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Agroecology and Sustainable Food Systems

ISSN 2168-3565 (Print) 2168-3573 (Online) Journal homepage httpwwwtandfonlinecomloiwjsa21

Pervasive presence of transgenes and glyphosatein maize-derived food in Mexico

E Gonzaacutelez-Ortega A Pintildeeyro-Nelson E Goacutemez-Hernaacutendez EMonterrubio-Vaacutezquez M Arleo J Daacutevila-Velderrain C Martiacutenez-Debat ampER Aacutelvarez-Buylla

To cite this article E Gonzaacutelez-Ortega A Pintildeeyro-Nelson E Goacutemez-Hernaacutendez EMonterrubio-Vaacutezquez M Arleo J Daacutevila-Velderrain C Martiacutenez-Debat amp ER Aacutelvarez-Buylla(2017) Pervasive presence of transgenes and glyphosate in maize-derived food in MexicoAgroecology and Sustainable Food Systems DOI 1010802168356520171372841

To link to this article httpdxdoiorg1010802168356520171372841

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Accepted author version posted online 31Aug 2017Published online 31 Aug 2017

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Pervasive presence of transgenes and glyphosate inmaize-derived food in MexicoE Gonzaacutelez-Ortegaab A Pintildeeyro-Nelsonac E Goacutemez-HernaacutendezbE Monterrubio-Vaacutezquezb M Arleod J Daacutevila-Velderrainae C Martiacutenez-Debatdand ER Aacutelvarez-Buyllaab

aCentro de Ciencias de la Complejidad (C3) Programa de Biologiacutea de Sistemas Universidad NacionalAutoacutenoma de Meacutexico Cd Universitaria Meacutexico Mexico City Meacutexico bInstituto de Ecologiacutea UniversidadNacional Autoacutenoma de Meacutexico Cd Universitaria Mexico City Meacutexico cDepartamento de ProduccioacutenAgriacutecola y Animal Universidad Autoacutenoma Metropolitana Unidad Xochimilco Mexico City MexicodLaboratorio de Trazabilidad Molecular Alimentaria Seccioacuten Bioquiacutemica Facultad de CienciasUniversidad de la Repuacuteblica Iguaacute 4225 Montevideo Uruguay eMassachusetts Institute of TechnologyComputer Science and Artificial Intelligence Laboratory MIT Stata Center Cambridge MA USA

ABSTRACTIn Mexico the Center of origin and diversity of maize a dailyper capita average of 05 kg of maize-based foods are con-sumed Approximately 10 million tons of maize is produced insmall peasant holdings Nevertheless a greater proportion ofindustrially produced maize-derived products have started toappear in recent years We traced the presence of transgenesand glyphosate in maize-based foods collected in Mexico andelsewhere as a means to describe the landscape of distributionabundance and types of transgenes in maize-derived food inMexico Transgenic sequences were present in overall 82 ofassayed food categories while the most widely form of maizeconsumption in Mexico tortillas had recombinant sequencesin 904 of the samples Furthermore we found statisticallyindistinguishable frequency distributions of transgenes in sam-ples from Mexico and other countries Additionally glyphosatewas detected in approximately 277 of the samples renderedpositive for transgenic events tolerant to this herbicide

KEYWORDSMexico food maize tortillatransgenes glyphosate

Introduction

Mexico is the Center of Origin and Diversification (COD) for many crops(Acevedo Gassman et al 2009) Currently peasant agriculture maintains existingvarieties of crop plants while generating new varieties (Turrent-Fernaacutendez Wiseand Garvey 2012) This is particularly true for maize the countryrsquos staple foodMaize originated in Mexico more than 6000 years ago (Kato et al 2009) and waslater integrated as a core component of an agroecological multi-cropping system

CONTACT Aacutelvarez-Buylla RE eabuyllagmailcom Instituto de Ecologiacutea Universidad Nacional Autoacutenomade Meacutexico Cd Universitaria Mexico City 04510 Meacutexico Centro de Ciencias de la Complejidad (C3) UniversidadNacional Autoacutenoma de Meacutexico Cd Universitaria Meacutexico Mexico City 04510 Meacutexico

Supplemental data for this article can be accessed on the publisherrsquos websiteColor versions of one or more of the figures in the article can be found online at wwwtandfonlinecomwjsa

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMShttpsdoiorg1010802168356520171372841

copy 2017 Taylor amp Francis

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called ldquoMilpardquo that continues to be used in a significant part of Mexico (Bourges2013) The dynamic and constant breeding of maize landraces within this agroe-cological context is known to be critical for the adaptation of maize varieties tochanging environments (Mercer et al 2012 Ureta et al 2012 Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A 2013)

Maize is sown across contrasting agroecological conditions and farmingsystems in approximately half of the arable land inMexico with a total estimatedproduction volume of 267 million tons in the 20162017 season (FIRA 2016)Approximately 10 million tons of maize per year are produced under indus-trialized farming systems predominantly in Northern states while another 10million tons are produced in small peasant holdings (Puyana 2012) An addi-tional 10million tons are importedmainly from the USA (Puyana 2012)Mexicothus represents a unique scenario where one of the worldrsquos most important cropsis sown in diverse agroecological systems in some areas while being planted asmassive industrial monocultures in others Additionally since 2003 a steadilyincreasing amount of maize grain is imported each year mainly for feed andindustrial uses (FIRA 2016 Otero 2011) How much of this maize reaches theMexican food chain is uncertain

Maize production processing distribution and consumption are equallycomplex in Mexico Of the current national maize production approximately6 million tons derived mostly from peasant production do not enter themarket and are instead used for self-consumption by peasant families(Ministry of Economy 2012) Maize food produced domestically under indus-trial conditions is primarily distributed in urban areas where it is consumedprincipally as tortillas Imported maize by contrast is often highly processedand incorporated into industrial food feed and as a raw material for thechemical industry (Ministry of Economy 2012)

A well-established economic agenda that discourages peasant agriculture andcreates unfavorable local conditions as well as unfavorable market prices fortraditional crops has forced migration into large cities and abroad (Otero 2011)Nonetheless centuries-old resilience of peasant and indigenous communitiescontinue to use landraces for maize production (Perales and Golicher 2014)Recently a growing interest in ldquoorganicrdquo and ldquoGMO-Freerdquo products in thehighest-value market sectors has also led to increases in the prevalence ofagroecological production and distribution pathways (Sourcebook 2017)

Despite the importance of understanding the complex dynamics amongmaize production processing and distribution at its COD but also at regio-nal and international levels studies in the past have relied on derivativeestimations and models to predict where local maize production and importsare ending with little or no direct empirical evidence Here we use twomarkers that provide a novel approach to the study of the maize-food chaintransformation industry focused on providing food for human consumptionin Mexico Rather than focusing on establishing the source of the maize used

2 E GONZAacuteLEZ-ORTEGA ET AL

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for food production we directly evaluated the presenceabsence of eighttransgenic DNA markers as well as the chemical herbicide glyphosate inboth industrial and artisan maize-derived food items These DNA markerswere created through genetic engineering of maize destined for use inindustrial agricultural systems and glyphosate has become the most-usedherbicide since the implementation of transgenic crops in agriculture(Benbrook 2016) Such an industrial production regime contrasts with theagroecological context of peasant indigenous andor artisan maize produc-tion and transformation in Mexico Hence transgenes can be used to eval-uate putative alternative pathways of imported grain leading to food itemsdestined for human consumption We focused our sampling in the region ofthe Mexican Altiplano Central where the majority of the countryrsquos popula-tion lives but also where the various pathways of maize production andprocessing intersect and comingle most intensely We compared foods fromthis region with food products from other areas in Mexico and abroad

We hypothesized that transgenic DNA markers and glyphosate should befound in low frequencies in food samples because a) transgenic maize is notauthorized for commercial planting in Mexico although previous reportshave confirmed the existence of (trans) gene flow into native varieties insome localities (Dyer et al 2009 Pintildeeyro-Nelson et al 2009 Serratos et al2007 Quist and Chapela 2001) and b) imported maize grain is derived fromyellow maize varieties which are supposed to be used primarily for animalfeed and industrial uses (Ministry of Economy 2012)

In contrast we found presence of transgenic DNA markers in 82 of thesamples analyzed as well as glyphosate in 277 of samples positive for DNAmarkers that confer resistance to this herbicide Furthermore the distribution ofpositive samples across food categories surveyed here was indistinguishablefrom the distribution found in maize-based food samples produced abroad

Materials and methods

Samples

Between the years 2013ndash2015 367 different maize-based food items were col-lected in Mexico and elsewhere in the Americas and Europe Sampling wasfocused on representing the highly complex maize food market in the MexicoCity Metropolitan area comprising maize-based food sold by large food retai-lers small neighborhood tortilla factories (tortilleriacuteas) as well as small-scaleArtisan food manufacturers Our sampling strategy was designed with twomain goals 1) to provide a picture of the food landscape as actually experiencedby consumers in the most populated area of Mexico and 2) as a way of accessinga food system expected to have mostly mixed maize inputs derived from bothMexican industrial agriculture as well as agroecological systems To be included

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 3

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in the sampling the food items had to contain maize as the main (gt90 byweight) ingredient In order to have a representative sampling of products wecollected samples from 5 different food categories Cereal (n = 34) Flour(n = 30) Snack (n = 64) Tortilla (n = 209) and Tostadas (n = 30) (Table 1)The bias for tortilla samples a thin unleavened baked food item was introducedbecause this is by far the most common food item through which Mexicansconsume maize Approximately 82 of Mexican households include tortillas intheir daily diet (INEGI 2010) Tostada is the name for an oven-toasted or deep-fried tortilla In addition maize-based food items were also collected from 9different countries in the Americas and Europe (Table 3)

