Dispersive liquid–liquid microextraction followed by microwave-assisted silylation and gas chromatography-mass spectrometry analysis for simultaneous trace quantification of bisphenol A and 13 ultraviolet filters in wastewaters

Accepted Manuscript Title: Dispersive liquid-liquid microextraction followed by microwave-assisted silylation and GC-MS analysis for simultaneous trace quantification of bisphenol A and 13 UV Filters in wastewaters Author: S.C. Cunha A. Pena J.O. Fernandes PII: DOI: Reference:

S0021-9673(15)01087-0 http://dx.doi.org/doi:10.1016/j.chroma.2015.07.099 CHROMA 356726

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

2-6-2015 23-7-2015 24-7-2015

Please cite this article as: S.C. Cunha, A. Pena, J.O. Fernandes, Dispersive liquid-liquid microextraction followed by microwave-assisted silylation and GC-MS analysis for simultaneous trace quantification of bisphenol A and 13 UV Filters in wastewaters, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.07.099 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Highlights (for review)

Novel DLLME procedure for extraction of multi-residues (BPA and 13 UV-filters) Fast and reliable microwave-assisted silylation Quantification of 14 analytes in 15 wastewater treatment plants effluent and influent

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Eighth analytes were positively identified in most of the samples of 15 WWTPs

Page 1 of 36

*Manuscript Click here to view linked References

1

Dispersive liquid-liquid microextraction followed by microwave-assisted

2

silylation and GC-MS analysis for simultaneous trace quantification of

3

bisphenol A and 13 UV Filters in wastewaters

4

S.C. Cunha1*, A. Pena2, J.O. Fernandes1 1

LAQV-REQUIMTE, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy,

6 7

University of Porto, Rua Jorge de Viterbo Ferreira 228 4050-313 Porto, Portugal 2

LAQV-REQUIMTE, Group of Bromatology, Pharmacognosy and Analytical Sciences, Faculty of Pharmacy, University of Coimbra, Polo III, Azinhaga de Stª Comba, 3000-

9

548 Coimbra, Portugal

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*Correspondence: Sara Cunha; Laboratório de Bromatologia e Hidrologia, Faculdade

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de Farmácia, Universidade do Porto, Rua Jorge de Viterbo Ferreira 228, 4050-313

13

Porto, Portugal

14

Phone: +351 220428639

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Fax: +351 226093390

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Email: [email protected]

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Abstract

19

A novel multi-residue gas chromatography-mass spectrometry (GC-MS) method was

20

validated for the simultaneous determination of trace levels (ng/L) of 13 UV-filters and

21

bisphenol A (BPA) in wastewater samples. It was based on dispersive liquid-liquid

22

microextraction (DLMME) followed by rapid microwave-assisted silylation of the

23

analytes. Several parameters of both extraction and derivatization steps such as type

24

of extractive and dispersive solvents, solvent volumes, pH, salt addition, time and

25

power of microwave were evaluated to achieve the highest yield and to attain the

26

lowest detection limits. Optimized DLLME consisted in the formation of a cloudy

27

solution promoted by the fast addition to the sample (10 ml) of a mixture of

28

tetrachloroethylene (50 µL, extraction solvent) in acetone (1 mL, dispersive solvent).

29

The sedimented phase obtained was evaporated and further silylated under the

30

irradiation of 600 W microwave for 5 min, being the derivatization yields similar to those

31

obtained after a conventional heating process for 30 min at 75°C. Limits of detection

32

and quantification of the method using real samples were 2 ng/L and 10 ng/L,

33

respectively. Mean extraction efficiency of 82% for three concentrations were achieved,

34

supporting the accuracy of the method. Intraday and interday repeatability of

35

measurements (expressed as relative standard deviation) were lower than 22%. The

36

method was successfully applied to the determination of UV-filters and BPA in samples

37

collected from 15 wastewater treatment plants (WWTPs) in Portugal. Eight analytes

38

were detected, among which 2-Hydroxy-4-methoxybenzophenone, 2-ethylhexyl-4-

39

(dimethylamino)benzoate, and octocrylene as well as , and BPA were consistently

40

found in the three seasons of collection.

41

Key Words: Bisphenol A, UV-filters, DLLME, Contaminants, endocrine-disruptors,

42

Wastewater

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Page 3 of 36

Introduction

46

In recent decades, a tremendous increase in chemicals production has taken place as

47

a result of growing population pressure and inherently increasing demand of consumer

48

goods. The global chemical output has grown from US$171 billion in 1970 to US$4.12

49

trillion in 2010, and the predictions are that these figures will increase around 3% per

50

year until 2050 (OECD, 2012). After usage most of synthetic chemicals finally end up in

51

the different environmental compartments, where they can remain active for long

52

periods of time, being a source of environmental stress, yielding risks for living

53

organisms, humans included, at all stages of their life cycle.

