Fate of Glyphosate during Production and Processing of Glyphosate

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Food Safety and Toxicology

Fate of Glyphosate during Production and Processing of Glyphosate-Resistant Sugar Beet (Beta vulgaris) Abigail Barker, and Franck E. Dayan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05672 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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Journal of Agricultural and Food Chemistry

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Barker—Glyphosate and sugar beet

Fate of Glyphosate during Production and Processing of Glyphosate-Resistant Sugar Beet (Beta vulgaris) Abigail L. Barker and Franck E. Dayan* Department of Bioagricultural Sciences and Pest Management, Colorado State University, 1177 Campus Delivery, Fort Collins, CO 80523

*Corresponding author: Tel: +1 (662) 816-6214; Fax: +1 (970) 491-3562; email: [email protected] ORCID Franck E. Dayan: 0000-0001-6964-2499 Abigail L. Barker: 0000-0003-2806-4148

ACS Paragon Plus Environment


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Barker—Glyphosate and sugar beet 1

Keywords: Roundup Ready, glyphosate-resistant, sugar beet, glyphosate, residue,

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ABSTRACT:

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Glyphosate is a widely used herbicide in commercial crop production for both conventional and

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herbicide-resistant crops. Herbicide-resistant crops, like glyphosate-resistant sugar beet, are often

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exposed to multiple applications of glyphosate during the growing season. The fate of this

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herbicide in resistant crops has not been publically documented. We investigated the fate of

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glyphosate and main metabolite AMPA in glyphosate-resistant sugar beet grown in Northern

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Colorado. Glyphosate residues were measured via directed UHPLC-MS/MS analysis of sugar

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beet shoots and roots throughout the growing season, from samples collected at various steps

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during sugar beet processing, and from flow-through samples of greenhouse-grown beets. Sugar

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beet rapidly absorbed glyphosate after foliar application, and subsequently translocated the

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herbicide to its roots, with between 2 to 3 µg/g FW measured in both tissue types within one

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week of application. However, only trace amounts of glyphosate remain in either the shoots or

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the roots two weeks after application. Analysis of irrigation flow-through in pot assays

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confirmed that the herbicide readily exuded out of the roots. Processing of the beets removed

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glyphosate and herbicide levels were below the limit of detection in the crystalline sugar final

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product.


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INTRODUCTION

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Genetically modified (GM) crops have become an integral part of production agriculture in the

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United States and around the world. There is a large gap in understanding and acceptance of GM

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crops that exists between the consumer and scientific community.1 A recent National Academy

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of Sciences meta-analysis determined proper use of herbicide-resistant crops resulted in no

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negative environmental impact and there was no evidence that consumption of products derived

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from herbicide-tolerant crops had negative health impacts on humans or animals, including any

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as a result of gut microbiota perturbations.2 Despite this, certain advocacy groups have recently

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expressed concerns about the possibility of pesticide residues in crops and subsequently food.3-4

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As of 2015, there are ten commercially available GM crops expressing a variety of traits,

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including herbicide resistance, insect resistance, or browning reduction in fruit that took up 12%

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of the worldwide cropland in 2015.2 One such crop is glyphosate-resistant (GR) sugar beet (Beta

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vulgaris L. subsp. vulgaris).

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Sugar beet is an industrial crop grown commercially as a hybrid, with sucrose from the root as

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the primary plant constituent of interest. Sugar beet is a biennial plant that takes two years to

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complete its biological lifecycle and produce seed, but is commercially harvested after the first

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year when the sucrose content is highest.5 These are grown primarily in Minnesota, Idaho, North

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Dakota, Michigan, Montana, Nebraska, Wyoming, California, Colorado and Oregon and

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collectively produce nearly 50% of the raw sugar consumed in the US. The remainder is obtained

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from cane sugar produced in the US or imported from tropical countries. Weed management is

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one of the major challenges in sugar beet production.6 Sugar beet is a slow growing crop and is

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sensitive to weed competition. Consequently, complex programs were developed for managing

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weeds in conventional sugar beet, with over 120 herbicides using 19 active ingredients registered



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for use in this crop. In conventional sugar beet production, herbicide mixtures containing up to

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five active ingredients were used, together with mechanical weeding in 50% of the crop.5 Post-

