Identification of sulfonated and hydroxy-sulfonated PCB metabolites in

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Characterization of Natural and Affected Environments

Identification of sulfonated and hydroxy-sulfonated PCB metabolites in soil: new classes of intermediate products of PCB degradation? Renzo Bagnati, Elisa Terzaghi, Alice Passoni, Enrico Davoli, E. Fattore, Angelo Maspero, Giovanni Palmisano, Elisabetta Zanardini, Sara Borin, and Antonio Di Guardo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03010 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Environmental Science & Technology

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Identification of sulfonated and hydroxy-sulfonated PCB metabolites in soil: new classes of intermediate products of PCB degradation?

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Renzo Bagnatia, Elisa Terzaghib, Alice Passonia, Enrico Davolia, Elena Fattorea, Angelo Masperob, Giovanni Palmisanob, Elisabetta Zanardinib, Sara Borinc, Antonio Di Guardob 

5 6

a

7

b

Department of Science and High Technology, University of Insubria, Via Valleggio 11, 22100 Como, Italy

8

c

Department of Food, Environmental and Nutritional Sciences, University of Milan, Via Celoria 2, 20133 Milan, Italy

9

TOC art

Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche “Mario Negri” IRCCS, Via Mario Negri 2, 20156 Milan, Italy

10 11

Abstract

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In this paper we describe the identification of two classes of contaminants: sulfonated-PCBs and

13

hydroxy-sulfonated-PCBs. This is the first published report of the detection of these chemicals in

14

soil. They were found, along with hydroxy-PCBs, in soil samples coming from a site historically

15

contaminated by the industrial production of PCBs and in background soils. Sulfonated-PCB levels

16

were approximately 0.4-0.8% of the native PCB levels in soils, and about twice the levels of

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hydroxy-sulfonated-PCBs and hydroxy-PCBs. The identification of sulfonated-PCBs was

18

confirmed by the chemical synthesis of reference standards, obtained through the sulfonation of

19

an industrial mixture of PCBs. We then reviewed the literature to investigate for the potential

20

agents responsible for the sulfonation. Furthermore, we predicted their physico-chemical



Corresponding author e-mail: [email protected],  Shared 1st authors

ACS Paragon Plus Environment


21

properties and indicate that, given the low pKa of sulfonated- and hydroxy-sulfonated-PCBs, they

22

possess negligible volatility, supporting the case for in situ formation from PCBs. This study shows

23

the need of understanding their origin, their role in the degradation path of PCBs and their fate,

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as well as their (still unknown) toxicological and ecotoxicological properties.

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Keywords: Contaminated Site; sulfonated-PCBs; hydroxy-sulfonated-PCBs; soil; environmental fate; risk assessment

27 28 29

Introduction

30

Polychlorinated biphenyls (PCBs) are an important class of ubiquitous persistent organic

31

pollutants. Although they are not produced anymore, their relevance lies in their environmental

32

persistence, bioaccumulation and their important toxicological1,2 and ecotoxicological

33

characteristics3. In Italy PCBs were produced and sold as mixtures (Fenclor, Fenclor DK and

34

Apirolio) by Caffaro S.p.A. in Brescia (Northern Italy) until 1984, when their production was

35

stopped4. More than 80 Ha of agricultural areas close to the factory were found to be heavily

36

contaminated by PCBs (including PCB 209, the decachlorinated congener), present at up to some

37

mg/kg of soil (dry weight)4, but also PCDDs, PCDFs, DDTs and their isomers/metabolites and

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metals. This contamination was related to the use of factory outlet contaminated waters to

39

irrigate the adjacent agricultural fields. Since these areas are now part of a National Priority Site

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(SIN) for Remediation (SIN Brescia-Caffaro site), a number of tools were employed and activities

41

were started to plan the soil bioremediation of the site4–11, including the present study.

42

Biodegradation of PCBs was demonstrated to proceed, in laboratory and in field situations, under

43

aerobic and anaerobic conditions via two distinct mechanisms. PCBs can be used as carbon and


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energy source or electron acceptors, respectively12. Aerobic bacteria preferentially degrade less

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chlorinated PCB congeners through different catabolic pathways responsible for the

46

transformation of PCBs into dihydroxychlorobiphenyls [PCB-(OH)2] and chlorobenzoates13,14 and

47

further mineralized to aliphatic acids. In anoxic environmental niches, anaerobic bacteria were

48

shown to degrade highly chlorinated PCB congeners, through reductive dechlorination,

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preferentially removing meta and para chlorines and releasing low chlorinated ortho substituted

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PCB congeners which are more easily degradable by aerobic bacteria5,15,16. Fungi, especially white

51

rot fungi, were also shown to be involved in PCB degradation5,17,18. Recently, much attention is

52

devoted to the presence in the environment of polar metabolites deriving from the initial

53

degradation of PCBs, such as those containing an hydroxy group (-OH) i.e. OH-PCBs 19, and those

54

with a sulfate (-OSO3H) group, i.e. “sulfated-PCBs”

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today increasingly considered as a new class of environmental contaminants, possessing specific

56

chemical, physical, and biological properties which are not shared with the parent PCBs20, but

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little is currently known about their biodegradability, distribution and behavior in the

58

environment 23. OH-PCB were found in plants24–28, rain, snow, surface waters, sewage treatment

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plants23,29. OH-PCBs were also recently detected in surficial sediments, as well as in five original

60

Monsanto Aroclors, suggesting that in this case they can be present as degradation products, but

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also as by-products in PCB mixtures19). Among other polar metabolites discovered, some

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monochloro sulfated-PCBs were found in plants, animals and humans22,26,30 and showed the

63

capability of being transformed to OH-PCB. Recently, Liu et al31 found in polar bear blood a

64

number of new halogenated contaminants, among these sulfonated-PCBs were discovered.

