Interactions of 15N-Sulfadiazine and Soil Components As Evidenced

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Article 15

Interactions of N-sulfadiazine and soil components as evidenced by N-CPMAS NMR 15

Anne Elisabeth E. Berns, Herbert Philipp, Hans Lewandowski, Jeong-Heui Choi, Marc Lamshöft, and Hans-Dieter Narres Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06164 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Environmental Science & Technology

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Interactions of 15N-sulfadiazine and soil components as

2

evidenced by 15N-CPMAS NMR

3

Anne E. Berns,*,† Herbert Philipp,† Hans Lewandowski,† Jeong-Heui Choi,‡,§

4

Marc Lamshöft‡,|| and Hans-Dieter Narres†,⊥

5

†

6

52425 Jülich, Germany.

7

‡

8

Strasse 6, 44227 Dortmund, Germany.

9

* Corresponding author ([email protected])

Institute of Bio- and Geosciences (IBG-3) - Agrosphere, Forschungszentrum Jülich GmbH,

Institute of Environmental Research (INFU), Dortmund University of Technology, Otto-Hahn-

10

§

National Institute of Environmental Research, Hwangyeong-ro 42, Seo-gu, 22689 Incheon,

Republic of Korea. ||

Bayer CropScience, Alfred-Nobel-Str. 50, 40789 Monheim am Rhein, Germany.

⊥

Venloerstr. 738, 50827 Köln, Germany.

1 Environment ACS Paragon Plus


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Abstract

12

The extensive use of sulfonamides (SNs) in animal husbandry has led to an

13

unintentional widespread occurrence in several environmental compartments. The

14

implementation of regulations and management recommendations to reduce the

15

potential risk of development of antibiotic resistances necessitates detailed knowledge

16

on their fate in soil. We present results from two independent incubation studies of 15N-

17

labeled sulfadiazines (SDZ) which focused on identifying binding types in bound

18

residues. In the first study

19

isolated humic acids in the presence and absence of Trametes versicolor laccase, while

20

in the second study

21

Luvisol and isolated the humic acid fraction after sequential extraction of the soil. The

22

freeze-dried humic acid fractions of both studies were then analyzed by

23

NMR and compared with the

24

studies amide bonds and Michael adducts were identified, while formation of imine

25

bonds could be excluded. In the humic acid study, where less harsh extraction methods

26

were applied, possible formation of H-bridging and sequestration were detected

27

additionally.

15

15

N-amino labeled SDZ was incubated with two previously

N-double-labeled SDZ was incubated with a typical agricultural

15

N-CPMAS

15

N-spectra of synthesized model compounds. In both

28


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Introduction

30

Sulfonamides (SNs) are the oldest class of synthetic antibiotics in use and act as

31

competitive inhibitors of p-aminobenzoic acid in the folate synthesis in bacteria.1 Their

32

extensive use in animal husbandry has led to an unintentional widespread occurrence in

33

several environmental compartments like surface waters, ground waters and soils.2-4

34

Baran et al.2 estimated that worldwide over 20,000 Mg of SNs are introduced into the

35

biosphere every year. The potential risk of development of antibiotic resistances in soil

36

microorganisms through this permanent exposure to low levels of antibiotics, including

37

a possible subsequent transfer of the resistance genes to pathogens,5-8 and induced

38

alterations in soil microbial community structures9-14 are main environmental concerns.

39

Furthermore, several studies demonstrated that SNs were able to leach into deeper soil

40

layers and could hence potentially reach the groundwater table.15-18

41

Possible routes of attenuation in soil are biodegradation,19-21 photodegradation22,

23

42

and sorption to the soil matrix24-28 with subsequent formation of bound residues.29,

30

43

Due to their amphoteric character, the sorption affinity of SNs to different mineral or

44

organic soil constituents is higher at pH values below pKa2 and sorption decreases at

45

elevated pH values.31 In general sorption was found to be higher for organic sorbents

46

than for inorganic soil components25,

47

desorption.31,

48

transport experiments in soil columns17,

49

sorption site. Several studies, investigating the fate of SNs in soil, identified a number

50

of metabolites37-39 and more or less large amounts of what is often termed non-

51

extractable or bound residues.40,

52

(SDZ) occuring in soil were hydroxylation of the pyrimidyl moiety, acetylation of the

53

aniline moiety and cleavage of the molecule at the sulfonamid bond23 (also see Figure

34

32, 33

and often hysteresis was observed during

Modeling of long-term sorption/desorption experiments31 and of SN

41

35, 36

required the inclusion of an irreversible

The main reported transformations of sulfadiazine



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54

S1 in SI section). Specific identification of the structure of the unknown residues is

55

usually deemed necessary for problematic xenobiotics as a true incorporation into the

56

soil organic matter, through chemical reaction of reactive groups of the parent with

57

functional groups from the soil matrix, constitutes a definite removal of the parent and

58

hence reduces its potential environmental risk.

59

Several excellent studies on the possible chemical interactions of SNs with soil

60

organic matter have been published. Bialk et al.42 incubated different SNs with model

61

humic constituents in the presence of different enzymes or manganese oxide in aqueous

62

solutions and recorded the decline in SN concentration. With solution-state NMR

63

spectroscopy, they were able to demonstrate the formation of covalent imine bonds

64

(Schiff bases) between

65

acid in the presence of the peroxidase Arthromyces ramosus (ARP) and

66

hydrogenperoxide. Bialk et al.43 could additionally identify a Michael adduct between

67

15

68

Trametes versicolor laccase in the presence of oxygen, which oxidized the

69

protocatechuic acid in a first step to an ortho-quinone. The authors hypothesized that

70

Michael adducts were more likely to persist in soils as Schiff bases can be hydrolyzed in

71

aqueous environments. Bialk and Pedersen44 presented the reaction of

72

sulfamethazine and 15N-labeled sulfapyridine with Elliot soil humic acid in the presence

73

of A. ramosus peroxidase. The reaction products were analyzed with solution-state 13C-

74

and

75

coupling products of 13C-sulfamethazine indicated a covalent linkage through the anilic

76

nitrogen. 1H-15N heteronuclear multiple-bond correlation (HMBC) experiments on the

77

products of 15N-sulfapyridine were consistent with Michael adduct formation. Enamine

78

and imine structures were not detected.