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emergent herbicidal active ingredients included phenmedipham, clopyralid, ethofumesate,

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desmedipham, clethodim and triflusulfuron-methyl.7

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GR sugar beet was introduced in 2007 and has been the most rapidly adopted GM technology to

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date, accounting for nearly 100 percent of sugar beet acres now grown in the US 8-9. These

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varieties express the glyphosate-insensitive CP4 microbial gene for 5-enolpyruvylshikimate-3-

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phosphate synthase (EPSPS), the target of the herbicide.10-12 Following the introduction of GR

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sugar beet, growers have relied primarily on glyphosate for broad-spectrum weed control.9, 13

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These simple management programs include one to three applications of glyphosate for season-

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long weed control in GR sugar beet.14 Along with superior weed management practices and

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greatly reduced crop injury, this GM technology has additional environmental and production

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benefits. These benefits include: a 5-fold using GR sugar beets, and the environmental impact

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quotient has been reduced over 90%, environmentally superior production practices such as

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conservation tillage that has reduced carbon emission 83%, reduced fuel usage and its associated

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emissions by 50%, reduction of water usage by over 30% and increased land use efficiency

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(increased yields of more than 30%).5, 7, 9

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Glyphosate [N-(phosphonomethyl)glycine] was developed by Monsanto in 1970.15 The herbicide

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inhibits EPSPS, the protein which catalyzes the formation of 5-enolpyruvylshikimate-3-

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phosphate from shikimate-3-phosphate and phosphoenolpyruvate. This inhibition disrupts

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synthesis of the aromatic amino acids—phenylalanine, tyrosine and tryptophan—and causes

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plant death. Plants turn chlorotic within a few days and eventually die after a few weeks. EPSPS

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is an attractive target for herbicides because the shikimate pathway is absent from mammals.16-17


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Additionally, glyphosate does not persist in most soils, due to rapid adsorption to the soil organic

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matter and degradation by soil microbes, giving it a particularly favorable environmental

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profile.18

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Physiochemical properties of glyphosate enable it to move to metabolic sinks along with

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photosynthates19-21 and it is not subject to rapid metabolic degradation in plants.22-24 While

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translocation of glyphosate in sugar beet was self-limiting in conventional sugar beet due to the

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rapid phytotoxic response of the herbicide on this species,25-30 this is no longer a limiting factor

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in the GR sugar beet since these plants are no longer affected by the herbicide. Consequently, a

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significant amount of glyphosate can potentially accumulate in sugar beet roots and remain as the

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parent molecule for an extended period of time.

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Glyphosate residues in crops and food products have not been reported extensively in the public

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domain. The most extensive published research has been on residues in GR soybean (Glycine

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max) and canola (Brassica napus), where small levels of glyphosate accumulated in the beans, as

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the metabolic sink of the plant.31-33 These plants also metabolized some of the glyphosate into the

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main metabolite aminomethylphosphonic acid (AMPA), but this metabolic degradation in other

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plants is generally slow or nonexistent.33-34 A recent study in maize found no accumulation of

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glyphosate in the seed, but did find a small amount of glyphosate degradation into AMPA.35

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Before GM crops can be grown commercially in the United States (US) they must pass rigorous

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testing and follow the guidelines for pesticide residues set by the EPA under the Federal Food,

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Drug, and Cosmetic Act (21 U.S.C. § 346a (2015)) and the Code of Federal Regulations (40

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C.F.R. § 180.364 (2014)). Under the EPA, the maximum residue levels (MRLs) of glyphosate in

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beet shoots and roots is 10 µg/g fresh weight (FW) and 25 µg/g FW in beet pulp. Beet molasses



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is not specified in the document. Acceptable consumable limit of glyphosate set by the EPA is

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1.75 mg/kg body weight. The European Commission on Food Safety established similar

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guidelines with glyphosate MRLs in sugar beet set at 15 µg/g FW (Regulation (EC) No

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396/2005), and an acute reference dose (consumable limit) of 0.5 mg/kg body weight (European

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Food Safety Authority, 2015; DOI: 10.2903/j.efsa.2015.4302).

