality and the biotope or vegetation type.
The mappings can be analysed in terms of the spatial distribution of the localities and
the temporal development during the study years.

The documentation includes a description of the methods and all data acquired in
the field and from other investigations. Floristic mapping data gathered in accordance
with this guideline should be permanently archived in an appropriate database. In
Guideline VDI 4330 Part 1, Section 6, the general requirements for documentation
and data management are specified.

General discussion and conclusions

Monitoring the environmental effects of GM organisms comprises measurements and
observations of elements of communities, habitats and landscapes in regular spatiotem-
poral sequences that are designed to achieve accurate results on the state and changes
of those elements. A suitable monitoring employs scientific methods and is directed to-
wards nature conservation and environmental protection objectives (Sukopp and Wed-
deling 2007). In the case of viable GM plants entering the environment during cultiva-
tion, transport, handling or processing, one important issue of the monitoring are effects

100 Ulrich Sukopp & Ulf Schmitz / BioRisk 8: 89-110 (2013)

on the local and regional flora. Thus, floristic mapping provides an overview of the
frequency and spatial pattern of spread and persistence of GM plants as well as of their
potential crossing partners and hybrid offspring. Areas to be mapped are those where
the before mentioned plants can spontaneously or subspontaneously occur (Bleeker et
aleO1I!

If GM plants are cultivated on arable land, GM volunteers appearing on the fields
can have the same effects on the local and regional flora as the formerly cultivated
plants (Munier et al. 2012). This particularly applies to cultivated GM oilseed rape
that can build up a persistent seed bank. Several studies in Canada and the USA have
observed volunteers of GM herbicide resistant oilseed rape that can contribute to sub-
sequent gene flow to other sexually compatible plants in the surrounding area (Knispel
et al. 2008, Beckie and Warwick 2010, Schafer et al. 2011, Munier et al. 2012).

The magnitude of potential spread, persistence and outcrossing events strongly
depends, among other important factors, on the area cultivated with GM plants and
on the amount of GM plants in imported commodities to be processed for further
use, e.g. as human food or animal feed. Therefore, it is reasonable to focus the floristic
mapping primarily on localities and regions where cultivation, transport, handling
or processing takes place (Ziighart et al. 2005, 2008, BEN et al. 2011). On the other
hand, there is evidence for human-mediated unintentional long distance transport of
viable seeds (von der Lippe and Kowarik 2007a, 2007b, Wichmann et al. 2009) that
can result in spontaneous growth of GM plants in regions more or less remote from
the original localities of cultivation or processing.

Directive 2001/18/EC (EC 2001) and Regulation (EC) No 1829/2003 (EC 2003)
stipulate monitoring activities in order to identify adverse effects on human health or
the environment caused by GM organisms during commercial use. Therefore, the re-
sults of the analysis of monitoring data have to be accurately assessed in order to deter-
mine whether observed environmental changes may be classified as adverse effects. For
this purpose, a sound concept of environmental damages based on predefined criteria
has to be applied (Heink et al. 2007, 2012, Kowarik et al. 2008, Bartz et al. 2009). Ad-
verse environmental effects can only be determined if they are related to certain relevant
protection goals (e.g. protection of a natural resource such as a population of an endan-
gered species) (Heink et al. 2007, Kowarik et al. 2008). Descriptions of environmental
changes have to be clearly separated from the evaluation of such changes. ‘The first issue
is part of a scientific approach, the latter issue depends on normative settings beyond
science. It is a matter of politics and society to enact a legal framework and detailed
regulations defining relevant environmental and nature protection goals, as it was done
e.g. by the Directive 2004/35/EC on environmental liability with regard to the preven-
tion and remedying of environmental damage (EC 2004). Furthermore, criteria have to
be agreed on, the application of which leads to a decision whether a certain protection
goal is significantly impaired through environmental impacts of GM plant use.

Both approaches, the scientific description of change and the normative assess-
ment of monitoring results, are often erroneously mixed up creating false conclusions.
The review of Devos et al. (2012) on feral GM herbicide tolerant oilseed rape from

How to track genetically modified (GM) plants in the field? The VDI standard method... 101

seed import spills raises the question if concerns about environmental damages are
scientifically justified. The question and the attempt of an answer fail to separate sci-
entific analysis from evaluation. On the contrary, Wilkinson and Ford (2007) present
a much more sound concept of environmental hazard identification when they intro-
duce so-called endpoint species of conservational significance that could be negatively
impacted upon by certain effects of spread, persistence or outcrossing of GM plants.

