Quantitative Evaluation of Noncovalent Interactions between

Article pubs.acs.org/est

Quantitative Evaluation of Noncovalent Interactions between Glyphosate and Dissolved Humic Substances by NMR Spectroscopy Pierluigi Mazzei†,‡ and Alessandro Piccolo*,† †

Centro Interdipartimentale per la Risonanza Magnetica Nucleare (CERMANU), Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy ‡ Istituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche (CNR), Via Salaria Km 29.300, 00016 Monterotondo Stazione, Italy S Supporting Information *

ABSTRACT: Interactions of glyphosate (N-phosphonomethylglycine) herbicide (GLY) with soluble fulvic acids (FAs) and humic acids (HAs) at pH 5.2 and 7 were studied by 1H and 31P NMR spectroscopy. Increasing concentrations of soluble humic matter determined broadening and chemical shift drifts of proton and phosphorus GLY signals, thereby indicating the occurrence of weak interactions between GLY and humic superstructures. Binding was larger for FAs and pH 5.2 than for HAs and pH 7, thus suggesting formation of hydrogen bonds between GLY carboxyl and phosphonate groups and protonated oxygen functions in humic matter. Changes in relaxation and correlation times of 1H and 31P signals and saturation transfer difference NMR experiments confirmed the noncovalent nature of GLY−humic interactions. Diffusion-ordered NMR spectra allowed calculation of the glyphosate fraction bound to humic superstructures and association constants (Ka) and Gibbs free energies of transfer for GLY−humic complex formation at both pH values. These values showed that noncovalent interactions occurred most effectively with FAs and at pH 5.2. Our findings indicated that glyphosate may spontaneously and significantly bind to soluble humic matter by noncovalent interactions at slightly acidic pH and, thus, potentially pollute natural water bodies by moving through soil profiles in complexes with dissolved humus.

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environmental pollution.3,10 While the microbial persistence is due to the carbon-to-phosphorus bond, which is highly resistant to biodegradation,11 the GLY environmental transport is attributed to its strong interactions with fine organomineral soil particles12,13 and soluble humus fractions.12−15 Despite the environmental importance of humic matter, its chemical complexity, as supramolecular association of small heterogeneous molecular bioproducts,16 makes investigation of the molecular interactions with herbicides difficult. In fact, it is the strength of these interactions that controls not only the transport of agrochemicals through soil profiles to groundwater, but also their bioavailability to microorganisms.17 Moreover, the thermodynamic evaluation of GLY−humic interactions is further complicated by the difficulty in determining low

INTRODUCTION N-Phosphonomethylglycine is the active compound of the popular broad-spectrum and relatively nonselective herbicide commonly referred to as glyphosate (GLY) and commercialized under trade names such as Roundup, Rodeo, Glyfonox, and Glycel.1 Glyphosate represents today up to 60% of the global “broad-spectrum” herbicide sales, reaching over 70 000 t year−1 of technical acid.2 Furthermore, the 1997 introduction of Roundup Ready crops, such as cotton, maize, and soybeans, which incorporated a glyphosate-resistant protein, added popularity to the synthetic herbicide.3 The high efficiency of GLY herbicide is due to its specific inhibition of 5enolpyruvylshikimate 3-phosphate (EPSP) synthase,4 an enzyme of the shikimate pathway that governs the synthesis of aromatic amino compounds in higher plants, algae, bacteria, and fungi.5−8 Although GLY is less toxic than other herbicides,9 its relative persistence in soil and its potential leaching from soil surfaces into groundwater may still represent a major threat for a wider © 2012 American Chemical Society

Received: Revised: Accepted: Published: 5939

January 20, 2012 May 16, 2012 May 17, 2012 May 17, 2012 dx.doi.org/10.1021/es300265a | Environ. Sci. Technol. 2012, 46, 5939−5946