20–22

For example, OH-PCB metabolites are



65

The aims of this study were to identify two new classes of polar PCB metabolites in soil,

66

sulfonated PCBs (-SO3H, from now on “sulfonated-PCBs”) and their hydroxy derivatives (-OH,-

67

SO3H, from now on “OH-sulfonated-PCBs”) and develop an analytical method for their

68

approximate quantification. This, to our knowledge, is the first report of their presence in soil

69

and for OH-sulfonated-PCBs the first evidence in any sample. We also estimated the metabolite

70

physico-chemical properties to evaluate how they could affect their environmental fate

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compared to PCBs and highlight their implications for human and ecological risk assessment.

72

Materials and methods

73

Site description and sample collection

74

About 1.5 m3 of surficial soil (first 40 cm) was collected from the contaminated site in an area

75

representative of average concentrations 4,32. The soil was then thoroughly mixed in the field and,

76

to additionally reduce the heterogeneity of the naturally weathered soil, it was homogenized

77

using the Japanese Slab Technique33. More than 200 samples were obtained for a greenhouse

78

rhizoremediation experiment32. One representative sample of the initial soil employed in the

79

experiment was used to obtain the concentrations of PCBs and metabolites in the contaminated

80

site (from now on “SIN sample”). Additional soil samples (from now on called “background

81

samples”) were taken in two sites (one for each site), 2 km away from the contaminated site in

82

opposite directions (South West, SW and North East, NE) (Map SI-1). Each sample was a

83

composite sample of 5 subsamples taken within 1 m up to a depth of 10 cm. The 2 km SW sample

84

received partially contaminated irrigation water in the past, while the 2 km NE sample did not

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receive any contaminated irrigation water from the factory, therefore the contamination could


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be presumably only related to deposition of PCBs from air. The agricultural areas in the

87

contaminated site are located at about 1.5 km south west from the center of the city of Brescia

88

and adjacent to a river crossing north south (River Mella). More information on the site can be

89

found in a recently published paper 4.

90

Chemicals and reagents

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Fuming sulfuric acid (20% free SO3, ACS reagent) and dichloromethane (ACS reagent, ≥ 99.8%)

92

were obtained from Aldrich. HPLC solvents and reagents were of pesticide or LC-MS grade: water

93

(in house Milli-Q apparatus), acetonitrile, acetone, formic acid and ammonium acetate (Carlo

94

Erba Reagents). Standards of PCBs and OH-PCBs (4'-hydroxy-2,2',3,3',4,5,5'-heptachlorobiphenyl,

95

OH-PCB-172; 4-hydroxy-2,2',3,4',5,5’,6-heptachlorobiphenyl, OH-PCB-187 and

96

2,2',3,4',5,5’,6-heptachlorobiphenyl,

97

Laboratories (Guelph, ON, Canada). Analytical standards of 4-chlorobenzoic acid (99%), 2,4-

98

dichlorobenzoic acid (96%), 3,4-dichlorobenzoic acid (99%), 2,4,6-trichlorobenzoic acid (99.5%)

99

and decachlorobiphenyl (PCB 209) were purchased from Sigma Aldrich. Anhydrous pyridine [KF

100

(H2O) ≤ 50 ppm], anhydrous NaOH and MeOH (99.8%) were also purchased from Sigma Aldrich.

101

A technical PCB blend (Askarel, Monsanto) was kindly provided by Sea Marconi Technologies SaS,

102

Collegno (Torino, Italy).

103

Synthesis of sulfonated-PCBs and OH-nonachloro-PCBs

104

Sulfonation of the Askarel PCB mixture was performed according to a procedure adapted from

105

Plotnikova et al.34. Askarel was employed as a starting point for sulfonation to compare the

106

sulfonated reaction products to the original commercial mixture. A 10-mL thick-walled glass tube

13C

12-OH-PCB-187)

13C

12-4-hydroxy-

were purchased from Wellington



107

with a Teflon screw seal at the top was sequentially loaded with fuming sulfuric acid (3.3 g) and

108

Askarel (1.1 mL) and stirred for 10 min, until a homogeneous mixture was obtained. This solution

109

was slowly heated to 150 °C and kept at that temperature for 6 h. The resulting brown viscous

110

mixture was allowed to cool to room temperature and cautiously poured onto ice-cold water (10

111

g). In addition to an oil, a solid product also appeared, floating on the surface of the mixture. The

112

mixture was then extracted with dichloromethane (3 x 10 mL), the combined organic phases were

113

dried (MgSO4), filtered and concentrated in vacuum to leave a dull yellow residue. The residue

114

was then dissolved in 1 mL of tetrahydrofuran and further diluted with acetonitrile to obtain

115

concentrations suitable for LC-HRMS analysis. The main reaction products were dichloro-,

116

trichloro- and tetrachloro-benzenesulfonic acids and a mixture of sulfonated PCBs, containing

117

congeners whose chlorine distribution was similar to the one found in the original Askarel

118

mixture (Figures SI-1 and SI-2). The estimated reaction yield, in terms of sulfonated PCBs, was

119

about 6.3 % (see Table SI-1). The identification of the sulfonated-PCB congeners was based on

120

the determination of the exact masses, MS2 fragmentations and on the matching of experimental

121

and theoretical isotopic profiles. NMR confirmations of the chemical structures were not possible

122

at this time, because of difficulties in single isomer purification by HPLC, starting from the

123

complex reaction mixture and mainly because of the limited amounts of pure substances that

124

could be obtained. Further studies will be focused on the chemical synthesis and characterization

125

of pure isomers of sulfonated-PCBs, which will be used as reference standards for accurate

126

quantitation purposes.

