Coexistence Between Transgenic MON 810 Maize and Hives: Pollen and Flour Flow by Pollinator Bees and Honey Labelling

Coexistence between transgenic MON 810 maize and hives: pollen and flour flow by pollinator bees and honey labelling Eugénia de Andrade Plant Health and GMO Laboratory Instituto Nacional de Investigação Agrária e Veterinária, I.P. [email protected] Maria Lopes Instituto Politécnico de Santarém Escola Superior Agrária de Santarém [email protected] Mónica Rodrigues Plant Health and GMO Laboratory Instituto Nacional de Investigação Agrária e Veterinária, I.P. [email protected] Ana Chegão Plant Health and GMO Laboratory Instituto Nacional de Investigação Agrária e Veterinária, I.P. [email protected] Fátima Quedas Instituto Politécnico de Santarém Escola Superior Agrária de Santarém [email protected] Amélia M. Lopes Plant Health and GMO Laboratory Instituto Nacional de Investigação Agrária e Veterinária, I.P. [email protected] Contributed paper prepared for presentation at the Seventh International Conference on Coexistence between Genetically Modified (GM) and non-GM based Agricultural Supply Chains (GMCC-15) Amsterdam, The Netherlands, November 17-20, 2015 Copyright 2015 by the authors. All rights reserved. Readers may make verbatim copies of this document for non-commercial purposes by any means, provided that this copyright notice appears on all such copies. Abstract: In Portugal, in 2012 and 2013, the cultivation of GM maize covered 9278 ha and 8202.2 ha, respectively, representing approximately 6-7% of the total maize sowing areas. The large-scale cultivation of GM maize is not uniformly distributed along the country. It has its highest expression in the Centre-South regions. Maize is a cross-pollinating species and, preferentially, its pollen is wind-dispersed although it may also be insect- dispersed. In 2011, the European Court of Justice decided upon the need to demonstrate that pollen is a natural constituent of honey rather than an ingredient. Later, in 2013, the European Parliament defined pollen as a natural constituent of honey. This decision avoided strong financial implications concerning the need of honey labelling whenever Genetically Modified (GM) pollen makes up more than 0.9% of the species pollen fraction, according to the Regulation (EC) 1829/2003. Contrarily, the presence of flour, which is not a natural component of honey but may occur when industrial mills are in the neighborhood of the hives, might be treated according to the legal labelling framework for ingredients. Therefore, there is a need to distinguish between pollen and flour. Currently, the detection/quantification of GM components in honey is done by real-time PCR. However, until now no procedure can distinguish pollen from other kinds of GM material. Our main goal was, therefore, to develop a reliable and accurate method to allow distinguishing pollen from flour in honey. We investigated the ability of quantitative real-time PCR together with plasmid calibrants, triploid maize seed endosperms and haploid maize pollen, to develop a new approach to differentiate between adventitious presence of GM pollen and adventitious presence of GM flour. Plasmid calibrant certified for a 1:1 copy number ratio (transgene copy number in relation to an endogenous gene copy number) allow for the distinction between triploid and haploid tissues. A seven point dilution series from a 2x106 plasmid copies/µL solution was used to establish two calibration curves, being one for the transgene and other for a species- specific gene. DNA was extracted from honey, originally obtained in a Portuguese GM free zone (Região Autónoma da Madeira), spiked either with pollen, flour or embryos from a hemizygous population having the female progenitor as the transgene donor. PCR efficiencies were of 93 and 94% for both reactions. As proof-of-concept, samples of national and imported honey, commercialized either at local producers or in the supermarkets, were analyzed and the quantification results compared with the spiked honey samples. With this information available, correct estimation of the relative transgene copy number allowed for the distinction between pollen and flour. We have adapted real-time PCR to fit into the requirements of GMO labelling regulations. This approach has considerable potential to evaluate escapes of maize flour and for the establishment of recommendations for the milling companies in order to minimize effective entry of flour into the hives. The procedure has been in-house validated for maize. Keywords: Honey, GMO, contaminant, labelling, real-time PCR, plasmid calibrant. JEL codes: Q18 1. Introduction In 2011, the European Court of Justice decided that beekeepers must prove that pollen is a natural constituent of honey and not an ingredient (Court of Justice of the European Union, 2011). Pollen, in general, is the male gametophytes of flowers and contains approximately 10% of nectar and commercial honey is composed of pellets of pollen moistened with bees’ mouth secretions which are composed of different enzymes (Campos et al., 2008). The presence of pollen in