Environmental Science & Technology

Article

were stirred for 10 min to guarantee complete analyte dissolution before NMR analysis. For the most sensitive STD-NMR spectra, the small ligand is required to be stoichiometrically in greater amount than the interacting macromolecule.26,27 By assuming a nominal molecular weight between 5000 and 10 000 for humic superstructures, four different samples were prepared by dissolving 8.45 mg (5 × 10−2 mmol) of GLY in deuterated solutions at either pH 5.2 or pH 7 that also contained 5 mg of either HAs or FAs. All samples were transferred into 5 mm NMR tubes and the solutions degassed by N2 flux for 5 min and sonicated for 15 min before NMR analysis. NMR Experiments. A 400 MHz Bruker Avance spectrometer, equipped with a 5 mm Bruker BBI (broad-band inverse) probe, was employed to conduct 31P and 1H NMR measurements at 161.81 and 400.13 MHz frequencies, respectively, and at 298 ± 1 K. 1H NMR spectra were acquired with a 2 s delay and a 90° pulse length ranging between 7.3 and 8.9 μs. 31P NMR spectra were acquired with a 7 s delay and a 90° pulse length ranging between 11.1 and 11.5 μs. A total of 100 transients and 32 768 time domain points were acquired for both 31P and 1H experiments. An inverse gated pulse sequence, with an 80 μs length Waltz16 decoupling scheme with a power level around 15.6 dB, was employed to decouple phosphorus from proton nuclei. An inversion recovery pulse sequence with 20 increments and variable delays from 0.01 to 10 s was adopted to measure both 1H and 31P longitudinal (spin−lattice) relaxation time constants (T1), while the transverse (spin−spin) relaxation time constants (T2) were measured using a Carr−Purcell− Meiboom−Gill (CPMG) pulse sequence using 20 increments and 2−2000 spin−echo repetitions, with a constant 1.5 ms spin−echo delay. A time domain of 16 384 points was set for all the relaxometric experiments. 1 H and 31P DOSY (diffusion-ordered spectroscopy) NMR spectra were obtained by choosing a stimulated echo pulse sequence with bipolar gradients and combined with two spoil gradients and an eddy current delay. This sequence was selected to reduce signal loss due to short spin−spin relaxation times. The acquisition was executed by setting, for 1H and 31P nuclei, respectively, 1600 and 2300 μs long sine-shaped gradients (δ) that linearly ranged from 0.674 to 32.030 G cm−1 in 32 increments and selecting a delay of 0.09 and 0.25 s (Δ) between the encoding and the decoding gradients. STD experiments were conducted according to previous indications26 and accumulation of 2048 scans. Selective saturation of humic substances was achieved by a train of 50 ms Gauss-shaped pulses, truncated at 1% and separated by a 50 μs delay. A total length of the saturation train of 5.005 s was achieved with 100 selective pulses. The best on-resonance irradiation for humic matter was found at 0.8 ppm, whereas offresonance irradiation was set at 30 ppm, where no proton signals were visible. Spectra were internally subtracted by phase cycling after each scan. Reference spectra were recorded using the same sequence without irradiation. The residual water signal was removed from 1H NMR spectra by the presaturation technique, whereas the Watergate 3−9−19 pulsed train sequence was preferred for DOSY experiments, and excitation sculpting was chosen for STD experiments by using a 2 ms selective rectangular pulse, defined by 1000 points and truncated to 1%. The spectral widths of 1H and 31P NMR spectra were 12 ppm (4.789 kHz) and 100 ppm (16.238 kHz), respectively. The free induction decays (FIDs)

