glyphosate and aminomethylphosphonic acid content in glyphosate

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Agricultural and Environmental Chemistry

Glyphosate and aminomethylphosphonic acid content in glyphosateresistant soybean leaves, stems and roots and associated phytotoxicity following a single glyphosate-based herbicide application Élise Smedbol, Marc Lucotte, Sophie Maccario, Marcelo Pedrosa Gomes, Serge Paquet, Matthieu Moingt, Lila Lucero Celis Mercier, Millaray Rayen Perez Sobarzo, and Marc-André Blouin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00949 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Journal of Agricultural and Food Chemistry

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GLYPHOSATE AND AMINOMETHYLPHOSPHONIC ACID CONTENT IN

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GLYPHOSATE-RESISTANT SOYBEAN LEAVES, STEMS AND ROOTS AND

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ASSOCIATED PHYTOTOXICITY FOLLOWING A SINGLE GLYPHOSATE-BASED

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HERBICIDE APPLICATION

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*ÉLISE SMEDBOL1, MARC LUCOTTE1, SOPHIE MACCARIO1, MARCELO

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PEDROSA GOMES2, SERGE PAQUET3, MATTHIEU MOINGT1, LILA LUCERO

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CELIS MERCIER3, MILLARAY RAYEN PEREZ SOBARZO4 , MARC-ANDRÉ

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BLOUIN4

1 Université

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du Québec à Montréal, GEOTOP & Institut des Sciences de

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l’environnement, 201, Avenue du Président-Kennedy, H2X 3Y7, Montréal, Québec,

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Canada

12

2 Universidade

biológicas, 80050-540, Curitiba, Paraná, Brazil

13 14

3

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Université du Québec à Montréal, Département des sciences biologiques, 141, Avenue du Président-Kennedy, H2X 1Y4, Montréal, Québec, Canada

15 16

Federal do Paraná, Departamento de Botânica, Setor de Ciências

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Université du Québec à Montréal, Département de Chimie, 2101, rue JeanneMance, H2X 2J6, Montréal, Québec, Canada

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*Correspondance: É. Smedbol, Université du Québec à Montréal, GEOTOP &

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Institut des Sciences de l’environnement, 201, Avenue du Président-Kennedy, H2X 3Y7,

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Montréal, Québec, Canada

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E-mail: [email protected]

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Abstract

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Glyphosate-based herbicides (GBH) applications were reported to induce physiological

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damages to glyphosate-resistant (GR) soybean, which were mainly attributed to

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aminomethylphosphonic acid (AMPA). In order to study glyphosate and AMPA dynamics

29

in plants and associated phytotoxic effects, a greenhouse experiment was set where GR

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soybeans were exposed to GBH (0.7 to 4.5 kg glyphosate ha-1) and sampled over time (2,

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7, 14 and 28 days after treatment (DAT)). Hydrogen peroxide content increased 2 DAT,

32

while a decrease was observed for the effective quantum yield (2, 7, 14 DAT), stomatal

33

conductance (2 DAT) and biomass (14 DAT). Glyphosate content was higher in leaves,

34

followed by stems, then roots. AMPA content tended to increase with time, especially in

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roots, and the amount of AMPA in roots was negatively correlated to mostly all

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phytotoxicity indicators. This finding is important since AMPA residues are measured in

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agricultural soils several months after GBH applications, which could impact productivity

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in GR crops.

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Keywords: glyphosate and AMPA toxicokinetics, glyphosate-based herbicides,

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glyphosate-resistant soybean, H2O2, stomatal conductance, biomass, effective quantum

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yield

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1.

Introduction

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Application of glyphosate-based herbicides (GBH) on glyphosate-resistant (GR) crops is

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now the main weed control strategy worldwide in field crops and, for example,

47

approximately 78% of all soybean crops are GR

48

(phosphonomethyl)glycine) has previously been described as slighlty mobile in plants,

49

residues of this molecule as well as aminomethylphosphonic acid (AMPA), its main

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metabolite, are measured in grains several weeks/months after GBH applications

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Glyphosate and AMPA are also detected in GR soybean leaves and stems at all growth

52

stages, following GBH applications 5. Glyphosate fate in plants after foliar application is

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relatively well documented, however, very little is known about that of AMPA.