Molecular analyses

We used PCR-based techniques to detect the presence or absence of eighttransgenic markers The primary markers where the 35S promoter from theCauliflower Mosaic Virus (CaMV) gene and the terminator from the Nopaline-synthase (T-NOS) gene DNA sequences Taken together these markers arepresent in more than 80 of the commercially available transgenic maize linesworldwide (James 2015) We also assayed for six maize-specific transgenicevents Bt11 Bt176 GA21 MON810 NK603 and TC1507 We used as positivecontrols certified reference materials consisting of maize flour developed by theInstitute of Reference Materials and Methods (IRMM Geel Belgium) Thematerials used were ERM-BF411 ERM-418 ERM-BF414 ERM-BF412 ERM-BF415 ERM-BF413 corresponding to specific transgenic events for maizeBt176 (SYN-EV176-9) TC1507 (DAS-Oslash15Oslash7-1) GA21 (MON- OslashOslash21-9)Bt11 (SYN-BTOslash11-1) NK603 (MON-OslashOslash6Oslash3-6) and MON810 (MON-OslashOslash81Oslash-6) respectively There are 68 transgenic maize events approved forconsumption in Mexico 48 events are events with stacked transgenes while 20are single transgene events Moreover 60 out of 68 (882) of the maizetransgenic events approved in Mexico contain the CaMV 35S promoterwhile 54 out of 68 (794) contain the T-NOS sequence (Center forEnvironmental Risk Assessment 2015) The six maize-specific transgenic eventsanalyzed here comprise the first events approved in Mexico (year of approval2002 for NK603 MON810 GA21 2003 for TC1507 and 2007 for Bt11)Currently these events and their combinations are present in ldquostacked trans-generdquo varieties thus allowing for the detection of 70 of transgenic maizeevents approved worldwide and all the transgenic maize events approved inMexico up to 2015 (CIBIOGEM 2015)

DNA extraction

Total DNA extraction was performed using the procedure described by Doyleand Doyle (Doyle and Doyle 1987) To avoid the possibility of cross-

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contamination DNA extraction of the Certified Reference Materials (positiveand negative controls) and food samples was performed in different days afterthorough cleaning and sterilization of the materials used for extraction DNAconcentration was quantified by assessing UV absorption at 260 nm and puritywas assessed evaluating the absorption ratio at 260 and 280 nm (A260A280)using a NanoDrop Lite UV-Vis Spectrophotometer (Thermo Scientific) Goodquality DNA had A260A280 values of around 18 (Querci M Jermini M Vanden Eede 2006) Extracted DNA was stored at minus20degC until further use

GMO screening strategy

Isolated DNA was screened for the presence of the CaMV 35S promoter andthe T-NOS using real-time PCR For both the CaMV 35S promoter andT-NOS assays we used DNA extracted from ERM-BF415 (NK603) maizeflour as a positive control Samples positive for either genetic element werefurther monitored by real-time PCR for the presence of the six maize-specifictransgenic events listed above (Bt11 Bt176 GA21 MON810 NK603 andTC1507) The possibility of having samples that could amplify event-specificmarkers in the absence of CaMV 35S and T-NOS was not pursued

Quantitative real-time PCR (qpcr)

qPCR reactions were performed in a StepOne Real Time PCR System(Applied Biosystems USA) using MicroAmpreg Fast Optical 48-WellReaction Plate (01mL) (Applied Biosystems USA) following the manufac-turerrsquos protocols Three PCR reactions per DNA sample for each primer setwere conducted All primers used in this study were synthesized exactly asdocumented in the EU Reference Methods for GMO Analysis described inthe GMO Detection Methods Database (Supplementary Table 1)(Supplementary Materials and Methods)

Determination of GM maize content in unknown samples

GM maize content in unknown samples was determined with relative quantifi-cation methods based on the absolute quantification of the endogenous gene(hmgA) (Querci Jermini and Van Den Eede 2006 Van Den Eede et al 2011)and a target-specific gene (CaMV 35S) For this assay the endogenous gene andthe target-specific gene were amplified in independent micro-tubes during thesame PCR run The relative percentage of transgenes found in a given samplewas calculated as follows We divided the concentration of the target-specifictransgene by that of the endogenous gene and then we multiplied this productby 100 (SupplementaryMaterials andMethods) Since the CaMV35S sequenceis still common to most commercially grown crops an internationally

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 5

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recognized quantitative method based on this DNAmarker has been substitutedfor assays based on the quantification of unique events (Fernandez et al 2005Holden et al 2010 Wu et al 2014) For the acceptance criteria for a positiveqPCR test see Supplementary Materials and Methods

Detection of glyphosate AMPA and glufosinate residues in food samples

A subset of positive samples for maize-specific transgenic events that areglyphosate tolerant were evaluated for the presence of this herbicide its meta-bolite Aminomethylfosfonic Acid (AMPA) and Ammonium GlufosinateAssays were performed at Eurofins GeneScan (Freiburg Germany) usingLiquid Chromatography with tandem Mass Spectrometry (LC MSMS) afteracidic extraction and derivatization of the samples (Supplementary Table 5)

Results

We detected transgene presence in a wide range of maize food productsconsumed in Mexico The CaMV 35S promoter sequence was present in 279samples (76 of all samples) while T-NOS was amplified in 301 samples(82 Table 1) The Tortilla category showed the highest percentage ofsamples positive for the CaMV 35S promoter and the T-NOS terminator(837 and 904 respectively) (Table 1) Within each food category ana-lyzed samples positive for both CaMV 35S promoter and T-NOS representedmore than 50 of all food items (Figure 1)

We quantified the presence of the CaMV 35S promoter in a subset of samplespositive for this marker (n = 107) as a means to estimate howmuch of themaizeused in the elaboration of these food products was transgenic (SupplementaryTable 3) We found that 46 out of 107 samples contained more than 5 oftransgenic material 30 out of such 46 samples corresponded to the Tortillacategory Some samples had a pattern of amplification consistent with concen-trations of transgenic material (ie CaMV 35S) above the experimental 14threshold (data not shown)

Table 1 Presence of the CaMV 35S promoter T-NOS and maize-specific transgenic events insampled foods grouped by category Numbers in brackets are percentages of the total sample

Foodcategory

Samples(n)

35Spromoter

(+)T-NOS(+)

Bt176(+)

NK603(+)

TC1507(+)

GA21(+)

Bt11(+)

MON810(+)

Tortilla 209 175 [837] 189 [904] 0 [-] 144 [689] 141 [675] 2 [096] 66 [316] 75 [359]Snack 64 41 [641] 47 [734] 0 [-] 30 [469] 18 [281] 9 [141] 16 [25] 23 [359]Flour 30 24 [80] 25 [833] 0 [-] 20 [667] 18 [60] 0 [-] 1 [33] 13 [433]Tostada 30 22 [733] 23 [767] 0 [-] 18 [60] 12 [40] 0 [-] 3 [10] 8 [267]Cereal 34 17 [50] 17 [50] 0 [-] 11 [324] 11 [324] 7 [206] 15 [441] 9 [265]TOTAL 367 279 [760] 301 [82] 0 [-] 223 [608] 200 [545] 18 [49] 101 [275] 128 [349]

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Figure 1 Frequency of the different transgenic markers detected across the entire data set(n = 367) without considering food classes (Bt176 was not detected in any food sample) (A)Frequency of the number of total events detected per instance ie the frequency of times agiven number of different markers was detected in a single food sample calculated over theentire data set (B)

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 7

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Identification of maize-specific transgenic events in food products

All samples positive for either the CaMV 35S promoter or T-NOS terminatorwere further assayed for the presence of six maize-specific transgenic eventsThe three most frequent events identified in this subsample were NK603TC1507 and MON810 with 608 545 and 349 respectively (Table 1)By contrast the least frequent event was GA21 which was found only in49 of all samples assayed for this event The transgenic event Bt176 was notdetected in any sample We surveyed this event due to reports concerning itspossible toxicity (Glockner and Seacuteralini 2016)

The distribution of positive samples for maize-specific transgenic eventswas not homogenous across food categories For instance NK603 was mostabundant in the Tortilla category with 689 of samples positive for thismarker while it was only present in 324 of samples in the Cereal category(Table 1) This heterogeneity was found in most other events (Figure 1 PanelB) When analyzing the total number of different maize-specific transgenicevents per food category the Tortilla category again had the highest numberof different events followed in descending order by the Flour Cereal Snackand Tostada categories The estimated mean values and confidence intervalsare shown in Supplementary Figure 1

Industrial maize-derived food has a greater chance of having transgenesthan artisan products

Another subsample compared 27 samples from ldquoartisanrdquo food products inthe Tortilla Flour and Tostada categories The ldquoartisanrdquo category used hererefers to food products that are putatively derived from local maize landraceswhich are further processed into food items in small facilities followingtraditional practices such as alcali-treatment of the kernels also known asldquonixtamalizationrdquo

Of the 27 artisan samples analyzed 556 resulted positive for the pre-sence of the CaMV 35S promoter while 593 resulted positive for T-NOS(Table 2) These results contrast with the rest of samples comprising indust-rially produced food products where the frequency of transgenic markerswas of 851 for the presence of CaMV 35S promoter and 913 for T-NOS(Table 2)

The artisan Tostada category encompassed the majority of samples positivefor CaMV 35S promoter (857) followed by the Tortilla category (60) Thetransgenic event NK603 was the most frequent transgenic event found in boththe industrial and artisan food products with 731 and 185 respectiveNevertheless the transgenic event Bt11 was the second most frequently trans-genic event detected in artisan food with 148 Notably the five artisan floursamples analyzed were negative for all transgenic markers

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 11

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 15

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for food production we directly evaluated the presenceabsence of eighttransgenic DNA markers as well as the chemical herbicide glyphosate inboth industrial and artisan maize-derived food items These DNA markerswere created through genetic engineering of maize destined for use inindustrial agricultural systems and glyphosate has become the most-usedherbicide since the implementation of transgenic crops in agriculture(Benbrook 2016) Such an industrial production regime contrasts with theagroecological context of peasant indigenous andor artisan maize produc-tion and transformation in Mexico Hence transgenes can be used to eval-uate putative alternative pathways of imported grain leading to food itemsdestined for human consumption We focused our sampling in the region ofthe Mexican Altiplano Central where the majority of the countryrsquos popula-tion lives but also where the various pathways of maize production andprocessing intersect and comingle most intensely We compared foods fromthis region with food products from other areas in Mexico and abroad

