1193 -glufosinate;NAFTA,North L FSANZ, Food Standards Australia -phosphinico-acetic acid; MPB, 4- ood and Agriculture Organisation; , Organisation for Economic Coop- 5-enolpyruvylshikimate-3-phosphate pean Food Safety Authority; EPA, En- ingen University and Research Centre, PP, 3-methyl-phosphinico-propanoic – Institute of Food Safety, Wageningen limentarius Commission; CCPR, Codex 2011 Society of Chemical Industry e; HRAC, Herbicide Resistance Action c -phosphinothricin N-acetyltransferase; PPO, L c ALS, acetolactate synthase; AMPA, amino- Chelmsford, Essex, UK Exponent International, Ltd, Harrogate, UK Abbreviations used: methylphosphonic acid; CAC, Codex A Committee on Pesticide Residues; DBHA,DNA, 3,5-dibromo-4-hydroxybenzoic deoxyribonucleic acid; acid; EFSA, Euro Private consultant, vironmental Protection Agency; EPSPS, synthase; EU, EuropeanFDA, Union; Food FAO, and F New Zealand; Drug GAT, Administration; ified; glyphosate GOX, N-acetyltransferase; glyphosate GM,Committee; oxidoreductas IUPAC, genetically International Union mod- for Pure andJoint Applied Chemistry; FAO/WHO JMPR, Meetings on Pesticide Residues;2-hydroxy-butanoic MHB, 4-methyl-phosphinico- acid; MPA,methylphosphinico-butanoic methyl acid;acid; M MRL, maximum residue limit; NAG, N-acetyl- Correspondence to: Gijs A Kleter, RIKILT University and Research Centre, BuildingWageningen, The 123, Netherlands. E-mail: Akkermaalsbos [email protected] 2, NL-6708 WB RIKILT – Institute of Food Safety,Wageningen, Wagen Holland American Free Tradeeration Agreement; and OECD Development; PAT, 4-methyl-phosphinico-2-oxo-butanoic acid; PRAPeR, Pesticide Risk Assessment Peer Review; STMR,Organisation. supervised trial median residue; WHO, World Health allowing for more flexible timing of applications withherbicide. the target ide resistance; pesticide residues; maximum residue limits; tant, for which it can be envisaged that the modification has ∗ c crops has rapidly increased since their introduction in the a b he herbicide resistance, the nature and levels of the residues • In sum, herbicidemanagement. resistance provides more flexibility in weed an be drawn concerning the nature and level of residues, which definitions and MRLs are still lacking for some herbicide–crop bitors, bromoxynil, glufosinate and glyphosate, are reviewed. mum residue limits (MRLs) defined by the Codex Alimentarius and Caroline A Harris b for example by: 2 John B Unsworth ∗ a : 1193–1210 www.soci.org The most important GM crops are soybean, 67 1 2011; herbicides; transgenic crops; genetic modification; herbic

obviating the need forweeds weed between crop tillage plants; orallowing directed spraying for on the usecertain of crop non-residual rotations herbicidesowing that to that the phytotoxicity would enable of residues otherwise ofin herbicides the be remaining soil; impossible replacing combinations of conventional post-emergencebicides her- targeting grassbroad-spectrum and herbicide; broadleaf weedsavoiding with crop a injury single post-emergence herbicides; that may be caused by conventional 2011 Society of Chemical Industry The main traits with which the major GM crops have been c • • • • Pest Manag Sci has to be done on a case-by-case basis. International residue formed and their impactCommission and on national the authorities. No residue general conclusions definition c and maxi combinations, and harmonisation is therefore recommended. Keywords: regulatory affairs; international harmonisation The topics considered are the molecular mechanism underlying t 1INTRODUCTION Since geneticallycommercially on a modified large scale inwith the (GM) these mid-1990s, crops the worldwide, area covered where crops as these well crops as are grown, were the hasarea number steadily reached increased. of 148 In introduced countries million 2010, ha, this nantly divided the over United States 29 (45%), countries, Brazil (17%), predomi- Argentina(6%), (15%), India Canada (6%), China (2%),South Paraguay Africa (2%), (1%). Pakistan (2%) and Abstract The global area coveredmid-1990s. Most with of these transgenic crops have (geneticallyan been impact rendered modified) herbicide on resis the profile andresistance, level including of herbicide resistance residues within to these acetolactate-synthase crops. In inhi this article, the four main categories of herbicide on standard setting for herbicide residues Gijs A Kleter, in transgenic herbicide-resistant crops (wileyonlinelibrary.com) DOI 10.1002/ps.2128 The impact of altered herbicide residues Review Received: 12 December 2010 Accepted: 4 January 2011 Published online in Wiley Online Library: cotton, maize and canola,papaya, while squash, other sugar beet, GM sweet crops, pepperto and such a tomato, as more are limited alfalfa, grown extent at present. modified are herbicide resistance and insectresistance resistance. Herbicide allows for ‘over-the-top’sprays of (direct herbicides that to could otherwise the be toxic growingresistance to traits the crop) introduced plant. The into GM herbicide-resistantfacilitate crops weed can management in various ways, EU Canad USA : 1193–1210 67 2011; -resistant and insect-resistant Pest Manag Sci Insect-resistant GM crops Insect-resistant Number of authorisations / completed consultations Number of authorisations 0 5 10 15 20 25 30 status of authorisations and completed consultations as of 1 8 itself) instead ofcontact with the directed crop; or sprays, avoiding herbicide conventional crop and not,the or GM in crop. different quantities, to Maize ted States) for the use of GM herbicide Potato Cotton Tomato (a) post-emergent over-the-top applications (i.e. on the crop herbicides can be used on these crops, for example: (b) pre-emergent and preharvest applications made to the altered on the basis of the nature of the modification. crop may have been altered,pesticide leading residues to overall. different exposure to Soybean In the present article, the background of the herbicide resistance 2. The residue profile of the3. applied pesticide The may have overall been pattern of pesticides applied to the particular traits will be highlightedthat have first, already received including authorisation themolecular for mechanisms by commercial traits which use. resistance has in The been achieved and GMthe crops impacts that theand modification profile may of crop have residues on willdefinition and the be appropriate indicated. residues metabolism levels The in GM issue and of non-GMwill crops residue be dealt with. 1. GM herbicide-resistant crops can change the way that 2Toprovideanimpressionoftherangeofcropsintowhichherbicide AUTHORISATIONS OF GM CROPS and insect resistance traitsbeen have been authorised introduced around andherbicide- that the and have world, insect-resistant Fig. crops 1for that commercial summarises have use been the as authorised in GM food Canada and and feedconsultation by the has regulatory been European completed authorities no Union in formal authorisation the (EU), of United a andThe States. GM food There for Food per is se and which inconsult Drug the the the United administration Administration States. before (FDA) commercialisation ofto urges GM ensure foods that developers all regulatory to matters arethe resolved. use In of the the following, terms ‘authorisation’ andrefers to ‘authorised’ these also completed consultations implicitly with the FDA, ifAs applicable. can be seensuch in as the soybean, graphs, maize, GM canola varieties and cotton of have commodity authorised crops uses m single-trait GM crops are combined have not been included as they are EU Can USA and Health Canada; 7 Another FDA 3,4 6 2011 Society of Chemical Industry . The proteins, c www.soci.org GA Kleter, JB Unsworth, CA Harris The present article 5 genetic modification. Herbicide-resistant crops Herbicide-resistant Bacillus thuringiensis proteins’, are toxic towards specific via Bt Number of authorisations / completed consultations Number of authorisations 0 5 10 15 20 25 Flax Rice Maize Alfalfa Potato Wheat Cotton Soybean Radicchio Sugar beet Summary of authorisations (Canada, EU) and completed consultations (Uni Canola (oilseed rape) Given that the herbicide and insect resistance traits aim at Theothermaintraitisinsectresistance,whichhasbeenachieved Further questions that may be posed with regard to the impact prospective study by thepotential present impacts authors’ on team pesticide focused usagewere on and glyphosate-resistant the environmental crops impact towas be concluded introduced that, inenvironmental Europe. under impacts It would certain be circumstances, feasible. favourable designated ‘Cry proteins’ or ‘ insect species butresistance trait not of insect-resistant GM to cropsthe may humans therefore applications replace of or insecticides domesticsensitive against towards the the animals. pest action of The insects thebeen that particular introduced are into Cry the protein crop that has facilitating the management ofreplacing crop or pests and supplementingpractices, weeds, other they thereby conventional arecrops. likely management Previous to research impactwas by on the pesticide carried use present outInternational on authors’ Union within these team, for which theexplored the Pure framework data and that of areof Applied available pesticide a on Chemistry that the are types project (IUPAC), conventional used and counterparts. on quantities of Because GM each crops the pesticideown specific as may effects, compared dose–effect have with relationships its and their behaviour, environmental the observed changes inon quantities these of pesticide crops used predicted were environmental impact also associated interpreted withThe outcomes these in of changes. this terms research showedthe a of quantities general of decrease pesticides the both used on in GM overall cropsGM compared crops with and non- in their predicted environmental impact. mainly through theinsecticidal introduction, proteins that via naturally occur geneticinclusions in of crystalline, the modification, parasporal soil bacterium of will further focus onbelow. GM herbicide-resistant crops, as explained of the altered pesticidebring usage on changes GM to crops crop ismight be. whether residue Several this factors levels, must may be and taken into what account these here: changes wileyonlinelibrary.com/journal/ps Figure 1. crops as food. Information sources: European Commission, January 2011. ‘Stacked’ versions of GM crops in which multiple traits fro commonly not authorised separately in the United States and Canada.