127

OH-nonachloro-PCBs were obtained by aromatic nucleophilic substitution: decachlorobiphenyl

128

(15 mg) and anhydrous NaOH (3 mg) were dissolved in dry pyridine (3 mL) containing MeOH (20


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μL) in a 5-mL flame-dried thick-walled glass tube (teflon screw sealed) under N2. This solution

130

was kept at 105°C for 6 h resulting in a color change from colorless to pale orange, allowed to

131

cool to room temperature and evaporated in vacuum. The oily residue was diluted with

132

dichloromethane (5 mL) and washed with 20% citric acid solution (2 x 5 mL). The organic layer

133

was dried over MgSO4 and evaporated in vacuum to give a pale-yellow foam.

134 135

Extraction and analysis of PCBs

136

SIN sample. PCBs were analyzed according to EPA 1668 C 2010 method35 by a commercial

137

laboratory. In brief, the sample was extracted with an Accelerated Solvent Extractor (ASE)

138

(Thermo Scientific DIONEX ASE 350) and analyzed by high-resolution GC-MS. 79 congeners,

139

including PCB 209 were determined. For more details on selected congeners and analytical

140

method please see Text SI-1 and Table SI-2 (A).

141

Background samples. Sample extraction was performed on a modified Velp Scientifica (Italy,

142

mod. SER 148) solvent extractor Soxtec and analyzed with a single quadrupole GC-MS. 82

143

congeners, including PCB 209 were determined. For more details on selected congeners and

144

analytical method please see Text SI-1 and Table SI-2 (B).

145

Extraction and analysis of PCB metabolites

146

Soil samples were weighed (1 g) in 10 mL glass tubes, then 4 mL of a mixture of acetone:water

147

(95:5 %, v/v), containing 1% formic acid and 5 ng of OH-PCB-187-13C12 (internal standard) were

148

added. The tubes were vortexed and placed on an ultrasonic bath for 30 min. After

149

centrifugation, the supernatant solvent mixture was recovered, and the extraction procedure



150

was repeated using 4 mL of dichloromethane. The joined supernatants were then evaporated

151

with nitrogen and reconstituted with 200 µL of acetonitrile. The calculated extraction recovery

152

of the internal standard from soil samples was 75 ± 10 % (mean ± SD, n=6) and the limit of

153

detection was 0.01 ng/g. We assumed the same values for all other compounds for which no

154

reference standards were available. Analysis of the extracts was performed by HPLC-HRMS, using

155

a 1200 LC system (Agilent Technologies) coupled to an Orbitrap Q Exactive mass spectrometer

156

(Thermo Fisher), operating in ESI negative ion full scan MS (120-1200 u) at 70000 resolution and

157

in data dependent MS2, at 17500 resolution and 20-60 NCE collision energies. The ionization

158

conditions were optimized for acidic compounds and resulted in the formation of deprotonated

159

molecules [M - H]- for OH-PCBs and other PCB metabolites. With these instrumental conditions,

160

unmodified PCBs were not ionized and consequently not detectable. The chromatographic

161

separation was performed with an XBrigde C18 column, 100x2.1 mm ID, 3.5 µm phase (Waters

162

Corp.), using a gradient of ammonium acetate, 10 mM in water (A) and acetonitrile (B) (1% to

163

99% of B in 36 min, at a flow rate of 200 µL/min). Semi-quantitative analyses of OH-PCBs and

164

sulfonated-PCBs were performed by extracting high resolution ion chromatograms (XIC) from full

165

scan data, with a 5 ppm window. Reference standards of OH-PCB-187, OH-PCB-172 and 13C12-OH-

166

PCB-187 were used to construct external calibration curves. Calculations were done integrating

167

the sum of all the chromatographic peaks of equally chlorinated congeners of PCB metabolites,

168

assuming they had an instrumental response equal to that of the compounds in the reference

169

standard mixture. The data obtained must be considered as an approximation of an accurate

170

quantitation. However, this is currently the only viable method, since no reference standards of

171

sulfonated-PCBs and OH-sulfonated-PCBs are currently available, and it is not possible to


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evaluate instrumental responses of the different congeners. Soil samples were analyzed twice

173

for PCB metabolites and calculations of concentrations were done using the mean of the results

174

obtained. More details on the extraction and analytical procedures are given in Text SI-2. In Table

175

SI-3 the exact masses of the ions of the different metabolite classes and chlorination families are

176

reported.

177

Quality assurance/Quality control (QA/QC)

178

The quality of the whole analytical procedure for PCB analysis was assessed using recovery

179

standards, instrumental and method blanks and a certified reference material. Refer to Text SI-3

180

for more details about QA/QC. For the analysis of PCB metabolites, no reference materials were

181

available yet and the quality of the analytical procedures relied on the use of instrumental and

182

method blanks and on the availability of some reference and synthesized standards.

183

Estimation of the physico-chemical properties

184

Physico-chemical properties of PCB and their metabolites were estimated with EPISUITE 4.11

185

program36. More detail in Text SI-4. The estimates must be considered approximate although

186

representative of each chlorination class.