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The main objective of this study was to determine the fate of glyphosate in GR sugar beet after

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common practice use of the herbicide during the growing season, and during the processing of

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the beet roots into sugar. To meet this goal, glyphosate levels in shoots and roots were

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determined before and after each glyphosate application and directly before harvest, and in

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samples obtained during the processing of the roots into sugar from a processing plant in

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Colorado. The ability of sugar beet roots to exude glyphosate was verified by quantifying

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glyphosate in irrigation flow-through collected from experiments performed in pots.

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EXPERIMENTAL PROCEDURES

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Field study. Two fields in Northern Colorado located at 40.229981° -105.015767° (Field 1) and

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40.189568° -105.024268° (Field 2) were selected for this study. Field 1 was planted with HM

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9617 beets on 15 April 2017. Glyphosate, 825 g ai/ha and clopyralid, 110 g ai/ha were applied on

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1 June 2017 when beets were at a 4-6 leaf stage. Glyphosate, 870 g ai/ha and metolachlor, 1.07

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kg ai/ha were applied on 28 June 2017 to a small portion of the field that was missed with the

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initial application. Nitrogen (78.5 kg/ha) was side dressed in late June 2017. Difenoconazole and

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propiconazole at 130 g ai/ha each were applied in early August 2017 for powdery mildew

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protection.


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Field 2 was planted with Crystal W322NT on April 17th 2017. Glyphosate, 1.26 kg ai/ha with

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clethodim, 140 g ai/ha was applied on 30 May 2017 when beets were at a 4-6 leaf stage.

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Glyphosate, 1.26 kg ai/ha with Intensity clethodim, 140 g ai/ha and metolachlor, 1.07 kg ai/ha

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was applied 22 June 2017. Azoxystrobin was band-applied at 0.15 g ai/ha on 7 June 2017.

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Nitrogen (112 kg/ha) was top-dressed with 45 kg/ha of phosphate in late June 2017. The

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fungicides difenoconazole and propiconazole at 130 g ai/ha each were applied in early August

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2017 for powdery mildew protection.

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Samples were collected on 26 May, 6 June, 21 June, and 28 June 2017, before and after each

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treatment (Fig. 1). Additional samples were collected midseason on 12 July 2017 and on 6

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October 2017 directly before harvest. At each collection date 6 random plants were collected

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from each field, except for the 21 June and 28 June dates when only Field 2 was used for

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collection. The samples were separated into shoots and roots then directly put on dry ice, and

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then moved to a -80 C freezer within 4 h until further processing.

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Flow-through experiment. Black plastic “cone-tainers” (6.5 cm diam.) were filled with steam

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pasteurized Fafard custom potting soil (45-55% peat moss, vermiculite, bark, and dolomite lime;

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Sungro Horticulture, Agawam, MA) and pre-watered to adequately moisten soil. Crystal

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W322NT sugar beet seed(s) were planted with 1 seed per cone-tainer at approximately 1 cm

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below the soil. Seedlings were then grown in a greenhouse at 27 ± 2 °C, 50% relative humidity,

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and 16 hrs lighting for 4-5 weeks or until the plants have 2-3 fully expanded leaves. Plants were

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fertilized every 2-3 weeks with Peters Professional 20-20-20 General Purpose fertilizer (The

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Scotts Company, Marysville, OH) at a rate of 200 µg/mL. When plants reached 5 weeks they

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were transferred to new cone-tainers containing Profile Field & Fairway inert medium (Profile,

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Buffalo Grove, IL). This medium is free of any organic matter, and was selected to facilitate the



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recovery of any glyphosate exuded from the roots. They were transferred to a greenhouse

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maintained between 21 and 28 C with 12 h daylight. Beets were sprayed with glyphosate, 1.26

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kg ai/ha with the potting medium covered. Plants were then watered daily directly to the potting

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medium with 100 mL of ¼ strength Hoagland’s solution and the flow-through collected.

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Sample processing. Thawed roots from the field collected sugar beet were cut into 2.5 cm

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pieces, placed in a blender and homogenized with D.I. water (1:1 w/v) for 4 min at highest

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speed. The homogenate was filtered through 4 layers of cheesecloth to separate the juice from

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the pulp. The filtrate (diffusion juice) was collected. Shoots were ground with mortar and pestle

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under liquid nitrogen. Ground tissue (2.5 g) were mixed with 5 mL of D.I. water and shaken for

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10 min. The mixture was then centrifuged at 4,000 g for 5 min, and the supernatant was used

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for analysis.