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Glyphosate, the active molecule of GBH, is a phosphonic acid, usually sprayed in a

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watersoluble form (isopropylamine salt) and is best absorbed by plants with the addition

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of surfactants contained in the commercial formulations 6. After foliar application, it is

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proposed that glyphosate is absorbed through cuticular uptake and/or stomatal infiltration

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at varying rates following the species tested, the applied concentration and the surfactant

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concentration 7. Different degrees of glyphosate absorption are reported in the literature,

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ranging from 4 to 70% of the applied dose 7-9. About 40-45% of the absorbed glyphosate

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is then systematically translocated in the entire plant 7. After reaching the vascular tissues,

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glyphosate is preferably distributed towards specific tissues following the photoassimilates

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pathway

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stems is estimated at 5.39 and 4.02 DAT in plants exposed to glyphosate doses of 0.8 and

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1.6 kg ha-1 11. Glyphosate dissipation in plants can occur by its metabolization into AMPA,

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particulary so in the second generation of GR plants expressing glyphosate oxidoreductase

10.

1.

While glyphosate (N-

2-4.

The half-time dissipation of glyphosate measured in GR maize leaves and



12.

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(GOX) enzyme, isolated from Ochrobactrum anthropi

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resistant to GBH through its metabolization into AMPA, by the expression of GOX which

69

cleaves glyphosate C-N bond

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residues are expected to be present. The second resistance pathway in GR crops is the

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introduction of the CP4 gene isolated from Agrobacterium sp., coding for an insensitive 5-

72

enolpyruvolshikimate 3-phosphate synthase (EPSPS) enzyme

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insentitive EPSPS, while some crops have both an insensitive EPSPS and express GOX 15.

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However, AMPA residues are also measured in GR plants not expressing GOX but only

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an insensitive EPSPS, as well as in non-GR plants

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for glyphosate metabolization into AMPA in plants. Glyphosate dissipation in plants could

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also occur via its excretion in root exudates, which is estimated to be in the order of 8-12

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% of the applied glyphosate dose 16. Moreover, non-treated plants may absorb glyphosate

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via the rhizosphere from treated plants17. Whether or not this phenomenon may also occur

80

for AMPA is actually unknown.

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Since GR plants are not resistant to AMPA, it is suggested that glyphosate degradation into

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AMPA in plant tissues could be responsible for the phytotoxity observed in GR crops, such

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as reduced photosynthesis, stomatal conductance and transpiration

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chlorophyll content and biomass 19, occuring after GBH applications. Similar phytotoxic

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effects are also reported in non-GR plants following GBH applications, with for example,

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reduced photosynthesis and chlorophyll content in phytoplankton

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assimilation in non-GR soybean

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oxygenase (Rubisco) activity

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effects on photosynthesis and carbon metabolism can occur in both GR and non-GR

13.

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Indeed, these GR crops are

Thus, in the case of GR plants expressing GOX, AMPA

22,

9, 13,

14.

All GR crops have an

which suggests another pathway

18

20, 21,

or reduced

reduced CO2

reduced ribulose 1,5-biphosphate carboxylase

or carbohydrates accumulation in leaves

24.

Since these


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species, the phytotoxicity observed after GBH applications could result either from the

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effects of glyphosate, AMPA or both molecules simultaneously. In non-GR plants, it was

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proposed that AMPA could reduce chlorophyll biosynthesis by competitioning with δ-

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aminolevulinic acid (ALA) synthetase or glycine, while glyphosate seemed to induce

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chlorophyll degradation through the enhanced formation of reactive oxygen species 25. This

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suggests that glyphosate and AMPA may interfere with different mecanisms both resulting

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in a decrease of the chlorophyll content. The main objective of this study is to investigate

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glyphosate degradation into AMPA in GR soybean, as well as to relate the distribution of

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both molecules among plant tissues with observed phytotoxic effects upon GBH

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application. The effects of GBH applications on phytotoxicity indicators such as ΦPSII,

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H2O2, biomass, chlorophyll content and stomatal conductance, have been evaluated over

101

the first month following a single GBH application along with the determination of

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glyphosate and AMPA content.

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2.