1194 1195 ). EU 7,8 10 Canada ). The post-emergence applica- What these herbicides have in istant crops as food. Same references Year 12 . 9 wileyonlinelibrary.com/journal/ps et al Insect-resistant crops Insect-resistant USA Interestingly, with regard to pyrithiobac, various 11 It therefore appears that the ALS inhibitor resistance of GM 1994 1996 1998 2000 2002 2004 2006 2008 2010 13 herbicide-resistant and insect-res

5 0

25 20 15 10 For one of the two GM soybean lines, it is mentioned that The documentation provided by companies that have devel- Resistance to ALS inhibitors in crops has been achieved through Crops publications in the scientific literature mentiongenerally that pyrithiobac considered is safe for post-emergencein use non-GM ‘over cotton, the and top’ cotton also towards the describe phytotoxic the actionexperiments resistance of (e.g. pyrithiobac of Harrison observed non-GM in common is theirvert inhibition pyruvate of plant to2-hydroxbutyrate. ALS 2-acetolactate, enzymes, This or which constitutes 2-ketobutyratebiosynthesis con- to of an 2-aceto- several importantisoleucine, as branched well step as amino leucine. While in differentsuch acids, as groups imidazolinones of and the i.e. herbicides, sulfonylureas, act on valine ALS, thisnecessarily does not mean and that resistancecross-resistance to to one another family chemical (e.g. family p. 203 confers of Duke tion of pyrithiobac tolabel. cotton is also described on the herbicide the ALS resistancemarker during trait the has creation only of been the GM used crop, as while a not selection being cotton allows for the additional inclusion ofas thifensulfuron active methyl ingredient in post-emergence herbicide sprays. oped these GM cropsherbicides describe the that usage of herbicides are containing specific ALS resistantinhibitors, towards of which ALS-inhibiting most areemergence sulfonylureas. applications of For the GM sulfonylurea thifensulfuron cotton, methyl (synonymous post- to M6316) andare another described. ALS inhibitor, pyrithiobac, 3.1.1 ALS inhibitorALS resistance inhibitors fallAction into category Committee’s B (HRAC)bicides of classification, the which of Herbicideidine, includes Resistance her- the pyrimidinyl(thio)benzoatetriazolinone imidazolinone, chemical and families. sulfonylurea, sulfonylaminocarbonyl- triazolopyrim- mutation of ALSresidues, enzymes in yielding one enzymestowards or that more are the of stillGM inhibiting their active and amino action but acid GMfirst insensitive of crops through with somaclonal the mutated variationexample, herbicides. or ALS and chemical have the Both mutagenesis, latter been for ALS-encoding non- through created, genes introduction the of viaCanadian new, recombinant mutated- regulations DNA techniques.include on Under foods ‘novel derivedimidazoline-resistant from foods’, canola, ‘plants for lentil, withwheat have example, maize, novel received favourable traits’, rice, decisions which forALS-inhibitor-resistant non-GM marketing sunflower as GM foods. and crops thatdecisions include have flax, maize and received soybean in favourable Canada, the as United States well and as resistant cotton in the United States. EU 2011 Society of Chemical Industry c Canada Year USA : 1193–1210 Herbicide-resistant crops Herbicide-resistant 67 2011; Chronology of the authorisations/completed consultations for use of GM 1994 1996 1998 2000 2002 2004 2006 2008 2010

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25 20 15 10 Crops Another means of conferring herbicide resistance is by intro- These authorisations of GM crops do not include the au- Impact of altered herbicide residues in transgenic crops on residue standard setting www.soci.org as for Fig. 1. Pest Manag Sci 3.1 Overview ofThere herbicide are resistance mechanisms basically two mechanismsherbicides through has which been resistance to achieved inthat GM target crops. an Firstly, enzyme in forenzyme plant herbicides can cells, be an introduced insensitive by variantplant genetic cells of to modification, function this in allowing the the presence of otherwiseof inhibitory levels herbicide. This insensitive varianta of mutant the of enzyme the can either nativeor more be enzyme, amino which acid carries residues that aaction are of mutation involved the in with herbicide, the one or inhibitory organism, an analogous such enzyme from as afrom different soil herbicide-resistant or other microorganisms environments that contain isolated of environmental the levels pertinent herbicide. ducing an enzyme that detoxifiesit to the a herbicide less by phytotoxic or metabolising enzymes non-phytotoxic have compound. been Usually, discovered such in microorganisms, andencoding the these genes enzymes have subsequently beencloned elucidated and into GM plantsthe in herbicide. order to render them resistant against 3 HERBICIDE-RESISTANTWith CROPS regard toauthorised the for herbicide-resistant foodhave use, crops been the rendered that resistant herbicidesnamely have fall to the into been which acetolactate fourglufosinate synthase these different and glyphosate. categories, (ALS) crops inhibitors, bromoxynil, thorisation of theherbicide-resistant target crops; herbicide thislation usually to that, falls be in under some appliedAlso, situations, parallel for is to the legis- handled intrinsic the by ‘pesticide’tion, a introduced additional GM assessments separate via may agency. genetic be needed, modifica- whichwith may the be linked assessment of themental Protection GM Agency crop, (EPA) such and the as AustralianVeterinary by Pesticides Medicines and Authority. the US Environ- in all of the threecrops, sets of such legislation, as while a potato,United number States have and of Canada. other only Although the GM number beenappears of to authorisations authorised be higher so in the United far Stateswith and in Canada as the the compared EU,the the main timeline of partCanada authorisations of took (Fig. the 2) place authorisations in showsauthorisations in in the that the last 1990s, the 10 years whilelegislations. United appear to the converge States for annual all and three numbers of Figure 2. 23,24 ). 10 : 1193–1210 Mutations of 67 19 thereby causing 19 9 2011; and by Duke 20 . N S O et al C 3 from bromoxynil in soil from OO S O O H S O O O Pest Manag Sci C NH 3 H ozaenae NH O var. In addition, bromoxynil-resistant canola O NH NH N Rimsulfuron N 7,8,21,22 N O Tribenuron methyl -homoalanin-4-yl (methyl)phosphonic acid] (Fig. 5), DL N O C 3 H N C O 3 H C 3 C Klebsiella pneumonia Genes coding for the bromoxynil nitrilase enzyme have been H 3 H has been givenbromoxynil-resistant cotton authorisation can be grown for in the United States. cultivation in Canada, while the ammonium salt of which is usually included in herbicide the formation of reactive oxygen species. A bromoxynil-degrading enzyme, i.e. bromoxynil nitrilase, was identified in bacterial isolates of previous use. This enzyme convertsmoxynil the into cyano a (CN) carboxyl (COOH) group group, of therebybicide bro- rendering inactive. the The her- putative naturalcontribution function to of the this degradation of enzyme aldoxime is compounds its by secreted plants into soils (reviewed by Kleter As explained in Section 3.3.2, the marketing ofin seeds of the these United crops States and Canada has been discontinued. 3.1.3 Glufosinate resistance Glufosinate [ 3.1.2 Bromoxynil resistance Bromoxynil (3,5-bromo-4-hydroxybenzonitrile) (Fig.rived 4) esters are cyano and (nitrile)-group-containing active substances de- of herbicides that belong to the HRAChibiting group plant C3 photosynthesis of at compounds photosystem in- II, introduced into canola and cotton, which have been authorisedfood for use in Canada, the United States,among Australia and others. New Zealand, and have subsequently been commercialised. various specific amino acid residuesactive site that within the are ALS located enzyme contribute around toALS resistance inhibitors the towards in these conventionally bred crops. With + 14 S 17 ts applied to GM ALS-inhibitor-resistant maize and soy. O C 2011 Society of Chemical Industry 3 c H O www.soci.org GA Kleter, JB Unsworth, CA Harris O S O O S O O O Similar to this soybean NH 15 NH C 3 These sulfonylurea-resistant O H O 18 16 NH NH N N Chlorimuron ethyl Thifensulfuron methyl N O N O N C 3 C H 3 C 3 H H Cl Structures of ALS-inhibiting herbicide active ingredien Besides GM crops modified with ALS inhibitor resistance trait, a While the ALS inhibitor resistance in GM cotton, maize and regard to theinhibitors other and soybean glyphosate. Accordingdeveloped line, to this it the line, is companycan various resistant that sulfonylurea-containing be has to herbicides appliedthis both soybean either ALS line. pre-emergenceherbicides The include or sulfonylurea chlorimuron ethyl, active post-emergence rimsulfuron,methyl ingredients thifensulfuron to and of tribenuron these methyl (Fig. 3). range of conventional crops with thissulfonylurea-resistant trait also exist. soybeans For example, haveexposure of soybean been seeds to a obtained mutagenicquent selection compound of and through seeds subse- and subsequent generationsresistance of plants towards for sulfonylureas. soybean enables the applicationthe of pertinent ALS-inhibiting herbicides crop, to grown the in resistance the inALS presence GM inhibitors of flax resulting the enablescrop otherwise from it grown toxic to application previously soil be ofdeveloper residues in of herbicides of this the to GMof the rotation. the flax sulfonylureas triasulfuron mentions More and resistance metsulfuron specifically, methyl. against the residues wileyonlinelibrary.com/journal/ps intended as a herbicide resistance trait for field use. Figure 3. soybeans were subsequently brought to the market.range In of imidazolinone-resistant addition, crops, a including brassica (canola), maize, rice, sunflower andbreeding wheat, of have lines been containingtions obtained or either through naturally mutagenesis-derived occurring muta- mutations selected in wild relatives, line, a GM maize linefeaturing has both been developed the by ALS the inhibitorwhich same and company, glyphosate can resistance traits, besulfonylureas combined as used with on soybean. herbicides containing the same