We hypothesized that transgenic DNA markers and glyphosate should befound in low frequencies in food samples because a) transgenic maize is notauthorized for commercial planting in Mexico although previous reportshave confirmed the existence of (trans) gene flow into native varieties insome localities (Dyer et al 2009 Pintildeeyro-Nelson et al 2009 Serratos et al2007 Quist and Chapela 2001) and b) imported maize grain is derived fromyellow maize varieties which are supposed to be used primarily for animalfeed and industrial uses (Ministry of Economy 2012)

In contrast we found presence of transgenic DNA markers in 82 of thesamples analyzed as well as glyphosate in 277 of samples positive for DNAmarkers that confer resistance to this herbicide Furthermore the distribution ofpositive samples across food categories surveyed here was indistinguishablefrom the distribution found in maize-based food samples produced abroad

Materials and methods

Samples

Between the years 2013ndash2015 367 different maize-based food items were col-lected in Mexico and elsewhere in the Americas and Europe Sampling wasfocused on representing the highly complex maize food market in the MexicoCity Metropolitan area comprising maize-based food sold by large food retai-lers small neighborhood tortilla factories (tortilleriacuteas) as well as small-scaleArtisan food manufacturers Our sampling strategy was designed with twomain goals 1) to provide a picture of the food landscape as actually experiencedby consumers in the most populated area of Mexico and 2) as a way of accessinga food system expected to have mostly mixed maize inputs derived from bothMexican industrial agriculture as well as agroecological systems To be included

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 3

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in the sampling the food items had to contain maize as the main (gt90 byweight) ingredient In order to have a representative sampling of products wecollected samples from 5 different food categories Cereal (n = 34) Flour(n = 30) Snack (n = 64) Tortilla (n = 209) and Tostadas (n = 30) (Table 1)The bias for tortilla samples a thin unleavened baked food item was introducedbecause this is by far the most common food item through which Mexicansconsume maize Approximately 82 of Mexican households include tortillas intheir daily diet (INEGI 2010) Tostada is the name for an oven-toasted or deep-fried tortilla In addition maize-based food items were also collected from 9different countries in the Americas and Europe (Table 3)

Molecular analyses

We used PCR-based techniques to detect the presence or absence of eighttransgenic markers The primary markers where the 35S promoter from theCauliflower Mosaic Virus (CaMV) gene and the terminator from the Nopaline-synthase (T-NOS) gene DNA sequences Taken together these markers arepresent in more than 80 of the commercially available transgenic maize linesworldwide (James 2015) We also assayed for six maize-specific transgenicevents Bt11 Bt176 GA21 MON810 NK603 and TC1507 We used as positivecontrols certified reference materials consisting of maize flour developed by theInstitute of Reference Materials and Methods (IRMM Geel Belgium) Thematerials used were ERM-BF411 ERM-418 ERM-BF414 ERM-BF412 ERM-BF415 ERM-BF413 corresponding to specific transgenic events for maizeBt176 (SYN-EV176-9) TC1507 (DAS-Oslash15Oslash7-1) GA21 (MON- OslashOslash21-9)Bt11 (SYN-BTOslash11-1) NK603 (MON-OslashOslash6Oslash3-6) and MON810 (MON-OslashOslash81Oslash-6) respectively There are 68 transgenic maize events approved forconsumption in Mexico 48 events are events with stacked transgenes while 20are single transgene events Moreover 60 out of 68 (882) of the maizetransgenic events approved in Mexico contain the CaMV 35S promoterwhile 54 out of 68 (794) contain the T-NOS sequence (Center forEnvironmental Risk Assessment 2015) The six maize-specific transgenic eventsanalyzed here comprise the first events approved in Mexico (year of approval2002 for NK603 MON810 GA21 2003 for TC1507 and 2007 for Bt11)Currently these events and their combinations are present in ldquostacked trans-generdquo varieties thus allowing for the detection of 70 of transgenic maizeevents approved worldwide and all the transgenic maize events approved inMexico up to 2015 (CIBIOGEM 2015)

DNA extraction

Total DNA extraction was performed using the procedure described by Doyleand Doyle (Doyle and Doyle 1987) To avoid the possibility of cross-

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contamination DNA extraction of the Certified Reference Materials (positiveand negative controls) and food samples was performed in different days afterthorough cleaning and sterilization of the materials used for extraction DNAconcentration was quantified by assessing UV absorption at 260 nm and puritywas assessed evaluating the absorption ratio at 260 and 280 nm (A260A280)using a NanoDrop Lite UV-Vis Spectrophotometer (Thermo Scientific) Goodquality DNA had A260A280 values of around 18 (Querci M Jermini M Vanden Eede 2006) Extracted DNA was stored at minus20degC until further use

GMO screening strategy

Isolated DNA was screened for the presence of the CaMV 35S promoter andthe T-NOS using real-time PCR For both the CaMV 35S promoter andT-NOS assays we used DNA extracted from ERM-BF415 (NK603) maizeflour as a positive control Samples positive for either genetic element werefurther monitored by real-time PCR for the presence of the six maize-specifictransgenic events listed above (Bt11 Bt176 GA21 MON810 NK603 andTC1507) The possibility of having samples that could amplify event-specificmarkers in the absence of CaMV 35S and T-NOS was not pursued

Quantitative real-time PCR (qpcr)

qPCR reactions were performed in a StepOne Real Time PCR System(Applied Biosystems USA) using MicroAmpreg Fast Optical 48-WellReaction Plate (01mL) (Applied Biosystems USA) following the manufac-turerrsquos protocols Three PCR reactions per DNA sample for each primer setwere conducted All primers used in this study were synthesized exactly asdocumented in the EU Reference Methods for GMO Analysis described inthe GMO Detection Methods Database (Supplementary Table 1)(Supplementary Materials and Methods)

Determination of GM maize content in unknown samples

GM maize content in unknown samples was determined with relative quantifi-cation methods based on the absolute quantification of the endogenous gene(hmgA) (Querci Jermini and Van Den Eede 2006 Van Den Eede et al 2011)and a target-specific gene (CaMV 35S) For this assay the endogenous gene andthe target-specific gene were amplified in independent micro-tubes during thesame PCR run The relative percentage of transgenes found in a given samplewas calculated as follows We divided the concentration of the target-specifictransgene by that of the endogenous gene and then we multiplied this productby 100 (SupplementaryMaterials andMethods) Since the CaMV35S sequenceis still common to most commercially grown crops an internationally

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 5

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recognized quantitative method based on this DNAmarker has been substitutedfor assays based on the quantification of unique events (Fernandez et al 2005Holden et al 2010 Wu et al 2014) For the acceptance criteria for a positiveqPCR test see Supplementary Materials and Methods

Detection of glyphosate AMPA and glufosinate residues in food samples

A subset of positive samples for maize-specific transgenic events that areglyphosate tolerant were evaluated for the presence of this herbicide its meta-bolite Aminomethylfosfonic Acid (AMPA) and Ammonium GlufosinateAssays were performed at Eurofins GeneScan (Freiburg Germany) usingLiquid Chromatography with tandem Mass Spectrometry (LC MSMS) afteracidic extraction and derivatization of the samples (Supplementary Table 5)

Results

We detected transgene presence in a wide range of maize food productsconsumed in Mexico The CaMV 35S promoter sequence was present in 279samples (76 of all samples) while T-NOS was amplified in 301 samples(82 Table 1) The Tortilla category showed the highest percentage ofsamples positive for the CaMV 35S promoter and the T-NOS terminator(837 and 904 respectively) (Table 1) Within each food category ana-lyzed samples positive for both CaMV 35S promoter and T-NOS representedmore than 50 of all food items (Figure 1)

We quantified the presence of the CaMV 35S promoter in a subset of samplespositive for this marker (n = 107) as a means to estimate howmuch of themaizeused in the elaboration of these food products was transgenic (SupplementaryTable 3) We found that 46 out of 107 samples contained more than 5 oftransgenic material 30 out of such 46 samples corresponded to the Tortillacategory Some samples had a pattern of amplification consistent with concen-trations of transgenic material (ie CaMV 35S) above the experimental 14threshold (data not shown)

Table 1 Presence of the CaMV 35S promoter T-NOS and maize-specific transgenic events insampled foods grouped by category Numbers in brackets are percentages of the total sample

Foodcategory

Samples(n)

35Spromoter

(+)T-NOS(+)

Bt176(+)

NK603(+)

TC1507(+)

GA21(+)

Bt11(+)

MON810(+)

Tortilla 209 175 [837] 189 [904] 0 [-] 144 [689] 141 [675] 2 [096] 66 [316] 75 [359]Snack 64 41 [641] 47 [734] 0 [-] 30 [469] 18 [281] 9 [141] 16 [25] 23 [359]Flour 30 24 [80] 25 [833] 0 [-] 20 [667] 18 [60] 0 [-] 1 [33] 13 [433]Tostada 30 22 [733] 23 [767] 0 [-] 18 [60] 12 [40] 0 [-] 3 [10] 8 [267]Cereal 34 17 [50] 17 [50] 0 [-] 11 [324] 11 [324] 7 [206] 15 [441] 9 [265]TOTAL 367 279 [760] 301 [82] 0 [-] 223 [608] 200 [545] 18 [49] 101 [275] 128 [349]

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Figure 1 Frequency of the different transgenic markers detected across the entire data set(n = 367) without considering food classes (Bt176 was not detected in any food sample) (A)Frequency of the number of total events detected per instance ie the frequency of times agiven number of different markers was detected in a single food sample calculated over theentire data set (B)

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Identification of maize-specific transgenic events in food products

All samples positive for either the CaMV 35S promoter or T-NOS terminatorwere further assayed for the presence of six maize-specific transgenic eventsThe three most frequent events identified in this subsample were NK603TC1507 and MON810 with 608 545 and 349 respectively (Table 1)By contrast the least frequent event was GA21 which was found only in49 of all samples assayed for this event The transgenic event Bt176 was notdetected in any sample We surveyed this event due to reports concerning itspossible toxicity (Glockner and Seacuteralini 2016)