187 188

Results and discussion

189

Identification of OH-PCBs, sulfonated-PCBs and OH-sulfonated-PCBs

190

Twelve samples were analyzed in the contaminated and in the background areas, showing similar

191

contamination patterns (with the presence of sulfonated-, OH-sulfonated- and OH-PCBs), three

192

representative samples were selected to compare such patterns and describe the new



193

metabolites. Soil samples were initially analyzed to search for polar PCB metabolites, expecting

194

to find OH-PCBs and possibly other lower molecular weight compounds (e.g., chlorobenzoic

195

acids) resulting from partial microbial degradation that reasonably occurs in these soils with such

196

a long contamination history. For this reason, we set up an extraction method suitable for acidic

197

compounds, using a mixture of solvents less apolar than those normally used for the extraction

198

of unmodified PCBs and with the addition of a strong acid (1 % formic acid). The analytical

199

conditions, both for chromatography and for mass spectrometry were also optimized using the

200

available standards of OH-PCBs and chlorobenzoic acids. Chlorobenzoic acids were not found at

201

detectable levels, while we found significant amounts of OH-PCBs, starting from trichloro- up to

202

nonachloro- congeners, with complex chromatographic profiles, due to the high number of

203

possible isomers (Figures SI-3 to SI-7). The peaks characterized by larger abundances were those

204

of hexachloro- and pentachloro- congeners, and we also found an unusually high concentration

205

of nonachloro- congeners. These findings are in accordance with the data obtained from the

206

analysis of unmodified PCBs and reflect the contamination of the soil by the industrial production

207

of PCB mixtures and decachloro-PCB. The identification of OH-PCBs, other than the ones

208

contained in the reference standard, was based on their exact mass and isotopic abundances.

209

With our instrumental conditions all the congeners did not show significant fragmentation in

210

their MS2 spectra. For the three isomers of nonachloro-hydroxy-PCBs, we also confirmed their

211

identification by retention time matching with an ad hoc synthesized standard. Since the mass

212

spectrometric detection was performed with a high resolution Orbitrap instrument and was

213

based on full scan MS and MS2 acquisitions, we found, by manually reviewing the data, the

214

presence of several other peaks of chlorinated compounds, which had different masses, but


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chromatographic profiles and mass spectra similar to those of the OH-PCBs. After investigations

216

based on the exact masses, isotopic abundances and fragmentations found on the MS2 spectra,

217

we identified two classes of sulfur containing PCB congeners, one consisting of the generic

218

molecular formula C12H(10-x)ClxO3S and one consisting of the generic molecular formula C12H(10-

219

x)ClxO4S.

220

The mass spectrometric properties of the compounds of the first class were consistent with the

221

structures of sulfonated-PCBs and contained tetrachloro- to nonachloro- congeners, with a

222

higher presence of hexachloro-congeners. The MS2 spectra showed fragment ions corresponding

223

to [SO3]-, [SO3Cl]-, [M]- - SO2 and [M]- - SO2 - HCl (Figure 1 and Figures SI-8 to SI-13) and the

224

experimental isotopic abundances of the deprotonated molecules matched the ones calculated

225

from the proposed molecular formulas. These data are consistent with those reported by Liu et

226

al. for sulfonated-PCBs found in the serum of polar bears and mice31.



227 228 229 230

Figure 1 - Negative ion Extracted Ion Chromatograms of hexachloro congeners of sulfonatedPCBs from a contaminated soil sample (SIN) [A] and from a sulfonation reaction of a technical PCB mixture [B]. ESI-MS [C] and ESI-MS2 [D] spectra of the congeners were identical in the two


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231 232 233 234


samples. ESI-MS and ESI-MS2 spectra were acquired at 70000 and 17500 resolutions, respectively, (Orbitrap Q Exactive) and chromatograms were extracted with a 5 ppm window. Experimental ESI-MS spectra are shown in comparison with the theoretical calculated isotopic profile (error=0.91 ppm for m/z=438.7914).

235 236

The identity of these compounds was finally confirmed by the analysis, in the same conditions,

237

of the reaction mixture obtained by the sulfonation reaction of the industrial Askarel PCB mixture

238

(see Materials and methods section). The chromatographic profiles of the synthesized

239

sulfonated-PCBs were similar (although with differences in the relative abundances of the large

240

number of possible isomers) and the isotopic abundances and MS2 spectra were identical to

241

those of the compounds found in soil samples (Figure 1 and SI-8 to SI-13). The congeners of the

242

second class can be assigned either to the structures of OH-sulfonated-PCBs or sulfated-PCBs (or

243

a mixture of both). The MS2 spectra showed main fragment ions corresponding to [SO3]-, [SO3Cl]-,

244

[M]- - HCl, [M]- - SO3, [M] - - SO3 - HCl and [M] - - SO3 - 2HCl - C2O. This fragmentation pattern was

245

also found by Liu et al.31 for PCB-sulfates and for other PCB metabolites; however, they did not

246

find OH-sulfonated-PCBs (Figure SI-14 to SI-16). A detailed discussion about MS2 fragmentation

247

of PCB metabolites can be found in the cited article, however, with this method and with our

248

instrumentation and analytical conditions, it was not possible to clearly distinguish between the

249

two proposed structures. Additional observations showed that the chromatographic profiles of

250

these substances had the same complexity of the sulfonated-PCBs, and contained trichloro- to