1196 1197 - D The gene or 25 pat (encoded by (encoded by the 26 O 3 -phosphinothricin has been L P CH OH O 3 P CH OH wileyonlinelibrary.com/journal/ps -glufosinate leads to accumulation of PPO L MPA -acetyltransferase (PAT) enzymes. Two O OH N Also, for GM glufosinate-resistant radicchio, OH O Streptomyces hygroscopicus Streptomyces viridochromogenes 6–8 O -glufosinate and -glufosinate) is also part of technical glufosinate- L D -enantiomers, but is not herbicidally active. -phosphinothricin/glufosinate. L L gene). These enzymes share a high degree (85%) of O gene have been authorised in the United States, Canada -phosphinothricin/glufosinate molecule, with a relatively 3 L O 3 -and -phosphinothricin P CH OH pat gene) and in -phosphinothricin or bar L D L Phosphinic acids such as glufosinate and bialaphos fall A number of glufosinate-resistant GM crops that have been Resistance to P CH OH andtheEU.Thesecropsincludecanola,cotton,maizeandsoybean,while also rice and sugar beetStates have and been Canada. authorised in the United into HRAC group HGlutamine of synthase glutamine-synthase-inhibiting is herbicides. anmetabolism enzyme of nitrogen with in prokaryotes an and important eukaryotes.synthase Glutamine role in catalyses the theglutamic condensation acid into of glutamine.by ammonium Inhibition ions of glutamine and synthase ingredient of broad-spectrumas herbicides, preharvest desiccant while for a it number of is crops. also used modified with the PAT enzyme encodedthe by either the high affinity compared with other acetyltransferases,coenzyme using A acetyl as a cosubstrate,activity thereby of inactivating the herbicidal identical residues whenaligned both using computer their algorithms. They amino catalyseof the the acid acetylation sequences are PAT enzymes haveoccur naturally been in introducedbar intothe GM crops, which containing formulations containing the racemic mixture with both the ammonium ions and depletionacids in of the glutamine plantphotosynthetic and cell, other activity which amino and ultimately other leads to phytotoxic a effects. decrease in achieved in GM crops through the introduction offor genes encoding enantiomer (i.e. MPP NH 3 OH Br CH O OH O N-acetyl-glufosinate O O C 3 H OH 2011 Society of Chemical Industry c DBHA HO -phosphinothricin L O O Br 3 O 3 3 P CH OH Br P CH OH P CH OH 3 O CH N O 2 MPB MHB Bromoxynil heptanoate OH NH OH DL-glufosinate OH Br OH O O O : 1193–1210 67 Br Br 2011; O -Phosphinothricin is a component of natural antibiotics L Bromoxynil octanoate N O Structure of glufosinate and its metabolites NAG, PPO, MHB, MPP, MPA and MPB. Structure of bromoxynil, its heptanoate and octanoate esters N OH Bromoxynil Br Br Impact of altered herbicide residues in transgenic crops on residue standard setting www.soci.org such as bialaphos andwhich phosalacin produced are by soil streptomycetes, microorganisms. Glufosinate ammonium is an Figure 5. Pest Manag Sci and its metabolite DBHA. formulations, is a synthetic analogue of the Figure 4. molecule. : 1193–1210 31 67 30 2011; Pest Manag Sci Sulfonylureas comprise a broad range of substances, most Bromoxynil within currently marketed herbicide formulations For imidazolinones, for example, the most important reactions of which containi.e. an an aromatic aromaticatoms, six-atom pyrimidinyl respectively, ring linked or including tolinkage. two triazinyl Metabolic the reactions or group, urea that three are side observedin nitrogen with of plants sulfonylureas include: the hydroxylation sulfonylurea ofatoms aliphatic and followed aromatic carbon byof alkoxy conjugation groups withester to glycosides; groups; form demethylation dealkylation hydroxyl ofwith groups; nitrogen (N) glutathione; de-esterification atoms; of hydrolysis conjugation contraction/rearrangement of the of sulfonylurea group. the sulfonylurea linkage; and is either present as such, which is also referred to as the ‘phenol’ 3.2.2 Bromoxynil A limited numbermetabolism of and residue scientific formation of reportsthat bromoxynil in have bromoxynil crops. Given appeared istowards on its used toxicity, many the in of thesethe investigations many residues have formed focused cereals, in on these which crops, in particular are wheat. resistant in plants insubstituent general of the include:to the aromatic the imidazolinone ring hydroxylation molecule;this the (e.g. of hydroxyl subsequent phenyl group the conjugationthe imidazolinone of with ring) alkyl group a itself; attached andcondensation glycosyl of the the cyclisation group; carboxylate following group the the (attachedring) with to a hydrolysis the nitrogen aromatic in of the imidazolinone ring. 3.2 Thein profile herbicide-resistant crops of herbicide residuesIt and can their be metabolites envisaged thatto herbicide resistance, sustain which allows exposure crops target to herbicides, will affect foliar not only or theof levels residual but the also the applications residues nature of withtissues. Besides the various the research herbicide studies published active inliterature, the ingredient additional scientific residue in data the fromproducts crop’s of field herbicide-resistant trials and non-resistant and crops have processed consideredbyorganisationssuchastheOrganisationforEconomic been Cooperation and Development (OECD)Agriculture and Organisation (FAO)/World the Health Organisation Joint (WHO) Meetings Food and on PesticideCodex Alimentarius Residues Commission (CAC) and (JMPR). in particular theCommittee The Codex on JMPR Pesticide advise Residuesresidue (CCPR) the issues, dealing with includingresidue pesticide the limits (MRLs). establishment This section ofon summarises the CAC the data levels maximum available andwhich commercialised metabolites GM of cropsi.e. residues have of imidazolinones, been the sulfonylureas, renderedglyphosate. herbicides resistant, bromoxynil, to glufosinate and 3.2.1 ALS inhibitors The group ofcomprises a ALS-inhibiting broad range active of3.1.1 above. substances, The ingredients main as two largest mentioned categories of of in theseare ALS Section inhibitors herbicides the imidazolinoneThe ability and of sulfonylureasubstances conventional often types crops relates rapidlyphytotoxic of to to to the substance. particular metabolise selectivity weedsWhereas these the but of nature non-phytotoxic and herbicides importance to of being awith the given an metabolic crop. imidazolinone reaction or sulfonylureathe in plants specific depends active on ingredient both andapplied, the it is plant still species possible to to which infer generalisations. it is 7,8 3 -(phos Achro- -acetyl- OH CH N N although strain CP4, 6 P O OH NH 2011 Society of Chemical Industry c AMPA have been used www.soci.org GA Kleter, JB Unsworth, CA Harris sarcosine -acetyl-glyphosate OH N N 2 H O Agrobacterium GAT-expressing crops that 27 29 10 -acetyltransferase (GAT), which N ). Also, a mutated EPSPS from maize Bacillus licheniformis OH OH 20 . P P O OH O OH et al 3 A relatively novel example of this strategy is CH 10,20 NH N glyphosate O LBAA isolated from sludge. The latter enzyme catalyses Structure of glyphosate and its metabolites AMPA, OH and by Kleter OH N-acetyl-glyphosate 28 O O The second strategy involves the enzymatic inactivation of Two strategies have been followed by developers of GM crops mobacter breakage of the carbon–nitrogen (C–N) bonding in glyphosate, yield- amino-methylphosphonic acid (AMPA)acid (Fig. as 6) products. and glyoxylic has been used for genetic modification of cotton and maize. glyphosate. For example, GM canola thatCP4 has EPSPS been also contains modified glyphosate with oxidoreductasewas (GOX), which identified in the glyphosate-degrading bacterium the company developing thisnot GM commercialise crop these GM has varieties. indicated that it will have been introduced intocanola, a cotton, range maize, GM potato,and, crops, for soybean, including lawns and sugar alfalfa, golf beetDuke courses, creeping and bentgrass (reviewed wheat, by to confer glyphosate resistance toinvolves the these introduction, crops. through The genetic modification, firstenzymes of strategy EPSPS that are insensitiveFor this towards purpose, inhibition genes bya encoding glyphosate-degrading glyphosate. the soil bacterium, CP4 EPSPS enzymes from have been authorised for food use include maize and soybean. wileyonlinelibrary.com/journal/ps 3.1.4 Glyphosate resistance The herbicide active ingredient glyphosate [ a consultation has been concluded in the United States, Figure 6. phonomethyl)-glycine] (Fig. 6) is a glycine derivativeHRAC that group falls G of into compounds that inhibit3-phosphate 5-enolpyruvylshikimate- synthase (EPSPS).of EPSPS organisms, occurs including inplants. microorganisms a It (bacteria, wideenolpyruvylshikimate-3-phosphate is range fungi) from an and shikimate-3-phosphate and enzyme phosphoenolpyruvate. This is one of that the steps in thepathway’, catalyses ‘shikimate which the is responsibleprecursors formation for for the of compounds biosynthesistyrosine, of such 5- phenylalanine) and aromatic as lignin. aromatic amino acids (e.g. theuseoftheenzymeglyphosate glyphosate and sarcosine. catalyses the acetylation of glyphosate into for ‘DNA shuffling’ in order to create novelcatalytic sequences activity with towards optimal glyphosate. (Fig. 6), which is non-herbicidal.GAT The enzymes isolated gene from sequences coding for

1198 1199 45 These 48 Thelatteris 42 In its assessment ). 21,22 22 -glufosinate (NAG) (Fig. 5), L studied the metabolism of 47 48 . -acetyl- wileyonlinelibrary.com/journal/ps N p. 8 of FSANZ et al For bromoxynil in onion, residue field 21 46 ¨ oge-Laser ). Besides bromoxynil, DBHA has also been included in 44 43 C]-radiolabelled glufosinate applied to plant leaves. One of these two routes is specifically linked with the Food Standards Australia New Zealand (FSANZ) has also Similarly to the situation in the United States, bromoxynil The second route, which is linked to glufosinate metabolism -acetylated metabolite corresponds to the enzymatic activity of 14 the residue definition for animal-derived productsoccur because it in may feed commoditiesanimal products from after which intake of it these(p. commodities can 37 by of be the EPA animals transferred to modification of GM cropsappearance with of the a metabolite PAT enzyme, which causes the authors observed that two parallelglufosinate routes in exist plants. for metabolism of garlic, grass, onion,cereals, peppermint, including spearmint barley,When and maize, defining oat, the a residue, rye, the numberthese EPA sorghum other makes of crops a and and distinction bromoxynil-resistant wheat. between cotton. evaluated the safety of imported foodlinters products, derived including from oil GM and bromoxynil-resistantGM cotton and bromoxynil-resistant oil brassica from (canola). trial data indicated thatthe the limit of residues quantitation of (0.02 ppm). bromoxynil were below has also been registeredherbicides that in can be Canada applied as toflax, garlic, a the grass range grown for of active seed crops, and ingredient a suchbarley, number as of of maize, cereals, alfalfa, millet, including oats, rye, sorghumhas been and registered wheat. for In use addition, on it GM bromoxynil-resistant canola. in non-modifiedglufosinate plants, to involves 4-methyl-phosphinico-2-oxo-butanoic acid(Fig. the 5), (PPO) which initial wasFor deamination found example, to PPO of was be found prone to to be further further metabolism. decarboxylated to besides the parent compound glufosinate.N This formation of the the PAT enzyme. With regard to the definition of the residues ofthis bromoxynil in is crops, defined by Health Canada asfor bromoxynil dry plus bulb DBHA, onions, except milletonly and includes sorghum, bromoxynil. for which the definition 3.2.3 Glufosinate In laboratory-growncarrots experimental and plants, alfalfaPAT including that enzyme, had tobacco, been Dr genetically modified with a of the GM cotton products,amounts FSANZ of also DBHA mentions can that be significant whereas present it in was bromoxynil-resistant not cotton, clear toin what linters extent and residues could oil alsoGM derived occur canola, from it notes this that cotton.did residue not In field contain the trials quantifiable showed assessment levels thatDBHA, of of which the residues were seed just of above bromoxynil the limit and was of detectable detection, and in that processed none canolaassessments fractions (oil by and FSANZ meal). Both ofMRL has cotton been and set for canolatherefore bromoxynil also residues in in these note these commodities, that products andlevels should that no (pp. be 9–10 below of detectable FSANZ; designated only as cotton in the Code of Federal Regulations,this while obviously