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Samples of sugar beet fractions processed at the Western Sugar Cooperative factory in Fort

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Morgan, CO were also collected and stored at -80 C until analysis (Fig. 1). Samples included

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pressed pulp, diffusion juice, thin juice, evaporated thick juice, crystalline sugar and molasses

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(Fig. 1). Sugar and molasses samples were diluted 25 mg/mL in distilled water. Diffusion juice

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and thin juice were analyzed as is. Pressed pulp was mixed 1:3 (w/v) with distilled water and

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homogenized in a blender for 4 min at highest speed, then the homogenate was filtered through a

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4 layers of cheesecloth to separate the liquid which was used for analysis. Evaporated thick juice

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was diluted in distilled water at a 1:1 ratio. Flow-through samples were processed without

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dilution.

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Glyphosate analysis. Glyphosate and AMPA were quantified in the beet samples via

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derivatization with fluorenylmethyloxycarbonyl (FMOC) chloride as described in Takano et al.37


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Briefly, a 400 µL aliquot of 5% borate solution was added to 800 µL of sample in a 2 mL

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Eppendorf tube and vortexed. A 400 µL aliquot of derivatization agent (10 mg of FMOC-Cl

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dissolved in 1 mL of acetonitrile) was added to the tubes and the mixture was vortexed. The

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samples were incubated at room temperature for 4-16 h. Standard curves of glyphosate and its

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primary metabolite AMPA were made from a derivatized 5 µg/mL stock and diluted in water to

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concentrations of 0.005, 0.01, 0.05, 0.1, 0.5, 1 µg/mL stock solutions. The samples were

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centrifuged at 10,000g for 5 min and filtered through a 0.25 µm nylon syringe membrane filter

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prior to analysis on a Shimadzu LCMS 8040 (Shimadzu Scientific Instruments, Columbia, MD

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21046).

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The system consisted of a Nexera X2 UPLC with 2 LC-30AD pumps, a SIL-30AC MP

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autosampler, a DGU-20A5 Prominence degasser, a CTO-30A column oven, and SPD-M30A

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diode array detector coupled to an 8040 quadrupole mass-spectrometer. For glyphosate, the MS

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was in negative mode with a MRM of 390>168 and set for 100 ms dwell time with a Q1 pre-bias

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of 26.0V, a collision energy of 11.0V and a Q3 pre-bias of 30V. For AMPA, the MS was in

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negative mode with a MRM of 331.9>110 and set for 100 ms dwell time with a Q1 pre-bias of

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21.0V, a collision energy of 5.0V and a Q3 pre-bias of 20V. The samples were chromatographed

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on a 100x4.6 mm Phenomenex kinetex 2.6 µm Polar C18 100 Å column maintained at 40°C.

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Solvent A consisted of water with 0.01 M ammonium acetate and solvent B was acetonitrile. The

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gradient started at 10% B for the first min, increased linearly to 99% B until 5 min. The mobile

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phase remained at 99% B until 8 min, then returned to 10% B at 8.5 min and maintained at 10%

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until the end of the run at 11 min. The flow rate was set at 0.4 mL/min and each sample was

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analyzed as 2 µL injection volumes. Retention times for AMPA and glyphosate were 4.99 and

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4.72, respectively. The extraction efficiencies were 90% for AMPA and 99% for glyphosate.



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LOD and LOQ for glyphosate were 2.76 pg/µL and 8.36 pg/µL, LOD and LOQ for AMPA were

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7.4 pg/µL and 22 pg/µL calculated based on the standard deviation of the response and the slope

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of the standard curve.38

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Statistical Analysis. Multivariate factorial ANOVA was performed, and comparison of means

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was calculated using the emmeans package in R (v.3.3.1). Analysis of root and shoot data and

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factory samples were performed separately. In all cases the response variable was the amount of

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detected glyphosate. For root and shoot data the variables were collection date and field. For

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factory samples variables were type of sample and factory. For field samples n=6, for factory

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samples n=3.

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RESULTS AND DISCUSSION

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In the 2017 field season in Colorado, the weed pressure was lighter than average. Field 1

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required only one glyphosate application while Field 2 required two applications to control weed

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species; normally fields receive 2-3 applications per year. As indicated in Figure 1, samples were

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collected from the fields approximately 5 d before and after each application. Two additional

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collections were made in the middle of the season and directly before harvest.