Material and Methods

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2.1

Growth conditions and glyphosate applications

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GR soybean seeds (5091RR2Y, ÉliteMD) were planted in two gallon (7.57 l) pots filled with

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the surface soil (0-30 cm) of an eutric Brunisol

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Canada). The Brunisol had a sandy loam texture with 3% of organic matter, pH of 7.59 ±

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0.35 and mineral content of 4980 ± 172 µg g-1 for Ca; 124 ± 48 µg g-1 for Al; 118 ± 3 µg

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g-1 for Fe; 90 ± 4. µg g-1 for Mg; 75 ± 3 µg g-1 for K; 40 ± 2 µg g-1 for Mn; 1.6 ± 0.1 µg g-

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1

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34-0-0), but not inoculated with diazotrophs, one week prior to the GBH applications. GBH

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(Factor 540®, Interprovincial Cooperative Limited, Saskatoon, Canada) was applied

26

collected in Boisbriand (Québec,

for Cu; 1.4 ± 0.2 µg g-1 for Zn. Soybeans were fertilized with ammonium nitrate (N-P-K:



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manually to V3 stage soybeans (three trifoliate leaves) at doses of glyphosate of 0.7; 1.8;

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2.3; 3.6 and 4.5 kg ha-1. For each treatment, three plants (pseudoreplicates) were grown

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in three pots. Soybeans were harvested 2, 7, 14 and 28 DAT and plants from each pot were

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pooled (n=3). A similar number of plants not treated with glyphosate were used as controls

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(n=3). Pots were randomly placed (random number generator) in the greenhouse under

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natural light conditions complemented with sodium vapor lamps to maintain a 12h:12h

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light/dark cycle and mean photosynthetic active radiation (PAR) level of 850 µmol photons

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m-2 s-1. Temperature was maintained between 20-25 °C. In vivo measurements were

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performed on the third fully expanded leaf on at least two plants from each pot at four time

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points (2; 7; 14 and 28 DAT). All plants were then harvested (within a 3-hour period) and

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rinsed, and fresh weight (FW) measured. The third fully expanded leaves were frozen in

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liquid nitrogen and stored at -80 °C while the rest of the plants was frozen at -20 °C for

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further measurements. Dry weight (DW) measurement were performed later by drying

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them to a constant weight at 60 °C.

2.2

Photosynthesis and pigment evaluations

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Chlorophyll fluorescence measurements were performed using a portable chlorophyll

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fluorometer PAM-2500 (Waltz, Effeltrich, Germany). Samples were dark acclimated for

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15 minutes then exposed to ten actinic light intensities (11, 30, 47, 71, 113, 168, 257, 380,

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570, 846 µmol photons m-2 s-1), for 20 seconds intervals, with a saturating pulse at the end

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of each interval. The effective quantum yield was calculated as:

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ΦPSII = (Fm’ - Ft) / Fm’

(1)


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where Fm’ = maximal fluorescence in light and Ft = fluorescence level before light

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illumination 27.

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Chlorophyll content was measured using an atLEAF PLUS chlorophyll meter (atLeaf,

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Delaware, United States). Data were converted in SPAD units

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determination (r2) of 0.9999, then in chlorophyll content (μg cm-2) with a r2 of 0.9883.

28

with a coefficient of

140 141

2.3

Gas exchange

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Stomatal conductance (gs) measurements were performed using a SC-1 leaf porometer

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(Decagon Devices Inc., Washington, United States) and expressed in mmol m-2 s-1. The

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accuracy of the leaf porometer was of ± 10% of measurement. Three measurements were

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performed on each leaf and the mean value was used as the stomatal conductance value.

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The porometer was calibrated under the greenhouse conditions prior to measurements for

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each timepoint.

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2.4

Oxidative stress marker

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Hydrogen peroxide (H2O2) content in soybeans was measured following Velikova et al.

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(2000). Briefly, 200 mg of frozen leaves were grounded on ice with 2 ml of trichloroacetic

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acid (0.1%, w/v), then centrifuged for 15 minutes (12 000 x g) at 4 °C. 0.5 ml of the

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supernatant was added to 0.5 ml of 10 mM phosphate buffer (KH2PO4 + K2HPO4, pH 7)

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and 1 ml of potassium iodide 1 M. Absorbance was read at 390 nm and values where

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reported on a standard curve with H2O2 concentrations ranging from 0 to 9 µg ml-1.

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2.5

Glyphosate and AMPA content

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Glyphosate and AMPA content were determined following the slighly modified method

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described in Gomes et al. (2016). In short, 50 mg of fresh material (leaves, stems and roots)

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were weighed and homogenized in a 50 ml Falcon tube containing 10 ml of deionized

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water, 10 ml of HPLC grade methanol and 5 ml of GC grade dichloromethane for 1 minute

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(25 000 rpm) using a T18 digital ULTRA-TURRAX® (IKA, North Carolina, USA). Falcon

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tubes were then centrifuged 20 minutes at 3400 rpm. 50 µl (leaves and stems) or 100 µl

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(roots) of supernatant were transferred and evaporated to dryness under nitrogen flow.