applies toother bromoxynil-resistant than cotton. cotton, MRLs Forthe (‘tolerances’) crops residue have of been bromoxynil, while established forand for cotton hulls, as seeds, well gin as for byproducts a numberhave of animal-derived been products, MRLs established for theand sum DBHA. of the residues of bromoxynil [ 37 ,are .Cummins 2011 Society of Chemical Industry 32 in planta c who observed 36 ., et al 39–41 The authors concluded -glucoside formation. O 32 Also, other studies specifically 34 38 Among the metabolites found in barley In wheat seedling leaves, a wide range 35 36 : 1193–1210 67 2011; isolated an esterase enzyme with a molecular mass of 32 33 . WhereasJMPRhasnotevaluateddataonresiduesofbromoxynil, With regard to the formation of residues in the crop parts used In line with the observation by Schaller The metabolism of bromoxynil was found to be more extensive Impact of altered herbicide residues in transgenic crops on residue standard setting www.soci.org 45 kDa and anwheat seedlings. isoelectric While this pointbromoxynil esterase of was octanoate, capable pI the of 4.6 authorscould hydrolysing only estimated from account that for the this 0.4%substrate shoots of in enzyme wheat the of extracts. total In activity atowards hydrolysing bromoxynil follow-up octanoate this study, as esterase a activity substrateextracts was of observed various in crop the species, in particular maizeflax, and rice, also sorghum alfalfa, and soybean, besides a numbershowed of weeds. that It also the repertoire ofplant esterase species, enzymes based differed on among their sensitivity the towards inhibition. activity of separated enzymes and of metabolites wasbromoxynil octanoate observed and besides bromoxynil. the parent compounds the authorities ofcrops two are allowed nations onto the where market,have the GM United States done bromoxynil-resistant and Canada, thishas at registered a the nationaloctanoate use level. esters of The bromoxynil ona US and GM EPA, range its bromoxynil-resistant for of heptanoate cotton, example, and other besides food and feed crops, such as alfalfa, flax, as food, various experimentsare indicate present that in littlelittle the or radioactivity crops no was residues that present,been for have harvested example, been from inapplicationofradiolabelledbromoxyniloctanoateat3–4timesthe field-grown studied. grains wheat No that plants or had recommended 4 very application months rate. after in non-GM resistant plant species, suchspecies, as such barley, as than tomato. in sensitive Pest Manag Sci form, or asoctanoic an acid. ester Regulatory authorities of haveingredients evaluated the these within active phenolic the group frameworkis with of based a heptanoic on single or form the evaluation, of bromoxynil which rapid and the hydrolysis organicexample, acids. of 0.4% In of the wheat radiolabelled seedlings, esters for bromoxyniltheseseedlingsremainedunhydrolysedwithin7 octanoate to applied the to days. ‘phenol’ seedlings were thehydroquinones parent and compound,hydroquinone. glycosides differently The of substituted presencethe both of nitrile (cyano) the hydroquinones group of indicatedby parent bromoxynil must a that have and hydroxyl been replaced group. the that bromoxynil and its metabolites are glycosylated the outcomes of astrawberry. study A on recombinant form a ofable glycosyltransferase this to enzyme glycosylate enzyme 3,5-dichloro-4-hydroxybenzoic was from acid, found whichchlorine is to a analogue be of 3,5-dibromo-4-hydroxybenzoic acid(Fig. (DBHA) 4), one of the mainwas metabolites observed of to bromoxynil. take Glycosylation placeformation, both and at at the the phenol carboxyl group, i.e. group, i.e. ester that the nature of the majorreactions, metabolites was including indicative of the various a substitution carboxyl of or thea amide nitrile hydroxyl group, group group thethe or with metabolites substitution a containing a hydrogen of carboxylgroup. atom group bromide replacing and the with nitrile decarboxylation of et al analysing the content of bromoxynilshow in its seeds presence, of such cerealspost-emergence as failed applications in to with triticale bromoxynil esters, and whichmade wheat were 2.5 following months before early harvest in wheat. or 66 Moreover, : 1193–1210 69 67 These authors 58,59 Residues of both There are several 2011; 60–63 64 70,71 57 although it appears to be absent Pest Manag Sci 67 Experiments with greenhouse-grown 70,71 and trees, 65,66 -methylglycine) (Fig. 6) is another metabolite N However, these results should be interpreted with care, The levels of AMPA and glyphosate were found to be 68 71 54 With regard to metabolism of glyphosate in GM glyphosate- After the completion of the toxicological evaluation in 1999, Sarcosine ( from crop plants, i.e. glyphosate-treated, GMcanola. glyphosate-resistant required new MRLs.residue The as JMPR the thenexpressed proposed sum as to of redefine glufosinate-ammonium, freedefinition the with MPP glufosinate. NAG and The wasis NAG based a extension on major two ofresistant metabolite considerations: the crops; formed (1) NAG (2) from residue NAGchemical derivative glufosinate and that is in glufosinate prepared for glufosinate- the giveresidues. This analysis newly of rise proposed glufosinate definition to could not yetthen, the be pending adopted the same completion ofNAG. the evaluation of the toxicity of resistant crops, AMPA was detected in seedsgrownGMglyphosate-resistantsoybeancarryingagenecodingfor harvested from field- the glyphosate-insensitive CP4 EPSPS enzyme, which had received post-emergence applications of glyphosate. the JMPR adopted the newlyincluded proposed glufosinate-ammonium, residue NAG and definition, MPP which beingas expressed free glufosinate. Moreover, JMPRbeing suitable considered for this MRL setting definition and for as estimating theof dietary glufosinate intake and its metabolites. 3.2.4 Glyphosate Until the 1990s, anot metabolised generally by held crops beliefmetabolism to which of was it glyphosate that is to glyphosate phytotoxic,well AMPA described whereas is by by the soil that microorganisms time.1990s was Several showed that scientific AMPA reports was in formed from themetabolite glyphosate early in as its sterilised main cultures ofa crop possible cells or contribution tissues, of ruling microbiological out activity. variable, depending on the timing and/or numberwith of applications, later applicationsAMPA generally and giving glyphosate. rise to higher levels of as the sarcosine measured in plants mayfrom have contaminations been derived of either the applied glyphosate preparation accounts of the presence of sarcosine in glyphosate-treated plants, including weeds also mentioned that,other in studies, such several as strawberry, different no formationobserved. of crops AMPA Obviously, had investigated been the in through formation decarboxylation is caused of by an AMPA intrinsicin GOX-like activity crop from plants, glyphosate the exact sourcebeen elucidated. and nature of which have not yet a group of Canadianrange authors of experimentally showed field-grown conventional, that non-GMto AMPA which crops glyphosate occurred had been in applied as a a including preharvest barley, application, brassica (canola),and crested wheat. wheatgrass, AMPA levels field in the pea seeds,crops straw generally and/or was foliage a of fraction these of theapproximately levels 7% of glyphosate, in i.e. seed up or to by straw and of large barley. The followedwith levels the of regard same AMPA to pattern their asapplication those dependency and other of on environmental glyphosate application factors. rates, stage of from microbiological C–P lyase activity within plants. compounds were also detected in stems and leavesplants. of the soybean that ismicroorganisms formed isolated from from soilcleavage and glyphosate of water. It the by can C–P involve various the bond in environmental glyphosate. In 53 . et al By that 49,52,53 56 -glufosinate 2011 Society of Chemical Industry D -glufosinate is 53 c -glufosinate is -glufosinate is L D L www.soci.org GA Kleter, JB Unsworth, CA Harris -glufosinate and/or DL The outcomes of these 55 54 56 These studies show that, in the treated leaves In an experiment in which maize and brassica 49–53 53 -Glufosinate applied to maize was only found to D 48 -glufosinate to leaves of either crop (and L In the preamble to its report of 1998, JMPR noted that the A report that had previously been published by JMPR in 1991 The JMPR’s evaluation of glufosinate that was published in 1998 In line with these findings are other reports in the scientific The authors further concluded that both routes compete with -and -glufosinate by microorganisms under the experimental outdoor -glufosinate in experimentally grown crop plantlets, including rapidly metabolised by the plants to NAG, while metabolised to a significant extent.to NAG has be been demonstrated the main metabolite of the parent compound. different agricultural practice and residue patternsresistant in glufosinate- crops that had been newly developed by that time concluded that, in conventionalMPP non-glufosinate-resistant is crops, the main metabolite of the parent compound. includes a summary of studies on theglufosinate-resistant metabolism of glufosinate plants, in includingsoybean, brassica sugar (canola), beet maize, and tomato. literature that describe the metabolism of be absorbed tometabolised a to limited bothorganic extent, NAG acids. after and which methylphosphinico-substituted it was partially studies areliterature consistent discussed with above, those as reported they show in that the scientific wileyonlinelibrary.com/journal/ps DL L also reported thewell as additional 2-methyl-phosphinico-acetic presencemethylphosphinico-butanoic acid acid of (MPA) (MPB). MPP For (Fig.authors 5) MPA concluded and that and and they MPB, could MHB, 4- not the excludethese the as possibility metabolites that are possibly derived from the metabolism of L 3-methyl-phosphinico-propanoic acid (MPP)also found (Fig. 5). tometabolism. PPO re-form An glufosinate additional was metabolite through consistingform amination of the by of hydroxyl plant glufosinate,acid (MHB) 4-methyl-phosphinico-2-hydroxy-butanoic (Fig. 5), was alsoplants. found to be present in non-modified each other forexample, the account metabolism for ofroute the linked glufosinate. observation with This intrinsic thatand could, plant metabolites MPP, metabolism, are for of still such observed the asPAT in activity. PPO, GM MHB plants with comparatively low and other parts of glufosinate-resistant plants, to maize), comparativelythe parent minor compound, NAG levels andorganic acids, methylphosphinico-substituted occurred of in the residues, grains of these including crops. (canola) plants had been harvested 4 months after application of conditions. glufosinate-resistant brassica (canola), maize, cotton andexpressing soybean PAT. glufosinate-resistant maize and brassica (canola), Ruhland relatively stable. Inbesides many the parent cases, compound and the NAG, additionalglufosinate outcomes metabolites of are also present show withinmethylphosphinico-substituted that, plant organic tissues, acids, including MHB inand sometimes and particular also MPP MPA and MPB.residue MPP is in the most grain predominant relatively of important GM metabolite glufosinate-resistantglufosinate-resistant of maize, soybean. NAG glufosinate and is an in also importantseed, residue beans a grains in or of the fruits GM frommaize, soybean, glufosinate-resistant sugar brassica beet (canola), and tomato. time, JMPR defined theas residue the used sum for of theexpressed setting as the free of residues glufosinate. the of MRL glufosinate-ammonium and MPP