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Glyphosate levels in field-grown sugar beet. Glyphosate was below the limit of detection

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within the leaves and roots of sugar beet (data not significantly different from zero, p < 0.05)

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prior to glyphosate application. Consistent with other plant species, sugar beet readily absorbed

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glyphosate 5 d after foliar application, with between 2 and 4 µg glyphosate/g FW present in the

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shoot of beets in both fields (Fig. 2A). The active ingredient translocated to the roots with

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between 2 and 4 µg glyphosate/g FW in the roots (Fig. 2B). This massive translocation of

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glyphosate is associated with its unique ability to move along photosynthates to metabolic


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sinks.39-40 By the next collection date in field 2, 15 d later, most of the glyphosate had left the

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entire plant. A similar pattern of absorption, translocation and elimination was observed in the

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samples from the field that received a second application of glyphosate. Subsequently,

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glyphosate levels continued to decrease in the roots until harvest, with concentrations of 1.5 pg/g

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FW in Field 1 (one application) and 32 pg/g FW in Field 2 (two application) at harvest. Levels

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were significantly different between collection dates and between fields at the final two

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collection dates (p < 0.05), reflecting the difference in the number of glyphosate application each

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field received.

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AMPA was below the limit of detection in all of the samples, which was not surprising as rapid

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degradation of glyphosate into AMPA has only been shown in a few species.22-24, 33, 41 To date,

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exudation of glyphosate from sugar beet roots has not been reported. However, there is evidence

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that glyphosate can exude from the roots of some plants (e.g., wheat, soybean, canola, cotton,

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corn, and field horsetail).42-44 Therefore, we postulated that the rapid disappearance of glyphosate

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from the tissues of treated sugar beet plants was associated with exudation from the root into the

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soil as was similarly shown in wheat,43 where it is rapidly degraded by soil microbes.18

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Glyphosate exudation from the beet. To determine if glyphosate was exuded from the sugar

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beet roots, flow-through from watering was collected from beets for 21 d after a foliar

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application of glyphosate. Watering solution was added directly to the inert potting medium to

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eliminate flow off of the leaves. Glyphosate was detected at fairly uniform levels in the collected

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flow-through from the daily watering for the first 11 d, with an average of 218±40.5 µg of

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glyphosate per day. After 11 d detection rapidly tapered to below the limit of detection. This

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process takes longer in the field, evidenced by the amount of glyphosate still in the root one

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week after treatment (Fig. 2B). The differences in timing for glyphosate exudation in the flow



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through trial and the field trial, 11 days for the greenhouse trial and up to 21 days in the field to

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exude most of the glyphosate, likely due to the differences in water applied to the roots. In the

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greenhouse the beets were over-watered daily, so the glyphosate around the roots was removed

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more rapidly. In the field watering is less frequent and exuded glyphosate would adhere to

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organic matter around the root.

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Glyphosate levels during processing of sugar beet roots. The fate of glyphosate during the

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processing of sugar beet into crystalline sugar was monitored. The process involves

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homogenizing the roots and filtering the homogenate to separate the pressed pulp from the

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diffusion juice. This juice is processed via a carbonation step to remove proteins and produce the

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thin juice, which is concentrated into evaporated thick juice. Sugar is subsequently crystallized

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from the thick juice and separated from the remaining molasses (Fig. 1). The highest level of

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glyphosate occurred in the diffusion juice, the first product of processing, at 223 pg/mL (Fig. 3).

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The carbonation step successfully removed most of the glyphosate residue, with 3.8 pg/mL

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remaining in the thin juice. This value was not statistically different from zero (p < 0.05). Further

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processing of the juice removed all trace of the herbicide, with no glyphosate being observed in

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the thin juice, evaporated thick juice and granulated sugar (Fig. 3). Trace of glyphosate was

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measured in the pressed pulp, at 102 pg/g, which is at most 200 times lower than the 25 mg/kg

237

minimum allowed established by the US Code of Federal Regulation (40 C.F.R. § 180.364

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(2014)). The amount measured in molasses was 36.3 pg/g, which is at most 800 times lower than

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that approved for sugarcane molasses and the value was not statistically different from zero (p

Fate of Glyphosate during Production and Processing of Glyphosate

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