glyphosate concentrations, which cannot yet be achieved by straightforward analytical methods.18−21 Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying both the qualitative and quantitative relations among different organic molecules, and it is also applied to investigate those occurring between environmental pollutants and natural organic matter.17,22,23 Earlier works have attempted to investigate humus−glyphosate interactions by means of infrared spectrometry and adsorption isotherms.14,15 However, their conclusions on the importance of hydrogen bonds and dispersive forces in the adsorption of GLY on humic matter were only indirect, and the techniques did not allow reliable calculations of the thermodynamic parameters involved in such interactions. Direct NMR information has already been obtained to understand the glyphosate role in physiological and ecotoxicological processes in human and plant biology.1,11,24 However, 1H or 31P NMR spectroscopy has never been employed to assess the extent of GLY−humic bindings. In this work, we applied 1H and 31P NMR techniques to study the occurrence and type of interactions between glyphosate and water-soluble humic substances and calculate the corresponding thermodynamic parameters. On the basis of previous approaches,22,23,25 the adopted NMR techniques consisted of measurements of relaxation times to extrapolate nuclear correlation times and of self-diffusion values to calculate the association constants for humic−glyphosate complexes. Moreover, the homonuclear proton saturation transfer difference (STD) technique26,27 was employed here for the first time to prove the formation of noncovalent host−guest complexes between relatively large humic associations and the small glyphosate ligand.

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MATERIALS AND METHODS Humic Substances. Fulvic acids (FAs) and humic acids (HAs) were isolated, as previously described,28 from a volcanic soil (Typic Fulvuland, Lazio, Italy) and a North Dakota Leonardite (Mammoth International Chemical Co.), respectively. HAs were purified of coextracted inorganic particles first with three cycles of dissolution in 0.5 M NaOH, followed by flocculation in 6 M HCl, and then by shaking the precipitate twice in a 0.25 M HF/HCl solution for 24 h. HAs were redissolved in 0.5 M NaOH and passed through a strong cation-exchange resin (Dowex 50) to eliminate residual di- and trivalent metals. The eluate was precipitated at pH 1, dialyzed, and freeze-dried. After homogenization, 30 mg of HAs was suspended in H2O, titrated to pH 7, and freeze-dried again. FAs were adsorbed on an XAD-8 column to eliminate soluble hydrophilic impurities (carbohydrates and proteins) and then eluted by a 1 M NaOH solution, immediately neutralized, dialyzed against water, and freeze-dried. The total organic carbon content was 23.8% and 45.9% in FAs and HAs, respectively. Sample Preparations. A 1 mg sample of GLY free acid (99% pure, Dr. Ehrenstorfer, Germany) was dissolved in 1 mL each of two deuterated water (99.8% D2O/H2O, Armar Chemicals) solutions, and the resulting solutions were kept at a pH of either 5.2 or 7 and prepared with 0.2 M acetate and 0.2 M phosphate buffer solutions, respectively. Different amounts (0, 5, 10, 15, 20, and 25 mg) of either FAs or HAs were dissolved into the two GLY solutions, without observation of any precipitation at both pH values. Control humic solutions were similarly prepared but without GLY addition. All solutions 5940

dx.doi.org/10.1021/es300265a | Environ. Sci. Technol. 2012, 46, 5939−5946


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of 1H and 31P spectra were multiplied by 1 and 4 Hz exponential factors, respectively, without zero filling. All spectra were baseline corrected and processed by both Bruker Topspin software (v.1.3) and MestReC NMR processing software (v. 4.9.9.9). Viscosity Measurements. Possible artifacts in calculation arising from a change in viscosity29 were accounted for by measuring dynamic viscosity by a Bohlin Advanced Rheometer (Bohlin Instruments Ltd., Gloucestershire, U.K.) using a coaxial cylinder geometry with a gap size of 150 μm. All measurements were performed in triplicate, at 25 °C, and under a constant shear stress of 0.1 Pa.

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RESULTS AND DISCUSSION Chemical Shift Drifts and Signal Broadenings. Glyphosate is a weak acid with three dissociation constants in aqueous solution, as protons are released from carboxyl, phosphonate, and ammonium groups at increasing pH.30 In the 5.2−7.0 pH range, the phosphonate dissociation equilibrium (Figure 1) dictates the concentrations of GLY dianion

Figure 2. 1H (A, B) and 31P (C, D) spectra of the herbicide glyphosate as a function of the FA concentration (0, 5, 15, and 25 mg mL−1) at two different pH values.