1200 1201 - - A N N 82 -acetyl- N 77 -acetyl-AMPA N AMPA. × -acetyl-glyphosate -acetyl-glyphosate N N -acetyl-glyphosate is -acetyl-AMPAinGAT- -acetyl-glyphosate in 1.5 N N N + 79 Based on weight of evidence -acetyl-AMPA) that occur as -acetyl-AMPA, in all plant and this metabolite of glyphosate N 81 80,81 N ). 80 -acetyl-glyphosate was the major wileyonlinelibrary.com/journal/ps N -acetyl-glyphosate residues in GAT- The US definition thus includes both N -acetyl-glyphosate and N -acetyl-glyphosate is probably less toxic 78,79 N -acetyl-glyphosateand N -acetyl-glyphosate in these agricultural products. -acetyl-glyphosate. In order to take account of N N -acetyl-glyphosate, with the definition covering N 82 -acetyl-AMPA, as well as the outcomes of residue trials with N -acetyl-AMPA, another metabolite formed in GAT-expressing N In the EU, the issue of the occurrence of As mentioned in Section 3.1.4, recently approved lines of GM Thepresenceof -acetyl-glyphosate, AMPA and distinction was made betweenrisk the assessment residue and for definitionsFor enforcement used risk assessment, of for it pesticide was considered residueglyphosate, necessary MRLs. to the cover, metabolites besides formedN from glyphosate, including animal food commodities.changes For to the enforcement, residue definition three ofand glyphosate soybean options residues were in for offered maize tonot risk to managers. change the The current first definitionwhile option comprising was the only glyphosate, second optionthe was definition to of include glyphosate, i.e. the sum of glyphosate and maize and soybeanglyphosate express to the GATthe enzyme, potential which presence of converts comprising toxicological data provided for multiplication by a factor of 1.5, and subsequentlyto has the to level be of added glyphosate, i.e. glyphosate glyphosate and This is because theequally US toxic as EPA glyphosate, while assumes noting that that assumption this is a because conservative than glyphosate. AMPA is not included in thebecause US the residue US definition EPA has previouslytoxicological concern. concluded Moreover, that the AMPA US is EPA considers ofof the no toxicity acetyl-glyphosate. The third optionMRL was for to establish a separate and other metabolites in GMhas maize and been soybean expressing considered GAT (EFSA) by the Pesticide European Risk Food Safety Assessment Authority’s Peer Review (PRAPeR). expressing maize andStates, soybean the that US are EPAinto its has residue grown definition included for in glyphosatewell in the maize as and meat United soybean, and as milk. expressing maize and soybean has also been consideredin by the FSANZ safety assessment of these GM crops, importwhich commodities were of notified for marketingZealand approval (pp. in 86–105 Australia and of New FSANZ glyphosate, glyphosate, AMPA, residues in GAT-expressing crops. the GAT-expressing crops, thetwo assessments metabolites conclude of that glyphosate these areand therefore not do toxicologically not have significant to beThe included in Australian the residue residue definition. definitionbecause does glyphosate not is include considered AMPAsignificant to either, be compound the only among toxicologically the four substances ( and crops, to be low and of limited concern. acetyl-glyphosate only. EFSA noted that glyphosatewould residues not only be a goodit marker for had monitoring compliance been because observed that residue in maize grain andGM soybean seeds crops. of On the GAT-expressing residue the definitions other for maize hand, and soybeanin it could enforcement laboratories. create Similarly noted confusion to EPA, that EFSA concludedthe that creating metabolites separate AMPA, are of no higher toxicological concerntherefore, than for risk glyphosate assessment itself, purposes, and the ADIbe of used. glyphosate can 72 The These 74 75,76 2011 Society of Chemical Industry 75,76 c Also, trace levels of 75 Contrary to the way that 75,76 AMPA reaches substantial levels in 76 The JMPR also noted that this was in line 77 : 1193–1210 67 AMPA was not detected in maize plantlets, which also contained GOX, the enzyme producing AMPA 2011; 72 . 72–74 et al 75,76 With regard to the residue definition of glyphosate, the JMPR’s The evaluations that JMPR carried out in 1997 and 2005 also The JMPR evaluated data on the metabolism of radiolabelled Impact of altered herbicide residues in transgenic crops on residue standard setting www.soci.org crops included coffee, cotton, maize,as soybean and mixtures wheat, of as grass well extended and upon legumes the (i.e. wide alfalfain range and soil of clover), that crops had which exposed alreadydata to been evaluated glyphosate considered by in 1986. thecompound In present JMPR in general, non-GM show the crops that toat which glyphosate subherbicidal it has doses, is been while applied the itslimited metabolite main extent. AMPA An is exception was present noted to fora a maize forage, relatively in which highcomparable level with of that AMPA of glyphosate.glyphosate had Furthermore, from been the soil, uptake observed, of which,emergence at for applications, a had example, level only isvery been limited possible extent. observed after Further to metabolism pre- of occurincludes glyphosate the to and conjugation AMPA a of AMPA withas natural organic compounds, acids, such as wellthat are as incorporated degradation into to intrinsic crop one-carbon compounds. elements report that was published inwould 2005 continue concluded to that include this thecompliance definition with parent MRLs. compound glyphosate for considered data on glyphosate-resistant crops,maize, including soybean cotton, and sugar beet. glyphosate in acrops in number 2005, following of a previous non-GM, evaluation in non-glyphosate-resistant 1997. GM glyphosate-resistant brassicaNandula (canola) plantlets studied by Pest Manag Sci young plantlets of conventional non-resistant and GM glyphosate- resistant soybean and brassica (canola) alsoof showed the AMPA presence besides glyphosateapplication. in these plantlets after glyphosate had received relatively low application rates of glyphosate. from glyphosate, besides CP4these EPSPS. plantlets The were not AMPA particularly levels highglyphosate-resistant compared found with soybean those in without in GOX.to This conclude led that, in the canola, authors AMPA must be further metabolised. with the definition thataddition, is generally it used was in concludedthe national that residues systems. both of In glyphosate toxicological andthat concern, AMPA AMPA based are on is the thethe observation main estimation metabolite of ofestimated the glyphosate dietary supervised in intake, trial plants.sum the median For of JMPR residue the levels defined and oflevel the the glyphosate of and residue AMPA AMPA. has as to For be this the converted purpose, to the ‘glyphosate equivalents’ by conjugates of glyphosate and AMPA havecrops. been detected in these glyphosate is applied in-crop to conventional, non-resistant plants, itcanbeappliedtoresistantGMcropsathigherdosageanddirectlyto the crop plants. After pre-emergence application ofresidue glyphosate, levels in GMobserved in crop glyphosate-sensitive tissues non-GM crops. areemergence Following low, application post- similar of toGM glyphosate, the crops the levels contain glyphosate-resistant glyphosateIn and the AMPA various asbeet, the tissues the main of level residues. of resistantof AMPA glyphosate, cotton, is except relatively soybean forexceeds minor and the glyphosate compared seed sugar levels. with of that soybean, in which AMPA glyphosate-resistant maize thatwith has both been a genetically glyphosate-insensitivethelatterconvertingglyphosatetoAMPA. EPSPS modified and the GOX enzyme, ) 1 ), 1 1 − − − 91,92 : 1193–1210 67 The same MRLs A comparison of ) in maize grain and 2011; 1 − 91–94 , which is not a specific 84–89 1 ) and tribenuron methyl − 1 The product label of the − 90 01 mg kg . 0 Pest Manag Sci 84–86 ), these MRLs were sustained. The < 1 − ) in linseed, the latter being lower than the MRL ) were thus recommended by the US EPA for these 1 1 − − 90 In Section 3.1.1 it is noted that assessment reports on GM MRLs that corresponded to the limit of quantification herbicide formulation containinghowever, both mention the sulfonylureas use of does this not, herbicide on flax but on cereals to the proposed usage of thesebelow the sulfonylureas, limit their of quantitation residues ( were (0.01 mg kg have been established in Canada, whileMRLs the EU but has has set no takenfor specific the thifensulfuron lower methyl (0.05 limit mg of kg analytical determination exceeds the EU threshold of 0.05 mg kg ALS-inhibitor-resistant crops also mentionthifensulfuron the methyl suggested on resistant use cotton, of andand that of metsulfuron triasulfuron methyl inbeen established resistant by flax. EPA Whereasin for cotton, the an the usage MRL proposal of has use for thifensulfuron of this methyl MRL this only compound,sensitive mentions which and the may GM preplant be ALS-inhibitor-resistantpost-emergence possible cotton, ‘over-the-top’ in rather use both than in non-GM the resistant cotton alone. (0.01 mg kg national MRLs for these three sulfonylureasshows in that maize and the soybean MRL for rimsulfuron in maize grain (0.1 mg kg in both cases, which wasmethyl then used and as the tribenuron MRL methyl for thifensulfuron in linseed. Whereas triasulfuron and metsulfuron methylsoil were residues envisaged in fields as where GMbegrownsubsequentlyintherotation,nospecificMRLsinflaxhave ALS-inhibitor-resistant flax was to been set for these sulfonylureas byInstead, Canada and both the United nations States. havemethyl established and MRLs tribenuron for methylTribenuron thifensulfuron methyl in acts flax as (linseed awhich it grown precursor is initially of metabolised for by metsulfuron plants fibre). that methyl, havesupporting absorbed to it. data The for theproducing petition that company was filed forpost-emergence by the the broadcast herbicide- setting applications ofmethyl of and MRLs tribenuron both by methyl thifensulfuron toat EPA an GM early describe ALS-inhibitor-resistant stage flax of cropseed development. (linseed) No above residues were the found limit in of quantification, i.e. 0.02 mg kg soybean seeds. Residues were detectableanimal in feeding, such plant as parts maize usedand soybean and for hay. soybean Moreover, processing forage, studies showed maize thatsulfonylureas stover these were concentrated inof the both aspirated crops. Rimsulfuron grain and tribenuron fractions methyl wereto also concentrate found in soybean hulls. latter is relevantFree within Trade Agreement the (NAFTA) framework among theand of United Mexico, States, which the Canada also strive North towards harmonisingMRLs American MRLs. were Specific recommended formaize each forage, of maize stover, the soybean hay, three soybean forage,seed sulfonylureas soybean and in aspirated grain fractions,tribenuron as methyl well in as soybean for hulls. rimsulfuronno Based and on additional studies MRLs on were animals, consideredproducts necessary including for edible meat, animal milk and eggs. and that for rimsulfuron in fielding maize MRL grain in with Mexico the (0.1 correspond- mg kg MRL but