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Samples were derivatized by the addition of 1 ml of trifluoroacetic anhydride (TFAA) and

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500 µl of trifluoroethanol (TFE), prior to heating at 100 oC for one hour. After cooling to

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room temperature for 15 minutes, samples were evaporated to dryness under nitrogen flow,

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then resuspended with 1 ml of ethyl acetate, prior to injection (0.5 µl). A Varian CP 3800

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gas chromatograph coupled with an electron capture detector and equipped with a Rxi®-

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5Sil MS column (Restek, Pennsylvania, USA) (30 m x 0.25mm x 0.25 μm) was used for

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glyphosate and AMPA quantification. Injector and detector were held at 280 °C and 300

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°C, respectively. Hydrogen was used as carrier gas with a column flow of 1.8 ml min-1.

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The oven temperature program was an initial temperature of 70 °C held for 0.8 min, a 5 °C

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min-1 increase up to 130 °C held for 5 min, followed by a 60 °C min-1 increase up to 250

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°C held for 12 min, for a total run time of 31.8 min.

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2.6

Statistical analyzes

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Analyzes of variance were performed followed by multiple mean comparisons for ΦPSII,

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H2O2 content, stomatal conductance, chlorophyll content and biomass (FW and DW), using


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Dunnett’s parametric test (DW, H2O2) or Steel’s non-parametric test when data did not

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fulfill the conditions of residuals normality (FW, ΦPSII, chlorophyll content, stomatal

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conductance), for comparison with the control treatment (0 kg ha-1).

183 184

Two-factor analyzes of variance (ANOVA) were also performed (DAT, Plant tissue

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[leaves, stems, roots], DAT*Plant tissue), followed by Tukey’s post hoc tests with

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glyphosate and AMPA content in soybean leaves, stems and roots, for all GBH doses. Data

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were previously logarithmically (ln) converted in order to fulfill the conditions of residuals

188

normality. When the interaction between DAT and plant compartment was non-significant,

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the two-factor ANOVA was repeated without the interaction variable (DAT*Plant tissue).

190 191

Spearman’s rank correlations were performed with parameters measured in leaves (ΦPSII,

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H2O2 content, chlorophyll content, stomatal conductance and glyphosate and AMPA

193

content), glyphosate and AMPA content in roots, as well as biomass (DW). The Bonferroni

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correction was performed and p-value was adjusted to 0.01 (0.05 / 5 variables), the tests

195

being significant when p-value < 0.01. All other statistical analyzes were significant when

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p-value < 0.05 and all analyzes were performed using the JMP 14 software from SAS.

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3

Results

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3.1

Physiological indicators

200

The effective quantum yield was significantly reduced 2 DAT, for GBH applications with

201

doses of 1.8 to 4.5 kg glyphosate ha-1 (Figure 1A). Φ PSII was also significantly reduced 7

202

DAT and 14 DAT at the higher dose of 4.5 kg glyphosate ha-1 (Figure 1B, 1C). No 9 ACS Paragon Plus Environment


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significant difference was observed 28 DAT (Figure 1D). Plant biomass was significantly

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reduced 14 DAT, when compared to the control treatment, at the dose of 4.5 kg glyphosate

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ha-1 for fresh weight and dry weight (Figure 1G, 1K). Plants fresh weight increased 7 and

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28 DAT at doses of 3.6 and 0.7 kg glyphosate ha-1, respectively (Figure 1F, 1H). There

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was no other significant difference regarding biomass (FW and DW) for all other

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timepoints (Figure 1E, 1I, 1J, 1L). GBH applications significantly reduced stomatal

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conductance 2 DAT at the highest dose (4.5 kg glyphosate ha-1) (Figure 2I). This was

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followed by a significant increase of the stomatal conductance 14 DAT, occurring at doses

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of 0.7, 1.8 and 4.5 kg glyphosate ha-1 (Figure 2K), as well as an increase 28 DAT at the

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two highest doses of 3.6 and 4.5 kg glyphosate ha-1 (Figure 2L). There was no significant

213

differences 7 DAT (Figure 2J). H2O2 content was significantly increased 2 DAT for the

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two highest doses (3.6 and 4.5 kg ha-1) (Figure 2E), but there was no signifiant differences

215

further in the experiment (7, 14 and 28 DAT) (Figure 2F, 2G, 2H). Finally, regardless of

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the time of exposure and glyphosate doses, chlorophyll concentrations were not affected

217

by treatments (Figure 2A, 2B, 2C, 2D).