54

A group of chemicals which have gained increasing interest in recent years in the field

55

of environmental research are the so-called emerging contaminants, a structurally

56

diverse and heterogeneous group of chemicals not covered by current legislation,

57

which are believed to pose a threat to environmental ecosystems (Farré et al., 2008).

58

Among others, pharmaceuticals (e.g. hormones, pain relievers, antibiotics), personal

59

care products (e.g. antiseptics, UV-filters/sunscreen components of cosmetics),

60

plasticizers such as phthalates and bisphenol A (BPA), and brominated compounds,

61

are included in this group.

62

UV-filters can be components of industrial products, such as house paints, plastics, or

63

textiles to prevent photodegradation of polymers and pigments (Gago-Ferrero et al.,

64

2012), but they are specially used in sunscreens and other personal care products to

65

absorb, scatter or reflect UV radiations. In EU there are currently authorized 26 UV-

66

filters, 25 of which are organic chemical compounds, being only one, TiO2, an

67

inorganic UV-filter (EC No 1223/2009), while in USA there are only 17 approved UV-

68

filters, of which 15 are of organic nature (FDA, 1999). Approved compounds comprise

69

various chemical classes, being the most common para-amino-benzoates, cinnamates,

70

salycilates

71

benzimidazoles (Shaath, 2010; Santos et al., 2012). The hydrophobicity of many of

72

these analytes indicates their potential for bioaccumulation in aquatic environmental

73

compartments (Díaz-Cruz et al., 2008). Therefore, UV-filters released to the

74

environment directly via wash-off from the skin or industrial discharges, or indirectly via

75

waste water domestic discharges or releasing by sewage treatment plants, have been

76

detected in surface waters, sediment and biota (Balmer et al., 2005; Remberger et al.,

77

2011; Zhang et al., 2011), drinking water (Rodil et al., 2012), seawater (Sánchez

78

Rodríguez et al. 2015), sludge and effluent water from waste treatment plant (Balmer et

79

al., 2005; Zhang et al., 2011) and even in breast milk (Schlumpf et al. 2010) and

80

human urine (Kunisue et al. 2012). The continuous inputs into the environment may

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45

dibenzoylmethanes,

camphor

derivatives,

and

Ac

benzophenones,

3

Page 4 of 36

lead to toxic effects in human and wildlife, mainly because most of these compounds

82

are considered as potential endocrine disruptors.

83

Other emerging contaminant that have been detected in landfill leachates, wastewater,

84

ground water, and surface water is BPA, a synthetic compound commonly employed

85

as a key monomer in the manufacturing of polycarbonate plastic and epoxy resins,

86

used in thermal paper, baby bottles, or as coating on the inside of many food and

87

beverage metallic cans (Fromme et al., 2002; Cunha et al., 2011a; Song et al., 2014).

88

BPA have potential toxicity towards humans and animals, and is suspected to possess

89

carcinogenic and endocrine disrupting activity even at low concentration (Swedenborg

90

et al. 2009).

91

When studying the occurrence, behavior and fate of environmental emerging

92

contaminants, multiresidue methods are understandably preferred taking into account

93

the huge amount of compounds of interest and the broad coverage they could provide.

94

Multiresidue methods for UV filter determination in water are usually based on LC-

95

MS/MS (Diaz-Cruz et al. 2008; Rodil et al. 2009a; Zhang et al. 2011; Magi et al. 2012;

96

Rodil et al. 2012; Gago-Ferrero et al. 2013a; Capriotti et al. 2014), GC-MS (Balmer et

97

al. 2005; Jeon et al. 2006; Cuderman and Heath, 2007; Zenker et al. 2008; Negreira et

98

al. 2010; Diaz-Cruz et al. 2012; Zhang and Lee 2012; Pintado-Herrera et al. 2013;

99

Benedé et al. 2014), or GC-MS/MS (Liu et al. 2011; Ho and Ding 2012; Silva et al.

100

2015), depending the choice of the physico-chemical properties of the compounds to

101

be analyzed and, in many situations, of the equipment availability.