The distribution of positive samples for maize-specific transgenic eventswas not homogenous across food categories For instance NK603 was mostabundant in the Tortilla category with 689 of samples positive for thismarker while it was only present in 324 of samples in the Cereal category(Table 1) This heterogeneity was found in most other events (Figure 1 PanelB) When analyzing the total number of different maize-specific transgenicevents per food category the Tortilla category again had the highest numberof different events followed in descending order by the Flour Cereal Snackand Tostada categories The estimated mean values and confidence intervalsare shown in Supplementary Figure 1

Industrial maize-derived food has a greater chance of having transgenesthan artisan products

Another subsample compared 27 samples from ldquoartisanrdquo food products inthe Tortilla Flour and Tostada categories The ldquoartisanrdquo category used hererefers to food products that are putatively derived from local maize landraceswhich are further processed into food items in small facilities followingtraditional practices such as alcali-treatment of the kernels also known asldquonixtamalizationrdquo

Of the 27 artisan samples analyzed 556 resulted positive for the pre-sence of the CaMV 35S promoter while 593 resulted positive for T-NOS(Table 2) These results contrast with the rest of samples comprising indust-rially produced food products where the frequency of transgenic markerswas of 851 for the presence of CaMV 35S promoter and 913 for T-NOS(Table 2)

The artisan Tostada category encompassed the majority of samples positivefor CaMV 35S promoter (857) followed by the Tortilla category (60) Thetransgenic event NK603 was the most frequent transgenic event found in boththe industrial and artisan food products with 731 and 185 respectiveNevertheless the transgenic event Bt11 was the second most frequently trans-genic event detected in artisan food with 148 Notably the five artisan floursamples analyzed were negative for all transgenic markers

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 9

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 11

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 15

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in the sampling the food items had to contain maize as the main (gt90 byweight) ingredient In order to have a representative sampling of products wecollected samples from 5 different food categories Cereal (n = 34) Flour(n = 30) Snack (n = 64) Tortilla (n = 209) and Tostadas (n = 30) (Table 1)The bias for tortilla samples a thin unleavened baked food item was introducedbecause this is by far the most common food item through which Mexicansconsume maize Approximately 82 of Mexican households include tortillas intheir daily diet (INEGI 2010) Tostada is the name for an oven-toasted or deep-fried tortilla In addition maize-based food items were also collected from 9different countries in the Americas and Europe (Table 3)

Molecular analyses

We used PCR-based techniques to detect the presence or absence of eighttransgenic markers The primary markers where the 35S promoter from theCauliflower Mosaic Virus (CaMV) gene and the terminator from the Nopaline-synthase (T-NOS) gene DNA sequences Taken together these markers arepresent in more than 80 of the commercially available transgenic maize linesworldwide (James 2015) We also assayed for six maize-specific transgenicevents Bt11 Bt176 GA21 MON810 NK603 and TC1507 We used as positivecontrols certified reference materials consisting of maize flour developed by theInstitute of Reference Materials and Methods (IRMM Geel Belgium) Thematerials used were ERM-BF411 ERM-418 ERM-BF414 ERM-BF412 ERM-BF415 ERM-BF413 corresponding to specific transgenic events for maizeBt176 (SYN-EV176-9) TC1507 (DAS-Oslash15Oslash7-1) GA21 (MON- OslashOslash21-9)Bt11 (SYN-BTOslash11-1) NK603 (MON-OslashOslash6Oslash3-6) and MON810 (MON-OslashOslash81Oslash-6) respectively There are 68 transgenic maize events approved forconsumption in Mexico 48 events are events with stacked transgenes while 20are single transgene events Moreover 60 out of 68 (882) of the maizetransgenic events approved in Mexico contain the CaMV 35S promoterwhile 54 out of 68 (794) contain the T-NOS sequence (Center forEnvironmental Risk Assessment 2015) The six maize-specific transgenic eventsanalyzed here comprise the first events approved in Mexico (year of approval2002 for NK603 MON810 GA21 2003 for TC1507 and 2007 for Bt11)Currently these events and their combinations are present in ldquostacked trans-generdquo varieties thus allowing for the detection of 70 of transgenic maizeevents approved worldwide and all the transgenic maize events approved inMexico up to 2015 (CIBIOGEM 2015)

DNA extraction

Total DNA extraction was performed using the procedure described by Doyleand Doyle (Doyle and Doyle 1987) To avoid the possibility of cross-

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contamination DNA extraction of the Certified Reference Materials (positiveand negative controls) and food samples was performed in different days afterthorough cleaning and sterilization of the materials used for extraction DNAconcentration was quantified by assessing UV absorption at 260 nm and puritywas assessed evaluating the absorption ratio at 260 and 280 nm (A260A280)using a NanoDrop Lite UV-Vis Spectrophotometer (Thermo Scientific) Goodquality DNA had A260A280 values of around 18 (Querci M Jermini M Vanden Eede 2006) Extracted DNA was stored at minus20degC until further use

GMO screening strategy

Isolated DNA was screened for the presence of the CaMV 35S promoter andthe T-NOS using real-time PCR For both the CaMV 35S promoter andT-NOS assays we used DNA extracted from ERM-BF415 (NK603) maizeflour as a positive control Samples positive for either genetic element werefurther monitored by real-time PCR for the presence of the six maize-specifictransgenic events listed above (Bt11 Bt176 GA21 MON810 NK603 andTC1507) The possibility of having samples that could amplify event-specificmarkers in the absence of CaMV 35S and T-NOS was not pursued

Quantitative real-time PCR (qpcr)

qPCR reactions were performed in a StepOne Real Time PCR System(Applied Biosystems USA) using MicroAmpreg Fast Optical 48-WellReaction Plate (01mL) (Applied Biosystems USA) following the manufac-turerrsquos protocols Three PCR reactions per DNA sample for each primer setwere conducted All primers used in this study were synthesized exactly asdocumented in the EU Reference Methods for GMO Analysis described inthe GMO Detection Methods Database (Supplementary Table 1)(Supplementary Materials and Methods)

Determination of GM maize content in unknown samples

GM maize content in unknown samples was determined with relative quantifi-cation methods based on the absolute quantification of the endogenous gene(hmgA) (Querci Jermini and Van Den Eede 2006 Van Den Eede et al 2011)and a target-specific gene (CaMV 35S) For this assay the endogenous gene andthe target-specific gene were amplified in independent micro-tubes during thesame PCR run The relative percentage of transgenes found in a given samplewas calculated as follows We divided the concentration of the target-specifictransgene by that of the endogenous gene and then we multiplied this productby 100 (SupplementaryMaterials andMethods) Since the CaMV35S sequenceis still common to most commercially grown crops an internationally

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 5

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recognized quantitative method based on this DNAmarker has been substitutedfor assays based on the quantification of unique events (Fernandez et al 2005Holden et al 2010 Wu et al 2014) For the acceptance criteria for a positiveqPCR test see Supplementary Materials and Methods

Detection of glyphosate AMPA and glufosinate residues in food samples

A subset of positive samples for maize-specific transgenic events that areglyphosate tolerant were evaluated for the presence of this herbicide its meta-bolite Aminomethylfosfonic Acid (AMPA) and Ammonium GlufosinateAssays were performed at Eurofins GeneScan (Freiburg Germany) usingLiquid Chromatography with tandem Mass Spectrometry (LC MSMS) afteracidic extraction and derivatization of the samples (Supplementary Table 5)

Results

We detected transgene presence in a wide range of maize food productsconsumed in Mexico The CaMV 35S promoter sequence was present in 279samples (76 of all samples) while T-NOS was amplified in 301 samples(82 Table 1) The Tortilla category showed the highest percentage ofsamples positive for the CaMV 35S promoter and the T-NOS terminator(837 and 904 respectively) (Table 1) Within each food category ana-lyzed samples positive for both CaMV 35S promoter and T-NOS representedmore than 50 of all food items (Figure 1)

We quantified the presence of the CaMV 35S promoter in a subset of samplespositive for this marker (n = 107) as a means to estimate howmuch of themaizeused in the elaboration of these food products was transgenic (SupplementaryTable 3) We found that 46 out of 107 samples contained more than 5 oftransgenic material 30 out of such 46 samples corresponded to the Tortillacategory Some samples had a pattern of amplification consistent with concen-trations of transgenic material (ie CaMV 35S) above the experimental 14threshold (data not shown)

Table 1 Presence of the CaMV 35S promoter T-NOS and maize-specific transgenic events insampled foods grouped by category Numbers in brackets are percentages of the total sample

Foodcategory

Samples(n)

35Spromoter

(+)T-NOS(+)

Bt176(+)

NK603(+)

TC1507(+)

GA21(+)

Bt11(+)

MON810(+)

Tortilla 209 175 [837] 189 [904] 0 [-] 144 [689] 141 [675] 2 [096] 66 [316] 75 [359]Snack 64 41 [641] 47 [734] 0 [-] 30 [469] 18 [281] 9 [141] 16 [25] 23 [359]Flour 30 24 [80] 25 [833] 0 [-] 20 [667] 18 [60] 0 [-] 1 [33] 13 [433]Tostada 30 22 [733] 23 [767] 0 [-] 18 [60] 12 [40] 0 [-] 3 [10] 8 [267]Cereal 34 17 [50] 17 [50] 0 [-] 11 [324] 11 [324] 7 [206] 15 [441] 9 [265]TOTAL 367 279 [760] 301 [82] 0 [-] 223 [608] 200 [545] 18 [49] 101 [275] 128 [349]

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Figure 1 Frequency of the different transgenic markers detected across the entire data set(n = 367) without considering food classes (Bt176 was not detected in any food sample) (A)Frequency of the number of total events detected per instance ie the frequency of times agiven number of different markers was detected in a single food sample calculated over theentire data set (B)

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Identification of maize-specific transgenic events in food products

All samples positive for either the CaMV 35S promoter or T-NOS terminatorwere further assayed for the presence of six maize-specific transgenic eventsThe three most frequent events identified in this subsample were NK603TC1507 and MON810 with 608 545 and 349 respectively (Table 1)By contrast the least frequent event was GA21 which was found only in49 of all samples assayed for this event The transgenic event Bt176 was notdetected in any sample We surveyed this event due to reports concerning itspossible toxicity (Glockner and Seacuteralini 2016)