FAs, and thus, the P resonance was still neatly visible even at the largest HA concentration. These observations are explained with the establishment of weak interactions between GLY and humic molecular superstructures and represent evidence of host−guest complex formation.22,23 In particular, the hydrogen bond appears mostly responsible for the host−guest interaction, since both the GLY dianion (I) form (Figure 1) and humic matter are more protonated at pH 5.2 than pH 7, and a larger number of Hbonds may be formed among complementary functions.14 The consequence of complex formation is a restricted molecular mobility and, thus, tumbling rate, which implies a progressive signal broadening for the involved GLY nuclei with increasing humic concentration. Furthermore, a downfield signal shift was observed at pH 7 in both 31P and 1H spectra at increasing FA concentrations (Figure 2). At 25 mg mL−1 FAs, the H-2 signal shifted from 2.93 to 2.98 ppm, while the 31P signal moved from 7 to 7.75 ppm. These shifts further support the occurrence of hydrogen bonds between GLY and complementary functional groups of FAs, which had been previously shown by other indirect methods.14 HA additions (Figure S2, Supporting Information) produced a similar, though smaller, downfield shift of the H-1 signal at both pH 5.2 and pH 7. However, no chemical shift drift was observed for the 31P signal. Saturation Transfer Difference. The STD pulse sequence was introduced31 to evaluate the extent of noncovalent protein−ligand interactions by “through-space” polarization transfer. This technique had already been employed to prove weak interactions occurring between humic substances and some pesticides,27 since only the pesticide actually interacting with humic matter is visible in STD-NMR spectra. The soluble large-sized humic association16 was first allowed to interact with the relatively small glyphosate ligand, and then the humic matter frequency was saturated by an on-resonance selective shape pulse irradiation. The GLY ligand interacting with the irradiated molecular aggregate receives a partial saturation transfer through the nuclear Overhauser effect (NOE) phenomenon, and it is NMR detected. A second

Figure 1. Dissociation equilibrium of glyphosate in water within the pH range of interest.

(I) and trianion (II) forms.14 Dianion I at pH 5.2 still has an acidic proton on an oxygen atom, and it is slightly more abundant than trianion II, whereas the latter largely predominates at pH 7. The 1H NMR spectra of HA and FA solutions (15 mg mL−1) are shown in Figure S1, Supporting Information. The large broadening of 1H signals in these spectra is characteristic of humic molecules in superstructures, whereby fast relaxation times and signal flattening are observed. HAs and FAs appear different in conformational structure since alkyl (0.5−2.4 ppm) and hydroxyalkyl (2.4−4.7 ppm) proton signals were more intense in FA than in HA spectra, possibly because of the lower hydrophobicity and, thus, looser conformation in FAs.16 The 1 H and 31 P spectra of GLY at different FA concentrations are reported in Figure 2 at both pH 5.2 and pH 7, while the corresponding GLY spectra with different HA additions are shown in Figure S2, Supporting Information. Both humic materials progressively broadened the 1H and 31P signals, although the extent of changes varied with the humic type and concentration, being larger for FAs and at pH 5.2. Signals resonating between 3.75 and 2.95 ppm are attributed to GLY protons in methylene groups (Figure 2). The singlet peak (H-1) corresponds to protons next to the GLY carboxyl group, while the doublet (H-2) derives from proton splitting (J = 11.67 Hz) due to geminal coupling with the P nucleus (Figure 1). Increasing FA additions produced progressively larger broadening of GLY proton signals at both pH values. Also for 31P spectra, FA concentrations of 15 and 25 mg mL−1 progressively flattened the 7.75 ppm signal at pH 7, while at pH 5.2 the P signal was broadened to disappearance for the largest FA addition. Though a similar behavior was noted in 1H and 31 P spectra of GLY treated with HAs (Figure S2, Supporting Information), signal broadening was significantly lower than for 5941