102

Often, the main drawback of GC-MS methods lies on the need to an additional time-

103

consuming derivatization step, although essential to improve volatility, thermal stability,

104

and other desirable chromatographic features of the target analytes. This is the case

105

for some of the compounds here studied, namely BPA, benzophenone and salicylic

106

acid derivatives, which have phenolic hydroxyls in their structure, so requiring the

107

transformation in less polar derivatives, typically by a silylation reaction, in order to

108

enhance their chromatographic behaviourbehavior. Most of the described derivatization

109

procedures for the GC-MS analysis of the above mentioned UV-filters are time-

110

consuming, typically, 30-60 min at a temperature between 60 and 90°C (Zhang and

111

Lee, 2012). One way to overcome this inconvenience consists of using microwave

112

assisted derivatization procedures, which have been proven to allow better outcomes.

113

When compared to conventional derivatization, protocols involving conductive heating

114

have demonstrated to be able to decrease the time required for derivatization to a few

115

minutes, and can thus very effectively shorten the overall analysis time (Söderholm et

116

al 2010).

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4

Page 5 of 36

Before chromatographic analysis a well-designed sample preparation step is required,

118

being of utmost importance to achieve enriched extracts, due the very low levels (ng.L-

119

1 range) usually found. Besides the use of well-established extraction techniques such

120

as liquid-liquid extraction (LLE) and solid phase extraction (SPE) (Balmer et al. 2005;

121

Rodil et al. 2009a; Zhang et al. 2011; Gago-Ferrero et al. 2013b), other more novel

122

techniques have been also applied like solid phase micro-extraction (SPME) (Liu et al.

123

2010), stir bar sorptive extraction (SBSE) (Almeida et al. 2013), single drop micro-

124

extraction (SDME), and membrane assisted liquid-liquid extraction (MALLE) (Rodil et

125

al. 2009b). Compared with the common procedures, these novel techniques can

126

provide less consumption of organic solvents and higher yield enrichment. Dispersive

127

liquid-liquid micro-extraction (DLLME) is another recently developed extraction

128

technique presenting unique features in what concerning simplicity of operation,

129

amount of organic solvent extractor (only a few microliters), quickness, and high

130

enrichment factor (Cunha et al. 2011b). Overall, DLLME is a very suitable technique for

131

the extraction/enrichment of compounds with some hydrophobicity prior to their

132

determination by GC. DLLME was firstly used in UV filters analysis by Tarazona et al.

133

2010 for the determination of hydroxylated benzophenones in seawater by GC-MS

134

after derivatization with BSTFA, and by Negreira et al. 2010 who have quantified 8 UV

135

filters belonging to different classes in environmental water samples by GC-MS without

136

derivatization.

137

respectively, as extractant/dispersive solvents. The first proposal was later used by the

138

same group for the determination of 8 UV filters in both soluble and particulate fractions

139

of seawaters (Benedé et al. 2014). Meanwhile different DLLME approaches have been

140

also proposed, namely magnetic stirring-assisted DLLME, a technique using a special

141

designed flask in which the dispersive solvent is replaced by magnetic agitation (Zhang

142

et al. 2011), vortex-assisted DLLME, wherein the dispersive solvent is replaced by

143

vigorous vortex agitation (Zhang et al. 2012) and ultrasound-assisted DLLME with

144

simultaneous derivatization (Wu et al. 2013).

145

The work presented here deals with the development of a simple, fast and reliable GC-

146

MS method that enables the simultaneous measurement of BPA and 13 of the most

147

prevalent UV-filters pertaining to different chemical classes (Table S1, supporting

148

information) at trace level (ng/L) in waste waters. Analytes were extracted by an

149

optimized DLLME procedure and , consisting in the addition to 10 mL of sample of a

150

mixture of 50 µL of tetrachloroethylene dissolved in 1 mL of acetone. The sedimented

151

phase obtained (~ 38 µL) was evaporated and further silylated with 40 μL of

152

BSTFA/TMCS (99:1) during 5 min in a household microwave at 600 watt.

153

Quantification was achieved by GC-MS in selective ion monitoring mode, using

and

chlorobenzene/acetone

were

used,

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Chloroform/acetone

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117

5

Page 6 of 36

154

deuterated BPA and deuterated benzophenone as internal standards. The developed

155

method was validated and further applied in the analysis of influent and effluent waste

156

water samples collected from 15 different wastewaters treatment plants (WWTP)

157

distributed over Portugal, in three different seasons of 2013.

158

In recent decades, a tremendous increase in chemicals production has taken

160

place as a result of growing population pressure and inherently increasing

161

demand of consumer goods. According with the Organization for Economic

162

Cooperation and Development (OECD), the global chemical output has grown

163

from US$171 billion in 1970 to US$4.12 trillion in 2010, and the predictions are

164

that global chemical sales will increase around 3% per year until 2050 (OECD

165

2012). Of course, it is well known that usage and disposal of synthetic

166

chemicals are source of environmental stress, yielding risks for living

167

organisms, humans included, at all stages of their life cycle; moreover most of

168

chemicals finally end up in the different environmental compartments, where

169

they can remain active for long periods of time.