13

C-labeled sulfamethazine and the substituted phenol syringic

N-amino labeled sulfapyridine and protocatechuic acid. The reaction was catalyzed by

15

13

C-labeled

N-NMR. 1H-13C heteronuclear single quantum coherence (HSQC) spectra of the


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79

Schwarz et al.45 performed a series of reaction experiments with three differently

80

substituted SNs (sulfanilamide, sulfapyridine and sulfadimethoxine) and three

81

substituted phenols (catechol, guaiacol and vanillin) as typical model fragments of soil

82

humic substances. The authors found that depending on the substituted phenol and SN

83

used nonenzymatic reaction could lead to a decrease of free SN in solution. When the

84

different SN and phenol mixtures were incubated with laccase the decrease was

85

significantly stronger and faster. The coupling product of vanillin and sulfapyridine

86

formed in the presence of laccase was analyzed by 15N-CPMAS NMR and the authors

87

recorded a signal at -260 ppm, which they assigned to amide-N. The lack of a 15N-label

88

prevented further signal resolution.

89

Gulkowska et al.46-48 studied the reaction of SNs with model humic constituents and

90

synthetic and natural humic acids. Reaction with model humic constituents revealed

91

that, although aromatic amines bind to quinones through 1,2- and 1,4-additions (to form

92

imines and anilinoquinones, respectively), SNs are relatively weak nucleophiles, which

93

need reactive quinones for addition.46 A second study evidenced that the reactivity

94

toward nucleophilic attacks was mainly depending on the redox state of the organic

95

matter. However, experiments with natural humic acid indicated that the pool of

96

reactive quinones is much smaller than the total number of quinones in SOM and that

97

most quinones in soil have a low electrophilicity. The authors demonstrated that laccase

98

converts unreactive hydroquinones to reactive quinones and that subsequently the

99

formed quinones can form covalent bonds with SNs.47 In a third study they concluded

100

that the number of reactive quinones was the limiting factor for the formation of non-

101

extractable SN residues in soils.48

102

As soil incubation studies often lead to samples with very low signal-to-noise ratios

103

or no recordable signal in solid-state NMR, most bound residue studies involving NMR



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104

are done on soil fractions like humic acids. This approach, however, bears the risk of

105

generating reaction products which are not necessarily formed in soil. We aimed at

106

identifying binding types of SDZ with different humic acids and estimating whether

107

results from such humic acid studies are also found in soil incubation studies. The

108

present paper combines two independently conducted incubation studies with

109

labeled SDZ and links the results of a relatively artificial humic acid approach to those

110

of a more realistic incubation with natural soil. Hence in one study, 15N-amino labeled

111

SDZ was reacted with two previously isolated humic acids in the presence and absence

112

of Trametes versicolor laccase and changes in the chemical environment of the labeled

113

amino group were identified through 15N-CPMAS NMR. In a second study, 15N-double-

114

labeled SDZ was incubated with a typical agricultural Luvisol. After incubation and

115

extraction of the still extractable antibiotic fraction the humic acid fraction was isolated

116

and analyzed by 15N-CPMAS NMR.

117

Materials and Methods

118

Chemicals for the humic acid study 15

119

15

N-

N-amino labeled sulfadiazine (15N-SDZ) was synthesized starting from 15N-labeled 15

120

aniline (synthesis is described in the Supporting Information (SI) section).

121

aniline (99 %

122

was checked with HPLC and the structure with liquid state 1H and

123

spectroscopy. All other chemicals were purchased in synthetic grade (p.a.) and used as

124

is.

15

N-labeled

N) was purchased from Isotec (Miamisburg, Ohio, USA). The purity 13

C NMR

125

The syntheses of the 15N-labeled model compounds are described in the SI.

126

Laccase C (EC 1.10.3.2) from the white-rot fungus Trametes versicolor was

127

purchased from ASA Spezialenzyme GmbH (Braunschweig, Germany) and had a


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128

laccase activity of 615.3 U g-1.49 Heat-inactivated laccase was used as a control. For

129

application a suspension containing 20.5 U ml-1 (33.3 mg mL-1) was prepared.

130

Two humic acids (HAs) were used in the present study. The first HA originated from

131

the Ap horizon (0–30 cm) of a field site at Krauthausen near Jülich, Germany,50

132

(formerly classified as gleyic Planosol,51 now considered a gleyic Stagnosol52). The

133

extraction and clean-up procedures are described in Witte et al.53 and Berns et al.49 The

134

second HA was the standard IHSS Elliot soil HA (1S102H), which was purchased from

135

IHSS and used as is. The elemental compositions of the HAs are summarized in Table

136

S1. Reaction between humic acid and 15N-SDZ

137 138

The reaction of

15

N-SDZ with HA was carried out as described in Berns et al.49

139

Briefly, we added 300 mg HA, 15 mg SDZ (i.e. 50 µg SDZ/mg HA) and 20.5 U of

140

active laccase from T. versicolor per preparation. The pH value of the

141

suspension was 6.0 to avoid precipitation of the HAs and remained at this pH during the

142

experiment. The suspension was stirred in a temperature-controlled glass vessel at 25°C

143

in the dark and the decrease of free SDZ in solution was monitored via HPLC (see SI)

144

until a constant value was reached (approx. 30 days). Free, non-reacted or loosely

145

sorbed SDZ was removed through repetitive dialysis against deionized water until the

146

concentration of SDZ in the dialysis water, determined by HPLC, dropped below the

147

detection limit. The 15N-SDZ-HA adducts, remaining in the dialysis bags, were freeze-

148

dried. Preparations without SDZ (i.e., only HA and laccase), without humic acid (i.e.,

149

only SDZ and laccase) and with heat deactivated laccase (i.e., HA, SDZ and heat

150

deactivated laccase) were run as control experiments.