251

octachloro- congeners, with a higher presence of pentachloro- congeners (Figure 2 and SI-14 to

252

SI-16). This indicates that their origin involves the loss of a further chlorine atom, with respect to

253

sulfonated-PCBs, which is more in accordance with structures including both a hydroxyl and a



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254

sulfonate group, rather than a single sulfate group. In the absence and given the difficulty to

255

obtain suitable OH-sulfonated-PCBs reference standards, we then tried a different approach to

256

distinguish between the two possible structures, based on the report by Grimm et al. 22 , who

257

found that a sulfate metabolite of PCB 11 could be rapidly hydrolyzed to the corresponding OH-

258

PCB in the presence of sulfuric acid. Using the hydrolysis conditions there reported to treat soil

259

extracts (33 % sulfuric acid, for 6 h, at 25 °C) and other conditions (1 N HCl for 1 h, at 80 °C) we

260

did not find significant alterations of the chromatographic profiles of PCB metabolites. This result

261

suggests that the compounds found were, at least at a very large extent, OH-sulfonated-PCBs

262

because, if they were sulfated-PCBs, they would have disappeared in these conditions, with a

263

corresponding increase of OH-PCBs. Sulfonated-PCBs remained unaffected, as confirmed by

264

experiments using the synthesized standards. A scheme of “confidence levels” for qualifying the

265

results obtained with a high resolution mass spectrometer was recently proposed

266

classification goes from level 5, where just the exact mass is measured to level 1 where the

267

structure is confirmed by a reference standard. Looking at our results the confidence level (CL)

268

for sulfonated-PCBs could be set as “level 1b” (although not defined in the cited paper, we can

269

define it as “confirmed by the synthesis of a mixture of isomers”) while the CL for OH-sulfonated-

270

PCBs could be set as level 3 (“tentative candidates”). However, a final confirmation of the

271

identification of OH-sulfonated-PCBs would require further studies and the use of original

272

standards. Figure 4 shows the general structures of the three chemical classes measured.


37.

Their

Page 15 of 34


Clm-1

Clm-1

Clm

SO3H

OH Cln

Cln

Cln

Clm-2

Clm-1

Cln

Cln-1

and/or

SO3H

273 274 275

HO SO3H

HO

Figure 2 - General structures of the PCB metabolite classes measured. From left to right, top to bottom: PCBs, OH-PCBS, sulfonated-PCBs, OH-sulfonated-PCBs. Please note that m + n: 1 → 10.

276 277

Other hydroxylated, dihydroxylated and/or sulfur containing metabolites of PCBs (including

278

methylsulfone-PCBs) were not found in soil samples. It must be also remarked that none of the

279

PCB metabolites found in soil samples were found in blank samples and in the industrial PCB

280

mixture and standard (Askarel and PCB-209). This contrasts with what was reported19 for OH-

281

PCBs, which were also found in standard Aroclor mixtures.

282

Concentrations of native PCBs and metabolites in SIN and background samples

283

PCBs. Total PCB concentration in SIN sample was around 12,000 ng g-1 dw. Soil from the

284

contaminated site shows a typical highly chlorinated PCB fingerprint, possibly deriving by highly

285

chlorinated mixtures and/or weathering of the initial mixture4. Figure 3a shows the total

286

concentrations for each chlorination class while Figure 4a shows the relative percent fingerprint.

287

The most abundant congeners were penta- and hexa-PCB representing about the 25% each of



288

the sum of PCBs. This is also true considering the number of congeners found among penta- and

289

hexa-PCBs (Table SI-4). Tetra- and hepta-PCBs represented about 15% each, di-, tri- and octa-

290

PCBs less than 10%, while mono- and nona-PCBs less than 1% of the sum of PCBs. PCB 209 (a

291

peculiar product of the Caffaro plant) represented about 12% of the sum of PCBs. Background

292

samples collected at about 2 km distance from the SIN showed lower concentrations, of about

293

250 ng g-1 dw. They presented a high chlorinated PCB fingerprint like the SIN sample. The most

294

abundant congeners in the SW and NE samples were penta- (13% and 23% respectively), hexa-

295

(34% and 28% respectively), hepta- (23%-14% respectively) and deca-PCBs (21%-27%

296

respectively); tetra-, octa- and nona-PCBs represented less than 5%, while tri-PCBs less than 1%.

297

Mono- and di-PCBs were below the LOQ (see SI for more details).

298

Sulfonated-PCBs. Total sulfonated-PCBs were about 50 ng g-1 dw, 2 ng g-1 dw and 1 ng g-1 dw in

299

the SIN, 2 km-SW and 2 km-NE samples respectively (Figure 3b). It must be noted that the total

300

sulfonated-PCB concentration in the SIN sample is close to the legal threshold for residential

301

areas for PCBs in Italy (60 ng g-1 dw). The fingerprint of SIN sample is dominated by hexa-, penta-

302

and hepta-chloro congeners of sulfonated-PCBs representing the 26%, 12%, 7% respectively of

303

the sum of sulfonated-PCBs (Figures 3b and 4b). Similarly, these congeners predominated in the

304

background samples: -hexa- represented 71% (2 km-SW) and 65% (2 km-NE) of the sum of the

305

sulfonated congeners, while penta- are about 8% (2 km-SW) and 16% (2 km-NE) and hepta- about

306

19% (2 km-SW) and 17% (2 km-NE) (Figures 2b and 3b).