honey seems not to be controversial. Several studies were conducted on comparison of honey composition on the basis of pollen content, botanical origin, quality, antibacterial activity among other characteristics (Mercan et al., 2007; Hermosín et al., 2003). Pollen is expected to represent only a very small fraction of the honey matrix. Depending on the author, total pollen content in honey varies from 0.003% to 0.104% (Piazza and Oddo, 2004; Kleinjans et al., 2012; Davison and Kershen, 2014). After the first cultivation of GM crops in Europe, one of the earliest concerns of the European Commission (EC) was to enable consumers to choose between GM and non- GM organic or conventional products. This led to the establishment of labelling policies and procedures for GM food and feed. Labelling is mandatory except when adventitious or technically unavoidable presence of a GMO in a given ingredient remains below 0.9% of that ingredient (European Commission, 2003). The official requirement issued by the European Court of Justice rose from the need to substantiate the exemption from label of honey containing genetically modified (GM) pollen, either authorized or non-authorized for food purposes, what, if approved, would have cost hundreds of thousands of Euros to bee keepers and to the final consumers. Indeed, if pollen was considered as an ingredient, according to the Regulation 1829/2003, all GM pollen would need to be labelled if making up more than 0.9% of the total pollen fraction or of the pollen with the same botanical origin (European Commission, 2003). Considering pollen as a natural component of honey then the reference matrix for labelling purposes is the honey itself. In November 2013, contrarily to the first decision of European Court, it was published the final verdict of the European Parliament that defined pollen as natural constituent of honey (European Recommendation (EC) 787/2004). Consequently and accordingly to the 1 rules regarding labelling of GM food, any GM pollen produced from plants containing authorized events, has not to be labelled even if it makes up more than 0.9% of the pollen fraction as this will never be above 0.104% of the honey fraction (Piazza and Oddo, 2004; Kleinjans et al., 2012; Davison and Kershen, 2014). However, if the genetically modified organism (GMO) is not authorized or is still undergoing an authorization process, it is not considered as approved for human consumption and therefore, the honey cannot be marked in Europe. Additionally, if the GM material present in honey is flour rather than pollen, then it has to be considered as an ingredient or contaminant. In this case the labelling regulation has to be applied (European Commission, 2003; European Parliament, 2013). In Portugal, in 2012 and 2013, the cultivation of GM maize covered 9278 ha and 8202.2 ha, respectively, representing approximately 6-7% of the total maize sowing areas (Carvalho & Mourão, 2012). The large-scale cultivation of GM maize is not uniformly distributed along the country. It has its highest expression in the Centre-South regions. In fact, 2215.3 ha were localized in Lisbon and Tagus Valley region and 5041.5 ha in the Alentejo province (MAM, 2013). The cultivation of GM maize in Portugal follows, in first instance, the European rules. Therefore, only the MON 810 maize event is authorized. MON 810 transgene codes for the CRY1Ab pro-toxin, a Bacillus thuringiensis derived protein that has insecticide properties against the European corn borer insects Ostrinia nubilalis (Hübner) and Sesamia nonagrioides (Lefebvre). Additionally, the Portuguese decree-law 160/2005, from 21st September, regulating genetically modified varieties, ensuring coexistence with conventional crops and with organic production, describes the mandatory rules of coexistence to be implemented by those who are interested in cultivating GM MON 810 maize. In detail, the coexistence law explains the technical standards to avoid cross- contamination of non-GM fields, to mitigate the selection of more resistant insects, to trace and label the production. In addition to the use of certified seeds, the most important measure is the distance of isolation. A GM maize field must be 200 m far from a conventional maize production field and 300 m from an organic production field (Quedas and de Andrade, 2013). These distances proved to be adequate for this purpose. However, no reference distance was stated in order preventing the presence of pollen in hives and/or in honey as the mechanisms of pollen dispersal may vary (Arrit et al., 2007). Maize is a cross-pollinating species and, preferentially, its pollen is wind-dispersed although it may also be insect-dispersed. Honeybees, Apis mellifera L., are the most important pollinators. Several studies indicate that maize pollen may be present in honey, especially when the hives are localized in the neighbourhood of large fields and when maize blooms earlier or later than other plants with more attractive pollen (Wróblewska et al., 2010). Honeybees may forage up to 2 Km (Ramsay et al., 1999).

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