The distribution of positive samples for maize-specific transgenic eventswas not homogenous across food categories For instance NK603 was mostabundant in the Tortilla category with 689 of samples positive for thismarker while it was only present in 324 of samples in the Cereal category(Table 1) This heterogeneity was found in most other events (Figure 1 PanelB) When analyzing the total number of different maize-specific transgenicevents per food category the Tortilla category again had the highest numberof different events followed in descending order by the Flour Cereal Snackand Tostada categories The estimated mean values and confidence intervalsare shown in Supplementary Figure 1

Industrial maize-derived food has a greater chance of having transgenesthan artisan products

Another subsample compared 27 samples from ldquoartisanrdquo food products inthe Tortilla Flour and Tostada categories The ldquoartisanrdquo category used hererefers to food products that are putatively derived from local maize landraceswhich are further processed into food items in small facilities followingtraditional practices such as alcali-treatment of the kernels also known asldquonixtamalizationrdquo

Of the 27 artisan samples analyzed 556 resulted positive for the pre-sence of the CaMV 35S promoter while 593 resulted positive for T-NOS(Table 2) These results contrast with the rest of samples comprising indust-rially produced food products where the frequency of transgenic markerswas of 851 for the presence of CaMV 35S promoter and 913 for T-NOS(Table 2)

The artisan Tostada category encompassed the majority of samples positivefor CaMV 35S promoter (857) followed by the Tortilla category (60) Thetransgenic event NK603 was the most frequent transgenic event found in boththe industrial and artisan food products with 731 and 185 respectiveNevertheless the transgenic event Bt11 was the second most frequently trans-genic event detected in artisan food with 148 Notably the five artisan floursamples analyzed were negative for all transgenic markers

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 9

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 11

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 13

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

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contamination DNA extraction of the Certified Reference Materials (positiveand negative controls) and food samples was performed in different days afterthorough cleaning and sterilization of the materials used for extraction DNAconcentration was quantified by assessing UV absorption at 260 nm and puritywas assessed evaluating the absorption ratio at 260 and 280 nm (A260A280)using a NanoDrop Lite UV-Vis Spectrophotometer (Thermo Scientific) Goodquality DNA had A260A280 values of around 18 (Querci M Jermini M Vanden Eede 2006) Extracted DNA was stored at minus20degC until further use

GMO screening strategy

Isolated DNA was screened for the presence of the CaMV 35S promoter andthe T-NOS using real-time PCR For both the CaMV 35S promoter andT-NOS assays we used DNA extracted from ERM-BF415 (NK603) maizeflour as a positive control Samples positive for either genetic element werefurther monitored by real-time PCR for the presence of the six maize-specifictransgenic events listed above (Bt11 Bt176 GA21 MON810 NK603 andTC1507) The possibility of having samples that could amplify event-specificmarkers in the absence of CaMV 35S and T-NOS was not pursued

Quantitative real-time PCR (qpcr)

qPCR reactions were performed in a StepOne Real Time PCR System(Applied Biosystems USA) using MicroAmpreg Fast Optical 48-WellReaction Plate (01mL) (Applied Biosystems USA) following the manufac-turerrsquos protocols Three PCR reactions per DNA sample for each primer setwere conducted All primers used in this study were synthesized exactly asdocumented in the EU Reference Methods for GMO Analysis described inthe GMO Detection Methods Database (Supplementary Table 1)(Supplementary Materials and Methods)

Determination of GM maize content in unknown samples

GM maize content in unknown samples was determined with relative quantifi-cation methods based on the absolute quantification of the endogenous gene(hmgA) (Querci Jermini and Van Den Eede 2006 Van Den Eede et al 2011)and a target-specific gene (CaMV 35S) For this assay the endogenous gene andthe target-specific gene were amplified in independent micro-tubes during thesame PCR run The relative percentage of transgenes found in a given samplewas calculated as follows We divided the concentration of the target-specifictransgene by that of the endogenous gene and then we multiplied this productby 100 (SupplementaryMaterials andMethods) Since the CaMV35S sequenceis still common to most commercially grown crops an internationally

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 5

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recognized quantitative method based on this DNAmarker has been substitutedfor assays based on the quantification of unique events (Fernandez et al 2005Holden et al 2010 Wu et al 2014) For the acceptance criteria for a positiveqPCR test see Supplementary Materials and Methods

Detection of glyphosate AMPA and glufosinate residues in food samples

A subset of positive samples for maize-specific transgenic events that areglyphosate tolerant were evaluated for the presence of this herbicide its meta-bolite Aminomethylfosfonic Acid (AMPA) and Ammonium GlufosinateAssays were performed at Eurofins GeneScan (Freiburg Germany) usingLiquid Chromatography with tandem Mass Spectrometry (LC MSMS) afteracidic extraction and derivatization of the samples (Supplementary Table 5)

Results

We detected transgene presence in a wide range of maize food productsconsumed in Mexico The CaMV 35S promoter sequence was present in 279samples (76 of all samples) while T-NOS was amplified in 301 samples(82 Table 1) The Tortilla category showed the highest percentage ofsamples positive for the CaMV 35S promoter and the T-NOS terminator(837 and 904 respectively) (Table 1) Within each food category ana-lyzed samples positive for both CaMV 35S promoter and T-NOS representedmore than 50 of all food items (Figure 1)

We quantified the presence of the CaMV 35S promoter in a subset of samplespositive for this marker (n = 107) as a means to estimate howmuch of themaizeused in the elaboration of these food products was transgenic (SupplementaryTable 3) We found that 46 out of 107 samples contained more than 5 oftransgenic material 30 out of such 46 samples corresponded to the Tortillacategory Some samples had a pattern of amplification consistent with concen-trations of transgenic material (ie CaMV 35S) above the experimental 14threshold (data not shown)

Table 1 Presence of the CaMV 35S promoter T-NOS and maize-specific transgenic events insampled foods grouped by category Numbers in brackets are percentages of the total sample

Foodcategory

Samples(n)

35Spromoter

(+)T-NOS(+)

Bt176(+)

NK603(+)

TC1507(+)

GA21(+)

Bt11(+)

MON810(+)

Tortilla 209 175 [837] 189 [904] 0 [-] 144 [689] 141 [675] 2 [096] 66 [316] 75 [359]Snack 64 41 [641] 47 [734] 0 [-] 30 [469] 18 [281] 9 [141] 16 [25] 23 [359]Flour 30 24 [80] 25 [833] 0 [-] 20 [667] 18 [60] 0 [-] 1 [33] 13 [433]Tostada 30 22 [733] 23 [767] 0 [-] 18 [60] 12 [40] 0 [-] 3 [10] 8 [267]Cereal 34 17 [50] 17 [50] 0 [-] 11 [324] 11 [324] 7 [206] 15 [441] 9 [265]TOTAL 367 279 [760] 301 [82] 0 [-] 223 [608] 200 [545] 18 [49] 101 [275] 128 [349]

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Figure 1 Frequency of the different transgenic markers detected across the entire data set(n = 367) without considering food classes (Bt176 was not detected in any food sample) (A)Frequency of the number of total events detected per instance ie the frequency of times agiven number of different markers was detected in a single food sample calculated over theentire data set (B)

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Identification of maize-specific transgenic events in food products

All samples positive for either the CaMV 35S promoter or T-NOS terminatorwere further assayed for the presence of six maize-specific transgenic eventsThe three most frequent events identified in this subsample were NK603TC1507 and MON810 with 608 545 and 349 respectively (Table 1)By contrast the least frequent event was GA21 which was found only in49 of all samples assayed for this event The transgenic event Bt176 was notdetected in any sample We surveyed this event due to reports concerning itspossible toxicity (Glockner and Seacuteralini 2016)

The distribution of positive samples for maize-specific transgenic eventswas not homogenous across food categories For instance NK603 was mostabundant in the Tortilla category with 689 of samples positive for thismarker while it was only present in 324 of samples in the Cereal category(Table 1) This heterogeneity was found in most other events (Figure 1 PanelB) When analyzing the total number of different maize-specific transgenicevents per food category the Tortilla category again had the highest numberof different events followed in descending order by the Flour Cereal Snackand Tostada categories The estimated mean values and confidence intervalsare shown in Supplementary Figure 1

Industrial maize-derived food has a greater chance of having transgenesthan artisan products

Another subsample compared 27 samples from ldquoartisanrdquo food products inthe Tortilla Flour and Tostada categories The ldquoartisanrdquo category used hererefers to food products that are putatively derived from local maize landraceswhich are further processed into food items in small facilities followingtraditional practices such as alcali-treatment of the kernels also known asldquonixtamalizationrdquo

Of the 27 artisan samples analyzed 556 resulted positive for the pre-sence of the CaMV 35S promoter while 593 resulted positive for T-NOS(Table 2) These results contrast with the rest of samples comprising indust-rially produced food products where the frequency of transgenic markerswas of 851 for the presence of CaMV 35S promoter and 913 for T-NOS(Table 2)

The artisan Tostada category encompassed the majority of samples positivefor CaMV 35S promoter (857) followed by the Tortilla category (60) Thetransgenic event NK603 was the most frequent transgenic event found in boththe industrial and artisan food products with 731 and 185 respectiveNevertheless the transgenic event Bt11 was the second most frequently trans-genic event detected in artisan food with 148 Notably the five artisan floursamples analyzed were negative for all transgenic markers

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 9

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 11

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 13

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

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recognized quantitative method based on this DNAmarker has been substitutedfor assays based on the quantification of unique events (Fernandez et al 2005Holden et al 2010 Wu et al 2014) For the acceptance criteria for a positiveqPCR test see Supplementary Materials and Methods

Detection of glyphosate AMPA and glufosinate residues in food samples

A subset of positive samples for maize-specific transgenic events that areglyphosate tolerant were evaluated for the presence of this herbicide its meta-bolite Aminomethylfosfonic Acid (AMPA) and Ammonium GlufosinateAssays were performed at Eurofins GeneScan (Freiburg Germany) usingLiquid Chromatography with tandem Mass Spectrometry (LC MSMS) afteracidic extraction and derivatization of the samples (Supplementary Table 5)