307

OH-sulfonated-PCBs. Total OH-sulfonated-PCBs were about 30 ng g-1 dw, 3 ng g-1 dw and 2 ng g-

308

1

dw in the SIN, 2 km-SW and 2 km-NE samples, respectively (Figure 3c). The fingerprint of SIN


Page 16 of 34

Page 17 of 34


309

sample is dominated by penta- (about 50% of the sum of the congeners) followed by tetra-and

310

hexa- (about 25% each one) (Figures 3c and 4c). Background samples showed a similar pattern

311

with penta- representing about 60%, hexa- about the 30% and tetra- about 10% in each sample

312

(Figures 3c and 4c).

313

OH-PCBs. Total OH-PCBs were about 30 ng g-1 dw, 0.7 ng g-1 dw and 0.3 ng g-1 dw in the SIN, 2

314

km-SW and 2 km-NE samples, respectively (Figure 3d). The most abundant congeners in SIN

315

sample were penta- and hexa- representing each one about 25% of the sum of congeners; other

316

important congeners are tetra-, hepta- and nona- representing each one about 15% of the sum

317

of the congeners (Figure 4d). Background samples showed both high levels of penta- and hexa-

318

(about 15% in 2 km-SW and about 60% in 2 km-NE for penta- and about 30% in both samples for

319

hexa-); however, while in 2 km-SW nona- represented about 50% of the congener sum, in 2 km-

320

NE sample was about 10%.

321

Total PCB metabolite concentrations were much lower (although not negligible) than the

322

concentrations of native PCBs, representing between the 0.1% and 2.2% of native PCB

323

concentrations; in general PCBs > sulfonated-PCBs > OH-sulfonated PCBs > OH-PCBs in SIN

324

sample, while PCBs > OH-sulfonated-PCBs > sulfonated PCBs > OH-PCBs in background samples.

325

SIN and background samples showed similar fingerprint (Figure 3) for PCBs, sulfonated-PCBs and

326

OH-sulfonated-PCBs, but they differed for OH-PCBs. More specifically, while penta- and hexa-

327

dominated the fingerprint of SIN sample and in the 2 km-NE sample, nona- and hexa- were the

328

most abundant congeners in 2 km-SW sample. This may indicate a contribution from the native

329

PCB mixture (containing also OH-PCB) arrived via irrigation water in the SIN and/or the detection



330

of low chlorinated OH-PCBs in the SIN sample due only to the higher contamination and higher

331

detection limits in the SIN. The appearance of high OH-nonachloro-PCBs in the 2 km SW sample

332

could indicate an additional loading (through contaminated irrigation water) of the decachloro

333

PCB with the consequent increased level of the OH-derivatives. The choice of analyzing

334

background samples was made because no originally Caffaro produced PCB mixtures were

335

available to be analyzed, in order to show that the sulfonated and hydroxy-sulfonated PCB

336

metabolites were not a by-product of PCB or other chemical production, incidentally arriving at

337

the agricultural areas. Proving that sulfonated and hydroxy-sulfonated PCB metabolite

338

fingerprints were comparable would therefore represent an indication of new formation as

339

degradation products of the parent PCBs. When looking at the fingerprints of sulfonated-PCB and

340

OH-sulfonated-PCB metabolites (Figure 3b and 3c), they appear very much comparable in all

341

samples and with ratios comparable to the respective PCBs. This would indicate an in-situ

342

formation of these metabolites rather than their transport and deposition from the source. Being

343

the metabolites practically involatile (see the section “Implications for risk assessment”)

344

especially due to the low pKas, one could expect that in case of aerial transport, the most volatile

345

congeners would predominate the fingerprint of the most distant samples. However, since these

346

data should be considered as semi-quantitative, in the absence of reference standards for the

347

sulfur containing PCB metabolites, more quantitative relationships among families and samples

348

cannot be currently performed.

349


Page 18 of 34

Page 19 of 34


SIN sample

Background sample – 2 km SW

2500 1,751

2000

1,493

1,331

1500 1000

727

487

705 121

59

80

60 35

40 20

3

4

5

6

7

8

9

10

1

2

3

4

25 20

11.52

15

6.42

10 5

0.01 1.28

0 2

3

4

0.41 0.12 5

6

7

8

PCB+SO3 soil conc. (ng g-1 dw)

ng g-1 dw

PCB+ SO3 soil conc. (ng g-1 dw)

(B) sulfonated-PCBs

30

OH-PCB+SO3 soil conc. (ng g-1 dw)

0.88

0.79 4

5

9 8 7 6 5 4 3 2 1 0

6

7

0.08 8

1.02 3

4.04

3.27

3.95

0.60 4

5

6

40

40 14

20

0.4

10

1

2

3

4

0.31

0.5

0.14 3

4

5

* *9

0.03 0.001

0.0 2

6

7

8

9

5

6

7

7

3

8

9

10

n° of chlorines

1.0

7

8

2.5 2.0 1.5

0.54

1.0 0.14

0.5

0.14

* *9

0.01 0.001

0.0 1

2

3

4

5

6

7

8

n° of chlorines

2.5 1.55

2.0 1.5

0.80

1.0 0.5

0.22

0.03

0.0 1

2

3

4

5

6

7

8

n° of chlorines

0.4

6.89 7.10

2

9

1.5

1

OH-PCB soil conc. (ng g-1 dw)

dw ng


6.42

3

8

2.5

7.37

2

7

75

60

n° of chlorines

14.68

1

6

1.17

2.0

9

n° of chlorines


g-1

18 16 14 12 10 8 6 4 2 0

5

2.5

n° of chlorines

(C) OH-sulfonatedPCBs ng g-1 dw

(D) OH-PCBs

25.82

35

76 65

80

n° of chlorines

PCB+SO3 soil conc. (ng g-1 dw)

