Degradation of Glyphosate by Mn-Oxide May Bypass Sarcosine and

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Degradation of glyphosate by Mn#oxide may bypass sarcosine and form glycine directly after C#N bond cleavage Hui Li, Adam F. Wallace, Mingjing Sun, Patrick N. Reardon, and Deb P. Jaisi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03692 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Environmental Science & Technology

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Degradation of glyphosate by Mn‒oxide may bypass sarcosine and form glycine directly

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after C‒N bond cleavage

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Hui Li1, Adam F. Wallace2, Mingjing Sun1, Patrick Reardon3, and Deb P. Jaisi1*

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Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716 United States

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2

Department of Geological Sciences, University of Delaware, Newark, Delaware 19716, United States

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3

NMR Facility, Oregon State University, Corvallis, Oregon 97331 United States

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* Corresponding author: Deb P. Jaisi; Email: [email protected]; Phone: (302) 831‒1376.

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Revised for Environmental Science & Technology

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December 16, 2017

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ACS Paragon Plus Environment


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ABSTRACT

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Glyphosate is the active ingredient of the common herbicide Roundup®. The increasing presence

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of glyphosate and its byproducts has raised concerns about its potential impact on the

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environment and human health. In this research, we investigated abiotic pathways of glyphosate

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degradation as catalyzed by birnessite under aerobic and neutral pH conditions to determine

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whether certain pathways have the potential to generate less harmful intermediate products.

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Nuclear magnetic resonance spectroscopy (NMR) and high-performance liquid chromatography

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(HPLC) were utilized to identify and quantify reaction products, and density functional theory

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(DFT) calculations were used to investigate the bond critical point (BCP) properties of the C‒N

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bond in glyphosate and Mn(IV)‒complexed glyphosate. We found that sarcosine, the commonly

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recognized precursor to glycine, was not present at detectable levels in any of our experiments

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despite the fact that its half-life (~13.6 h) was greater than our sampling intervals. Abiotic

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degradation of glyphosate largely followed the glycine pathway rather than the AMPA

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(aminomethylphosphonic acid) pathway. Preferential cleavage of the phosphonate adjacent C‒N

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bond to form glycine directly was also supported by our BCP analysis, which revealed that this

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C‒N bond was disproportionately affected by the interaction of glyphosate with Mn(IV).

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Overall, these results provide useful insights into the potential pathways through which

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glyphosate may degrade via relatively benign intermediates.

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INTRODUCTION

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Glyphosate (N‒phosphonomethyl glycine) was first introduced into the herbicidal

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industry by John Franz of Monsanto in the 1970s to kill broadleaf plants and grasses.1 The

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agricultural innovation of genetically engineered glyphosate-tolerant crops that followed

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dramatically enhanced the applicability of glyphosate as a non-selective, post-emergence

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herbicide on agricultural fields. As a result, the application rate of glyphosate skyrocketed from

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less than 9×106 Kg yr-1 in the U.S. in 1992 to 127×106 Kg yr-1 in 2015 with more than 70 %

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applied to corn and soybean crops.2 The widespread usage of glyphosate in agriculture resulted

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in increased environmental contamination by glyphosate and its intermediate products [e.g.,

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aminomethylphosphonic acid (AMPA)] in soils, sediments, surface water, and ground water.3, 4

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This alarmed the public and prompted the need for studies on the environmental and health

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impacts of glyphosate. Glyphosate kills plants by blocking the shikimic acid pathway, which is

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necessary for the synthesis of vital aromatic amino acids in plants and some microorganisms.5

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Several toxicity studies suggested that the risk of adverse health effects due to glyphosate

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exposure were negligible in animals and humans;6,

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negative health effects in zebrafish, navigational honey bees (influencing navigational

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capabilities), and potential risks to soil nematodes (such as Caenorhabditis elegans), thus putting

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the safety of glyphosate and its widespread application under greater scrutiny.8-10 The United

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States Environmental Protection Agency (EPA) is evaluating the potential carcinogenicity of

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glyphosate but has not yet reached a conclusion regarding its toxicity.11

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however, recent studies have identified

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The degradation of glyphosate in soils is driven by primarily biotic pathways mediated by

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bacteria and fungi.12,13 However, abiotic degradation may also occur in the presence of

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manganese oxides and/or light (photolysis).14-16 In each instance, glyphosate degradation occurs

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either through the cleavage of a C‒N bond, forming AMPA and glyoxylic acid as a consequence

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(the AMPA pathway), or through the cleavage of a C‒P bond, forming sarcosine and then

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glycine (the sarcosine pathway).1, 12, 13 The focus of most past studies on the biotic degradation of

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glyphosate has been restricted to the two major degradation pathways. Biotic degradation of

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glyphosate in soils has been found to accumulate AMPA.1,

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commonly detected as a product of biotic glyphosate degradation. For instance, the apparent

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formation of glycine was detected in the absence of sarcosine during biotic degradation of

3


12, 17

Sarcosine, however, is less 14

C


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labeled glyphosate in soils.12 Glycine formation without sarcosine was also observed during the

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incubation of Achromobacter sp. MPS 12 in the presence of sarcosine oxidase inhibitor.18

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Similarly, sarcosine was not detected as a precursor to glycine in abiotic glyphosate degradation

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experiments catalyzed by either manganese oxide,16 or photo-electrochemically generated

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hydroxyl radicals on a TiO2 surface under alkaline conditions.19 These studies generally

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attributed the absence of sarcosine to its rapid degradation rate, and did not consider the

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possibility that glycine might also form as a direct degradation product of glyphosate. However,

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one solid-state NMR study of Pseudomonas sp. culture suggested that glycine might form

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directly.20 These findings have elevated to the fore the notion that the sarcosine pathway may be

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circumvented by a seldom recognized direct glycine pathway However, due to the focus of most

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studies on biogenic pathways (using soil bacteria or microorganisms with suppressed genes

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expression) and the lack of unequivocal evidence substantiating the absence of sarcosine in the

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reaction mixture, more direct evidence of the absence of sarcosine and the presence of the

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glycine pathway (through direct C‒N bond cleavage) is needed. In fact, glyphosate degradation

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may proceed through one of the two C‒N bond positions; cleavage of the first C‒N bond

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position results in the formation of AMPA, which is known to be more persistent and more toxic

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than glyphosate,14,

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comparatively benign reaction products (e.g. glycine). Therefore, the existence of this less toxic

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abiotic glyphosate degradation pathway carries both scientific and environmental significance.