170

A group of chemicals which have gained increasing interest in recent years in

171

the field of environmental research are the so-called emerging contaminants, a

172

structurally diverse and heterogeneous group of chemicals not covered by

173

current regulations or legislation, which are believed to pose a threat to

174

environmental ecosystems (Farré et al., 2008). Among others, pharmaceuticals

175

(e.g. hormones, pain relievers, antibiotics), personal care products (e.g.

176

antiseptics, UV-filters/sunscreen components, cosmetics), plasticizers such as

177

phthalates and bisphenol A (BPA), and brominated compounds, are included in

178

this group.

179

UV-filters are among the components of industrial products, such as house

180

paints, plastics, or textiles to prevent photodegradation of polymers and

181

pigments (Gago-Ferrero et al., 2012), but they are specially used in sunscreens

182

and other personal care products to absorb, scatter or reflect UV radiations

183

(320-400 nm for UVA and 290-320 for UVB). In European Union (EU) there are

184

currently approved by the Cosmetic Directive (from July 2013 by the Cosmetic

185

Products Regulation), 28 26 UV-filters, 275 of which are organic chemical

186

compounds, being only one, TiO2, an inorganic UV-filter (EC No 1223/2009). In

187

USA there are only 17 approved UV-filters, of which 15 are organic chemical

Ac

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159

6

Page 7 of 36

188

UV-filters and two, TiO2 and ZnO, are from inorganic nature (FDA, 1999). There

189

are 55 compounds in the world approved to be used as UV-filters, comprising

190

various chemical classes, being the most common para-amino-benzoates,

191

cinnamates,

192

derivatives, and benzimidazoles (Shaath, 2010; Santos et al., 2012). The

193

hydrophobicity of many of these analytes indicates their potential for

194

bioaccumulation in aquatic and other environmental compartments (Díaz-Cruz

195

et al., 2008). Therefore, UV-filters released to the environment directly via

196

wash-off from the skin or industrial discharges, or indirectly via waste water

197

domestic discharges or releasing by sewage treatment plants, have been

198

detected in surface water, sediment and biota (Balmer et al., 2005; Remberger

199

et al., 2011; Zhang et al., 2011), drinking water (Rodil et al., 2012), seawater

200

(Sánchez Rodríguez et al. 2015), sludge and effluent water from waste

201

treatment plant (Balmer et al., 2005; Zhang et al., 2011) and even in breast milk

202

and human urine (Kunisue et al., 2012; Schlumpf et al., 2010). The continuous

203

inputs into the environment may lead to toxic effect in human and wildlife,

204

mainly because most of these compounds are considered as potential

205

endocrine disruptors.

206

Other emerging contaminant that have been detected in landfill leachates,

207

wastewater, ground water, and surface water is BPA a synthetic compound

208

commonly employed as a key monomer in the manufacturing of polycarbonate

209

plastic and epoxy resins, used in thermal paper, baby bottles, or as coatings on

210

the inside of many food and beverage metallic cans (Fromme et al., 2002; Song

211

et al., 2014). BPA have potential toxicity towards humans and animals, and is

212

suspected to possess carcinogenic and endocrine disrupting activity even at low

213

concentration (Swedenborg et al. 2009).

214

One of the first challenges when studying the occurrence, behavior and fate of

215

environmental emerging contaminants is the development of reliable analytical

216

methods for its rapid, sensitive, accurateand selective quantification. Multi

217

residue methods are understandably preferred taking into account the huge

218

amount of compounds of interest and the broad coverage they could provide.

219

Multi residue methods for UV filter determination in waters are usually based on

220

LC-MS/MS techniques (Rodil et al., 2009a; Díaz-Cruz et al., 2008; Zhang et al.,

221

2011; Rodil et al., 2012; Gago-Ferrero et al, 2013) or GC-MS (Poiger et al.

benzophenones,

dibenzoylmethanes,

camphor

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salycilates

7

Page 8 of 36

2004; Pintado-Herrera et al. 2013;), depending the choice of the physico-

223

chemical properties of the compounds to be analyzed and, in many situations,

224

of the equipment availability. Before chromatographic analysis several sample

225

pre-treatment steps like filtration, pH adjustment, isolation from matrix, clean-up

226

and extract concentration are usually required, depending of the type of matrix

227

and the kind of compounds to be analysed. In most cases such as in surface

228

and wastewater samples, concentration of analytes is usually achieved along

229

with extraction/clean-up procedures. Besides the use of well-established

230

extraction techniques such as liquid-liquid extraction (LLE) and solid-phase

231

extraction (SPE) (Balmer et al., 2005; Rodil et al. 2009a; Zhang et al., 2011;

232

Mohapatra et al. 2011), other more novel techniques have been also applied

233

like solid phase micro-extraction (SPME), stir bar sorptive extraction (SBSE)

234

(Quintana et al., 2007), single drop micro-extraction (SDME), and membrane

235

assisted liquid-liquid extraction (MALLE) (Rodil et al., 2009b). Compared with

236

the common procedures, these novel techniques can provide faster sample

237

preparation, less consumption of organic solvents, and higher yield enrichment.