the lower limitcally of to analytical ensure determination that used allstandard. specifi- sulfonylureas are regulated to a common in Canada and the United States. sulfonylureas present in maize grains andthe soybean existing seed. Because higher MRLaligned for with chlorimuron the ethyl corresponding in MRL soybean in seed Canada (0.05 mg kg 83 2011 Society of Chemical Industry In particular, c 83 www.soci.org GA Kleter, JB Unsworth, CA Harris Currently, neither a review by JMPR nor For the fourth sulfonylurea, i.e. thifensulfuron 15,16 84–86 The residue definition of chlorimuron ethyl, rimsulfuron and As explained in Section 3.2, the altered metabolism of herbicide herbicide-resistant crops were considered. The experience of three members, namely the Unitedreviewed. States, This showed Canada that and theseherbicides Mexico, three applied were nations to approached GM the way herbicide-resistant as crops they approached incrops. the the For herbicides same example, applied an to in-depthherbicide conventional study in on the the resistant crop metabolismto will of be be the required different ifaddition this from is the to expected metabolism dataresidues in from of conventional the supervised crops, herbicidedefinition field in and would trials its have on metabolites. toany specific be Also, the metabolites of broadened the concern levels that in are residue formed order incrop of the but resistant to not incorporate in thefrom conventional the one. Because resistant theconventional crop commodities counterpart may in be practice,and a MRL mingled for single a given with residue herbicide will definition those bei.e. applied to no from both types distinction of the crop, willherbicide-susceptible be made crops. between In herbicide-resistant aconcluded and more that there general was sense, generalshould agreement the not that be CCPR separate elaborated MRLs for GM and conventional crops. methyl, no such petitionthifensulfuron was methyl apparently in made, maizeestablished while by and that MRLs time. soybean for had already been tribenuron methyl includes the parent compoundsdata only. on The new field trials withALS-inhibitor-resistant maize and these soybean showed three that, according sulfonylureas applied to GM 3.3.1 ALS inhibitors As mentioned in Section 3.1.1,GM maize the and company soybean that that are has resistantingredients developed towards that herbicide inhibit active the ALSof enzyme combining mentions the these possibility cropscontaining with herbicides. The the active use ingredientsinclude of of chlorimuron ethyl, these various rimsulfuron, herbicides thifensulfuron sulfonylurea- methyl and tribenuron methyl. residues in various GM herbicide-resistantgovernments crops and/or has led CAC national definition to of the revise, residue so in asof to a interest accommodate that novel number are metabolites specifically of formed fromto the cases, herbicides applied these the crops.a Besides theoretical the possibility definitionherbicide that of and also the its themaximally residue, metabolites tolerable level there may levels of of have is residuescommodities, residues increased present i.e. within MRLs, so of agricultural need that to the the be the safety adjusted, taking of into these account introductionofGMherbicide-resistantcropshashadonthesetting residues. In this section,of the MRLs by impact CAC that is the MRLs explored in for more a detail. given In herbicide, thehave the been absence reviewed. national of CAC MRLs set by the US EPA wileyonlinelibrary.com/journal/ps 3.3 Impact ofIn GM herbicide-resistant crops 2001, on MRL setting theseparate MRLs CCPR for pesticide considered residues in the GM crops. possibility of establishing a CAC MRL isassessed available for whether these new ALSto inhibitors. or The cover amended US the EPA MRLsinhibitor-resistant has use maize should of and be soybean. threefollowed adopted The a of US petition EPA’s these by assessment the sulfonylureasMRLs company for for on amendment the GM of pertinentand pesticide ALS- soybean. use of the ALS inhibitors on GM maize

1202 1203 45 99 43,102 103 at most. 7 − )butthat 1 10 − × No risks of non- 3 . In 1999, JMPR was 98 54 , the EPA considered 6 − The MRLs thus established 10 ,or5.0kgha 1 × − 90 100,101 wileyonlinelibrary.com/journal/ps ) that was lower than the field-tested 1 98 − At that time, in 1998, the adoption of bromoxynil- 99 , and that from drinking water intake 6 , or 1.7 kg ha 6 After that, its share declined, before eventually being 1 − − 10 100 × In Canada, the use of bromoxynil is permitted on GM A new risk assessment was also performed by the EPA in 1998, 5 . application rate (4.5 lbs AI acre corresponded to thepetition rate for for which registration.drinking the Also, water applicant to the had consumers’ contributionas filed exposure well by its was as taken residues the into safety in account, for infants and children. worst-case scenario used inin 1995. 1997, In anticipated residues the were determined scenario onthat that the assumption 3% was used of theAI crop acre was treated at an application rate (1.5 lbs bromoxynil-resistant brassica (canola)Canadian (see authorities Sectionbromoxynil have 3.1.2). in canola, The defined as well(eggs, an as meat in and a MRL milk). numbervalid In of for of products animal addition, for products a which 0.1 no ppm general specific MRL MRL has for of been 0.1 specified. ppm is basedonnewresiduedatathathadbeenprovidedbytheapplicantafter its petition, and alsoexpand because the acreage of of the cotton where applicant’s bromoxynilfrom proposal could 400 to be 000 applied acres (161The 875 latter ha) area corresponded to to 10% 1 of 300which the US 000 was cotton acres area also in (526 considered 1998, 093 inassessments. ha). the new scenario used for the risk the cancer risksuncertainties to inherent be in the negligible, extrapolationfactors of also used, data as taking and well as the into the safety overestimationsfood account of and the drinking the residue water. levels in Outside the United States, Mexico hasfor also bromoxynil set an in MRL of cottonseed,have 1.5 defined ppm while an MRL the of EU, 0.1limit ppm, of Norway which analytical corresponds determination. and to the Israel lower Outside Canada, nobromoxynil in specific brassica (canola) MRLs inhas the have set United the States, threshold been at while the the lower established limiti.e. EU of analytical for determination, atBromoxynil-resistant 0.1 ppm, canola was which2001, on after which aligns the it was Canadian discontinued with for market economic reasons. until the Canadian3.3.3 MRL. Glufosinate As explained above inresidues Section of 3.2.3, glufosinate the ammonium JMPR presentof in reconsidered new crops the on data the fromof basis the GM changes glufosinate-resistant that cropsthe JMPR considered in residue necessary 1998. definition, was One glufosinate to which and broaden at MPP that expressedalso as time considered free included it glufosinate, the necessary withto to parent NAG. cover change It the a altered levels numbercrops. of of residues Because MRLs in the GM so review glufosinate-resistant as completed of toxicity at data that on time,made NAG for no could these changes formal not by be JMPR recommendations to could the CAC. be carcinogenictoxicityintheshortandthelong termwereidentified.The upperbound cancer risk from food intake1 was estimated to be resistant cotton was indeed onwhen the rise, 7.8% reaching its of peakcrop. in the 1999 total US cotton area was planted with this pulled from the market in 2005. Based on these values,reference which of were close negligible to risk the EPA’s of common 1 in 1998 differed fromdelinted those cottonseed proposed was in set 1997.byproducts at The and 1.5 hulls new ppm, were MRL while set in the at 7.0 MRLs and for 5.0 gin ppm respectively. in vitro Such a 97 2011 Society of Chemical Industry c This coincided 96 95 The question as to whether the MRLs 42 : 1193–1210 67 . Bromoxynil had been classified as a group C 2011; 98 ). in vivo 44 The new risk assessments for the various kinds of potential Following this interim decision, EPA published a rule on At that time, an additional study on the carcinogenicity of Impact of altered herbicide residues in transgenic crops on residue standard setting www.soci.org toxicity in 1997 were based on a different scenario than the for animal products hadthe inclusion to of cotton be gin byproducts changedthe in was cancer animal feed. risk considered, In assessment addition, given alsosecond considered carcinogenicity study the in mice, findings which showed from the formation the of carcinomas in livers inof both EPA male and female mice (pp. 14–15 study had previously been requested bythe EPA petition independently from for thebromoxynil MRL that had in been cotton, used in extending aformation study the had in been mice dose identified in in livers which range of tumour male of animals.had Bromoxynil also beenmutagenicity/genotoxicity studies, tested while not positive beingin carcinogenic in rats several but not all bromoxynil in 1997, incompared which with a number the ofof previous changes bromoxynil decision were in as cotton. made bromoxynil a New in MRLs result cottonseed, were of while setMRLs the the was for also range enlarged the usage to of include residues two commodities cottonseed of byproductsadditional with and animal products (EPA,definition 1997b). for Moreover, the bromoxynilextended residue in with DBHA cotton (see andindicated Section that it wanted 3.2.2). animal to expand The the products acreage applicant forof which the had was usage bromoxynil also was registered400 from 000 200 acres 000 acres (161plus (80 875 DBHA ha). 937 in ha) undelinted The to cottonseed7 proposed was ppm, higher MRL than in previously for 1997, set namely MRLs for bromoxynil bromoxynil were in also cottonseed in proposedand 1995. for for cotton hulls gin at 21 byproducts ppm. at 50 ppm carcinogen, which isthere EPA’s is category limited evidence for of carcinogenicityin carcinogens the based absence on for of human animal data. which data EPA alsorule, mentioned, in that its proposed residues ofcottonseed bromoxynil would at contribute the insignificantly, i.e. proposedestimated overall 1/1000th, MRL cancer to risk levels and the long-term in toxicitycaused of by bromoxynil aggregate exposure. Thebasis risks of were a estimated oncrop worst-case the foods scenario containing inpermissible residues which levels of (i.e. consumers MRLs) bromoxynil in consumed been 100% at registered. of its all crops maximally for which it had Pest Manag Sci 3.3.2 Bromoxynil As explained inhave Section been 3.2.2, defined noand by limits residue CAC for definitionbrassica its for and (canola) have residues bromoxynil, MRL been put on whileauthorities in respectively. bromoxynil-resistant place definitions The by cotton US the EPA US and for adopted and an Canadian a interim decision temporarybromoxynil-resistant MRL cotton, of valid 0.04 ppm until for 1998. bromoxynil in seeds of that are naturally resistant. Flaxthat is can mentioned be as one planted of afterminimum the crop the crops rotation herbicide interval has of 2 been months. applied, with a with the commercial introduction ofin bromoxynil-resistant US cotton cotton-growing areas inbe 1995, commercialised there. being The the further chronology first of GM theof regulation cotton MRLs to of bromoxynilthe in estimation cottonseed highlights of the cancerresidues criticality risk in of food derived and from drinking water. intake of bromoxynil bromoxynil tested in mice still had to be reviewed. ). 