17, 21

while cleavage of the second may result in the direct formation of

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In our previous studies, kinetics of glyphosate degradation, two major degradation

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pathways (via AMPA and sarcosine intermediates), and oxygen isotope effect during degradation

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and the relationship of sources and products were analyzed and mechanisms during abiotic

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degradation had been proposed.14, 22, 23 In this research communication, our primary objective is

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to identify and quantify degradation products, with a special focus on sarcosine, glycine, and

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AMPA, that form during the degradation of glyphosate in the presence of synthetic birnessite.

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One‒ and two–dimensional (1‒D and 2–D) nuclear magnetic resonance (NMR) spectroscopy,

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high-performance liquid chromatography (HPLC) and density function theory (DFT)

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calculations were utilized in conjunction with colorimetric methods to identify and quantify

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products. Our results allow us to comment on the relative significance of specific degradation

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pathways, and the potential for further investigation into the factors that govern the preferential

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selection of pathways that introduce less toxic products into the environment.

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MATERIALS AND METHODS

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Synthesis and Characterization of Birnessite. K+–birnessite [Kx(Mn4+, Mn3+)2O4; where x0.99, except for sarcosine (r2 > 0.97).

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The precision of the method used, expressed as relative standard deviation, was within 12%. The

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limits of detection for glyphosate, AMPA, glycine, and sarcosine in this method were 10, 5, 5

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and 5 µmol L-1, respectively.

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Density Function Theory (DFT) Electronic Structure Calculations. Electronic structure

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calculations were performed to quantify the influence of Mn(IV) complexation on the bond

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critical point (BCP) properties of the C–N bonds in glyphosate. All geometry optimizations and

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electron density distributions were performed or obtained with the Gaussian 09 suite of

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programs30 using the B3LYP hybrid exchange-correlation functional31,

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31G(d) all–electron Gaussian basis set.33-35 Given the size and composition of the theoretical

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system employed in this study, the B3LYP/6–31G(d) method represents a reasonable

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compromise between accuracy and performance. While some previous studies have utilized

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larger basis sets,36, 37 a more compact basis set is warranted in the present study due to the high

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level of explicitly represented solvent molecules (>100 solvent atoms) included in the

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computations. The B3LYP functional was chosen following a survey of the literature, which 7


32

and a standard 6–


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showed this approach to be the main method utilized by previous computational studies of

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glyphosate.36-39

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The C–N bond critical point properties were obtained by analyzing the electron density

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distributions generated from Gaussian 09 within the theory of Atoms in Molecules (AIM)40, 41 as

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implemented in the AIMAll software suite.42 Two configurations of glyphosate were investigated

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in the absence of manganese; both exhibited a total charge of -2 and differed only by the

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placement of a proton in association with either the phosphonate or amine moieties. In a third

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configuration containing Mn(IV), the total system charge was +2, and due to the association of

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manganese with the amine, the aforementioned proton was placed on the phosphonate group. To

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determine the optimal spin state of Mn(IV), several preliminary geometry optimizations were

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performed on Mn(IV)(6 H2O) in various electronic configurations. The lowest energy solution

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was taken as the optimal spin state and used in all calculations involving Mn(IV) [i.e. high spin

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Mn(IV) with three unpaired electrons (spin multiplicity = 4)].

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RESULTS AND DISCUSSION

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Degradation Kinetics of Glyphosate. The XRD pattern and SEM image (Figure S1) of the

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synthetic mineral revealed a single-phase hexagonal birnessite, composed of sheets of edge-

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sharing MnO6 octahedra,43 with uniform flower-like morphologies, in concert with turbostratic

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birnessite.44 The specific surface area (SSA) of synthetic birnessite was 41.3±0.3 m2 g-1. The

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particle size distribution of birnessite varied within a narrow range (143.4±4.5 nm), thus the

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separation of aqueous media from the solid mineral phase using 0.10 µm filters was considered

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appropriate. Additional information about the characterization of this synthetic birnessite (i.e. the

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phase identity, purity, SSA, and morphology) was presented in our previous publication.25

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Glyphosate degradation was initially rapid but decreased gradually (Figure 1). A potential

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reason for this could be the passivation of the birnessite surface, which may be attributed to the

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sorption of glyphosate and its degradation products (e.g. AMPA and orthophosphate) at reactive

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surface sites and passivated the reaction. In the experiment with 3000 µmol L-1 glyphosate in 5 g

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L-1 birnessite (GB3000, Table 1), more than 80% of glyphosate degraded to form primary amines

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(the sum of AMPA and glycine), which were released into the aqueous solution (Figures 1 and 2)

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with sorbed primary amines limited to 3%. The solution chemistry results showed that ~70% of

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original P was transformed into orthophosphate within 24 h and the fraction of orthophosphate

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retained in residual solid-phase minerals was very small and limited to 7% at the conclusion of

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the experiments. When the concentrations of glyphosate and birnessite changed (GB300 and

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GB150 Table 1), the degradation kinetics were similar overall (Figure S2) with glycine as a

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dominant product (Figure S3). Control experiments verified that glyphosate was stable under the

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experimental conditions. These results indicated that the degradation of glyphosate by birnessite

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was highly efficient. The higher rate and extent of primary amines (sum of glycine and AMPA)

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compared to that of orthophosphate production indicated either faster formation and/or slower

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breakdown of primary amines than orthophosphate. The higher rate of glycine production

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compared to AMPA (Figures 1 and 2) indicated that the glycine pathway is more dominant than

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the AMPA pathway. This is particularly noteworthy because the longer half–life of AMPA, ~50

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h under our experimental conditions14 and about 24 times higher than that of glyphosate (i.e., 2.1

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h), should have allowed its continued accumulation over time.