238

Dispersive liquid-liquid micro-extraction (DLLME) is another recently developed

239

extraction technique presenting unique features in what concerning simplicity of

240

operation, amount of organic solvent extractor (only a few microliters),

241

quickness, and high enrichment factor. It was based in the rapid injection to an

242

aqueous sample of a mixture of a carefully chosen pair of extractor and

243

dispersive solvents, leading to the instantaneous formation of a cloudy solution

244

formed by microbubbles of the extractor, which are easily sedimented by a

245

short-time centrifugation. Like in SDME, the extraction equilibrium in DLLME is

246

quickly achieved due the easy contact between the micro droplet(s) of

247

extractive solvent and the sample, therefore, the extraction time being

248

substantially reduced when compared with sorption techniques (Pintado-

249

Herrera et al. 2013; Cunha et al. 2011). The great enrichment factor provided by

250

the technique is of crucial importance for analytes of interest at ng.L-1 level, as

251

is the case of UV-filters in the different water samples. DLLME was already

252

used in the determination of different types of UV filters by some authors (Wille

253

et al. 2012; Zhang and Lee 2012) usually as extractive/concentration step

254

before GC-MS analysis.

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Page 9 of 36

Often, the main drawback of GC-MS methods lies on the need to an additional

256

time-consuming derivatization step, although essential to improve volatility,

257

thermal stability, and other desirable chromatographic features of the target

258

analytes. This is the case for some of the compoundsof this study, namely BPA,

259

benzophenone and salicylic acid derivatives. The presence of phenolic

260

hydroxyls in their structure, require the transformation in less polar derivatives,

261

usually by a silylation reaction, in order to enhance their chromatographic

262

behaviour. Most of the described derivatization procedures for the GC-MS

263

analysis of the above mentioned UV-filters are time-consuming, taking, 30-60

264

min at a temperature between 60 and 90°C (Zhang and Lee, 2012). One way to

265

overcome

266

derivatization procedures which is giving better results. When compared to

267

conventional derivatization, protocols involving conductive heating have

268

demonstrated decrease the derivatization reaction time to a few minutes, and

269

enabling a high-throughput in the analysis format (Söderholm et al., 2010).

270

The work presented here deals with the development of a simple, fast and

271

reliable GC-MS method that enables the simultaneous measurement of BPA

272

and 13 of the most prevalent UV-filters pertaining to 8 different chemical classes

273

(Table S1, supporting information) at trace level (ng/L) in waste waters. Despite

274

some

275

quantification, as far as we know none was has been applied in multi-residues

276

analysis. Analytes were extracted by an optimized DLLME procedure,

277

consisting in the addition to 10 mL of sample of a mixture of 50 µL of

278

tetrachloroethylene dissolved in 1 mL of acetone. The sedimented phase

279

obtained was evaporated and further silylated with 40 μL of BSTFA/TMCS

280

(99:1) during 5 min in a household microwave at 600 watt. Quantification was

281

achieved by GC-MS in selective ion monitoring mode, using deuterated BPA

282

and deuterated benzophenone as internal standards. The developed method

283

was validated and further applied in the analysis of influent and effluent waste

284

water samples collected from 15 different wastewaters treatment plants

285

(WWTP) distributed over Portugal, in three different seasons of 2013.

inconvenience

consists

of

using

microwave

assisted

M

an

us

this

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ip t

255

procedures

have

been

ed

DLLME

applied

in

UV-filters

Ac

ce pt

previous

286 287

Experimental

288

Reagents 9

Page 10 of 36

2-Hydroxy-4-methoxybenzophenone (BP3; 98% purity), 2,3,4-trihydroxybenzophenone

290

(THB; 98% purity) and 2-ethylhexyl 4-(dimethylamino)benzoate (EPABA; 98% purity)

291

were purchased from Alfa Aesar (Heysham, Lancashire, UK). 3,3,5-trimethylcyclohexyl

292

salicylate (HMS; 98% purity) 2,2′-dihydroxy-4,4′-dimethoxybenzophenone (DHMB, 99%