151

Chemicals for the soil study


15

N-SDZ/HA-


152

15

N-double-labeled sulfadiazine (15N(dl)-SDZ), labeled at the amino and sulfonamido

153

positions, was purchased from Quotient Bioresearch Ltd. (Cardiff, UK) (chemical purity

154

≥ 98%, isotopic abundance > 98%). All other chemicals were purchased in synthetic

155

grade (p.a.) and used as is.

156

The soil used for the incubation was a Luvisol derived from loess and originated

157

from the plough layer of a field site at Merzenhausen, Germany.54 The collected soil

158

was air-dried, homogenized, sieved to 2 mm, and stored at ambient temperature prior to

159

incubation. The textural class was silt loam with 6 % sand, 78 % silt and 16 % clay.

160

Organic carbon content, effective cation exchange capacity and pH were 1.22 %, 11.4

161

cmolc kg-1 dry weight and 6.3, respectively.

162

The manure used was supplied from the former study40 as a blank manure, and

163

kept at –70°C until the present incubation experiment without drying. Incubation of soil with 15N(dl)-SDZ

164 165

15

N(dl)-SDZ was mixed with manure at a concentration of 2.5 mg SDZ g-1 manure.

166

The contaminated manure was mixed with 150 g dry soil at a concentration of 4 g

167

manure per 100 g dry soil resulting in a SDZ-concentration of 100 mg SDZ kg-1 soil.

168

The mixture was homogenized for 2 h in a tumbling mixer and subsequently

169

moisturized to 40% of maximum WHC. Soil incubation was set up in duplicate and

170

incubated with aeration at 24°C in the dark for 180 days. Water content was regularly

171

checked by weighing and replacing evaporated water. After 28 days a 5 g aliquot was

172

taken and extracted twice with 0.01 M CaCl2 solution at a 1:2.5 soil/solution ratio and

173

passed through ASE extraction applying the optimal ASE conditions determined by

174

Stoob et al.55 The ASE-extracted soil residue was then air-dried, sieved (< 0.063 mm)

175

and washed with 0.1 M HCl by shaking for 1 h to remove fulvic acid. The soil pellet

176

derived by centrifugation (5000×g, 5 min) was extracted according to Dec et al.56 with


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177

minor modifications. Briefly, the washed soil pellet was suspended in 1 mL of 2M

178

NaOH and 20 mL of 0.1 M NaOH, and shaken for 12 h under nitrogen. The NaOH

179

extract (supernatant) was separated by centrifugation (9000×g, 10 min), and these steps

180

were replicated twice. The combined NaOH extract was acidified with 2 mL of 5M HCl

181

to pH < 1 and kept at 4°C overnight, and lastly centrifuged. The precipitated humic acid

182

fraction was refined by dissolution with 0.1 mL of 2 M NaOH and 3 mL of 0.1 M

183

NaOH, followed by centrifugation. The supernatant was acidified, stored and

184

centrifuged as mentioned before. The pellet (humic acid fraction) was finally washed

185

with distilled water twice and lyophilized. The lyophilized HA sample was then

186

analyzed by 15N-CPMAS NMR. CPMAS 15N-NMR Spectroscopy

187 188

A Varian INOVA™ unity NMR spectrometer operating at 60.815 MHz for 15N was

189

used to acquire all spectra. The spectrometer was equipped with a MAS narrow bore

190

probe with a 6 mm stator.

191

referencing and chemical shift values are given in parts per million relative to CH3NO2

192

(= 0 ppm). Chemical shift values referenced to NH4+ (= 0 ppm) are given in the SI

193

section.

15

NH415NO3 and

15

N-labeled glycine were used for

194

For each humic acid adduct and its corresponding background experiment 256 k free

195

induction decays (FID) were accumulated with a repetition time of 2 s with VNMRJ

196

software (Version 1.1 RevisionD, Varian Inc., Palo Alto, CA, USA). A non-ramped

197

cross-polarization sequence with a contact time of 2 ms was applied. Humic acid

198

adducts and their respective blanks were spun at 6 kHz and measured using the same

199

amount of material to be able to subtract the background spectra from the HA adduct

200

spectra. For each model compound and labeled reactant the appropriate delay time to

201

avoid signal saturation was determined and the scan number adjusted for good signal-



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202

to-noise ratio. The soil HA samples were spun at 8 kHz and recorded with a ramped CP

203

sequence and a contact time of 1 ms. About 160000 FIDs were recorded with a recycle

204

delay of 0.5 s.

205

Fourier transforms of the FIDs were carried out using MestReC (Version 4.9.9.9,

206

Mestrelab Research, Santiago de Compostela, Spain). All FIDs were transformed by

207

first applying a zero filling and then an exponential filter function with a line

208

broadening (LB) of 10-20 Hz for the model compounds and 50-100 Hz for the humic

209

acid adducts and soil humic acids.

210

DFT calculations

211

Density functional theory (DFT) calculations were performed using Gaussian 03.57

212

The geometries were fully optimized using the B3-LYP methods in Gaussian 03 with

213

‘tight’ convergence criteria until the root-mean-square forces were smaller than 1×10−5

214

hartree bohr−1. The basis set for the geometry optimization was 6-311+G(d,p) and 6-

215

31G(d,p) for the more complex benzoquinone structure. The

216

shielding tensors were calculated using the GIAO method and the B3LYP functional in

217

conjunction with the 6-311+G(d,p) basis set. As a reference for the 15N chemical shifts

218

as calculated we employed nitromethane. For the geometry optimization and shielding

219

tensor calculation of nitromethane the same method and basis set were used. The

220

reference value was -158 ppm calculated with the 6-311+G(d,p) basis set and -159 ppm

221

with the 6-31G(d,p) basis set. The calculation of the chemical shift of the hydrogen

222

bonds between SDZ and p-benzochinone were done with the program Turbomol and the

223

B3-LYP method and the TZVP basis set.