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Identification of Glyphosate Degradation Products by Using NMR Spectroscopy. The 1H,

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13

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experiment demonstrated the temporal changes in compositions and concentrations of

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degradation products (Figure 3). Pure glyphosate and its common intermediate products were

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measured separately as reference standards under identical conditions to facilitate accurate peak

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assignments and product identifications. Detailed chemical shifts for these standards are

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provided in Table S1. After 12 h of reaction, negligible glyphosate was detected in 1H, 13C, and

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31

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consistent with the kinetics of glyphosate degradation (Figure 1). The 1H and

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indicated that glycine, with characteristic peaks at ~3.4 (singlet) and ~41.0 (singlet) ppm,

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respectively, was the major product of glyphosate degradation (Figure 3a, b). The presence of

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AMPA, one of the common degradation products during biotic degradation,1, 12 was identified by

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1

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respectively (Figure 3a, c). The amount of AMPA, however, was much less than glycine based

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on 1H and 13C relative peak intensity, which was consistent with quantitation results from HPLC

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(Figure 1, 2). Formaldehyde (hydrated to form methylene glycol) was detected as a major

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product together with its oxidation product, formic acid (Figure 3a, b). The hydration of

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formaldehyde (HCHO): [HCHO + H2O

C, and

31

P NMR spectra of sub-samples collected at selected time points in the GB3000

P NMR spectra indicating almost complete degradation of glyphosate. This result was also

H and

31

13

C NMR spectra

P NMR spectra with chemical shifts at ~2.8 (doublet) and ~9.0 (singlet) ppm,

HOCH2OH] in liquid media leaves a very low 9



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concentration of the unhydrated species.45,

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formaldehyde are distinct from other products, which still validates the formation of

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formaldehyde. Please note that the polymerization of HCHO [nHOCH2OH

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(n-1)H2O] is only significant above 1 M, much higher than the range of concentrations (µM) in

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this study.45 Therefore, the effect of HCHO polymerization during NMR analysis is not

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anticipated. A persistent NMR peak of hydrated formaldehyde at and after 3 h of

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experimentation further suggested the continued formation of formaldehyde during the mid–and–

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late stages of glyphosate degradation.

However, the chemical shifts of the hydrated

HO(CH2O)nH +

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The composition of the degradation products generated under different concentrations of

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glyphosate or birnessite (GB300 and GB150) was generally similar, with glycine, AMPA, formic

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acid, and formaldehyde as major products (Figure S3a, b). At the later stages of the experiment,

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as well as for those experiments with low initial glyphosate concentrations (GB300 and GB150),

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the intensity of representative doublet peaks of AMPA appeared in the 1H NMR spectra,

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however, was too low for its presence to be confirmed reliably. Thus a positional

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glyphosate, on the third C atom counting from the carboxyl moiety in the molecule, was utilized

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in order to enhance the

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characteristic doublet peak representing the 3–C in glyphosate and AMPA due to isotope

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labeling, thus confirming the generation of AMPA during glyphosate degradation. However, the

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amount of generated AMPA was still low in all cases.

13

C NMR peak intensity. The

13

13

C labeled

C NMR spectra showed enhanced

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In order to further validate the presence of other less commonly reported products such as

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formaldehyde, the 2–D NMR spectra for samples at the conclusion of experiments (GB3000)

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were collected and shown in Figure 4. The singlet peak at 4.71 ppm in 1H NMR coupled with

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another singlet peak at 81.74 ppm in

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between H and C. Similarly, the singlet peak at 4.86 ppm in 1H NMR coupled with another

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singlet peak at 81.74 ppm in

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further confirmed the presence of hydrated formaldehyde in both experiments. In addition to

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these NMR spectra, the presence of formaldehyde was further validated using Nash assay

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method. Since formaldehyde readily oxidized into carbon dioxide in the presence of air, sunlight,

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or microorganisms, and should have oxidized in our aerobic experimental setup, its conspicuous

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persistence throughout our experiments was intriguing and most likely caused by the continuous

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formation of formaldehyde during the experiment.

13

C NMR indicated a direct single–bond connection

13

C NMR in the GB300 experiment (Figure S7). These results

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Summarizing the NMR results from the series of degradation experiments performed,

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major degradation products were identified included glycine, AMPA, formic acid, formaldehyde,

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and orthophosphate (~2.7 ppm in Figure 3c); these findings are consistent with a number of other

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studies.1, 12, 16, 18, 22 However, some other common degradation products often reported in the

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literature such as sarcosine, glyoxylic acid, and acetic acid were not detected. It is unclear

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whether these undetected products formed at levels below the detection limits or if they were

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otherwise rapidly oxidized after formation. Nonetheless, given that the amount of generated

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AMPA was low, the formation of glyoxylic acid, a byproduct of AMPA, was also expected to be

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low.

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Half–lives of Major Intermediate Products of Glyphosate. Results from separate experiments

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performed to investigate the half–lives of AMPA, sarcosine, glycine, methylamine, and

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glyoxylic acid, major potential intermediate products of glyphosate, showed their facile

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degradation. For example, NMR spectral analyses demonstrated that sarcosine degraded to form

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glycine as the major degradation product and formic acid and methylamine as minor products

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(Figure S6). The apparently conspicuous peak intensity of sarcosine in NMR spectra confirmed

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that a certain amount of sarcosine remained after 24 h of reaction, which was further validated

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quantitatively by HPLC results (Figures S4, S5). The calculated half–life of sarcosine was ~13.6

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h (Figure S4). Similarly, glyoxylic acid was readily degraded by birnessite (half–life < 1 h) to

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generate formic acid (Figure S8c). AMPA has much longer (~50 h) half-life.14 The other two

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important primary amines, glycine, and methylamine, were not degraded by birnessite within 24

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h (Figure S8a, b).

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Sarcosine Is Not a Necessary Intermediate Product. Sarcosine was previously recognized as

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an intermediate product of glyphosate degradation; it forms as a consequence of C–P bond

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dissociation, and subsequently acts as a precursor to glycine.13, 16 Intriguingly, this compound

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was not detected in any of NMR spectra or HPLC chromatograms obtained from the suite of

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experiments performed in this study. Please note also, that the presence of sarcosine was reported

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to be at or below detection in a number of previous studies.12, 16, 18-20 More often, the absence of

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sarcosine was attributed to its presumably rapid degradation rate.16 To ensure that the absence of

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sarcosine in our experiments is not due to its rapid oxidation, we chose to further examine the

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kinetics of sarcosine degradation. We found that the half-life of sarcosine under the present

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experimental conditions was 13.6 h (Figure S4). This time is much longer than the sampling 11



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intervals employed in our experimental protocols. Consequently, if we assume that glycine is the

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sole degradation product of sarcosine, the amount of sarcosine in our experiments should have

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been much higher than that of glycine, which is inconsistent with our observations. Therefore,

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the absence of sarcosine in our experiments does not appear to be due to its rapid degradation

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rate.

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In light of this result, we have pursued alternative hypotheses to explain the absence of

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sarcosine in our experiments. One of the reasons could be the adsorption of sarcosine onto the

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birnessite surface, causing no or low (below detection limit) sarcosine concentrations in solution.