293

purity) and isoamyl-4 methoxycinnamate (IMC, 95% purity) were purchased from TCI

294

(Haven, Zwijndrecht, Belgium). Octocrylene (OC, 98% purity), 2-ethylhexyl 4-

295

methoxycinnamate (EHMC, 100% purity), 2-ethylhexyl salicylate (EHS, 99% purity),

296

hexyl 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoate (DBENZO, 99% purity), 2,4-

297

dihydroxybenzophenone (BP1, 99% purity), 3-(4-methylbenzylidene)camphor (4-MBC,

298

98.5% purity), butylmethoxydibenzoylmethane (BMDM, 100% purity), and bisphenol A

299

(BPA; > 98% purity) were purchased from Sigma-Aldrich (West Chester, PA; USA).

300

The internal standards (IS) bisphenol B (BPB- IS1; > 98% purity), d16-bisphenol A

301

(BPAd16- IS2; 98 atom % D), and Benzophenone-d10 (BPd10-IS3, 99 atom % D) were

302

also purchased from Sigma-Aldrich.

303

Acetonitrile (MeCN), methanol (MeOH), acetone (AC) all HPLC grade were obtained

304

from

305

trichloroethylene

306

tetrachloroethylene (C2Cl4) were high purity solvents for GC analysis obtained from

307

Fluka (Neu-Ulm, Germany).

308

Derivatization

309

(MTBSTFA, >97% purity grade) and N,O-bis(trimethylsilyl)trifluoroacetamide with 1%

310

TMCS (BSTFA+1%TMCS, 99% purity grade) were obtained from Fluka. Hydrochloric

311

acid and pH test strips (0-14 pH resolution: 1.0 pH unit) were purchased from Sigma-

312

Aldrich.

313

Water was prepared by purifying demineralised water in a “Seradest LFM 20” system

314

from Seral (Ransbach-Baumbach, Germany).

315

Ultrahigh purity Helium (99.999%) for GC-MS was purchased from Gasin (Maia,

316

Portugal).

us

cr

(C2HCl3),

octanol

1,1,1-trichloroethane

(C8H18O),

chlorobenzene

(C2H3Cl3),

(C6H5Cl)

and

N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide,

ed

reagents:

solvents:

an

Extractive

M

Sigma-Aldrich.

ce pt

317

ip t

289

Standard solutions and validation

319

A stock solution of each UV-filter (1000 mg/L) was prepared by dissolving the analyte

320

in MeOH. A stock solution of BPA (2000 mg/L) was also prepared in MeOH. An

321

intermediate working mixture standard solution of all analytes at 20 mg/L was prepared

322

from the stock solutions by appropriate dilution in MeOH. Individual stock solutions of

323

BPB, BPAd16 and BPd10 (1000 mg/L) used as internal standards were also prepared in

324

MeOH. All the solutions were stored at -- 20 ºC when not in use.

Ac

318

10

Page 11 of 36

The method was validated in accordance with the internationally accepted criteria, such

326

as linearity, accuracy and precision. Validation criteria were adopted from SANCO

327

guidelines for residues analytical methods (EC 2005). Linearity was studied in blank

328

water samples (free of analytes) spiked at ten concentration levels. The Analyte

329

concentration of the samples was obtained by the internal standard method, using BPB

330

and BPAd16 as IS in the quantification of UV-filters and BPA, respectively. Additionally,

331

BPd10 (IS3) was used with internal quality control purposes, namely to monitorize GC

332

injection during optimization of DLLME extraction.

333

Recovery Extraction efficiency and intra-day precision determinations were performed

334

by adding to blank samples 50 µl of mixed standard spiking solutions of the UV-filters

335

and BPA prepared in ultrapure water and further submitted to the extraction procedure,

336

being; the final extract was placed into vials containing a mixed solution of all IS,

337

previously evaporated by a gentle stream of nitrogen. Three spiking levels were

338

selected and six replicates analysed at each level. Results obtained for each analyte

339

were compared with values obtained from similar samples added after extraction with

340

all analytes at same level,

341

precision was evaluated for one level of concentration. For that purpose, six spiked

342

samples were extracted and analysed in 3 different days for a period of 3 weeks.

343

Limits of detection (LODs) and quantification (LOQs) for the selected analytes were

344

assessed based on signal-to-noise ratio (S/N) determination.

345

To avoid cross contamination, all glassware used in extraction was put in a muffle at

346

500°C by 3 hours before use. Glassware and screw-capped tubes were held

347

separately and rinsed with distillated water, acetone and after drying with HPLC water.

348

Blank tests were performed to rule out any possible contamination along sampling and

349

storage, or coming from instrumentation. In order to comply with internal quality control

350

procedures, two solvent injections and two procedural blanks were inserted into each

351

analytical batch made up of ten samples.