224

Results and Discussion

225

Uptake of 15N-labeled SDZ


15

N NMR magnetic

Page 11 of 32


226

The initial reaction between HAs and SDZ was monitored by recording the decrease

227

of free SDZ in solution and considering the difference to the original concentration as

228

being taken up by the HAs not differentiating between underlying processes. The initial

229

uptake of 15N-labeled SDZ on both humic acids in the presence and absence of laccase

230

is shown in Figure 1. The molar uptake was similar on both humic acids and in a similar

231

order of magnitude as the amounts observed in a previously conducted

232

aminobenzothiazole study49. The presence of laccase caused an increased uptake of

233

about 3-4 times compared to the respective experiments without laccase. This is in line

234

with Gulkowska et al.48 and can be explained by the increased amounts of reactive

235

quinones generated by the action of the laccase. Blank experiments without humic acids

236

and without laccase (Figure S7 in SI) showed that the reaction vessel itself adsorbed

237

only minor amounts of SDZ. Control experiments without humic acids, but with laccase

238

(Figure S7 in SI), showed a loss of SDZ in solution, which represented roughly a fourth

239

of the amounts sorbed to HAs. In order to determine whether the carrier material of the

240

enzyme was responsible for substantial sorption, control experiments with heat-

241

deactivated enzyme were done (Figure 1 and S7 in SI). No significant uptake was

242

recorded in these experiments. Hence, the decline in free SDZ recorded in the control

243

experiments with SDZ and enzyme was due to enzymatic transformation of SDZ as

244

already reported by Schwarz et al.,19,

245

during interpretation of the 15N-spectra.

246 247 248

45

15

N-

a fact which needs to be taken into account

The uptake curves recorded in the presence of laccase and oxygene were best fitted by assuming two overlapping processes (eq 1) 49, 58: y(t) = a1 [1-exp(-k1t)]+ a2 [1-exp(-k2t)]0.5

(1)

249

The first term mirrors a fast (sorption or reaction) process, which dominates at the

250

beginning of the experiment, while the second term accounts for a slow diffusion-



Page 12 of 32

251

controlled process. Both uptake curves recorded without laccase could be fitted solely

252

with the second term. Plotting the uptake curves as a function of the square root of time

253

(see insert in Figure 1) shows that the uptake without laccase is a purely diffusion-

254

controlled process while in the presence of laccase diffusion becomes only dominant for

255

reaction times greater than 2 days. Interaction of 15N-SDZ with humic acid

256 257

It was already evidenced from literature that the amino group was the main reaction

258

site of SNs, the same site through which SNs bind to intracellular dihydropteroate

259

synthase and exert their bacteriostatic effect.1, 2, 42, 59 Hence, we used 15N-amino labeled

260

SDZ to be able to follow changes in the chemical environment of this group through

261

solid-state NMR. Figure 2 shows the background corrected 15N-CPMAS spectra of the

262

reaction products of

263

absence of laccase. The spectra were recorded after dialysis of the humic acid reaction

264

mixtures, which removed non-reacted excess

265

from the mixtures, which were subsequently freeze-dried. The isolated

266

adducts contained 36 to 37 weight-% of

267

prepared without laccase contained only 10 to 11 weight-% of

268

non-corrected spectra and their corresponding background spectra can be found in the

269

SI (Figure S8). To allow a rough comparison between signal intensities the spectra were

270

recorded on similar amounts of humic acids with identical scan numbers. Both

271

experiments conducted in the presence of laccase (Figure 2a) resulted in significantly

272

higher amounts of

273

laccase (Figure 2b). Both spectra (Figure 2a) contained two major signals centered

274

around -245 and -300 ppm. However, the ratios of these two signals differed. In

275

between these two main signals a large shoulder was present most likely generated by a

15

15

N-SDZ with two different humic acids in the presence and

15

15

N-SDZ and soluble reaction products

N-labeled SDZ. The

15

15

15

N-SDZ-HA

N-SDZ-HA adducts

N-labeled SDZ. The

N-SDZ-HA adducts than the corresponding experiments without


Page 13 of 32


276

third signal centered at around -265 ppm. According to Knicker and Lüdemann60 the

277

signals at -245 and -265 ppm can be assigned to amide bonds. The signal at -300 ppm

278

lies in the region of free amino groups, but does not correspond to the original signal of

279

free SDZ at -309.5 ppm. In both spectra there are, however, clear shoulders at -309.8

280

ppm, which indicate the presence of sequestered or sorbed SDZ (via the pyrimidinyl

281

moiety) able to withstand dialysis. The spectrum of the control experiment containing

282

only laccase and 15N-SDZ (corrected for the signal generated by the enzyme itself and

283

its carrier material) contained one prominent signal at -261 ppm (amide region) and a

284

weak signal at -303 ppm (amino group). The fact that the recorded compounds

285

withstood dialysis suggested that the formed products were reactive enough to either

286

bond or strongly sorb to the carrier material of the enzyme (even though SDZ itself does

287

not) or reacted among each other to form larger entities not able to pass the dialysis

288

membrane. Schwarz et al.19 described a series of possible transformation products of

289

sulfapyridine (SPY). However, only one of the proposed metabolites contained an

290

amide group and all would be small enough to be able to pass the dialysis membrane.

291

Furthermore, during HPLC control of the dialysis no metabolites were detected.

292

However, as the UV detector was set at the optimal wavelength for SDZ possible

293

metabolites with significantly altered structures might have been overlooked. In any

294

case the amount of signal due to reaction between laccase and SDZ was estimated to

295

make up only 10-20 % of the signal of the main experiment. As these reaction products

296

might not form in this particular way in the presence of HAs we refrained from treating

297

this spectrum as background spectrum and subtracting it from the signal of the main

298

experiment spectrum.