Results

We detected transgene presence in a wide range of maize food productsconsumed in Mexico The CaMV 35S promoter sequence was present in 279samples (76 of all samples) while T-NOS was amplified in 301 samples(82 Table 1) The Tortilla category showed the highest percentage ofsamples positive for the CaMV 35S promoter and the T-NOS terminator(837 and 904 respectively) (Table 1) Within each food category ana-lyzed samples positive for both CaMV 35S promoter and T-NOS representedmore than 50 of all food items (Figure 1)

We quantified the presence of the CaMV 35S promoter in a subset of samplespositive for this marker (n = 107) as a means to estimate howmuch of themaizeused in the elaboration of these food products was transgenic (SupplementaryTable 3) We found that 46 out of 107 samples contained more than 5 oftransgenic material 30 out of such 46 samples corresponded to the Tortillacategory Some samples had a pattern of amplification consistent with concen-trations of transgenic material (ie CaMV 35S) above the experimental 14threshold (data not shown)

Table 1 Presence of the CaMV 35S promoter T-NOS and maize-specific transgenic events insampled foods grouped by category Numbers in brackets are percentages of the total sample

Foodcategory

Samples(n)

35Spromoter

(+)T-NOS(+)

Bt176(+)

NK603(+)

TC1507(+)

GA21(+)

Bt11(+)

MON810(+)

Tortilla 209 175 [837] 189 [904] 0 [-] 144 [689] 141 [675] 2 [096] 66 [316] 75 [359]Snack 64 41 [641] 47 [734] 0 [-] 30 [469] 18 [281] 9 [141] 16 [25] 23 [359]Flour 30 24 [80] 25 [833] 0 [-] 20 [667] 18 [60] 0 [-] 1 [33] 13 [433]Tostada 30 22 [733] 23 [767] 0 [-] 18 [60] 12 [40] 0 [-] 3 [10] 8 [267]Cereal 34 17 [50] 17 [50] 0 [-] 11 [324] 11 [324] 7 [206] 15 [441] 9 [265]TOTAL 367 279 [760] 301 [82] 0 [-] 223 [608] 200 [545] 18 [49] 101 [275] 128 [349]

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Figure 1 Frequency of the different transgenic markers detected across the entire data set(n = 367) without considering food classes (Bt176 was not detected in any food sample) (A)Frequency of the number of total events detected per instance ie the frequency of times agiven number of different markers was detected in a single food sample calculated over theentire data set (B)

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Identification of maize-specific transgenic events in food products

All samples positive for either the CaMV 35S promoter or T-NOS terminatorwere further assayed for the presence of six maize-specific transgenic eventsThe three most frequent events identified in this subsample were NK603TC1507 and MON810 with 608 545 and 349 respectively (Table 1)By contrast the least frequent event was GA21 which was found only in49 of all samples assayed for this event The transgenic event Bt176 was notdetected in any sample We surveyed this event due to reports concerning itspossible toxicity (Glockner and Seacuteralini 2016)

The distribution of positive samples for maize-specific transgenic eventswas not homogenous across food categories For instance NK603 was mostabundant in the Tortilla category with 689 of samples positive for thismarker while it was only present in 324 of samples in the Cereal category(Table 1) This heterogeneity was found in most other events (Figure 1 PanelB) When analyzing the total number of different maize-specific transgenicevents per food category the Tortilla category again had the highest numberof different events followed in descending order by the Flour Cereal Snackand Tostada categories The estimated mean values and confidence intervalsare shown in Supplementary Figure 1

Industrial maize-derived food has a greater chance of having transgenesthan artisan products

Another subsample compared 27 samples from ldquoartisanrdquo food products inthe Tortilla Flour and Tostada categories The ldquoartisanrdquo category used hererefers to food products that are putatively derived from local maize landraceswhich are further processed into food items in small facilities followingtraditional practices such as alcali-treatment of the kernels also known asldquonixtamalizationrdquo

Of the 27 artisan samples analyzed 556 resulted positive for the pre-sence of the CaMV 35S promoter while 593 resulted positive for T-NOS(Table 2) These results contrast with the rest of samples comprising indust-rially produced food products where the frequency of transgenic markerswas of 851 for the presence of CaMV 35S promoter and 913 for T-NOS(Table 2)

The artisan Tostada category encompassed the majority of samples positivefor CaMV 35S promoter (857) followed by the Tortilla category (60) Thetransgenic event NK603 was the most frequent transgenic event found in boththe industrial and artisan food products with 731 and 185 respectiveNevertheless the transgenic event Bt11 was the second most frequently trans-genic event detected in artisan food with 148 Notably the five artisan floursamples analyzed were negative for all transgenic markers

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 9

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

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Figure 1 Frequency of the different transgenic markers detected across the entire data set(n = 367) without considering food classes (Bt176 was not detected in any food sample) (A)Frequency of the number of total events detected per instance ie the frequency of times agiven number of different markers was detected in a single food sample calculated over theentire data set (B)

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Identification of maize-specific transgenic events in food products

All samples positive for either the CaMV 35S promoter or T-NOS terminatorwere further assayed for the presence of six maize-specific transgenic eventsThe three most frequent events identified in this subsample were NK603TC1507 and MON810 with 608 545 and 349 respectively (Table 1)By contrast the least frequent event was GA21 which was found only in49 of all samples assayed for this event The transgenic event Bt176 was notdetected in any sample We surveyed this event due to reports concerning itspossible toxicity (Glockner and Seacuteralini 2016)

The distribution of positive samples for maize-specific transgenic eventswas not homogenous across food categories For instance NK603 was mostabundant in the Tortilla category with 689 of samples positive for thismarker while it was only present in 324 of samples in the Cereal category(Table 1) This heterogeneity was found in most other events (Figure 1 PanelB) When analyzing the total number of different maize-specific transgenicevents per food category the Tortilla category again had the highest numberof different events followed in descending order by the Flour Cereal Snackand Tostada categories The estimated mean values and confidence intervalsare shown in Supplementary Figure 1

Industrial maize-derived food has a greater chance of having transgenesthan artisan products

Another subsample compared 27 samples from ldquoartisanrdquo food products inthe Tortilla Flour and Tostada categories The ldquoartisanrdquo category used hererefers to food products that are putatively derived from local maize landraceswhich are further processed into food items in small facilities followingtraditional practices such as alcali-treatment of the kernels also known asldquonixtamalizationrdquo

Of the 27 artisan samples analyzed 556 resulted positive for the pre-sence of the CaMV 35S promoter while 593 resulted positive for T-NOS(Table 2) These results contrast with the rest of samples comprising indust-rially produced food products where the frequency of transgenic markerswas of 851 for the presence of CaMV 35S promoter and 913 for T-NOS(Table 2)

The artisan Tostada category encompassed the majority of samples positivefor CaMV 35S promoter (857) followed by the Tortilla category (60) Thetransgenic event NK603 was the most frequent transgenic event found in boththe industrial and artisan food products with 731 and 185 respectiveNevertheless the transgenic event Bt11 was the second most frequently trans-genic event detected in artisan food with 148 Notably the five artisan floursamples analyzed were negative for all transgenic markers

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 11

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 13

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

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Identification of maize-specific transgenic events in food products

All samples positive for either the CaMV 35S promoter or T-NOS terminatorwere further assayed for the presence of six maize-specific transgenic eventsThe three most frequent events identified in this subsample were NK603TC1507 and MON810 with 608 545 and 349 respectively (Table 1)By contrast the least frequent event was GA21 which was found only in49 of all samples assayed for this event The transgenic event Bt176 was notdetected in any sample We surveyed this event due to reports concerning itspossible toxicity (Glockner and Seacuteralini 2016)

The distribution of positive samples for maize-specific transgenic eventswas not homogenous across food categories For instance NK603 was mostabundant in the Tortilla category with 689 of samples positive for thismarker while it was only present in 324 of samples in the Cereal category(Table 1) This heterogeneity was found in most other events (Figure 1 PanelB) When analyzing the total number of different maize-specific transgenicevents per food category the Tortilla category again had the highest numberof different events followed in descending order by the Flour Cereal Snackand Tostada categories The estimated mean values and confidence intervalsare shown in Supplementary Figure 1

Industrial maize-derived food has a greater chance of having transgenesthan artisan products

Another subsample compared 27 samples from ldquoartisanrdquo food products inthe Tortilla Flour and Tostada categories The ldquoartisanrdquo category used hererefers to food products that are putatively derived from local maize landraceswhich are further processed into food items in small facilities followingtraditional practices such as alcali-treatment of the kernels also known asldquonixtamalizationrdquo

Of the 27 artisan samples analyzed 556 resulted positive for the pre-sence of the CaMV 35S promoter while 593 resulted positive for T-NOS(Table 2) These results contrast with the rest of samples comprising indust-rially produced food products where the frequency of transgenic markerswas of 851 for the presence of CaMV 35S promoter and 913 for T-NOS(Table 2)

The artisan Tostada category encompassed the majority of samples positivefor CaMV 35S promoter (857) followed by the Tortilla category (60) Thetransgenic event NK603 was the most frequent transgenic event found in boththe industrial and artisan food products with 731 and 185 respectiveNevertheless the transgenic event Bt11 was the second most frequently trans-genic event detected in artisan food with 148 Notably the five artisan floursamples analyzed were negative for all transgenic markers

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 9

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 11

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

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Industrial Mexican tortillas are more likely to have transgenes than anyother maize-derived food product

We analyzed the presence of transgenes between industrially produced Tortillaand artisan Tortillas (Table 2) The industrial Tortilla category encompassed themajority of samples positive for the CaMV 35S promoter and T-NOS markers(856 and 918 respectively) (Table 2) We tested whether the data presentedhere support the hypothesis that artisan Tortillas have a lower number oftransgenic events in comparison to industrial Tortillas Due to the size of thetwo samples being considerably different (Artisan n = 15 and Industrialn = 194) we performed a statistical resampling experiment (SupplementaryMaterials and Methods) We found that the mean number of transgenic eventsin artisan products is significantly lower than that observed in the same-sizedsamples simulated from the original industrial Tortilla sample thus supportingour original hypothesis In fact no single sampling event from the 10000simulated samples obtained for the industrial Tortilla group data displayed amean number of transgenic events equal or lower to that observed in the actualsample of artisan products (Supplementary Figure 2)