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3.2

Glyphosate and AMPA content

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Glyphosate and AMPA content in plant tissues (leaves, stems, roots)

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Differences regarding glyphosate content in plant tissues was the most significant variable

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of the two-way ANOVA model (Table 1). Glyphosate content in GR soybean was

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significantly higher in leaves than in stems and roots for all applied doses (0.7 to 4.5 kg ha-

224

1)

225

measured in roots was significantly lower at 0.7, 3.6 and 4.5 kg glyphosate ha-1 doses

(Figure 3A, 3B, 3C, 3D, 3E). When compared to glyphosate content in stems, the amount


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(Figure 3A, 3D, 3E). A significant difference was observed regarding AMPA content

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measured in the different plant tissues (leaves, stems and roots) but only at the two highest

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3.6 and 4.5 kg glyphosate ha-1 doses (Table 1), the content in roots being significantly lower

229

than in leaves and stems (Figure 3I, 3J). For the three lowest GBH doses (0.7, 1.8 and 2.3

230

kg glyphosate ha-1), the AMPA content measured in leaves, stems and roots was not

231

significantly different (Figure 3F, 3G, 3H).

232 233

Glyphosate and AMPA content over time (DAT)

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For the lowest GBH application (0.7 kg glyphosate ha-1), the glyphosate content measured

235

in plants (leaves + stems + roots) was significantly lower 14 and 28 DAT in comparison to

236

7 DAT (Figure 3A). With increasing glyphosate doses (2.3, 3.6 and 4.5 kg glyphosate ha-

237

1),

238

following time after application in comparison to 2 DAT (Figure 3C, 3D, 3E). AMPA

239

content was significantly higher 28 DAT when comparing to 2 DAT (Figure 3G-3J) at

240

doses ranging from 1.8 to 4.5 kg glyphosate ha-1.

the opposite effect was observed, glyphosate content being significantly higher

241 242

Glyphosate and AMPA content in plant tissues over time (DAT*Plant tissue)

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The interaction of glyphosate content in plant tissues over time was significant only at the

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lowest and highest doses (0.7 and 4.5 kg glyphosate ha-1) (Table 1). At the lowest dose (0.7

245

kg glyphosate ha-1), glyphosate content in leaves decreased over time, the amount

246

measured 28 DAT being significantly lower than 2 DAT (Figure 3A). Moreover,

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glyphosate content in stems, for all timepoints (2, 7, 14 and 28 DAT), was not different,

248

while glyphosate content in roots was lower 2 DAT when comparing to 7 DAT (Figure



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3A). Finally, the glyphosate content measured in roots 2 DAT was lower than in leaves at

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all time points (2-28 DAT) and in stems 2 and 7 DAT (Figure 3A). At the highest dose (4.5

251

kg glyphosate ha-1), there was only one significant difference regarding glyphosate content

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in leaves, which was significantly higher 28 DAT, when compared to 14 DAT (Figure 3E)

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In stems, the amount of glyphosate was significantly higher 28 DAT when comparing to 2

254

DAT, while there was no differences regarding roots (Figure 3E). Similarly to the lowest

255

GBH application, the amount of glyphosate measured in stems and roots was lower 2 DAT,

256

when comparing to the amount in leaves (Figure 3E). For AMPA, the interaction of AMPA

257

content in plant tissues over time was significant for all GBH applications (Table 1). From

258

1.8 to 4.5 kg glyphosate ha-1, the AMPA content measured in roots 28 DAT was almost

259

always significantly higher than the amount measured at 2 DAT (Figure 3G-3J), which was

260

not the case for the lowest dose of 0.7 kg glyphosate ha-1 (Figure 3F). The AMPA content

261

in leaves and stems were not really different over time for the different GBH applications.

262

Similarly to glyphosate, AMPA content in roots 2 DAT was significantly lower from

263

AMPA content in leaves and stems for the same timepoint, but only for the two highest

264

doses of 3.6 and 4.5 kg glyphosate ha-1 (Figure 3I-3J).

265 266

3.3

Correlations between glyphosate and AMPA content and physiological

267

indicators of GBH exposure

268

Glyphosate content in leaves was significantly correlated with ΦPSII (-), while AMPA

269

content in leaves was also correlated with ΦPSII (-), as well as with chlorophyll content (-)

270

(Table 2). Glyphosate content in roots was solely correlated with H2O2 content (+)

271

measured in leaves. On the other hand, AMPA content in roots was significantly correlated 12 ACS Paragon Plus Environment

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with all indicators, except for ΦPSII (Table 2). Solely DW was used as the biomass proxy

273

in the correlation matrix, since FW and DW values are not independent.