299

Contrastingly, the experiments without laccase generated adducts with greater

300

variability in signal intensity (Figure 2b). The spectrum of the IHSS HA adduct



Page 14 of 32

301

displayed three broad signal regions of similar intensity at around -235, -270 and -300

302

ppm. In the spectrum of the Krauthausen HA adduct several signals of very different

303

intensities were found with the signal at -245 ppm being the smallest and the ones at -

304

303 and -310 ppm the most intense. Both spectra contained a signal at -310 ppm

305

stemming from sequestered SDZ and in the Krauthausen spectrum this signal made up a

306

large portion of the whole signal. The different spectra from both HA adducts formed

307

without laccase showed that the amount of inherent reactive sites were different in both

308

HAs.

309

As chemical shift regions in

15

N-NMR have a stronger overlap than those in

13

C-

310

NMR and to be able to do a more detailed signal assignment of the recorded signals in

311

the NMR spectra, we synthesized model compounds representing possible reactions

312

between the 15N-amino labeled SDZ and the HAs.

313 314

Comparison with synthesized model compounds Figure 3 displays the synthesized model compounds which concurred with the 15

315

signals found in the spectra of the

316

model compounds which had no corresponding signals in the 15N-SDZ-HA spectra.

317

N-SDZ-HA adducts. Figure S9 in the SI shows

The bottom spectrum (Figure 3i) displays the original signal of the

15

N-amino

318

labeled SDZ with a signal at -309.5 ppm. Due to the low sensitivity of the 15N-nucleus

319

the other three nitrogen signals are not visible as they would require much larger scan

320

numbers to reach a reasonable signal-to-noise ratio. This also applies to the spectra of

321

synthesized model compounds (Figure 3c-h) which were all done with

322

labeled SDZ. All tested substituents at the amino group caused the chemical shift of this

323

group to move to lower resonance frequencies. This was caused by the negative

324

inductive effect of the tested substituents reducing the electronic density and thereby the

325

shielding of the amino group. The only exception was the SDZ hydrochloride (Figure


15

N-amino

Page 15 of 32


326

S9) which showed a shift to higher resonance frequencies (-321.9 ppm) in accordance

327

with the higher electronic density of the –NH3+ group.

328

The signal of the unaltered

15

N-SDZ can be found in all four adduct spectra at

329

different intensities. In the laccase mediated adducts the signal is present in the form of

330

a shoulder at around -310 ppm. Part of the

331

physically sorbed or bound to the HAs through the pyrimidinyl moiety. As a covalent

332

binding of the pyrimidinyl moiety would probably alter the chemical shift of the amino

333

group at least a little bit, the most likely argument for the signal at -310 ppm in the

334

adduct is a physical sorption through van-der-Waals binding either of the aromatic ring

335

with the hydrophobic regions of the HAs or through dipole-dipole interactions of the

336

pyrimidinyl moiety with the polar regions of the HAs. In the experiments without

337

laccase the signals of unaltered

338

unequal sorption capacities of both HAs. In the Krauthausen HA the SDZ shoulder in

339

the laccase experiment (Figure 2a) was only slightly higher than in the laccase-free

340

experiment (Figure 2b). In the IHSS HA the difference in the SDZ signal was much

341

larger between the two experiments, indicating that the IHSS HA had a less active

342

sorption surface than Krauthausen HA. Elemental analysis (Table S1) of both HAs

343

revealed that Krauthausen HA contained less carbon, but higher amounts of

344

heteronuclear elements like O, N and S stemming from functional groups, which could

345

explain the different behaviour of both HAs.

15

15

N-amino labeled SDZ is hence either

N-SDZ were very different in intensity indicating

346

The synthesized amide bonds had chemical shifts between -232 ppm (formyl-SDZ,

347

Figure 3c) and -250.5 ppm (trimethoxybenzoyl-SDZ, Figure 3f). The series of

348

substituents showed a decreasing negative inductive effect with larger substituents,

349

which reduced the electronegative pull of the oxygen in the amide bond. The largest

350

reduction in electronic density around the amino-N was achieved with the formyl-



351

group. Further reduction can most likely only be achieved with strong electronegative

352

substitutents like chlorine or through a change in the pyrimidinyl moiety. The model

353

substance acetyl-SDZ is also the most easily formed metabolite of SDZ in biological

354

systems. Hence, part of the signal around -245 ppm could also be due to simple

355

sequestration of acetyl-SDZ. However, we found no indication of metabolites during

356

HPLC-control during dialysis and it seems reasonable to assume that in case of

357

formation of larger amounts of acetyl-SDZ part of the metabolite would be soluble and

358

detectable at the same wavelength than SDZ. Furthermore, the signal at -245 ppm is

359

small in the spectra without laccase, which indicated that potentially only small amounts

360

of the metabolite were formed during the time span of our experiments.

361

Michael-addition of SDZ to benzoquinone generated a product with a chemical shift

362

of -261.5 ppm. Hence, Michael adducts of SDZ resonate in the region which is typical

363

for amide/peptide bonds. The signal corresponds to the main signals recorded in the

364

15

365

can be the breakdown of the SDZ molecule, we also considered the possibility of the

366

resulting aniline moiety reacting on its own. We estimated that the deshielding effect of

367

an unsubstituted phenyl ring on the amino-N would be weaker than that of the

368

substituted ring. Hence, to test the magnitude on the chemical shift change we also

369

synthesized a Michael adduct with 15N-labeled aniline and found a chemical shift which

370

was shifted approx. 16 ppm to the high-field side at -278 ppm. This chemical shift

371

matches the shift region between the two main signals and hence part of the recorded

372

signal might be due to reaction of the aniline moiety following SDZ breakdown.