Presence of transgenes in maize-derived food products labeled as lsquogmo-freersquo available in mexican supermarkets

Ten samples labeled as lsquoGenetically Modified Organisms-freersquo (ldquoGMO-freerdquo)by the supplier were collected for this study Seven samples were Snackswhile three samples were Tortillas Surprisingly we found that half of theseldquoGMO-freerdquo labeled samples analyzed were-PCR positive for the presence ofeither generic or event-specific transgenic markers (Supplementary Table 4)For the Snack category (n = 7) we detected a transgenic marker in foursamples We found the CaMV 35S promoter and T-NOS in two out of threeTortilla samples but we were unable to detect maize-specific transgenicevents (Supplementary Table 4)

Table 2 Transgenic sequences in Industrial and Artisan maize foods Numbers in brackets arepercent of total sample

IndustrialSamples

(n)

CaMV 35Spromoter

(+) T-NOS (+)Bt176(+) NK603 (+) TC1507 (+) GA21 (+) Bt11 (+)

MON810(+)

Tortilla 194 166 [856] 178 [918] 0 [-] 143 [737] 141 [727] 1 [05] 62 [320] 74 [381]Flour 25 24 [960] 25 [1000] 0 [-] 20 [800] 18 [720] 0 [-] 1 [40] 13 [520]Tostada 23 16 [696] 18 [783] 0 [-] 14 [609] 11 [478] 0 [-] 3 [130] 6 [261]Total 242 206 [851] 221 [913] 0 [-] 177 [731] 170 [702] 1 [04] 66 [273] 93 [384]ArtisanTortilla 15 9 [600] 11 [733] 0 [-] 1 [67] 0 [-] 1 [67] 4 [267] 1 [67]Flour 5 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-] 0 [-]Tostada 7 6 [857] 5 [714] 0 [-] 4 [571] 1 [143] 0 [-] 0 [-] 2 [286]Total 27 15 [556] 16 [593] 0 [-] 5 [185] 1 [37] 1 [37] 4 [148] 3 [111]

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 9

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 15

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Undistinguishable frequency distribution of transgenic events betweenmexican and international maize-derived foods

To test if the transgenic maize lines found in industrialized maize-derived foodmanufactured in Mexico were similar to those used elsewhere we compared thefrequency distribution of events among samples collected in Mexico with that ofsamples collected abroad For this purpose we analyzed an additional set of 39maize-derived food products produced in South America other North Americancountries as well as Europe (Table 3) Most of the samples available were snacksand cereals 59 of the imported samples were positive for CaMV 35S promoterandor T-NOS compared with 82 of Mexican food products that were positivefor CaMV 35S promoter andor T-NOS plus at least onemaize-specific transgenicevent Transgenic events MON810 Bt11 NK603 TC1507 and GA21 weredetected in 359 308 256 205 and 103 of the imported samplesanalyzed respectively (Table 3) Focusing on the samples positive for transgenicmarkers we compared the frequency distribution of maize-specific events insamples from Mexico and those produced abroad The imported productsincluded only Cereal (n = 19) Flour (n = 2) Snack (n = 16) and Tortilla (n = 2)items thus we included only these categories for this analysis The nationallyproduced foods included Cereal (n = 34) Flour (n = 30) Snack (n = 64) andTortilla (n = 209) items Figure 2 shows the frequency distribution of the differentmarkers detected in each class of food for national (top panel A) and importedproducts (middle panel B) Due to the limited number of observations for the caseof imported Flour and Tortilla products we did not consider these two classesfurther leaving for this comparison only Cereal and Snack samples from bothnational and international origin

We estimated the difference in frequency between national and importedproducts for each marker Panel C in Figure 2 shows the estimated differ-ences and confidence intervals for each class In all cases the confidenceinterval included 0 difference (red line) Thus our data support the hypoth-esis that the frequency distribution of individual markers for maize-specificevents in the two food categories analyzed is statistically indistinguishablebetween national and imported products This suggests that overall similarsources of transgenic maize are used in Mexico and other regions of theworld to produce cereals and snacks

To further analyze this possibility we quantified the statistical similaritybetween the frequency distributions of national and imported products in theSnack and Cereal groups through two tests of rank order correlation (Figure 2Supplementary Materials and Methods) The data support the hypotheses of asignificant correlation between the samples fromMexico and those from abroadand thus of a very similar frequency distribution of transgenic events amongcereals and snacks independently of where they are sold

10 E GONZAacuteLEZ-ORTEGA ET AL

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

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Glyphosate and AMPA are present in food samples that contain herbicide-tolerant NK603 and GA21 transgenic maize events

We analyzed the presence of glyphosate its derivative AminomethylfosfonicAcid (AMPA) as well as ammonium glufosinate in a subset of samples that werepositive for herbicide-tolerant events namely NK603 and GA21 (glyphosatetolerant) and TC1507 (glufosinate tolerant) The subset included FloursTostadas Cereals and Tortillas (both industrial and artisan) Glyphosate andor AMPAwere detected in 277 of this sample subset In two out of four maizeflour samples the ranges of glyphosate went from 001 to 0045mg kgminus1 in one of

Table 3 Detection of the CaMV 35S promoter Nos terminator (T-NOS) and maize-specifictransgenic events for maize food products imported from different countriesFood Category Origin CaMV 35S T-NOS Bt176 NK603 TC1507 GA21 Bt11 MON810

Snack France + + - - - - - +Snack France + + - - - + - +Snack France + + - - - - + -Snack USA + + - + - - + +Snack Argentina + + - + - - - +Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack Spain - - - - - - - -Snack Spain + + - - - - + +Snack Spain - - - - - - - -Snack USA + + - + + - - +Snack USA - + - - - - - -Snack Spain - - - - - - - -Snack USA - + - - - - - -Snack USA + + - + + - - +Snack USA - - - - - - - -Cereal Germany - - - - - - - -Cereal Germany - - - - - - - -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Brasil - - - - - - - -Cereal Chile - - - - - - - -Cereal USA + + - - - - - -Cereal Argentina + + - - - + + -Cereal Argentina - - - - - - - -Cereal Argentina - - - - - - - -Cereal Uruguay - - - - - - - -Cereal Uruguay + + - + + - + +Cereal Uruguay + + - + - + + +Cereal Argentina + + - + + - + +Cereal Argentina + + - - + - + -Cereal Argentina + + - - - - + -Cereal Germany - - - - - - - -Cereal Argentina + + - + + + + +Cereal Germany - - - - - - - -Flour Colombia + + - + + - + +Flour Colombia + + - + + - - -Tortilla France + - - - - - - +Tortilla EUA - + - - - - - -

Positive (+) or (-) negative for a transgenic marker or maize-specific event

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 13

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

14 E GONZAacuteLEZ-ORTEGA ET AL

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 15

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these samples AMPA was also detected at 0015 mg kgminus1 (SupplementaryTable 5) Glyphosate was detected in three out of five Snack samples with onealso positive for AMPA Glyphosate was found in two out of the three Cerealsamples analyzed and in two out of 11 Industrial Tortilla samples but neither

Figure 2 Food class distribution of markers in national and imported products Frequencydistribution of the different transgenic markers assayed within each class of food for national(A) and imported products (B) Estimated bootstrap mean difference between national andimported products of the observed frequency of each marker (blue dots) in addition to thecorresponding bootstrap estimated 95 confidence intervals (horizontal black solid line) Thevertical dotted line indicates zero difference and it is the basis to either support or reject asignificant difference based on whether it overlaps with the confidence interval or not (C)

12 E GONZAacuteLEZ-ORTEGA ET AL

Dow

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187

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Sept

embe

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 13

Dow

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187

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Sept

embe

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

14 E GONZAacuteLEZ-ORTEGA ET AL

Dow

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by [

187

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841

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Sept

embe

r 20

17

Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

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glyphosate AMPA or ammonium glufosinate could be detected in any of the 9artisan Tortilla samples assayed (Supplementary Table 5)

Discussion

As a means to explore the possibility of transgenic industrialized maize enteringthe food production chain intended for human consumption we analyzed thepresence of transgenemarkers and glyphosate residues in a large and diverse arrayof food samples Our results show high abundance of transgenic maize in theMexican food chain with 82 of surveyed samples yielding positive results for atleast one transgenic marker This was unexpected on two grounds first Mexicomaintains amoratorium on open-field sowing of transgenicmaize for commercialpurposes and second most of the demand for maize destined for human con-sumption in the country is supposed to be fulfilled domestically (Ministry ofEconomy 2012)

The Flour and Tortilla categories had the highest frequencies oftransgenes As maize flour forms the basis of many of the productssurveyed in this study a high percentage of flour samples positive fortransgenes could explain the high frequency of transgene presence inother products such as industrially produced Tortillas and TostadasOther industrial food products analyzed here (for example Snacks) areusually elaborated with other types of ingredients in addition to maizesoy cotton mill or oil and canola oil mainly Some of the latter couldalso be of transgenic origin harboring genetic markers such as theCaMV 35S promoter or T-NOS but maize-specific markers used inthis study indicate that overall at least 608 of all food samplesanalyzed contain transgenic maize (Table 1)

Artisan and ldquogmo-freerdquo products sold in urban areas do containtransgenes but at lower frequencies

Industrialized food bears a higher frequency of transgenic markers than artisancounterparts and since the artisan flour samples analyzed were negative for alltransgenic markers assayed the presence of transgenes in artisan Tortillas andTostadas may be due either to the use of transgenic industrialized maize flour ordue to the presence of transgenes in the maize native varieties from which thesefoods putatively derive While previous studies have documented transgene flowinto local maize varieties at some localities (Dyer et al 2009 Pintildeeyro-Nelson et al2009 Serratos et al 2007) the estimated transgene frequency was very lowsuggesting this second scenario to be unlikely Furthermore the high similarityin the maize-specific event combinations detected in the industrial flour and themajority of transgene-positive artisan Tortillas (data not shown) strongly suggeststhat the source of transgenes is the use of industrialized flour in artisan products