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hydrophilic FAs than with hydrophobic HAs. In fact, both H-1 and H-2 protons were visible in STD difference spectra, thus implying the involvement of both carboxyl and phosphonate groups in the GLY−humic interactions. Hence, the STD findings agree with the observed signal broadenings and downfield shifts in confirming the occurrence of noncovalent binding between GLY and humic matter. Relaxation Times. The T1 and T2 relaxation constants for both 1H and 31P were obtained by inversion recovery and CPMG experiments, respectively (Table 1). For protons, both constants became progressively smaller with increasing humic matter, as compared to GLY alone. At both pH values, the largest decrease of T1 and T2 values was observed with FA additions. In fact, the largest FA concentration (25 mg mL−1) determined a T1 decrease of 81.45% and 87.84% at pH 5.2 and 7, respectively, whereas the corresponding reduction was only 23.36% and 16.39% with HAs (the values are related to averaged H-1 and H-2 relaxation measurements). In the case of T2, FAs still produced the greatest shortening of the spin−spin relaxation time (−94.58% with respect to the control) for both proton signals and most significantly at pH 5.2 (Table 1). Conversely, not only did progressive addition of HAs decrease T2 values to a lower degree than FAs at the same pH, but T2 shortening was more evident for H-1 than for H-2. This suggests that the GLY carboxyl group was more directly involved than the phosphono group in the interactions with HA complementary groups. As for 31P, FAs decreased both T1 and T2 values more than HAs at both pH values (Table 1). Moreover, the effect of FAs made the P spin−lattice relaxation in GLY so fast that the consequent signal broadening prevented measurement of T1 at the largest FA concentration and calculation of T2 even at an FA concentration as low as 5 mg mL−1 (Table 1). These observations further support the suggestion previously inferred by signal broadening and shifting that the GLY phosphono group particularly contributes to the interactions with FA molecules. Both T1 and T2 values in solution depend on the correlation time (τc), which is defined as the effective average time needed for a molecule to rotate through 1 rad. Therefore, the larger the τc value, the slower the molecular motion.22 T1 and T2 values allowed calculation of correlation times32 for both H and P nuclei in GLY, which invariably increased with the humic concentration (Table 1). FAs were more effective than HAs and provided a 3.6-fold increase in τc compared to the control for the H-2 signal at pH 5.2. The strong effect of humic matter on the GLY tumbling rate could also be inferred by the less abundant τc measurements for 31P. In fact, 31P τc values already significantly increased by adding as little as 5 mg mL−1 FAs at pH 5.2 and from 5 to 15 mg mL−1 FAs at pH 7 (Table 1). Diffusion Experiments. According to the Einstein−Stokes description, the diffusivity of a molecule decreases when the hydrodynamic radius increases,33 and thus, the larger the molecular dimension, the smaller the self-diffusive constant. Moreover, by measuring molecular diffusivity, one may obtain quantitative thermodynamic information on intermolecular interactions.25 DOSY-NMR spectroscopy is a fast and reliable method to evaluate changes in diffusivity when small molecules become involved in noncovalent complexes with humic matter.22,23 The 1H 2D DOSY projection for glyphosate added with the largest FAs in comparison with that of GLY alone is reported in Figure 4. The self-diffusion constants of 1H (1H values being

similar pulse sequence is then applied by an off-resonance irradiation to avoid any saturation of humic matter. By subtracting the second spectrum from the first one, the difference spectrum shows signals of the only proton nuclei which are involved in the weak GLY−humic interactions. These protons are affected by the applied saturation irradiation that determines the polarization transfer between GLY and humic molecules, thus inducing a through-space increase of the NOE intensity. Therefore, signal intensities for the bound GLY are proportional to its spatial proximity to the interacting humic molecules. Conversely, if the GLY ligand did not interact with the saturated humic matter, no signals would result in the final difference spectrum. It is essential that the first on-resonance pulse be irradiated sufficiently away from the ligand signal (as a function of the shape pulse length and selectivity) to avoid an undesired ligand-selective irradiation, which may result in meaningless difference spectra and overestimation of binding. Contrary to very small molecules, whereby isotropic tumbling may average dipolar interactions, these become dominant in stable humic superstructures, thus providing the means for an efficient propagation of spin diffusion that disturbs the spin population throughout the entire humic matter.27 Control and difference STD spectra at pH 7 and 5.2 for GLY−humic interactions are shown in Figure 3. The intensities