373

Assuming a similar order of magnitude in chemical shift change for amide bonds

374

formed by the aniline moiety the amide signals (Figure 3c to 3f) would shift to a region

375

from approx. -250 to -270 ppm. As the most prominent signal is centered around -245

N-SDZ-HA adducts obtained in the presence of laccase. As one route of attenuation


Page 16 of 32

Page 17 of 32


376

ppm (Figure 3a) and amide bonds formed by aniline seem to be shifted to the high-field

377

side of this position, we can assume that the main signal is due to reaction products of

378

SDZ rather than aniline.

379

The spectra of the adducts generated without laccase had only a minor signal in the

380

region of amide bonds and Michael adducts. Hence, as already indicated by the uptake

381

rates discussed above, for the formation of amide bonds and Michael adducts the action

382

of an enzyme is necessary to create reactive sites on the HAs.

383

We could exclude the formation of Schiff bases (imines) (Figure S9), which was in

384

line with most studies.43-48 Only one studie using peroxidases to induce enzymatic

385

transformations between SNs and model humic components found imine formation.42

386

As SN Schiff bases might have higher antimicrobial activity than the parent,61 a lack of

387

imine formation reduces the environmental risk of SDZ.

388

None of the synthesized model compounds matched the large signal at -300 ppm in

389

the spectra of the laccase-mediated HA adducts. The down-field shift of only 10 ppm of

390

the original amino-N signal indicates a slight reduction of the electronic density around

391

this nucleus causing only a minor deshielding. All tested covalent bonds, however, had

392

a stronger pull on the electronic shielding and induced a larger downfield shift. Neat

393

aniline has a chemical shift of approx. -325 ppm. Hence, sequestered aniline stemming

394

from a breakdown of the SDZ molecule did also not explain the -300 ppm shift.

395

Comparison with chemical shifts from natural N-containing compounds (Figure S10)

396

and compounds used in previous studies (Figure S11) also revealed no functional

397

groups other than amino groups. Amino groups in aliphatic chains (Figure S10 a, d, e

398

and i), however, resonated at higher fields and amino groups attached to hetero-cycles

399

(Figures S10g and S11f) were shifted to the downfield side of -300 ppm.



Page 18 of 32

400

We hence hypothesized that this signal shift could be induced by weak H-bridging of

401

the amino-protons. The involvement of these protons in a shared H-bridge would most

402

likely reduce the electronic density, but have a less strong effect than a covalent bond. DFT calculations

403 404

As model compounds with H-bridging of the amino-N could not be synthesized, we

405

used density functional theory (DFT) calculations to compute the theoretical chemical

406

shift of such a compound. In a first step we calibrated the calculations with known

407

chemical shifts and obtained good linear correlations between the calculated and the

408

experimental chemical shifts (Figure S12 in SI). Table 1 shows the calculated and

409

experimental δ values used for calibration. An exemplary H-bridge between

410

benzoquinone and the SDZ-amino-N reduced the electronic shielding of the amino-N by

411

about 4 ppm and the δ value shifted slightly downfield, which is in agreement with and

412

hence supports our hypothesis of H-bridging. Interaction of SDZ with soil

413 414

As breakdown of the SDZ molecule would lead to two separate moieties the

415

incubation experiments with soil were done with SDZ labeled at the amino- and

416

sulfonamido-N in order to be able to follow both moieties. Figure 4 shows the spectrum

417

of a HA isolated from incubated soil after the extractable residues were removed

418

through the sequential extraction procedure. For comparison the background spectrum

419

of the HA and

420

amino sigmal at -309 ppm and a broad signal at -237.5 ppm with two side peaks at -

421

227.5 and -246.5 ppm. The amino signal of the acetyl-SDZ is found at -244.5 ppm,

422

which unfortunately overlaps with the relatively broad signal of the second label. The

423

spectrum of the incubated HA had one broad signal with a maximum at -242 ppm.

424

Contrastingly to the HA study, around -300 ppm only a small signal and almost no

15

N(dl)-SDZ are shown. The spectrum of


15

N(dl)-SDZ generated an

Page 19 of 32


425

amino-SDZ signal was visible. Considering that the HA in this study was exposed to

426

much harsher extraction conditions the lack of a signal in this region confirms our

427

assumption that this signal stems from weaker bonds like H-bridging or sequestered

428

SDZ. As the sulfonamido-N signal overlaps with amide bonds and Michael adducts

429

from the amino-N the broad signal is unfortunately difficult to interprete. However,

430

even if part of the signal is generated by the labeled pyrimidinyl moiety, the spectrum is

431

still comparable to the spectra recorded in the HA study and no additional signals

432

occurred. As for the HA study the study in soil suggested amide bond and Michael

433

adduct formation, but gave no indication of formation of imines. Hence, results from

434

studies peformed on HA rather than whole soil can be used for risk assessment of the

435

qualitative behaviour of SDZ in soil. For quantification the studies would need to be run

436

with an additional 14C-label.

437

Environmental implications

438

In a previous batch incubation study with 14C-SDZ in soil Sittig et al.28 extrapolated,

439

from a series of consecutive microwave assisted extraction steps, that the amount of

440

truly non-extractable residues (NER) increased to approx. 30% of the applied SDZ

441

within 30 days. Batch experiments with sterilized soil generated approx. 10% of NER.