2

n° of chlorines

350 351 352

4

100

0

n° of chlorines

1

12

10

0

1

1

54

60


0

89

100

0.21

0.32

0.3 0.11 0.2 0.1

0.04

0.0 1

2

3

4

5

6

n° of chlorines

7

0.01 8

9

2.0 1.04

1.5 1.0

0.53

0.5

0.21

0.01

0.0 1

2

3

4

5

6

7

8

n° of chlorines 0.4


500

120

PCB soil conc. (ng g-1 dw)

2,743 2,784

3000

Background sample – 2 km NE

120


ng g-1 dw

(A) PCBs


3500

0.15

0.3 0.2

0.08

0.1 0.02

0.01 0.0 1

2

3

4

5

6

7

8

9

n° of chlorines

Figure 3 – Concentrations of PCBs (A) and their metabolites (B, C, D) in SIN and background samples. Error bars represent analytical variability. * indicates ACS Paragon Plus Environment < LOD

Environmental Science & Technology MONO

DI

TRI

TETRA

PENTA

HEXA

HEPTA

Page 20 of 34

OCTA

NONA

DECA

100%

353

90%

354

70%

a

PCB fingerprint (%)

355

80% 60% 50% 40% 30% 20%

356

10% 0%

357

TRI

SIN TETRA

PENTA

2 KM - SW HEXA HEPTA

OCTA

2 KM - NE NONA

358 359

b

360 361

Sulfonated- PCB fingerprint (%)

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% SIN TRI TETRA

362

366

OH-sulfonated PCB fingerprint (%)

c

365

80% 70% 60% 50% 40% 30% 20% 10% 0%

367

371 372

SIN TETRA

PENTA

2 KM - NW HEXA HEPTA

OCTA

2 KM - SW NONA

100%

d

OH-PCB fingerprint (%)

90%

368

370

2 KM - NE OCTA

90%

TRI

369

2 KM - SW HEXA HEPTA

100%

363 364

PENTA

80% 70% 60% 50% 40% 30% 20% 10% 0% SIN

2 KM - SW

2 KM - NE

Figure 4- Fingerprint of PCBs (a), sulfonated-PCBs (b), OH-sulfonated-PCB (c) and OH-PCBs (d) ACS Paragon Plus Environment

Page 21 of 34


373

Evidence for sulfonation reactions in the environment

374

To our knowledge, there have been no publications about OH-sulfonated-PCBs levels in any sample

375

and sulfonated-PCBs in soil. Recently sulfonated -PCBs were found in polar bear blood31. However,

376

some research on sulfonated metabolites of different organic chemicals can be found in the

377

literature. For example, many sulfonated compounds were found in organic matter in soil

378

different sulfonated organic chemicals were identified in different organisms (actinomycetes, rats,

379

marine sponge, plants) and environmental matrices (sediments, soil, surface water and ground

380

water)39–50. Additionally, many enzymatic systems able to catalyze the formation of carbon-sulfur

381

bonds in microorganisms, plants and animals were identified.51 For example, the enzyme

382

glutathione S-transferase (GST) is responsible for glutathione (a small peptide containing sulfur)

383

conjugation with organic chemicals during detoxification processes in plants, microorganisms and

384

fungi, as well as in animals52–55. GST was shown to be also involved in PCB dechlorination in aerobic

385

conditions 56–58. While hydroxylation of PCBs is a common starting step in the microbial aerobic PCB

386

mineralization pathway encoded by the bph operon59, to our knowledge sulfonated PCB metabolites

387

were never described in microbial degrading strains. This might be due to their low concentrations

388

and to the difficulty in the direct detection of polar (such as sulfonated) compounds with traditional

389

extraction and analytical techniques (i.e., direct analysis of sulfonated-PCB by GC-MS is not

390

possible). While it was reported that glutathione conjugation is a potentially important

391

detoxification pathway carried out by soil microorganisms, plants and animals 31,50, we can deduce

392

that the microbial aerobic metabolism evolved mainly to mineralize PCB and use them as energy

393

and carbon source. Sulfonation in this case will act as a mobility enhancer, to improve

394

bioaccessibility of the chemicals. However, the confirmation of GST as actor in the formation of

395

sulfonated- and OH-sulfonated-PCBs even with highly chlorinated congeners (as would appear from

396

our data), would mean that aerobic biodegradation of such chemicals is feasible, although it is not

397

currently possible to evaluate the time required by this process. Liu et al. 31 have hypothesized that


38

and


Page 22 of 34

398

in polar bear after an initial oxidation of PCBs by Cytochrome P450, GST and other enzymatic

399

systems may be involved in the sulfur addition, followed by some oxidative processes leading to

400

sulfonates. Although it is not among the scopes of this study to report the precise metabolic

401

pathway to the sulfonated PCBs, one can conclude that admitting the relevance of these oxidative

402

processes substantially breaks the paradigm that degradation of highly chlorinated PCBs with more

403

of 4-5 chlorines would be possible only in anaerobic conditions60. Additionally, some relationships

404

among sulfonated-, OH-sulfonated and OH-PCBs are apparent (e.g. the most abundant class is

405

hexachlorinated for sulfonated-PCBs and pentachlorinated for OH-sulfonated-PCBs); however, it is

406

not currently known which compound is produced first and in which direction the degradation

407

proceeds (from sulfonated- to OH-sulfonated- to OH-PCBs or vice versa or a combination of the

408

two). Liu et al. 31 reported that the sulfonated-PCBs they found in polar bear blood belonged to tri

409

to hexachlorinated classes. Here we report for the first time also hepta, octa- and nonachlorinated

410

sulfonated-PCBs. Further research is therefore needed in order to explore the array and the

411

sequence of metabolites produced by microbial degradation of PCBs and to elucidate the metabolic

412

and chemical intermediates involved in the following degradation steps.