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 13

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 15

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Another unexpected result was the high frequency of samples positive for trans-genes (50) among foods labelled as ldquoGMO-freerdquo (Supplementary Table 4) Thisfinding suggests that protocols aimed to assure segregation between transgenic andconventional maize are not 100 effective

Globally used transgenic lines are reaching the maize food chain at theCOD of this grain

We found that the frequency distribution of transgenic events in samples fromMexico and those from abroad were statistically indistinguishable (Figure 2)Moreover the maize transgenic events monitored in this study encompass thetotality of transgenic varieties allowed for planting inMexico for experimental andpilot trials between 2009 and 2013 (CIBIOGEM 2015 Reviewed on April 2017)Such events are also sown in the countries fromwhichMexico importsmaizeUSAand South Africa (Departament of Agriculture Forestry and Fisheries 2013) Thelatter suggests that at least a fraction of grain imports is being used to produce foodin Mexico and that the same transgenic maize grain stocks are being used forindustrial food production in several countries

Glyphosate present in the maize food chain in mexico

Our results showed that events NK603 and TC1507 which confer tolerance toglyphosate and ammonium glufosinate respectively were the most commontransgenic events across all food samples analyzed and were present both indivi-dually or combined (Table 1) In line with this finding we found glyphosate andAMPA residues in 277 of samples assayed for herbicide presence

Glyphosate-based herbicides are themostwidely used agrochemicalsworldwide(Benbrook 2016) and residual glyphosate and its metabolite AMPA are allowedfor human consumption at concentrations which can be as high as 500 ppm(Mesnage et al 2015) To our knowledge in Mexico there are no set limits forglyphosate residues in processed food and the concentration of such herbicide isnot assayed by official entities This study suggests that given the high levels ofmaize consumption inMexico the latter issue should be further considered Otherpopulations exposed to similar levels of unprocessedmaize products live in BoliviaBrazil Bosnia-Herzegovina Colombia Costa Rica Egypt Guatemala HondurasLesotho Nicaragua Timor-Leste South Africa Zambia and Zimbabwe (RanumPentildea-Rosas and Garcia-Casal 2014)

Our results imply that transgenic maize varieties produced abroad underindustrial agriculture are finding their way into the food manufacturingnetworks in Mexico Another possibility is that domestic seed stocks thatare supposed to be free of transgenic materials contain at least some GMmaize which suggests that currently applied biosafety guidelines should beprofoundly revised at the COD of maize Mexico

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 15

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Acknowledgments

This research was supported by the National Council of Science and Technology from theMexican Government (CONACYT) [Grants 240180 2015-01-687] the National AutonomousUniversity of Mexico DGAPA-PAPIIT [Grants IN 208517] Ministry of Public Education[Grant SEP-PRODEP-UAM PTC-597] and by Comisioacuten Sectorial de Investigacioacuten Cientiacutefica(CSIC) Universidad de la Repuacuteblica Uruguay CSIC VUSP2 C641-348

References

Aacutelvarez-Buylla E and A Pintildeeyro-Nelson 2013 El maiacutez en peligro ante los transgeacutenicos unanaacutelisis integral sobre el caso de Meacutexico CEIICH-UNAM UCCS Mexico 568p

Acevedo Gasman F et al 2009 La bioseguridad en Meacutexico y los organismos geneacuteticamentemodificados coacutemo enfrentar un nuevo desafiacuteo en Capital natural de Meacutexico vol II Estadode conservacioacuten y tendencias de cambio Edited by CONABIO Meacutexico pp 319ndash353

BourgesH 2013 Elmaiacutez su importancia en la alimentacioacuten de la poblacioacutenmexicana InElmaiacutezen peligro ante los transgeacutenicos Un anaacutelisis integral sobre el caso de Meacutexico Aacutelvarez-Buylla Eand Pintildeeyro-Nelson A (Eds) CEIICH-UNAM UCCS Mexico 231ndash247

Bourges H and C Carrillo Trueba 2013 El maiacutez Su importancia en la alimentacioacuten de lapoblacioacuten mexicana In El maiacutez en peligro ante los transgeacutenicos Un anaacutelisis integral sobre elcaso Meacutexico 231ndash47 Mexico UNAM

Center for Environmental Risk Assessment (2015) GM crop database httpcera-gmcorgGMCropDatabase

CIBIOGEM 2015 Solicitudes de permisos de liberacioacuten 2015 Accessed March 1 2017 httpconacytgobmxcibiogemindexphpsolicitudespermisos-de-liberacionsolicitudes-de-permisos-de-liberacion-2015

Doyle J J and J L Doyle 1987 A rapid DNA isolation procedure for small quantities offresh leaf tissue Phytochemistry Bulletin 1911ndash15

Dyer G A J A Serratos-Hernandez H R Perales P Gepts A Pineyro-Nelson A Chavezand E R Alvarez-Buylla 2009 Dispersal of transgenes through maize seed systems inMexico PLoS One 4 (5)e5734 doi101371journalpone0005734

Fernandez S C Charles-Delobel A Geldreich G Berthier F Boyer C Collonnier and MRomaniuk 2005 Quantification of the 35S promoter in DNA extracts from geneticallymodified organisms using real-time polymerase chain reaction and specificity assessmenton various genetically modified organisms part I Operating procedure InternationalJournal of AOAC International 88 (2)547ndash57

FIRA 2016 Panorama Agroalimentario Tendencias en el entorno nacional e internacional delas principales redes de valor agroalimentarias Mexico httpswwwgobmxfiradocumentospanorama-agroalimentario

Glockner G and G E Seacuteralini 2016 Pathology reports on the first cows fed with Bt176maize (1997-2002) Scholarly Journal of Agricultural Science 61ndash8

Holden M J M Levine T Scholdberg R J Haynes and G R Jenkins 2010 The use of 35Sand Tnos expression elements in the measurement of genetically engineered plant materialsAnalytical and Bioanalytical Chemistry 396 (6)2175ndash87 doi101007s00216-009-3186-x

INEGI 2010 Poblacioacuten rural y urbana httpcuentameinegiorgmxpoblacionrur_urbaspxtema=P

James C 2015 Global Status of Commercialized BiotechGM Crops 2014 Ithaca InternationalService for the Acquisition of Agri-Biotech Applications USA

AGROECOLOGY AND SUSTAINABLE FOOD SYSTEMS 15

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Kato T C L M Mera J A Serratos and R T Bye 2009 Origen Y Diversificacioacuten DelMaiacutez Una Revisioacuten Analiacutetica Edited by UNAM-CONABIO Mexico

Mercer K L H R Perales and J D Wainwright 2012 Climate change and the transgenicadaptation strategy Smallholder livelihoods climate justice and maize landraces in MexicoGlobal Environmental Change 22 (2)495ndash504 doi101016jgloenvcha201201003

Mesnage Defarge N J Spiroux de Vendemois and G E Seacuteralini 2015 Potential toxiceffects of glyphosate and its commercial formulations below regulatory limits Food andChemical Toxicology 84133ndash53 doi101016jfct201508012

Ministry of Economy 2012 Analysis of the Maize-tortilla chain value Current Situation andfactors of local competence Mexico

The Non-GMO Sourcebook 2017 The Non-GMO sourcebook Accessed July 24 2017httpwwwnongmosourcebookcom

Otero G 2011 Neoliberal Globalization NAFTA and migration Mexicorsquos loss of food andlabor sovereignty Journal of Poverty 15 (October)384ndash402 doi101080108755492011614514

Perales H and D Golicher 2014 Mapping the diversity of maize races in Mexico PloS One9 (12) 1ndash20 e114657 doi101371journalpone0114657

Pintildeeyro-Nelson A J Van Heerwaarden H R Perales J A Serratos-Hernandez A RangelM B Hufford P Gepts A Garay Arroyo R Rivera-Bustamante and E R Aacutelvarez-Buylla2009 Transgenes in Mexican maize Molecular evidence and methodological considera-tions for GMO detection in landrace populations Molecular Ecology 18 (4)750ndash61doi101111j1365-294X200803993x

Puyana A 2012 Mexican Agriculture and NAFTA A 20 year sheet Review of AgriculturalStudies 2 (1) 1ndash43

Querci M M Jermini and G Van Den Eede 2006 The analysis of food samples for thepresence of genetically modified organisms httpgmo-crljrceceuropaeucapacitybuildingmanualsManual ENUser Manual EN fullpdf

Quist D and I Chapela 2001 Transgenic DNA introgressed into traditional maize land-races in Oaxaca Mexico Nature 29 (414)541ndash43 doi10103835107068

Ranum P J P Pentildea-Rosas and M N Garcia-Casal 2014 Global maize productionutilization and consumption Annals of the New York Academy of Sciences 1312 (1)105ndash12 doi101111nyas12396

Serratos-Hernaacutendez J A J L Goacutemez-Olivares N Salinas-Arreortua E Buendiacutea-Rodriacuteguez FIslas-Gutieacuterrez amp A de-Ita (2007) Transgenic proteins in maize in the soil conservationarea of Federal District Meacutexico Frontiers in Ecology and the Environment 5(5) 247ndash252

Turrent-Fernaacutendez A Wise T and Garvey Elise 2012 Achieving Mexicos maize potentialGlobal Development and Environment Institute Working paper No12-03 TuftsUniversity Medford Ma USA httpasetuftsedugdae

Ureta C E Martiacutenez-Meyer H R Perales and E R Aacutelvarez-Buylla 2012 Projecting theeffects of climate change on the distribution of maize races and their wild relatives inMexico Global Change Biology 18 (3)1073ndash82 doi101111j1365-2486201102607x

Van Den Eede G L Bonfini L Cengia C Iannini L Kluga and M Mazzara 2011Compendium of reference methods for GMO analysesPublications Office of the EuropeanUnion Luxembourg Luxembourg httppublicationsjrceceuropaeurepositoryhandle11111111115068

Wu Y Y Wang J Li W Li L Zhang Y Li and G Wu 2014 Development of a generalmethod for detection and quantification of the P35S promoter based on assessment ofexisting methods Scientific Reports 47358 doi101038srep07358

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