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The pKa values of sarcosine and glycine, analogous in molecular structures, are similar: 2.3 and

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10.1 for sarcosine and 2.4 and 9.7 for glycine.47 Thus at our experimental pH (7.00±0.05), both

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molecules should have zwitterionic character with a positive charge on the –NH functional group

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and negative charge on the –COOH moiety. Therefore, the adsorption behavior of these two

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compounds on birnessite is expected to be reasonably similar. Our separate sarcosine

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degradation experiment showed sarcosine remained almost entirely in solution. Given that a high

337

amount of glycine was released into solution and hence was detected in NMR spectra, sarcosine,

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if formed, should also have been present in the solution. Additional analyses using HPLC did not

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find any sorbed or aqueous phase sarcosine during glyphosate degradation (Figures 1 and 2).

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Based on the series of supporting and refuting evidence listed above, it is reasonable to

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conclude that sarcosine did not form during the abiotic degradation of glyphosate by birnessite.

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This implies that the first step of glyphosate degradation does not have to involve C–P bond

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cleavage or sarcosine formation. Furthermore, the preferential cleavage of C–N bond towards the

344

N–C–P structure and the direct formation of glycine is also supported by DFT results (see below).

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These lines of evidence provide a reasonable explanation for a switch-circuited sarcosine

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degradation pathway among the two common pathways addressed in our previous study.23

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Potential for Preferred C−N Bond Cleavage: DFT Results. At circumneutral pH, the

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glyphosate zwitterion oscillates between two primary states in which a proton either resides on

349

the phosphonate group as –PO2OH- or on the amine as –NH2+ (Figure 5). Therefore, we first

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evaluated the influence of the proton position on the BCP properties of the C–N bonds. As

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shown in Table 2, the position of the proton affected each of these bonds differently. When the

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proton transferred to the amine, both C–N bond lengths increased, with the phosphonate–

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associated bond being the most perturbed. Correspondingly, the concentration of the electron 12


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density also decreased at the bond critical points, as did the value of the Laplacian of the electron

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density, which indicated that the charge in the BCP region was locally depleted in the –NH2+

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state relative to the –PO2OH state. As with the bond length, the density and Laplacian values

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were most affected in the C–N bond adjacent to the phosphonate group.

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To assess the influence of Mn(IV) on these bonds, a Mn(IV) atom was added adjacent to

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the amine group in the –PO2OH- state, resulting in the formation of a Mn(IV)–NH bonded

360

interaction. During the structural relaxation, the proton transferred from the phosphonate group

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into solution, forming an H3O+ ion. The influence of the bound manganese on the C–N bonds

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was analogous to the effect observed with the proton that was transferred from the –PO2OH state

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to form –NH2+. That is, the C–N bond lengths increased and the electron density and Laplacian

364

values at the BCP decreased, all to a greater degree on the phosphoryl side of the molecule

365

(Table 2).

366

Calculations used above did not directly probe the C–N bond breaking events that would

367

lead to the formation of glycine or AMPA. But the BCP analysis did allow us to assess which

368

regions of the molecule were most influenced by manganese complexation. These results

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indicated that the C–N bond adjacent to the phosphonate group was most affected, which in light

370

of the experimental results presented above, suggests that the presence of Mn(IV) may facilitate

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glycine formation directly from glyphosate. Given that the observed changes in the critical point

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properties of the –NH2+ and Mn(IV)–NH states were similar relative to the –PO2OH state it

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could be assumed that the phosphonate adjacent C–N bond was equally susceptible to

374

dissociation in either case; however, given that the lifetime of the doubly protonated state is

375

likely to be very short due to its zwitterionic character compared to the lifetime of a glyphosate

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molecule adsorbed on a manganese oxide, the potential for Mn complexation to bias the

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decomposition pathway towards glycine would presumably be much greater.

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Glycine Pathway of Glyphosate Degradation by Birnessite. The two commonly recognized

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degradation pathways of glyphosate are the sarcosine and AMPA pathways, which are initiated

380

via C–N and C–P bond cleavage, respectively.1, 48 The proportions of orthophosphate, primary

381

amines, and other products generated in the two major pathways are distinct, however the

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explicit contribution of each pathway is obscured by the various rates of formation and

383

degradation of intermediate products. Based on the HPLC and NMR results in this study, we

384

observed glycine as the most significant product in contrast to past studies in which AMPA was 13



385

the dominant product.1,

13, 16, 48

386

representing the –CH2– moieties of glycine and AMPA in 1H NMR spectra (Figure 3a) and

387

concentrations in HPLC chromatograms (Figures 1 and 2), was about 3:1. The limited amount of

388

AMPA generated in this study could be explained as a result of the suppression of the AMPA

389

pathway of degradation. Once formed, AMPA, however, could not undergo rapid degradation

390

because of its long half–life (~50 h) under experimental conditions.14 This means that

391

concentrations of orthophosphate derived from AMPA degradation should also be low. These

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lines of reasoning suggested that the majority of orthophosphate should be derived from the

393

glycine pathway. In the glycine pathway, the C–N bond cleavage at the N–C–P position in the

394

glyphosate molecule also released a P containing compound, which could further be degraded to

395

orthophosphate. The identification of this compound in our experimental data has remained

396

elusive [see below on possible product, methylphosphonic acid (CH3–PO3H2)], however, the

397

direct release of orthophosphate following C–P bond cleavage at the N–C–P position seems

398

unlikely, as sarcosine and orthophosphate would both be observed as products, contrary to our

399

experimental observations.

The relative ratio of glycine:AMPA, based on the peak area

400

The formation and preference of intermediate products can be discussed from the redox

401

properties of Mn–oxide. Birnessite consists of multi–valence Mn, varying from +2 to +4 but

402

dominantly as +4. The measured average oxidation state of the synthetic birnessite used in this

403

study was 3.8 suggesting its strong oxidizing capability. The C–N bond of glyphosate becomes

404

more vulnerable, after forming a Mn–N bond (see DFT results above), due to the changes in the

405

electron density distribution.23 A similar mechanism was proposed in studies on the oxidation of

406

nitrilotrismethylene–phosphonic acid, in which C–N bond cleavage took place at the N–centered

407

radical, after electron transferred from N to Mn, followed by C–P bond cleavage at the C–

408

centered radical.49 However, separate tests (MB3000, Table 1) performed to test the degradation

409

of methylphosphonic acid (CH3–PO3H2), a potential product formed after C–N bond cleavage,19

410

by birnessite confirmed negative results from both colorimetric and NMR analyses (Figure S9).

411

In contrast, AMPA with one more amine moiety than methylphosphonic acid, was degraded by

412

birnessite.14 AMPA is positively charged at neutral pH conditions on its NH2– moiety, which

413

might play a key role in binding to the negatively charged reactive surface sites of birnessite, and

414

thus facilitates electron transfer and bond cleavage. The lack of methylphosphonic acid

415

degradation and high rate of orthophosphate generation conclusively suggest facile C–P bond

14


Page 14 of 39

Page 15 of 39


416

cleavage of the unknown intermediate product. Further study on the relative dominance of this

417

pathway in other abiotic degradation is required.