413

Implications for risk assessment

414

Sulfonated-PCBs are recently discovered31 metabolites and OH-sulfonated-PCBs are first described

415

in this article as novel chemicals; for them no CAS number nor description of their physicochemical

416

properties is available. Although information on Kow and pKa is available for OH-PCBs20,61, in order

417

to compare the effect of the addition of one or more polar groups (-SO3H; -SO3H and -OH; -OH) to

418

selected PCB families on some physico-chemical properties, Episuite version 4.1136 was used to

419

estimate approximate Log Kow, vapor pressure, water solubility, Henry’s Law constant and Log Koa

420

(Table SI-5) of the undissociated compounds. The addition of a -SO3H group to a PCB considerably

421

lowers the Kow and vapor pressure of the chemical, while increases the water solubility and Koa.


Page 23 of 34


422

For example, the Log Kow for PCB 209 (the decachloro substituted) drops from 10.2 to 6.4 for the

423

corresponding sulfonated nonachloro-PCB and 5.27 for the sulfonated and hydroxylated octachloro-

424

PCB, while for the hydroxylated only nonachloro-PCB the drop is limited to 9.08. Additionally, other

425

properties will change in a relevant way: vapor pressure will decrease of 5 to 6 orders of magnitude

426

for the corresponding sulfonated or OH-sulfonated PCBs; water solubility will increase of several

427

orders of magnitude; Henry’s Law constant will also drop considerably. The changes are even more

428

important for the less chlorinated congeners, significantly modifying their properties. These

429

modifications will considerably reduce the already scarce mobility of the metabolites towards the

430

air compartment and dramatically increase the mobility in the water phase. For example, sulfonated

431

octa-PCBs look like tri-PCBs in terms of hydrophobicity (Log Kow), while they show a vapor pressure

432

of 4 order of magnitude lower than that of deca-PCB and a water solubility similar to that of mono-

433

/di-PCBs. OH-sulfonated-octa-PCBs look like di-PCBs in terms of hydrophobicity (Log Kow), while

434

they show a vapor pressure 6 order of magnitude lower than that of deca-PCB and a water solubility

435

similar to that of mono-PCBs. Finally, the octa-hydroxy-PCB look like hepta-PCBs in terms of

436

hydrophobicity (Log Kow), while they show a vapor pressure similar to that of deca-PCB and a water

437

solubility similar to that of hexa-PCBs. The calculation of the pKa of sulfonated- and OH-

438

sulfonated_PCB shows that these chemicals are practically totally dissociated at environmental pHs.

439

This confirms the negligible volatility of these sulfonated metabolites and the potential enhanced

440

mobility in water. This, on one hand, could change the potential (from native PCBs) for leaching

441

towards groundwater or runoff to surface water (especially for the less chlorinated compounds).

442

The relative low amount in soil of sulfonated and OH-sulfonated PCBs may be a proof of their

443

mobility in soil water and their relocation to deeper layers. It could also be an indication of enhanced

444

degradation because of the increase of their bioavailability and bioaccessibility62 and therefore of

445

the potential microbial degradability. When looking at persistence in soil and in water, sulfonation



Page 24 of 34

446

is often considered a way to make aromatic chemicals more recalcitrant63 to biodegradation. In this

447

case, given the relatively long half-life of the parent compounds, sulfonation may not additionally

448

increase their persistence, given the added contribution in terms of bioavailability. Regarding

449

toxicological and ecotoxicological properties of sulfonated and OH-sulfonated-PCBs again no

450

information is available, although it was reported31 that that sulfonated-PCBs were among the

451

unknown chemicals in polar bear plasma suspected to be responsible for immune-suppression,

452

endocrine and thyroid hormone binding disruption. For relatively similar compounds such as

453

sulfated-PCBs and OH-PCB, recent publications show a considerable attention to some undesired

454

effects such as the formation of DNA adducts, interference with hormonal signaling, transport and

455

production21,64,65. Therefore, the significance and potential effects of the sulfonated and OH-

456

sulfonated-PCBs would require further and thorough investigation.

457 458

Supporting Information

459 460

Additional information regarding analytical methods, physico-chemical properties and metabolite detected. can be found in SI, available at….

461 462

Conflict of interest

463

The authors declare no competing financial interest

464 465

Acknowledgements

466

The authors would like to acknowledge the collaborators of the “Caffaro Working Group” and the

467

funding agency Regione Lombardia by means of Ente Regionale per i Servizi all'Agricoltura e alle

468

Foreste (ERSAF), Decreto ERSAF n. III/5426 del 09.12.2013. Sea Marconi Technologies SaS,

469

Collegno (Torino, Italy), is kindly acknowledged for providing a sample of Askarel technical PCB


Page 25 of 34


470

mixture. The Department of Science and High Technology of the University of Insubria is

471

acknowledged for funding part of the salary of Elisa Terzaghi.

472

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473

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