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IS1, and IS2 and analytes at same levels. Inter-day

352 353

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354 355

Sampling

356

Samples were collected in duplicate from 15 different Portuguese WWTP’s serving 13

357

large cities along the country (Figure S1, supporting information). For each plant,

358

samples were collected, in glass containers previously rinsed with bi-distilled water, as

359

timecorresponding each sample proportional to 24-h composite influent and effluent

Formatted: Not Highlight

360

samples. Samples were kept refrigerated (±4 °C) during the transport to the laboratory.

Formatted: Not Highlight

361

Upon reception, samples were stored at 4 °C until analysis. 11

Page 12 of 36

362

Two types of waters were analyzed: influent and effluent waste waters collected in

363

three sampling campaigns, carried out March, May and July of 2013. The codes of the

364

sampling points and characteristics of the WWTPs are given in (Table S2, supporting

365

information).

366 Optimized Sample Preparation

368

Sample preparation procedure entails the following steps: (i) weight 10 g of

369

homogenized and filtered water sample into a 40 mL screw-capped amber glass vial;

370

(ii) add 25 µL of BPB (IS1, 2 mg/L) and 25 µL of BPAd16 (IS2, 2 mg/L); (iii) add 0.1

371

mol/L HCl until reaching pH 3; (iv) transfer rapidly a mixture of 50 µL of C2H3Cl3 and

372

1000 µL of AC; (v) seal the tube and shake gently by hand for 30 s; (vi) centrifuge the

373

tube at 3500 g for 1 min; (vii) transfer 38 µL of the lower phase to an amber vial and

374

add 50 µL of BPd10 (IS3, 4 mg/L); (viii) evaporate to dryness under a gentle stream of

375

nitrogen; (ix) silylate the analytes by addition of 40 μL of BSTFA with 1% TMCS during

376

5 min in a household microwave (600 watt); (x) finally, 1 µL of the extract was injected

377

in the GC-MS system.

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378 Apparatus and GC-MS conditions

380

An gas chromatograph 6890 (Agilent, Little Falls, DE, USA) equipped with a Combi-

381

PAL autosampler (CTC Analytics, Zwingen, Switzerland) and an electronically

382

controlled split/splitless injection port was interfaced to a single quadrupole inert mass

383

selective detector (5975B, Agilent) with electron ionization (EI) chamber was used. GC

384

separation was performed on a DB-5MS column (30 m x 0.25 mm I.D. x 0.25 μm film

385

thickness; J&W Scientific, Folsom, CA, USA). Helium was the carrier gas with a

386

constant flow of 1 mL/min. The injection was made in splitless mode (purge-off time 60

387

s) at 250°C. The oven temperature program was as follows: 95°C held for 1 min,

388

ramped to 180°C at 40°C/min, ramped to 230°C at 5°C/min and then ramped to 290°C

389

at 25°C/min, and held for 6.47 min. Total run time was 22 min. The MS transfer line

390

was held at 280°C.

391

Mass spectrometric parameters were set as follows: electron ionization with 70 eV

392

energy; ion source temperature, 230°C; MS quadrupole temperature, 150°C. The MS

393

system was routinely set in selective ion monitoring (SIM) mode and each analyte was

394

quantified based on peak area using one target and two qualifier ion(s). Complete SIM

395

parameters and retention times of the analytes are shown in Table 31. Agilent

396

Chemstation was used for data collection/processing and GC-MS control.

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Page 13 of 36

Results and discussion

399

3.1. Optimization of sSample preparation

400

3.1.1. Optimization of DLLME

401

To evaluate the capacity of DLLME method to allow the accurate quantification of BPA

402

and 13 UV-filters several parameters were optimized including nature and amount of

403

the extractive solvent as well as the nature and amount of dispersive solvent.

404

In DLLME procedure the extractive solvent has to satisfy several requirements: i)

405

higher density than water, ii) immiscibility with water, iii) good extraction capability of

406

the analyte(s), and iv) chromatographic compatibility. Among the solvents with density

407

higher than water (mainly chlorinated solvents) C8H18O (density: 0.824, water

408

solubility 2.3 g/L)The following solvents, C2H3Cl3 (density 1.32; insoluble in water),

409

C2HCl3 (density 1.46; water solubility 1.28 g/L), C6H5Cl (density 1.11; water solubility

410

0.5 g/L), C2Cl4 (density 1.62; water solubility 0.17 g/L) and the mixture C2Cl4:C8H18O

411

(3:1) were tested in this study. Hence, 10 mL of water containing 1 mg/L of each

412

analyte was rapidly injected with 1000 µL of acetone containing 50 µL of extractive