442

These observations are in line with our observation that the presence of laccase caused 3

443

to 4 times larger amounts of adducts to be formed. The largest part of the enzyme-

444

enduced adducts in the HA study was formed by amide formation and Michael adducts

445

to quinone systems as already identified by Bialk et al.44 and Gulkowska et al.46, 47 The

446

second largest part was composed of unaltered sequestered SDZ and SDZ bound by H-

447

bridging of the amino group. These were also the dominating species in the experiments

448

without laccase. In the HA-adducts isolated from the soil incubation study the signals of

449

amide formation and Michael adducts dominated, while only a small signal was found



450

indicating sequestered or H-bound SDZ. Considering that the HA extraction in the soil

451

study was much harsher than the dialysis in the HA study, we assume that the residues

452

from the soil study are mostly free of loosly bound SDZ. Our study hence indicates that

453

true NER are composed of residues which are covalently bound to soil organic matter

454

and can be considered as bound residues which have become an integral part of the

455

organic matter and where the bioactivity of the compound has been neutralized. The

456

consecutive extraction steps of Sittig et al.28 showed that the fraction of extractable

457

residues cannot be extracted with a single extraction step and, depending on the initial

458

amount of SDZ applied and the soil type, up to 55 extraction steps can be potentially

459

needed to reach an exhaustive extraction of residues. This sticky but potentially

460

extractable fraction is most likely formed by sequestered SDZ and SDZ bound by H-

461

bridging and, even though it might have temporarily lost its antimicrobial activity due to

462

blocking of the amino side chain, still needs to be considered as a potential threat.

463

Sequestration and H-bridging does not require the presence of an enzyme and most

464

likely also no biological activity as these are purely physical and chemical processes

465

which only require the reaction partners to be close enough to one another.

466

We could not identify Schiff base formation in any of the analyzed adducts in our

467

study as found by Bialk et al.42 and Gulkowska et al.46 nor could we identify the

468

formation of N-heterocycles as hypothesized as subsequent incorporation into the soil

469

matrix by Gulkowska et al.46 As already pointed out by Bialk et al.43 imines can be

470

hydrolyzed in aqueous solutions and hence if they formed during our experiments they

471

might have been released into the extractable fraction.

472

The SDZ amount in the present HA study was approx. 2 to 3 orders of magnitude

473

higher (based on amount SDZ per mg soil organic carbon) and the amount applied in

474

the soil study was 1 to 2 orders of magnitude higher than the amounts in the batch study


Page 20 of 32

Page 21 of 32


475

of Sittig et al.28 Despite these necessary high amounts, in order to be able to record 15N-

476

CPMAS signals, we nevertheless estimate that the identified binding types also occur

477

under natural conditions as the required reaction sites are present in natural soils.44, 47, 63

478

Furthermore, the study was conducted with two different HAs, but generated

479

comparable signals in both. The results differed in the recorded signal strength, which

480

indicated different amounts being formed. Hence, we conclude that the identified

481

binding types will occur in different soils, but the amounts formed will differ depending

482

on the soil type.

483

Supporting Information

484

Text, table and figures on humic acids, HPLC conditions, reaction pathways of SDZ,

485

the synthesis of 15N-SDZ and the model compounds, control experiments, non-corrected

486

15

487

substances, correlation of calculated and experimental chemical shifts.

488

N-NMR spectra,

15

N-spectra of further model compounds and N-containing natural

Acknowledgments

489

We thank Sabine Willbold from ZEA-3 (FZ Jülich) for the elemental analyses of the

490

synthesized model compounds. We gratefully acknowledge the help of Thomas Müller

491

from JSC (FZ Jülich) with the DFT modeling of the

492

project was funded by the German Research Foundation (DFG) within the Research

493

Unit FOR566 “Veterinary medicines in soils: Basic research for risk assessment”.

15

N-chemical shifts. Part of the

494

References

495 496 497 498

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(51) Food and Agriculture Organization of the United Nations World reference base for soil resources 2006 - A framework for international classification, correlation and communication; FAO: Rome, Italy, 2006. (52) Food and Agriculture Organization of the United Nations World reference base for soil resources 2014 - International soil classification system for naming soils and creating legends for soil maps (Update 2015); FAO: Rome, Italy, 2014. (53) Witte, E. G.; Philipp, H.; Vereecken, H. Study of enzyme-catalysed and noncatalysed interactions between soil humic acid and C-13-labelled 2aminobenzothiazole using solid-state C-13 NMR spectroscopy. Org. Geochem. 2002, 33 (12), 1727-1735. (54) Kasteel, R.; Burkhardt, M.; Giesa, S.; Vereecken, H. Characterization of field tracer transport using high-resolution images. Vadose Zone J. 2005, 4 (1), 101-111. (55) Stoob, K.; Singer, H. P.; Stettler, S.; Hartmann, N.; Mueller, S. R.; Stamm, C. H. Exhaustive extraction of sulfonamide antibiotics from aged agricultural soils using pressurized liquid extraction. J. Chromatogr. A 2006, 1128 (1-2), 1-9. (56) Dec, J.; Haider, K.; Benesi, A.; Rangaswamy, V.; Schäffer, A.; Plücken, U.; Bollag, J. M. Analysis of soil-bound residues of C-13-labeled fungicide cyprodinil by NMR spectroscopy. Environ. Sci. Technol. 1997, 31 (4), 1128-1135. (57) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J., J. A. ; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B. B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revision B.03, Gaussian, Inc.: Pittsburgh PA, 2003. (58) Schlüpen, J.; Haegel, F. H.; Kuhlmann, J.; Geisler, H.; Schwuger, M. J. Sorption hysteresis of pyrene on zeolite. Colloid Surf. A-Physicochem. Eng. Asp. 1999, 156 (1-3), 335-347. (59) Sarmah, A. K.; Meyer, M. T.; Boxall, A. B. A. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 2006, 65 (5), 725-759. (60) Knicker, H.; Lüdemann, H. D. N-15 and C-13 CPMAS and solution NMR studies of N-15 enriched plant material during 600 days of microbial degradation. Org. Geochem. 1995, 23 (4), 329-341. (61) Mondal, S.; Mandal, S. M.; Mondal, T. K.; Sinha, C. Spectroscopic characterization, antimicrobial activity, DFT computation and docking studies of sulfonamide Schiff bases. J. Mol. Struct. 2017, 1127, 557-567. (62) Begtrup, M.; Balle, T.; Claramunt, R. M.; Sanz, D.; Jimenez, J. A.; Mo, O.; Yanez, M.; Elguero, J. GIAO ab initio calculations of nuclear shieldings of monosubstituted benzenes and N-substituted pyrazoles. Theochem-J. Mol. Struct. 1998, 453, 255-273. (63) Berns, A. E.; Knicker, H. Soil Organic Matter. eMagRes 2014, 3, 43-54.