1 109 − was 1 − )thanin 1 − : 1193–1210 108 67 Considering the 76 2011; 77 ) and maximum residue 1 − while EPA has established ) and rice grain (1.0 mg kg 106 1 − Pest Manag Sci ) and maximum residue (28 mg kg 1 109 − ) for glyphosate in cotton. An MRL of 10 mg kg for total residues in cottonseed. 1 1 − − The company that produced both the herbicide and the 107 Data on residues in glyphosate-resistant maize had been Subsequently, in 2005, the JMPR reconsidered the residues (6.0 mg kg transgenic crops filed a petition toof the bring reasons these for lists this together. was that One registration the company of wanted to glufosinate extend to the both preplant transgenic burndown and applications non-transgenic in varietieshad previously of been three listed as crops transgenic, that namelymaize brassica and (canola), soybean. In EPA’s finalcluded rule that on the this MRLs petition, did itpreplant was not applications, con- have given to that be the raisedduring total a because season of amount was these to retained be andapplications applied because would post-emergence cause foliar higher residueones. levels In than these the cases, preplant itdetermined was the therefore MRLs. the Both transgenic lists varieties werein that thus the next merged edition and of appeared the Code of Federal Regulations. MRLs for cottonseed (4.0 mg kg provided to the JMPR in 2005 but were notSTMR, maximum used residue for or establishing MRL an because the field trials were found of glyphosate inboth cotton. susceptible Residue andsusceptible data GM cotton had resistant were been cotton,therefore found received only while the on not the data to on data resistant comply on cotton with were considered. GAP and 3.3.4 Glyphosate In 1997, the JMPR evaluatedhad data been from carried supervised out field in trials thein that United both States conventional on cotton glyphosate and residues GMThe glyphosate-resistant Good cotton. Agricultural Practicecotton allowed (GAP) for in the pre-emergence, bothand directed preharvest non-GM applications post-emergence of glyphosate and to cotton. GM Inpost-emergence GM applications, cotton, i.e. over-the-top applications, were alsoallowed.TheJMPRnotedthatthepreharvestapplicationswere considerednottocontributetoresiduesinthecottonseedbecause of lack of translocationin of senescent the cotton plants. absorbed The glyphosate JMPRdata considered to from that both the the types residue seed oftherefore cotton combined showed these similar data distributions intrial order and median to residue derive the (STMR; supervised 2.0 mg kg The data on glyphosate-resistant cotton not only included thethat data had already been considered in 1997 butsupervised also the field outcomes trials of with the ‘secondresistant generation’ cotton of glyphosate- plants, toover which the glyphosate top at can a still latedamage stage be to of applied crop the development cotton withoutthese bolls. causing The second-generation tables plants with showis data that higher on the residues than level in that of in residues first-generation plants. thus established. Until 2007, these two MRLsCode were of part Federal of Regulations ato that list had transgenic of specifically MRLs crops, been inrice, assigned including the soybean US brassica and sugar (canola), beet.crops cotton, A not separate being maize, designated list as containedwas transgenic. MRLs the The same for residue for the definition MPP. crops Cottonseed in and both cotton lists, gin i.e. byproductslists glufosinate, were included of NAG in and both the non-transgenic andMRLs. transgenic crops with the same 1997, it was recommended40 mg that kg the MRL be raised from 10 to for residues in these three crops, combined data on glyphosate-resistant cotton, including aSTMR higher (5.2 mg kg 1 1 57 − − 54 These data 2011 Society of Chemical Industry 57,105 in 1999. c 104 1 www.soci.org GA Kleter, JB Unsworth, CA Harris − In 1994, following 56 that had previously been on the basis of field trial data in 1991. 1 1 1 − − − was therefore maintained. No MRLs have been established by CAC 1 − 7,8 . The MRL estimated by JMPR for glufosinate- 1 − 54 hadtoberaisedtoanestimatedvalueof5mgkg 1 55,105 . − 1 − 57 Besides brassica (canola), maize, soybean and sugar beet con- MRLs were also established for animal products (e.g. meat, milk In a processing study, sugar from roots of glufosinate-resistant Withregardtosoybean,anMRL forglufosinateusedasdesiccant Data on residues of glufosinate in glufosinate-resistant maize In 1999, the MRL that JMPR recommended for glufosinate in resistant maize was equivalentConversely, for to maize that forage, for0.02 the mg non-resistant kg previously maize. established MRL of abandonment of the use of glufosinate asthis a MRL desiccant was for lowered soybean, to 0.1 mg kg sidered by JMPR for residue chemistryvarieties, in GM glufosinate-resistant otherresistant crops through have genetic modification.crops also GM glufosinate-resistant that been have notistry rendered been and reviewed that glufosinate- have byand been JMPR authorised rice for for in residue food chem- thein use United the include cotton United States States. and Canada, as well as radicchio and eggs) at the respective lower limits ofin analytical determination 1999. sugar beets had beenof shown glufosinate, to while contain the noin residues detectable the appeared residues molasses. to Oilsdid be from concentrated not brassica contain (canola), detectable maizebyproducts, and levels such soybean of glufosinate, ascontain whereas residues. the some meal of these crops, were found to in soybeans was set at 0.5 mg kg from supervised field trialsconsidered by the in JMPR in North 1998, following America itsof previous data on assessment and glufosinate in non-resistant Europe maize in 1994. were Mostin residues grain of glufosinate-resistantdetermination, with maize a were few belowstill below values the the MRL reaching limit previously established above for of namely non-resistant this 0.1 maize, mg limit kg but seeds of glufosinate-resistant brassica (canola) wasfrom based on supervised data field trials in Canada. Its value of 0.3 mg kg wileyonlinelibrary.com/journal/ps because of thein fact forage that of glufosinatebelow glufosinate-resistant residues the limit maize, were of detectable whereasIn detection in 1998, it forage an had from additional10 non-resistant been mg maize. MRL kg was estimated for maize fodder at fell below the MRL of 5 mg kg able to complete itsthe assessment product of that the is toxicity formedintroduced of from NAG, in glufosinate which glufosinate-resistant by is crops. thethat, The PAT among enzyme JMPR others, thustoxicity the noted when administered lowest to animals levelthan in animal that of trials previously NAG was observed higher for thatthe glufosinate. Additional showed toxicity data on no of MPP had been provided as well. recommended, in 1994,and European on field trials the with non-resistanthigher basis MRL brassica of (canola). of 5 The mg kg data from Canadian in which glufosinate had been used as herbicide.considered In 1998, new the data JMPR on residues ofin glufosinate GM glufosinate-resistant used soybean as in supervised herbicide trials, which also had shorter preharvest intervals than glufosinatetional used non-resistant in conven- soybean. The outcomes,and median showing residue levels maximum that exceeded theJMPR existing to MRL, raise led the the MRL for glufosinate to 2 mg kg led the JMPR to concludecomparable that with the or toxicity of lessacceptable daily NAG than intake and (ADI) that MPP for glufosinate of was could be glufosinate, sustained. and that the

1204 1205 -acetyl- N -acetylase en- N -acetyl-glyphosate, and for N ) and for soybean seed (7 or 1 − wileyonlinelibrary.com/journal/ps -acetyl-glyphosate. PRAPeR also recommended -acetyl-glyphosate under this option, including N N 82 ). 1 − With regard to herbicide-insensitive mutants of enzymes In line with the approach followed by CAC, national authorities Four categories of herbicide resistance traits that have been -acetylated derivatives NAG and MRL values for separate MRL for inhibitedbytheherbicideactiveingredient,ALS-inhibitor-resistant crops expressing mutant ALScrops enzymes expressing and insensitive glyphosate-resistant formsa of bacterial EPSPS, either analogue, achemistry have mutant of been or the authorisedled residues for to formed food a change use. withininhibitor-resistant crops, in The the these distinction the between crops GM residue andcrops non-GM definitions. has may In not not be addition, justified, forcarry as ALS- ALS conventionally bred mutants crops that can confer also resistance to ALS inhibitors, such usually do not specifycrops, but specific for the MRLs particular crop for inof general. GM glufosinate The residue herbicide-resistant has definition beenNAG, amended by while JMPR other withbromoxynil authorities the and glyphosate addition have on the of amended basis ofbicides the the in metabolism GM of crops. definitions For her- example, of the USmoxynil residue definition in for cotton bro- includes DBHA,while besides that the for parent glyphosate compound, in maize andglyphosate, besides soybean glyphosate, includes based on the metabolism ofactive these ingredients of herbicides applied tocrops. GM For herbicide-resistant these cases involving herbicide-metabolisingit enzymes, can be concludedimpact on that the definition the of residues.of genetic Conversely, the the modification introduction GOX has enzymeway had into that an a the glyphosate range residue ofa is major crops defined metabolite because of has AMPA glyphosate not in is non-GM also changed crops. the glyphosate-resistant crops expressing glyphosate oxidoreductase, which converts glyphosate to AMPA. zymes deactivate the herbicide activeN ingredient by forming the introduced into GM cropstance have to been ALS inhibitors, reviewed, bromoxynil,Two including glufosinate resis- different and glyphosate. moleculartance, mechanisms namely the underlie introduction herbicideversion in resis- the of GM the cropherbicide of enzyme or (1) (2) a that an mutated enzymegredient is that to converts targeted less the toxic and herbicidefor active or inhibited in- non-toxic GM by forms. crops Asconvert the has containing the been enzymes observed herbicide ofmetabolites active the has ingredient, the latter ledauthorities formation to group, JMPR, redefine of the which which residue new within reportsservedforbromoxynil-resistantcrops,whichcontainDBHAformed crops. This has to been ob- CAC,from or bromoxynil by bromoxynil national nitrilase, forand glufosinate-resistant some glyphosate-resistant crops, in which 4CONCLUSIONS In the previous sections,carried the by GM herbicide herbicide-resistant resistance crops thatfor traits have been food that approved are use inbeen the reviewed United with States,nature regard Canada and and/or level to of thethe their residues EU rationales potential present behind have within this impactthat project food asynchronous on was crops. authorisations to of One the GM explorenational of crops legislations the and on possibility differences herbicide residues in couldbarriers lead to to trade potential in crop commodities. 10 mg kg one for maize grain (0.3 mg kg 1 76 − ). 6–8 1 − )and 1 ),sugar -acetyl- − 1 77 N − 77 2011 Society of Chemical Industry c NoCAC MRLshave ) and wheat bran 1 The supervised field in roots) and potato 109 − 106 1 76 − ) and maximum residue 1 − ) measured in the field trials. 