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4.

Discussion and conclusions

276

A reduction of ΦPSII was observed 2 DAT, 7 and 14 DAT. The lowest dose being

277

significantly different from the control treatment or lowest observed concentration effect

278

(LOEC), was compared for each DAT, giving 0.7; 4.5 and 4.5 kg glyphosate ha-1

279

respectively at 2, 7 and 14 DAT (Figure 1A, 1B, 1C). There was no LOEC value at 28

280

DAT suggesting that soybeans were able to recover from GBH applications over time.

281

Reduced ΦPSII in GR soybeans exposed to GBH was previously reported 30, as well as the

282

potential of recovery 30, 31. It was proposed that these effects on photosynthesis were linked

283

to AMPA, produced from the degradation of glyphosate in plants. Indeed, following the

284

application of AMPA alone, its content measured in plants was correleted to a reduction

285

of the chlorophyll content of GR soybeans and the associated decreased FW 13. In our case,

286

ΦPSII reduction was correlated with both AMPA and glyphosate content in leaves (Table

287

2).

288 289

A reduction in photosynthesis efficiency can lead to the production of reactive oxygen

290

species (ROS) and H2O2 accumulation which were already observed in non-GR plant

291

species such as phytoplankton 21, willow shrubs 25 and duckweed 32. In GR soybeans, H2O2

292

accumulation was observed in germinating seeds exposed to GBH 33. In our study, it seems

293

that ΦPSII reduction could have resulted in an increase of H2O2 content in leaves. Indeed,

294

H2O2 content was significantly higher 2 DAT in comparison with the control treatment for 13 ACS Paragon Plus Environment


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GBH applications at the highest 3.6 and 4.5 kg glyphosate ha-1 doses (Figure 2E). However,

296

as for ΦPSII, it seems that plants could recover from early stress following GBH

297

application, since there were no significant differences for H2O2 content later in the

298

experiment (7, 14 and 28 DAT) (Figure 2F-2H). Moreover, it seems that ΦPSII reduction

299

and the H2O2 increase were unsufficient to affect the chlorophyll content (Figure 2A-2D).

300

An effective antioxidant response could have prevented damages to chlorophyll and could

301

also explain the recovery observed in this experiment, regarding ΦPSII and H2O2 content

302

(Figure 1D and Figure 2F-2H). For example, increased superoxide dismutase, catalase and

303

ascorbate peroxidase activities were observed following GBH applications in a

304

phytoplankton community 21. In GR and non-GR soybeans, there was a slight increase of

305

oxidative stress and the antioxidant response was modulated by GBH applications.

306

However, lipid peroxidation did not occur, and it was suggested that the pool of amino

307

acids implicated in stress response (proline and asparagine), unaffected in GR soybeans

308

leaves and roots exposed to GBH, could have prevent damages 34. While the chlorophyll

309

content was not significantly different from the control treatment at all glyphosate doses

310

and time points, there was still a significant negative correlation between AMPA content

311

measured in leaves and roots, and the chlorophyll content. The absence of a similar

312

correlation between glyphosate and chlorophyll content (Table 2) seems to support the

313

hypothesis that the effects of GBH application on GR soybeans chlorophyll content are

314

mostly due to AMPA effects.

315 316

A significant biomass reduction was observed 14 DAT for both FW and DW. Bernal et al.

317

(2012) related a similar decrease in FW of GR maize 14 DAT to the effects of AMPA.


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Indeed, they observed that the AMPA to glyphosate ratio was higher over the first 14 DAT,

319

indicating that glyphosate degradation into AMPA could occur more rapidly during the

320

first two weeks after GBH applications, due to an increased GOX activity

321

soybeans, previous studies attempted to link the biomass reduction with AMPA foliar

322

applications. However, contradictory results were reported, i.e. decreased FW when

323

exposed to AMPA 14 DAT

324

present study, neither glyphosate content measured in leaves or roots was correlated with

325

biomass (Table 2). A positive correlation was solely observed between AMPA content

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measured in roots and DW. Otherwise, biomass did not appear negatively correlated with

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glyphosate nor AMPA content measured in plants. On the other hand, DW was highly

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correlated with all other parameters i.e. stomatal conductance (ρ= -0.7644; p-value=

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glyphosate and aminomethylphosphonic acid content in glyphosate

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