Figure 3. 1H STD spectra of glyphosate with humic matter at pH 5.2 and 7: (A) reference STD spectra acquired without irradiation, (B) STD difference spectra of a sample treated with FA, (C) STD difference spectra of a sample treated with HA. A vertical expansion (64×) was applied to the difference spectra.

of GLY signals in difference spectra were rather low at pH 7 for both FAs and HAs, though slightly more intense signals were obtained for FAs. Conversely, at pH 5.2, signal intensities in difference spectra were significantly greater than at pH 7, those for FAs still being more intense than those for HAs. Since the more intense the signal in STD difference spectra, the larger the degree of intermolecular noncovalent interactions, our results confirmed that GLY exhibited a greater affinity with humic matter at pH 5.2 than at pH 7. Moreover, STD spectra indicated that GLY had a larger noncovalent association with 5942



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Table 1. T1 and T2 Relaxation and Correlation (τc) Times for Glyphosate 1H and 31P Signals as a Function of the HA and FA Concentrations (mg mL−1) at 25 °C and pH 7 and 5.2a T1 (s) humic matter concn

a

H-1

H-2

τc (ns)

T2 (s) P

0 5 10 15 20 25

1.58 1.46 1.37 1.40 1.32 1.28

1.16 1.11 1.04 1.07 1.02 1.00

5.67 5.47 5.19 5.59 4.31 4.00

5 10 15 20 25

0.67 0.55 0.45 0.39 0.30

0.48 0.39 0.31 0.27 0.21

1.60 1.32 1.03 0.85 0.54

0 5 10 15 20 25

1.46 1.39 1.26 1.19 1.11 1.07

1.15 1.12 1.03 0.99 0.93 0.92

5.02 4.85 4.45 3.66 3.44 3.23

5 10 15 20 25

0.36 0.26 0.22 0.21 0.19

0.26 0.18 0.13 0.14 0.13

1.59 0.19 0.29 0.20 ND

H-1 pH 7 HAs 1.00 0.84 0.86 0.68 0.49 0.45 FAs 0.33 0.23 0.17 0.15 0.09 pH 5.2 HAs 1.12 0.81 0.57 0.58 0.42 0.39 FAs 0.19 0.12 0.09 0.08 0.07

H-2

P

H-1

H-2

P

0.74 0.69 0.69 0.57 0.43 0.39

1.24 1.10 0.99 0.79 0.65 0.57

0.23 0.28 0.24 0.35 0.50 0.54

0.23 0.24 0.22 0.31 0.44 0.47

2.68 2.89 3.03 3.69 3.61 3.78

0.34 0.23 0.16 0.11 0.08

0.33 0.17 0.12 ND ND

0.34 0.42 0.47 0.47 0.60

0.20 0.26 0.33 0.46 0.49

2.85 3.77 4.01 ND ND

0.87 0.67 0.52 0.51 0.38 0.36

0.96 0.77 0.69 0.55 0.51 0.45

0.17 0.27 0.38 0.35 0.47 0.50

0.17 0.26 0.33 0.32 0.42 0.46

2.94 3.37 3.42 3.51 3.54 3.75

0.18 0.11 0.07 0.05 0.04

0.18 ND ND ND ND

0.31 0.36 0.43 0.46 0.54

0.21 0.24 0.34 0.50 0.61

4.03 ND ND ND ND

H-1 and H-2 are referred to glyphosate singlet and doublet signals, respectively (see Figure 1). ND = not determined.