1 wheat in the United States. − 77 77 . respectively). 1 1 − − : 1193–1210 The levels of residues that the JMPR had 67 77 2011; ), brassica (rapeseed; 20 mg kg 1 ) observed in conventional maize grain obtained from − 1 − Besides the crops for which CAC has considered the residues in Studies with food-producing animals showed that glyphosate Whereas data on glyphosate-resistant sugar beet had been In 2005, the JMPR also summarised the data from processing With regard to soybean, the JMPR considered data on residues As described in Section 3.2.4, EFSA’s PRAPeR considered three Impact of altered herbicide residues in transgenic crops on residue standard setting www.soci.org beenestablishedforalfalfaseed(UnitedStates,0.5 mg kg the maximum residue (20 mg kg GM glyphosate-resistant varieties, i.e. cotton, maizevarious other and GM soybean, glyphosate-resistant crops have been authorised for use as food as well,in including the brassica United (canola) States, and Canada sugar andUnited beet the States EU, alfalfa and and potato Canada in and the was detectable in the kidneys and liverswhile of it cattle, was pigs and not poultry, detectable inof meat, milk glyphosate or eggs, at corresponding feedingThe levels to the JMPR estimated thereforeeggs dietary that recommended burden. corresponded to MRLs thedetermination, minimum for and threshold specific MRLs for meat, for analytical edibleother milk offal mammalians from and cattle, and poultry. pigs, presented to the JMPR in 2005, theserepresent data GAP were owing not to considered deviant to preharvest applications. studies on commodities containingfor example, glyphosate, that glyphosate which and AMPA showed, werederived not from detectable in various oil crops, suchand as soybean. cottonseed, maize, It rapeseed wasproducts such found as to bran of be cereals and concentrated hulls in of soybean. several other from supervised field trialsGM glyphosate-resistant with soybean. both While non-GMof the soybean GAP susceptible included for pre-emergence and and both preharvestthe types applications, GAP for glyphosate-resistantemergence soybean directed also sprays. Preharvest allowed sprays for weretocontributetoresidueformationowingtolimitedtranslocationof not post- supposed absorbed residues to seed during senescence. Theconsidered JMPR therefore those field trials that includedcrop pre-emergence sprays to and comply in- with GAP.of Because the both residue populations types ofcombined soybean for the were determination similar, of these the populations STMR were (5.0 mg kg options for theassessment residue of definition data on of GMthe glyphosate, glyphosate-resistant crops following containing GAT the glyphosate enzyme, is in formed. One which of these the options novel was to metabolite establish a beet (United States and Canada, 10 mg kg these field trials led the JMPR to propose an MRL of 5 mg kg (United States, vegetable, root and tuber, group 1, 0.2 mg kg trials that had been carried outGM with susceptible conventional maize, maize, were i.e. found non- thereforeusedforfurtherevaluationbytheJMPR.WhiletheGAPfor to comply with GAP andconventional were maize included pre-emergence, spot and preharvest treatments, it was concluded by the JMPRspot that treatments the would preharvest not and substantially contributein to the the residue crop. The STMR (0.12 mg kg Pest Manag Sci CAC has established500 mg kg MRLs for glyphosate in alfalfa (fodder; The JMPR thus concluded that these data confirmed theestablished previously MRL of 20 mg kg for total residues. not to comply with the GAPs for these crops. (3 mg kg previously considered for conventional andmaize in glyphosate-resistant 1997 would also fall below this MRL. andstraw(20and300 mg kg ., et al :65–72 2 . [Online]. Pest Manag : 1193–1210 ). 67 118 AgBioForum 2011; . [Online]. International org/resources/publications/ ble:http://ec.europa.eu/food/ ://www.agbioforum.org/v2n2/ -biotech Applications, Ithaca, NY :47–52 (2005). 61 Pest Manag Sci :479–488 (2008). en.cfm [9 May 2011]. 64 /default.asp [9 May 2011]. Pest Manag Sci register/index Pest Manag Sci Global Status of Commercialized Biotech/GM Crops: 2010 -acetyl-glyphosate from glyphosate, for which both :1107–1115 (2007). ) (Tables 5 and 6 of Harris and Gaston N 1 63 − (ISAAA Brief 42–2010), ExecutiveService for Summary the Acquisition of Agri (2011). Available:briefs/42/pressrelease http://www.isaaa. are adopting Roundup(1999). Ready [Online]. varieties. v2n2a02-carpenter.pdf [9 Available: May 2011]. http resistant canolamanagement. on the environmental impact of weed Directorate General Health andsion,Brussels,Belgium(2011).Availa Consumers, European Commis- dyna/gm Altered pesticide usegeneral on impact from transgenic anSci crops environmental perspective. and the associated herbicide regimes andeffects the of associated glyphosate-resistant crops potentialEurope. versus environmental what they replace in Community Register of Genetically Modified Food and Feed 2 Carpenter JandGianessi L,Herbicidetolerantsoybeans:whygrowers 3 Brimner TA, Gallivan GJ and Stephenson GR, Influence of herbicide- 1JamesC, 5 Kleter GA, Harris C, Stephenson G and Unsworth J, Comparison of 4 Kleter GA, Bhula R, Bodnaruk K, Carazo E, Felsot AS, Harris CA, 6 For a number of residues, including those of bromoxynil and It has also been observed that no CAC MRLs exist for a number monitoring in the United Kingdom were0.1 similar mg (i.e. kg median value ALS inhibitors, it hasNorth been American observed countries inNAFTA. the strive In addition, present it towards has review also harmonisation been that take noted that under into national authorities account otherapplied nation’s to evaluations GM of crops, such herbicides asproducing being for residues of GAT-expressing crops FSANZ and EFSA referred to the US EPA whenand assessing the residue toxicity data.international It harmonisation can under CAC, be differentthresholds envisaged, definitions and may however, still that,which, be without in employed turn, by mayrecommended lead food to that control impediments the inspections, indefinitions trade. international and It harmonisation thresholds is of therefore beherbicide–crop continued residue combinations to so extend as to theon cover range GM the herbicide-resistant use of crops. of herbicides of herbicide–crop combinations thatof are GM applicable herbicide-resistant crops to and corresponding a herbicides that number have been authorised by nationalpertain authorities. to Not ALS only inhibitors doesMRLs and this at bromoxynil, all, for but which alsoapplications CAC to has of certain no crops glufosinate thatresistant or can be glyphosate, rice combined such with andnumber as of glyphosate-resistant glufosinate- cases, national brassicaconsidered authorities, (canola). such whether as In the thechanged US to residue a cover EPA, the definition have usage on or the GM crop. MRL should be ACKNOWLEDGEMENTS Financial support fromApplied Chemistry the (IUPAC, project International numberthe Dutch Union 2006-015-3-600) Ministry of and Economic for Affairs, Agriculture and(project Pure Innovation number and 72296.01)coauthor Dr is Harris is gratefully involvedconsultancy in acknowledged. activities, a this range While paper of is commercialpublicly based scientific available on literature sources and of other received information, any commercial and funding in Dr relation Harris to this has publication. not REFERENCES ). 112 114 110 .Intofu,a 1 2011 Society of Chemical Industry − c www.soci.org GA Kleter, JB Unsworth, CA Harris While bromoxynil and 113 report that the residues in states’ national programmes, the Canadian Food Inspection 118 In soy milk and popcorn samples, 111 116 . 1 − Polenta, a food product derived from maize, Forglyphosateinthemaizeandsoybeanproducts 115 . 1 − 116,117 No general trend of an increase or decrease in the levels While residue data from experimental studies have been protein-rich soybean product, 11 out offor 60 samples glyphosate, were positive with1.1 mg residue kg levels ranging between 0.1 and maize grain reported in dossier applications and measured during no glyphosate residuesfound. at or above the reporting limit were Agency’s National Chemical Residue Monitoring Program wileyonlinelibrary.com/journal/ps which operate inmeasure parallel glyphosate to residues. theResidues Committee EU and In the coordinated Norwegian particular, National(Mattilsynet) Food programme, monitor Authority the glyphosate in UK cerealsUK and Pesticide infant Pesticide foods. Residue The foods Committee derived also from measured twomaize, glyphosate crops in in of the interest,glyphosate years namely residues soybean 2002 in and and these studies 2006. was The 0.1 mg limit kg of reporting for Interestingly, various EU member and the EU Coordinated Programme. as for a rangeand sulfonylurea-tolerant of soybean. Interestingly, commercialised herbicides of imidazolinone-resistant the latter crops category are appliedto at residues of low the applicationthe parent rates, limit compound of giving and quantification, rise and itsconcern. are Authorities metabolites therefore generally below tend of to low limitfor these toxicological the herbicides residue to the definition parent compound, notwithstanding the fact that the metabolites may also be toxic (pp. 22–23 of OECD was found toat contain a glyphosate level in of six 0.1 out mg kg of 23 samples, all of herbicide residues inobserved in supervised GM field trials herbicide-resistant performed within the crops framework of has herbicide been registration.generalisations on It the impact of is these GMare therefore crops to on be the not MRLs set that for possiblebut the this to residues has of to infer the bereflected considered herbicide on active in a ingredients, case-by-case the basis.herbicides changes This for is in also use MRLs onthe following GM MRLs were herbicide-resistant the recommended by crops. JMPR registration in Increasesfor a of glufosinate few in in cases, soybean for and example for glyphosatein in many cottonseed, other while cases this was not recommended. used to establish thecombinations residue tolerances described for thecompare above, herbicide–crop these it tolerancesthe with field, would i.e. what in beof commercially is produced interesting actually the foods. No measuredin to measurement herbicides in question of isfederal interest pesticide apparently in residue carriedthe United monitoring the out States programmes and by particularethyl, of rimsulfuron, Canada. thifensulfuron the The crop methyl the ALS and centralisedbromoxynil, tribenuron EU, foods inhibitors methyl), (chlorimuron or glufosinate andfeature glyphosate, on the for listsUS example, of FDA’s do the Pesticide not most Program, recent monitoring data of the chlorimuron ethyl areAgriculture’s measured Pesticide Data under Program, this is the done in US water only. Department of containing residues, no MRLs hadKingdom been established at in the that United residues time in (since then, food,established EU-wide including MRLs those under for of EUresulting pesticide glyphosate, from regulations). have consumption of become The thewas products estimated intake by containing the residues committee of todose be glyphosate of below the glyphosate acute and reference In therefore did addition, not Harris raise safety and concerns. Gaston

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