695

Table 1:

Page 26 of 32

Calculated shielding tensors σcalc and chemical shifts δcalc of SDZ and model substances and experimental chemical shifts δexp.

696 molecule

position

σcalc

δcalc = σref a - σcalc

δexp

[ppm]

[ppm]

[ppm]

calibration with known chemical shifts

697

SDZ

amino-N

181.7

-340.7

-309.5

acetyl-SDZ

amino-N

96.1

-255.1

-244.7

benzoyl-SDZ

amino-N

106.0

-265.0

-250.1

trimethoxybenzoyl-SDZ

amino-N

105.7

-264.7

-250.5

anilino-benzoquinone

amino-N

132.7

-290.7b

-277.8

SDZ-p-benzoquinone

amino-N

131.7

-289.7b

-261.5

benzoyl-SDZ

sulfonamido-N

77.7

-236.3

-240.8

benzoyl-SDZ

pyrimidinyl-N1

-28.2

-130.8

-133.6

benzoyl-SDZ

pyrimidinyl-N2

-32.5

-126.5

-128.1

a

shielding tensor of nitromethane  isσref = -159 ppm62 (except for b = -158 ppm)

698


Page 27 of 32


Figure captions

699 700

Figure 1:

Uptake of 15N-amino labeled SDZ by soil humic acids (HA) in the presence

701

and absence of laccase determined through the determination of free 15N-

702

SDZ in solution. The uptake in the presence of laccase is fitted with y(t) =

703

a1[1 - exp(-k1t)] + a2[1 - exp(-k2t)]0.5 and in the absence of laccase is fitted

704

with y(t) = a[1 - exp(-kt)]0.5. The insert is the square root plot.

705

Figure 2:

Background-corrected 15N-CPMAS NMR spectra of the 15N-SDZ-HA

706

adducts after dialysis. The spectra were recorded on similar amounts of

707

material and with identical scan numbers to allow a rough estimate of the

708

quantities. a) 15N-SDZ-HA adducts formed with laccase (grey: reaction

709

product formed in the absence of humic acid solely with 15N-SDZ and

710

laccase), b) 15N-SDZ-HA adducts formed with laccase. (° = spinning side

711

bands)

712

Figure 3:

Comparison of the 15N-CPMAS NMR spectra of the labeled HA adducts

713

and 15N-labeled synthesized model compounds.

714

a) 15N-SDZ-HA adducts in the presence of laccase (thick line: Krauthausen

715

HA; thin line: IHSS HA); b) 15N-SDZ-HA adducts in the absence of laccase

716

(thick line: Krauthausen HA; thin line: IHSS HA); c) 15N-formyl SDZ; d)

717

15

N-acetyl SDZ; e) 15N-benzoyl SDZ; f) 15N-trimethoxybenzoyl SDZ; g)

718

15

N-SDZ-benzochinon adduct; h) 15N-aniline-benzochinon adduct; i) 15N-

719

amino labeled SDZ. (* = 15N-label; ° = spinning side bands)

720

Figure 4:

15

N-CPMAS NMR spectra of 15N(dl)-SDZ and humic acids extracted from

721

soil incubation experiments with and without 15N(dl)-SDZ after 28 days of

722

incubation. (° = spinning side bands)



352x198mm (72 x 72 DPI)

ACS Paragon Plus Environment

Page 28 of 32

Page 29 of 32


Uptake of 15N-amino labeled SDZ by soil humic acids (HA) in the presence and absence of laccase determined through the determination of free 15N-SDZ in solution. The uptake in the presence of laccase is fitted with y(t) = a1[1 - exp(-k1t)] + a2[1 - exp(-k2t)]0.5 and in the absence of laccase is fitted with y(t) = a[1 - exp(-kt)]0.5. The insert is the square root plot. 1057x793mm (72 x 72 DPI)



Figure 2: Background-corrected 15N-CPMAS NMR spectra of the 15N-SDZ HA adducts after dialysis. The spectra were recorded on similar amounts of material and with identical scan numbers to allow a rough estimate of the quantities. a) 15N-SDZ-humic acid adducts formed with laccase (grey: reaction product formed in the absence of humic acid solely with 15N-SDZ and laccase, spectra are corrected for background of laccase and its carrier material, heights of spectra are adjusted to roughly fit the amount of laccase in the adduct spectra), b) 15N-SDZ-humic acid adducts formed without laccase. (° = spinning side bands) 1057x1583mm (72 x 72 DPI)


Page 30 of 32

Page 31 of 32


N-CPMAS NMR spectra of the labeled HA adducts and 15N-labeled synthesized model compounds. a) 15N-SDZ/HA adducts in the presence of laccase (thick line: Krauthausen HA; thin line: IHSS HA); b) 15NSDZ/HA adducts in the absence of laccase (thick line: Krauthausen HA; thin line: IHSS HA); c) 15N-formyl SDZ; d) 15N-acetyl SDZ; e) 15N-benzoyl SDZ; f) 15N-trimethoxybenzoyl SDZ; g) 15N-labeled SDZ/benzochinon adduct; h) 15N-labeled aniline/benzochinon adduct; i) 15N-amino labeled SDZ. (* = 15Nlabel; ° = spinning side bands) Figure 3: Comparison of the

15

1057x1668mm (72 x 72 DPI)



Figure 4: 15N-CPMAS NMR spectra of 15N-double-labeled SDZ and humic acids extracted from soil incubation experiments with and without 15N-double-labeled SDZ after 28 days of incubation. (° = spinning side bands) 1057x573mm (72 x 72 DPI)


Page 32 of 32

Interactions of 15N-Sulfadiazine and Soil Components As Evidenced

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