control at the largest humic additions (Figure 4). The extent of reduction in diffusion was larger for FAs than for HAs and much greater at pH 5.2 than at pH 7. This behavior is explained with formation of a GLY−humic complex and consequent progressive enhancement of its hydrodynamic radius.22,23 However, while hydrogen bonds are favored at lower pH and by the more acidic FA, weaker dispersive bonds are the stabilizing forces of the GLY−humic complex at higher pH and with more hydrophobic HAs. Humic matter serves as a host for the glyphosate guest in a host−guest complex formation, and thus, both the host and guest tend to diffuse at an equal rate and show the same diffusion constant. The self-diffusion values allowed calculation of the GLY fraction bound (ρ) to humic matter by assuming that a fast exchange occurs between free and humic-bound GLY molecules on the NMR scale:25 ρ= Figure 4. 1H 2D DOSY spectra highlighting diffusion projections at pH 5.2 for glyphosate singlet and doublet signals at two different FA concentrations (0 and 25 mg mL−1).

DGLY,obsd − DGLY,free Dcomplex − DGLY,free

where DGLY,obsd is the measured apparent (weight-averaged) diffusion constant of both free and humic-bound GLY, Dcomplex is the diffusion constant of either GLY−FA or GLY−HA complexes when GLY is fully bound to humic matter, and DGLY,free is the diffusion constant of the GLY molecule in the absence of humic matter. Since GLY does not fully form a complex with humic matter, the value of Dcomplex cannot be experimentally known. However, the GLY molecule being

the average of H-1 and H-2 self-diffusion) and 31P for all HA and FA additions at different pH values are reported in Table 2. The diffusion of glyphosate progressively decreased with increasing amount of humic matter (Table 2), thus becoming significantly lower (greater negative values) than that of the 5943



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Table 2. Diffusion Values (10−10 m2 s−1) for Signals of Humic Protons and 1H and 31P of Glyphosate Progressively Added with Humic Matter (mg mL−1) at 25 °C and Different pH Valuesa pH 5.2

humic matter concn 0 5 10 15 20 25 a

HA

FA

1

1

H

1.65 1.12 1.24 0.94 1.00

1

H

1.83 1.77 1.69 1.70 1.64

pH 7

GLY + HA H

5.62 5.58 5.19 4.47 3.66 3.25

31

GLY + FA 1

P

ND ND ND ND ND ND

31

H

5.62 5.60 5.14 4.42 3.54 3.18

ND ND ND ND ND ND

FA

1

1

H

1.51 1.47 1.46 1.45 1.34

H

1.90 1.86 1.83 1.83 1.85

GLY + HA 1

H

5.68 5.45 5.41 5.43 5.42 5.24

31

GLY + FA P

5.67 5.64 5.54 5.48 5.16 5.22

1

H

5.68 5.40 5.16 5.07 4.59 4.50

31

P

5.67 5.25 ND ND ND ND

The values were corrected for solution viscosity. ND = not determined.

smaller in size than humic superstructures, Dcomplex for the GLY−humic complex should have a value equal to either the FA or HA diffusion value, as previously assumed.22,23 While a low signal-to-noise ratio in 31P spectra prevented calculation of GLY diffusion constants for phosphorus, the ρ values for the GLY fraction bound to humic matter was thus achieved only from proton spectra at both pH 5.2 and pH 7 (Figure S3, Supporting Information). As already revealed by relaxometric, STD, and diffusion measurements, soluble humic materials provided the largest interactions with GLY at pH 5.2. While FA interaction with GLY was generally larger than for HA at both pH values, we found that, at pH 5.2, a 25 mg mL−1 concentration of HAs and FAs was able to bind 51.31% and 61.38% of GLY, respectively (Figure S4, Supporting Information). The fraction of bound GLY (ρ) measured by diffusion experiments was also employed to calculate the association constants (Ka) between GLY and both FAs and HAs. This was achieved by fitting ρ versus the concentrations of free HAs and FAs22,23,25 according to the following equation derived from the Langmuir adsorption equation:34 ρ=a