Persistence and spread of GM crop plants or of their hybrid offspring may also
lead to unwanted detrimental effects such as the decline or even replacement of popu-
lations of native species. The persistence of herbicide tolerant GM plants or of their
hybrid offspring on crop land can also aggravate weed control resulting in much higher
quantities of herbicide applications or the need to use mixtures of different herbicides
(EFSA 2010). On the long run, this may progressively impoverish the wild flora in
agricultural regions or may deteriorate ground water quality. A primarily economic
damage could be the impurity caused by GM crops if admixed with non-GM crops of
conventional or organic farming. This would induce high costs to implement coexis-
tence measures that guarantee the side-by-side operation of GM and non-GM crop-
ping systems (Devos et al. 2009). Furthermore, it may indirectly hamper environmen-
tally sustainable ways of agriculture and increase the use of herbicides posing higher
threats to wild plants and associated insects and other animals on crop land.

One important goal of standardisation is safeguarding the quality of procedures and
results. Standardisation of monitoring methods ensures a high level of reproducibility and
comparability of the results (Schréder et al. 1991, Berhorn et al. 2005, Beismann et al.
2007, Graef et al. 2007, BEN et al. 2011, Ziighart et al. 2013). In case of floristic map-
ping, this involves testing the plausibility of the mapping data both during collection and
on completion of the report. Quality assurance also includes a comparison of the results
of the study with those of similar studies. The competence of the field observer and the
correct use of the necessary resources and equipment are a fundamental requirement for
assuring work quality. The field observer must be able to demonstrate a detailed and up-
to-date knowledge of the flora of the ferns and flowering plants of the investigated coun-
try or region. The locality of any mapped plant must be precisely recorded. An indepen-
dent repetition of the mapping can be performed to verify the results (Bleeker et al. 2011).

Yet, we are far away from understanding the environmental impacts of GM plants.
Many case studies and even reviews are predominantly based on results deduced from
rather fragmentary data or just models about real processes in the landscape. Con-
sequently, more or less imprecise likelihoods substitute clear facts. Thus, data-based
evidence from comprehensive and reliable monitoring programmes should be achieved
in the future. Standardisation of relevant monitoring techniques can contribute to this
goal. The application of the Guideline VDI 4330 Part 10 on floristic mapping pro-

vides several advantages in the context of GM plant monitoring:

e ‘The sampling design and the mapping methods are state-of-the-art.
e ‘The mapping strategy focuses on spread, persistence and outcrossing of GM plants
originating from one known potential source (e.g. a field or a processing factory).

102 Ulrich Sukopp & Ulf Schmitz / BioRisk 8: 89-110 (2013)

e ‘The floristic mapping intends to get best results with reasonable effort.

e ‘The spatial and temporal organisation of the floristic mapping is designed to find
a balance between the effort in the field and the reliability of data on the presence
and — even more important — absence of the surveyed plants.

e ‘The application of standardised methods leads to accurate, comprehensive and
reproducible mapping results.

e ‘The guideline provides detailed instructions on data management and quality
assurance.

e ‘The application of the guideline contributes to a cost-efficient monitoring of envi-
ronmental effects of GM plants.

e ‘The guideline on floristic mapping (VDI 4330 Part 10, Bleeker et al. 2011) and
the guideline on collection and preparation of plant samples for molecular biologi-
cal analysis (VDI 4330 Part 5, Belter et al. 2010) are coordinated and should be
applied together in order to determine the genetic identity of mapped plants.

In the future, new developments of GM plants or of the way of using GM plants
(e.g. new species or varieties, new traits, use in new environments) can give reasons for
amending or adjusting the Guideline VDI 4330 Part 10. According to the VDI rules
of guideline development, updates can be done at the latest five years after publication
when the guideline will be subjected to a revision.

Acknowledgements

The project was funded by the Federal Ministry for the Environment, Nature Conserva-
tion and Nuclear Safety (BMU) and the Federal Agency for Nature Conservation (BfN).
We thank the VDI for the fruitful cooperation. We wish to express our gratitude to Dr. W.
Bleeker (Osnabriick), A. Benzler (Bonn), S. Demuth (Karlsruhe), Dr. H.S. Fischer (R6tten-
bach), Rudolf May (Bonn), Dr. H. Seitz (Diisseldorf) and Dr. J. Zipperle (Karlsruhe), who
contributed to the Guideline VDI 4330 Part 10. Prof. em. Dr. Dr. h.c. Herbert Sukopp

and two anonymous reviewers provided helpful comments on earlier drafts of this paper.

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