418

Environmental Implications of Multi–pathway of Glyphosate Degradation. In this study,

419

birnessite catalyzed glyphosate degradation was studied to unravel degradation pathways and

420

their relative contributions by identifying and quantifying intermediate products using NMR

421

spectroscopy, HPLC, and DFT calculations. The results suggested that the dominant abiotic

422

degradation pathway initiates from C–N bond cleavage and generates glycine, formaldehyde,

423

and orthophosphate, bypassing the formation of sarcosine. A subordinate pathway was C–N

424

bond cleavage forming AMPA and glyoxylic acid that ultimately degraded to form CO2, H2O,

425

NH3, and orthophosphate. Overall, our results identified a less commonly recognized degradation

426

pathway of glyphosate. As discussed above, the direct glycine formation was observed in one

427

study on biotic degradation18 but rarely in abiotic cases; however, the glycine pathway has not

428

been recognized.

429

The relative ratio of glyphosate to birnessite had a minor influence on the selection of

430

degradation pathways. Overall, this research provided supportive evidence that glyphosate

431

primarily degrades at the C–N bond position favoring the direct formation of glycine, which is

432

less toxic than the major product of the other pathway. The natural abundance of Mn in soils

433

varies within 20-3000 mg Kg-1. The Mn content in soil depends largely on the nature of parent

434

materials, cycling processes, and supplemental Mn applied to crop plants and uptake by plants.50

435

Though the natural abundance of Mn is low, our results can be a reference for the fate of

436

glyphosate sorbed to negatively charged clay minerals which are abundant in soils across the US

437

Corn Belt and for further investigation on partitioning glyphosate degradation pathways for

438

environmentally friendly products. Potential avenues include applying Mn(IV)-oxides as

439

supplemental Mn, an essential micronutrient to crops, as well to degrade residual glyphosate.

440

15



441 442

ACKNOWLEDGEMENTS This research was supported by research grants from the U.S. Department of Agriculture

443

(NIFA award 2017–05362). We would like to acknowledge Advanced Material Characterization

444

Laboratory at the University of Delaware for providing access to XRD, BET, and SEM analyses

445

of birnessite mineral.

446

16


Page 16 of 39

Page 17 of 39


447

REFERENCES

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496

1. Franz, J. E.; Mao, M. K.; Sikorski, J. A. Glyphosate: A unique global herbicide; American Chemical Society, 1997. 2. United States Geological Survey; http://water.usgs.gov/nawqa/pnsp/usage/maps/show_map.php?year=2014&map=GLYPHOSATE&hi lo=L&disp=Glyphosate. 2014. 3. Battaglin, W. A.; Meyer, M. T.; Kuivila, K. M.; Dietze, J. E. Glyphosate and Its degradation product AMPA occur frequently and widely in U.S. soils, surface water, groundwater, and precipitation. J. Am. Water Res. Assoc. 2014, 50, 275-290. 4. Kolpin, D. W.; Thurman, E. M.; Lee, E. A.; Meyer, M. T.; Furlong, E. T.; Glassmeyer, S. T. Urban contributions of glyphosate and its degradate AMPA to streams in the United States. Sci. Total Environ. 2006, 354, 191-197. 5. Haslam, E. The Shikimate Pathway; Butterworths, London, 1974. 6. Williams, G. M.; Kroes, R.; Munro, I. C. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regul. Toxicol. Pharmacol. 2000, 31, 117-165. 7. Kwiatkowska, M.; Jarosiewicz, P.; Michalowicz, J.; Koter-Michalak, M.; Huras, B.; Bukowska, B. The impact of glyphosate, its metabolites and impurities on viability, ATP level and morphological changes in human peripheral blood mononuclear cells. PLoS One 2016, 11, e0156946. 8. Roy, N. M.; Ochs, J.; Zambrzycka, E.; Anderson, A. Glyphosate induces cardiovascular toxicity in Danio rerio. Environ. Toxicol. Pharmacol. 2016, 46, 292-300. 9. McVey, K. A.; Snapp, I. B.; Johnson, M. B.; Negga, R.; Pressley, A. S.; Fitsanakis, V. A. Exposure of C. elegans eggs to a glyphosate-containing herbicide leads to abnormal neuronal morphology. Neurotoxicol. Teratol. 2016, 55, 23-31. 10. Balbuena, M. S.; Tison, L.; Hahn, M. L.; Greggers, U.; Menzel, R.; Farina, W. M. Effects of sublethal doses of glyphosate on honeybee navigation. J. Exp. Biol. 2015, 218, 2799-2805. 11. Glyphosate Issue Paper: Evaluation of Carcinogenic Potential. EPA-HQ-OPP-2016-0385-0094; EPA's Office of Pesticide Programs, 2016. 12. Rueppel, M. L.; Brightwell, B. B.; Schaefer, J.; Marvel, J. T. Metabolism and degradation of glyphosate in soil and water. J. Agric. Food Chem. 1977, 25, 517-528. 13. Sviridov, A. V.; Shushkova, T. V.; Ermakova, I. T.; Ivanova, E. V.; Epiktetov, D. O.; Leontievsky, A. A. Microbial degradation of glyphosate herbicides (Review). Appl. Biochem. Microbiol. 2015, 51, 188-195. 14. Li, H.; Joshi, S. R.; Jaisi, D. P. Degradation and isotope source tracking of glyphosate and aminomethylphosphonic acid. J. Agric. Food Chem. 2016, 64, 529-538. 15. Sandy, E. H.; Blake, R. E.; Chang, S. J.; Jun, Y.; Yu, C. Oxygen isotope signature of UV degradation of glyphosate and phosphonoacetate: tracing sources and cycling of phosphonates. J. Hazard. Mater. 2013, 260, 947-954. 16. Barrett, K. A.; McBride, M. B. Oxidative degradation of glyphosate and aminomethylphosphonate by manganese oxide. Environ. Sci. Technol. 2005, 39, 9223-9228. 17. Bento, C. P.; Yang, X.; Gort, G.; Xue, S.; van Dam, R.; Zomer, P.; Mol, H. G.; Ritsema, C. J.; Geissen, V. Persistence of glyphosate and aminomethylphosphonic acid in loess soil under different combinations of temperature, soil moisture and light/darkness. Sci. Total Environ. 2016, 572, 301311. 18. Sviridov, A. V.; Shushkova, T. V.; Zelenkova, N. F.; Vinokurova, N. G.; Morgunov, I. G.; Ermakova, I. T.; Leontievsky, A. A. Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp. Appl. Microbiol. Biotechnol. 2012, 93, 787-796. 19. Muneer, M.; Boxall, C. Photocatalyzed degradation of a pesticide derivative glyphosate in aqueous suspensions of titanium dioxide. Int. J. Photoenergy 2008, 2008, 1-7. 17