interactions with FAs and HAs (Table 3). Negative values of transfer ΔG were found for both humic materials only at pH 5.2, thus showing that, though a decrease in motion implies an entropy reduction, the association of GLY with humic matter at this pH is thermodynamically favorable. Such negative ΔG values confirm the affinity of GLY for both humic substances at pH 5.2, as also shown by the larger variations in correlation times and greater signal intensities detected in STD spectra at this pH. Conversely, the association of GLY to HAs and FAs at pH 7 produced positive ΔG° values, thereby suggesting a lower mutual affinity at this neutral pH. Different NMR methods were applied here to quantify the binding of the widely employed glyphosate herbicide to humic substances. Measurements of spin−lattice (T1) and spin−spin (T2) relaxation times allowed calculation of nuclear correlation times τc for GLY in interaction with different concentrations of humic matter. These data together with through-space correlation by STD-NMR experiments confirmed the occurrence of noncovalent GLY−humic complexes of the host− guest type. All NMR results indicate that the largest GLY binding was observed at pH 5.2 and with FAs. Chemical shift drifts in 1H and 31P NMR spectra called for a major role of hydrogen bonds in forming GLY−humic complexes. Especially at pH 5.2, both GLY carboxyl and phosphono groups appeared responsible for hydrogen bonds with complementary functions in humic matter. The enhanced GLY−humic interactions at pH 5.2 with respect to pH 7 is thus explained with a larger presence of the GLY dianion form (Figure 1) and a greater degree of protonation in humic acidic groups. The slightly acidic condition not only provided both interacting molecules with more complementary sites for hydrogen bond formation, but also lowered their reciprocal electronegative repulsion. Diffusion NMR experiments (DOSY) allowed calculations of the fraction of GLY bound to humic materials and, consequently, the thermodynamic parameters controlling association of GLY to both HAs and FAs, such as the equilibrium constant (Ka) and the free energy of transfer (ΔG). In this work we showed direct evidence of the occurrence of weak interactions between glyphosate and soil humic matter at both subacidic and neutral pH, though to different extents. These findings thus suggest that a potential transport and/or mobility of this herbicide through the soil profile into groundwater may occur as in host−guest associations to soluble humic matter.

K a[host]free 1 + K a[host]free

where [host]free is the concentration of the unbound humic matter (expressed as moles of organic carbon) and a is a constant. The optimal a value was empirically found, and the best fitting in all cases resulted for a = 1.5 (Figure S3, Supporting Informaiton). The Ka values are reported in Table 3 and indicate that the largest affinity was again at pH 5.2 for GLY−humic interactions. Furthermore, the Ka constants permitted the calculation of the Gibbs free energy of transfer (ΔG = −RT ln Ka) for GLY Table 3. Calculated Values for the Binding Constant (Ka) and Free Gibbs Energy (ΔG) and Standard Deviation for the Association of Glyphosate with FAs and HAs at 25 °C and at Different pH Values

a

P

HA

pH

Ka (M−1)

5.2 7.0

3.414 ± 0.347 0.046 ± 0.008

5.2 7.0

6.262 ± 0.291 0.502 ± 0.059

r2a HAs 0.993 0.976 FAs 0.995 0.963

ln Ka

ΔG (kJ mol−1)

1.228 −3.079

−3.04 ± 0.101 7.63 ± 0.43

1.834 −0.689

−4.55 ± 0.080 1.71 ± 0.263

Coefficient of determination for the curves fitting the data. 5944



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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of both humic substances, 1H and 31P NMR spectra of glyphosate with increasing amount of HAs and FAs, and fitting of the ρ fraction bound to humic matter versus hostfree (unbound humic molecules) concentrations. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 081 253 91 60; fax: +39 081 253 91 86; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.M. gratefully acknowledges his doctoral fellowship funded by the Istituto di Metodologie Chimiche, CNR.

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REFERENCES

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