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20. Jacob, G. S.; Schaefer, J.; Stejskal, E. O.; McKay, R. A. Solid-state NMR determination of glyphosate metabolism in a Pseudomonas sp. J. Biol. Chem. 1985, 260, 5899-5905. 21. Giesy, J. P.; Dobson, S.; Solomon, K. Ecotoxicological risk assessment for Roundup herbicide. Rev. Environ. Contam. Toxicol. 2000, 167, 35-120. 22. Paudel, P.; Negusse, A.; Jaisi, D. P. Birnessite-catalyzed degradation of glyphosate: A mechanistic study aided by kinetics batch studies and NMR spectroscopy. Soil Sci. Soc. Am. J. 2015, 79, 815-825. 23. Jaisi, D. P.; Li, H.; Wallace, A. F.; Paudel, P.; Sun, M.; Balakrishna, A.; Lerch, R. N. Mechanisms of bond cleavage during manganese oxide and UV degradation of glyphosate: Results from phosphate oxygen isotopes and molecular simulations. J. Agric. Food Chem. 2016, 64, 8474-8482. 24. McKenzie, R. M. The influence of cobalt on the reactivity of manganese dioxide. Aust. J. Soil Res. 1971, 9, 55-58. 25. Li, H.; Jaisi, D. P. An isotope labeling technique to investigate atom exchange during phosphate sorption and desorption. Soil Sci. Soc. Am. J. 2015, 79, 1340-1351. 26. Murphy, J.; Riley, J. P. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 1962, 27, 31-36. 27. Brown, H. H. A study of 2,4,6-trinitrobenzenesulfonic acid for automated amino acid chromatography. Clin. Chem. 1968, 14, 967-978. 28. Nash, T. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 1953, 55, 416-421. 29. Ibáñez, M.; Pozo, Ó. J.; Sancho, J. V.; López, F. J.; Hernández, F. Residue determination of glyphosate, glufosinate and aminomethylphosphonic acid in water and soil samples by liquid chromatography coupled to electrospray tandem mass spectrometry. J. Chromatogr. A 2005, 1081, 145-155. 30. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A. Gaussian 09, rev. D. 01. Gaussian Inc., Wallingford, CT, 2009. 31. Lee, C. T.; Yang, W. T.; Parr, R. G. Development of the colle-salvetti correlation-energy formula into a functional of the electron-density. Phys. Rev. B 1988, 37, 785-789. 32. Becke, A. D. Density-functional thermochemistry. 3. The role of exact exchange. J. Chem. Phys. 1993, 98, 5648-5652. 33. Hehre, W. J.; Ditchfield, R.; Pople, J. A. Self-consistent molecular-orbital methods. 12. Further extensions of Gaussian-type basis sets for use in molecular-orbital studies of organic-molecules. J. Chem. Phys. 1972, 56, 2257. 34. Ditchfield, R.; Hehre, W. J.; Pople, J. A. Self-consistent molecular-orbital methods. 9. Extended Gaussian-type basis for molecular-orbital studies of organic molecules. J. Chem. Phys. 1971, 54, 724. 35. Rassolov, V. A.; Pople, J. A.; Ratner, M. A.; Windus, T. L. 6-31G* basis set for atoms K through Zn. J. Chem. Phys. 1998, 109, 1223-1229. 36. Caetano, M. S.; Ramalho, T. C.; Botrel, D. F.; da Cunha, E. F. F.; de Mello, W. C. Understanding the inactivation process of organophosphorus herbicides: A DFT study of glyphosate metallic complexes with Zn2+, Ca2+, Mg2+, Cu2+, Co3+, Fe3+, Cr3+, and Al3+. Int. J. Quantum Chem. 2012, 112, 2752-2762. 37. Purgel, M.; Takács, Z.; Jonsson, C. M.; Nagy, L.; Andersson, I.; Bányai, I.; Pápai, I.; Persson, P.; Sjöberg, S.; Tóth, I. Glyphosate complexation to aluminium(III). An equilibrium and structural study in solution using potentiometry, multinuclear NMR, ATR-FTIR, ESI-MS and DFT calculations. J. Inorg. Biochem. 2009, 103, 1426-1438. 38. Ali, M. M. N.; Kaliannan, P.; Venuvanalingam, P. Ab initio computational modeling of glyphosate analogs: Conformational perspective. Struct. Chem. 2005, 16, 491-506. 39. Peixoto, M. M.; Bauerfeldt, G. F.; Herbst, M. H.; Pereira, M. S.; da Silva, C. O. Study of the stepwise deprotonation reactions of glyphosate and the corresponding pKa calues in aqueous solution. J. Phys. Chem. A 2015, 119, 5241-5249. 40. Bader, R. F. W. A quantum theory of molecular structure and its applications. Chem. Rev. 1991, 91, 893-928. 18


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41. Bader, R. F. W. Atoms in molecules. Acc. Chem. Res. 1985, 18, 9-15. 42. Keith, T. A., AIMAll Version 17.01.25. TK Gristmill Software, Overl. Park, KS, 2017. 43. Post, J. E.; Veblen, D. R. Crystal structure determination of synthetic sodium, magnesium, and potassium birnessite using TEM and the Rietveld method. Am. Mineral. 1990, 75, 477-489. 44. Yin, H.; Li, H.; Wang, Y.; Ginder-Vogel, M.; Qiu, G.; Feng, X. H.; Zheng, L.; Liu, F. Effects of Co and Ni co-doping on the structure and reactivity of hexagonal birnessite. Chem. Geol. 2014, 381, 1020. 45. Zavitsas, A. A.; Coffiner, M.; Wiseman, T.; Zavitsas, L. R. The reversible hydration of formaldehyde. Thermodynamic parameters. J. Phys. Chem. 1970, 74, 2746-2750. 46. Winkelman, J. G. M.; Voorsinde, O. K.; Ottens, M.; Beenackers, A. A. C. M.; Janssen, L. P. B. M. Kinetics and chemical equilibrium of the hydration of formaldehyde. Chem. Eng. Sci. 2002, 57, 40674076. 47. Leussing, D. L.; Hanna, E. M. Metal ion catalysis in transamination. III. Nickel (II) and Zinc (II) mixed complexes involving pyruvate and various substituted aliphatic amino acids. J. Am. Chem. Soc. 1966, 88, 693-696. 48. Szekacs, A.; Darvas, B. Forty years with glyphosate. In Herbicides-Properties, synthesis and control of weeds; Dr. Hasaneen, M. N., Ed.; InTech 2012. 49. Nowack, B.; Stone, A. T. Manganese-catalyzed degradation of phosphonic acids. Environ. Chem. Lett. 2003, 1, 24-31. 50. Reisenauer, H. M. Chapter 6. Determination of plant-available soil manganese. In Manganese in soils and plants; Graham, R. D. et al., Eds.; Kluwer Academic Publishers, 1988; pp 87-98.

19



NMR spectroscopy

HPLC

Glyphosate

571 572 573

TOC/ Abstract graphic

20


Page 20 of 39

Page 21 of 39

574 575 576 577


Table 1. Experimental set up for glyphosate degradation by birnessite. All experiments were performed at pH 7 (±0.05) and 10 mmol L-1 NaCl. Experiments

GB3000 GB300 GB150 AB3000 SB3000 GCB150 MB3000 GAB300 MAB300

Concentration (µmol L1) (g L1) Glyphosate Birnessite 3000 5 300 0.5 150 0.5 AMPA 3000 5 Sarcosine 3000 5 Glycine 150 0.5 Methylphosphonic Acid 3000 5 Glyoxylic Acid 300 0.5 Methylamine 300 0.5

578

21



579 580 581 582 583

Page 22 of 39

Table 2. Calculated bond critical point (CBP) properties and equilibrium C–N bond distances for the configurations shown in Figure 5. The symbols C–N (P) and C–N (C) indicate that the bond is proximal to the phosphonate and carbonate moieties in glyphosate respectively.

C–N (P) C–N (C)

C–N (P) C–N (C)

C–N (P) C–N (C)

PO2OH(ρ) (e/a05) ρ (e/a03) 0.264 -0.721 0.266 -0.744 + NH2 3 (ρ) (e/a05) ρ (e/a0 ) 0.234 -0.549 0.248 -0.658 Mn(IV)–NH 3 (ρ) (e/a05) ρ (e/a0 ) 0.238 -0.579 0.250 -0.664

584 585

22


rC-N (Å) 1.466 1.465 rC-N (Å) 1.500 1.490 rC-N (Å) 1.499 1.486

Page 23 of 39


586

Aqueous glyphosate Aqueous orthophosphate Aqueous glycine Aqueous AMPA Sorbed orthophosphate Sorbed AMPA+glycine

-1

Concentration (µmol L )

3500 3000 2500 2000 1500 1000 500 0 0

5

10

15

20

25

Time (h)

587 588

Figure 1. Kinetics of glyphosate (3000 µmol L-1) degradation by birnessite (5 g L-1). No sorbed

589

glyphosate or sarcosine were detected on the solid phase.

1000 24 h

800

mAU

12 h

600

7h

400

3h

200

1 min 1

0 10

15

20

2

4

3

25

STD

30

35

40

45

50

Time (min)

590 591

Figure 2. High performance liquid chromatography (HPLC) results of aqueous degradation

592

products of glyphosate, with glyphosate:birnessite ratio of 3000 µmol L-1:5 g L-1. Peaks are

593

FMOC derivatised glyphosate (1), AMPA (2), and glycine (3). The 4th peak is for unreacted

594

FMOC. No sarcosine was detected in either aqueous or solid phases.

595

23


Environmental Science & Technology a: Glyphosate b: AMPA c: Glycine d: Formaldehyde (hydrated) e: Formic acid f: Orthophosphate

Page 24 of 39

a)

596 b)

597 c)

598 599

Figure 3. Nuclear magnetic resonance (NMR) spectra of degradation products of glyphosate: a)

600

1

601

Peak labels correspond to glyphosate (a), AMPA (b), glycine (c), formaldehyde (hydrated) (d),

602

formic acid (e), and orthophosphate (f).

H, b) 13C, c) 31P spectra, with glyphosate (3000 µmol L-1):birnessite (5 g L-1) at pH 7 (±0.05).

603

24


Page 25 of 39


604 605

Figure 4. Heteronuclear single quantum correlation (HSQC) spectroscopy of degradation

606

products of glyphosate, with glyphosate (3000 µmol L-1):birnessite (5 g L-1), at 12 h. Two axes in

607

f1 and f2 represent 1H and

608

formaldehyde (d).

13

C nuclei, respectively. Peak labels correspond to glycine (c) and

609 610

25



Page 26 of 39

611 612 613 614 615 616 617 618 619 620

Figure 5. Minimum energy structures of glyphosate in two of its zwitterionic states (i.e. PO2OH-

621

and NH2+) and in the presence of manganese (Mn(IV)–NH) as predicted by DFT. An analysis of

622

the bond critical point properties of the C–N bonds (see Table 2) indicates that these bonds are

623

similarly perturbed by the transfer of a proton form the phosphonate to the amine as by Mn(IV)

624

complexation. In both cases the phosphonate adjacent C–N bond is the most affected. Although

625

the effects are similar, the zwitterionic NH2+ state is presumably much shorter lived than the

626

manganese complex.

26


Page 27 of 39


Supporting Information for

627 628 629

Degradation of glyphosate by Mn‒oxide may bypass sarcosine and form glycine directly

630

after C‒N bond cleavage

631 632 by

633 634

Hui Li1, Adam F. Wallace2, Mingjing Sun1, Patrick Reardon3, and Deb P. Jaisi1*

635 636 637 638

1

Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716 United States

639

2

Department of Geological Sciences, University of Delaware, Newark, Delaware 19716, United States

640

3

NMR Facility, Oregon State University, Corvallis, Oregon 97331 United States

641 642 643

* Corresponding author: Deb P. Jaisi, Email: [email protected]; Phone: (302) 831‒1376.

644 645

Summary: SI contains thirteen pages, one table, and nine figures.

646

S1



647 648

MATERIALS AND METHODS

649 650

Characterization of Birnessite. The mineralogy, crystallinity and micromorphology, the BET

651

(Brunauer–Emmett–Teller) specific surface area (SSA), and particle size distribution of

652

synthesized birnessite were characterized using X–ray diffraction (XRD), field emission capable

653

scanning electron microscope (JSM–7400F, JEOL Japan), Micromeritics ASAP 2020 Surface

654

Area and Porosity Analyzer (Georgia, USA), and Wyatt Mobius Dynamic Light Scattering

655

(DLS; Santa Barbara, California), respectively. The SSA of birnessite was determined by the

656

physical adsorption of nitrogen gas on the surface of birnessite at cryogenic (liquid nitrogen)

657

temperature. With information of birnessite mass and density, and the amount of adsorbed

658

nitrogen gas measured, SSA was calculated based on the assumption that a monomolecular layer

659

of nitrogen adsorption formed on birnessite. For measuring particle size distribution, birnessite

660

suspension of 1 mg/L in DI water was prepared and transformed in a quartz cuvette immediately

661

after sonication for 10 min. The particle size distribution was calculated with results of 10

662

measurements. The Mn average oxidation state (AOS) was determined using potentiometric

663

titration method.1

664

S2


Page 28 of 39

Page 29 of 39


665 666

Table S1. The 1H and 13C NMR chemical shifts of glyphosate and its major potential degradation products

667

obtained from pure chemicals (pH was maintained at 7.0 ±1.0).

668 1

Glyphosate AMPA Glyoxylic Acid Sarcosine Glycine Formaldehyde (hydrated) Formic Acid Methylamine Methylphosphonic Acid Methanol

H NMR (ppm) 3.76 (singlet); 3.12, 3.15 (doublet) 2.95, 2.99 (doublet) 5.06 (singlet) 3.44 (singlet); 2.57 (singlet) 3.44 (singlet) 4.83 (singlet)

Chemical shifts 13 C NMR (ppm) 171.43 (singlet); 50.75 (singlet); 46.08, 45.23 (doublet) 37.75, 36.89 (doublet) 87.35 (singlet); 179.50 (singlet) 171.43 (singlet); 50.75 (singlet); 32.49 (singlet) 172.30 (singlet); 41.33 (singlet) 81.73 (singlet)

8.41 (singlet) 2.25 (singlet) 1.39, 1.42 (doublet) 3.33 (singlet)

166.31 (singlet) 26.68 (singlet) 12.30, 11.43 (doublet) 48.73 (singlet)

669 670

S3



671 672 673

b)

Intensity (AU)

a)

10

20

30

40

50

60

70

80

Two-theta (degree)

674 675

Figure S1. X-ray diffraction patterns (a) and scanning electron microscope (SEM) images (b)

676

of synthetic birnessite.

677

S4


Page 30 of 39

Page 31 of 39


678

Primary amine Orthophosphate

-1


3000 2500

GB3000

2000

300 GB300

200 GB150

100 0 0

10

20

30

40

50

Time (h)

679 680 681

Figure S2. Degradation kinetics of glyphosate. Open squares and circles represent concentrations

682

of orthophosphate and primary amines, respectively. GB3000, GB300, and GB150 represent

683

experiments performed at glyphosate:birnessite ratio of 3000 µmol L-1:5 g L-1, 300 µmol L-1:0.5

684

g L-1, and 150 µmol L-1:0.5 g L-1, respectively.

685

S5



Page 32 of 39

686 687 a)

688 b)

689 690

Figure S3. NMR spectra of degradation products of glyphosate: a) 1H and b)

691

for experiments performed with glyphosate (300 or 150 µmol L-1):birnessite (0.5 g L-1).

692

Glyphosate was

693

PO3H2), labeled as 3C in the spectra. Peak labels correspond to glyphosate (a), AMPA (b),

694

glycine (c), formaldehyde (hydrated) (d), and formic acid (e).

13

13

C NMR spectra

C labeled on 3–13C counting form carboxyl group (HOOC–CH2–NH–13CH2–

695

S6


Page 33 of 39


Sarcosine Primary amines Glycine Formaldehyde

-1


3000 2500 2000 1500 1000 500 0 0

5

10

15

20

25

Time (h)

696 697

Figure S4. Kinetics of sarcosine (3000 µmol L-1) degradation by birnessite (5 g L-1).

698

S7



Page 34 of 39

mAU

500 400

24 h

300

12 h 7h

200 3h

100

0.5 h 3

5

4

0

STD

10

15

20

25

30

35

40

45

50

Time (min)

699 700

Figure S5. Representative HPLC chromatograms of aqueous degradation products of sarcosine,

701

in the experiment performed at sarcosine:birnessite ratio of 3000 µmol L-1:5 g L-1. Peaks: 3)

702

glycine; 4) FMOC; 5) sarcosine.

703

S8


Page 35 of 39


704 705 a)

706 b)

707 708 709

Figure S6. NMR spectra of degradation products of sarcosine: a) 1H and b)

710

with sarcosine (3000 µmol L-1):birnessite(5 g L-1) in pH range of 7±0.05 and 10 mmol L-1 NaCl

711

as ionic medium. Peak labels correspond to glycine (c), formic acid (e), sarcosine (g), and

712

methylamine (h).

713

S9


13

C NMR spectra,


714

715 716

Figure S7. Heteronuclear single quantum correlation (HSQC) spectroscopy of glyphosate

717

degradation products, with glyphosate (300 µmol L-1):birnessite (0.5 g L-1) at 24 h. Two axes

718

include f1 and f2 represent 1H nuclei and

719

formaldehyde (hydrated) (d).

13

C nuclei, respectively. Peak labels correspond to

720

S10


Page 36 of 39

Page 37 of 39


721 722

a)

Glycine

-1


200

150

100

50

0

5

10

15

20

25

Time (h)

723 b) -1


3500

Methylamine

3000 2500 2000 1500 1000 500 0

5

10

15

20

25

Time (h)

724 c)

725 726

Figure S8. The 1H NMR spectra of degradation products of: a) glycine; b) methylamine; c)

727

glyoxylic acid. Peak labels correspond to glycine (c), formic acid (e), methylamine (h), glyoxylic

728

acid (i). Concentrations of glycine and methylamine were measured using colorimetric method.

729

S11



730 731

a)

732 b)

733 c)

734 735

Figure S9. The 1H (a), 13C (b), and 31P (c) NMR spectra of degradation products of

736

methylphosphonic acid, in the experiment with methylphosphonic acid (3000 µmol L-

737

1

):birnessite (5 g L-1). Peak label corresponds to methylphosphonic acid (j).

S12


Page 38 of 39

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738

REFERENCES

739

1. Lingane, J. J.; Karplus, R. New method for determination of manganese. Ind. Eng. Chem. Anal.

740

Ed. 1946, 18, 191-194.

741

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Degradation of Glyphosate by Mn-Oxide May Bypass Sarcosine and

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