413

solvent. The average peak areas of the analytes, attained by triplicate analysis of an

414

aliquot of the 38 µL of sedimented phase obtained from each extraction followed by

415

derivatization with 50 µL of BSTFA (as described in 3.2.2), showed that C2Cl4 was the

416

best extractive solvent for most target analytes as can be seen in Figure 2 1., the high

417

density of this solvent and the relative low solubility in water could explain the high

418

capacity of extraction. Then, the extractive solvent volume was evaluated. It is well

419

known that lower volumes enhance the enrichment factor of the DLLME process,

420

although reducing the volume of the sedimented phase. For the purpose of the present

421

study, two replicates were investigated using 10 mL of water added with 1000 µL of

422

acetone containing three different volumes of C2Cl4: 50 µL, 100 µL and 150 µL. Lower

423

volumes than 50 µL, tested at an early stage, resulted in a very small volume of

424

sedimented phase and subsequently in a decrease of reproducibility of the method.

425

The increment of the volume of C2Cl4 from 50 to 150 µL resulted in an increased

426

volume of sedimented phase from ~38 to ~100 µL with the subsequent decrease of

427

enrichment factor and detection limits. Thus, 50 µL was selected as an optimum

428

volume of extractive solvent.

429

In the optimization of the proposed method it was also evaluated the nature of the

430

dispersive solvent, as well as the absence of dispersive solvent. Taking into account

431

the two main requirements of a dispersive solvent in a DLLME procedure, miscibility

432

with both sample phase and extractive solvent and capacity to decrease the interfacial

433

tension of extractive solvent to make the droplet size smaller, AC, MeOH and MeCN

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Page 14 of 36

were compared between them and with the absence of dispersive solvent. Initially

435

triplicate experiments were performed in 10 mL of water spiked with 1 mg/L of each

436

analyte, which was rapidly injected with 1000 µL of the different dispersive solvents

437

containing 50 µL of C2Cl4. The capacity of the extractive solvent alone was also

438

experimented. In this case the fine droplets were achieved by aspirating and injecting

439

the extraction solvent using the by pipette several 10 consecutive times. The results

440

showed that AC provided a cleaner sedimented phase with higher density of droplets,

441

and also a higher extraction yield than those obtained by MeCN and MeOH or in the

442

absence of dispersive solvent (Figure 32). Therefore, AC was chosen as dispersive

443

solvent. In the next step the effect of dispersive solvent volume was tested using 500

444

µL, 1000 µL and 1500 µL of AC. Peak areas of the analytes were enhanced by

445

increasing the volume of AC from 500 to 1000 µL, whereas the extraction efficiency

446

decreased with 1500 µL of AC. The reason was that a lower amount of AC could not

447

disperse C2Cl4 completely and a cloudy state was not well formed. On other hand, a

448

higher volume of AC increased the solubility of some analytes in aqueous sample,

449

thereby, the extraction abilities were reduced.

450

Commonly, pH of aqueous samples is an important parameter to consider in extraction

451

procedures because it can affect the existing forms of some analytes in solution, such

452

as EPABA (pKa=2.39), DHMB (pKa=6.99), THB (pKa=7.51), BP1 (pKa=7.53), BP3

453

(pKa=7.56), HMS (pKa=8.09), EHS (pKa=8.13) and BMDM (pKa=9.7). As it is well

454

known, this kind of analytes can be better extracted by organic solvents when they are

455

in their neutral forms. To investigate the effect of pH on extraction efficiency, different

456

water samples with pH in the range 2-7 were prepared by adding 0.1 mol/L

457

hydrochloric solution to adjust. As might be expected the results showed that extraction

458

efficiency decrease when the pH is higher than 4, being the better analytical signal

459

obtained for most of the analytes at pH 3. At lower pH the analytes exist mostly in their

460

neutral forms being the ionization suppressed, which is beneficial for their transfer to

461

the organic phase. At higher pH values the analytes underwent ionization, resulting in

462

decreases extraction yields.

463

Increased ionic strength and changes in ionic composition can lead to a decrease in

464

solubility of the analytes in the water solution, thus, extraction efficiency may be

465

enhanced. To investigate the effect of ionic strength on extraction efficiency, NaCl at

466

5%, 5% and 10% (w/v) were added into the aqueous solution, being the extraction

467

performed under the optimal conditions referred above. Results showed that addition of

468

salt decrease the analytical response for most lipophilic compounds and only a slight

469

increase in the analytical response of benzophenone compounds was observed. Due

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Page 15 of 36

470

to the negligible effect obtained in the analytical response for most of the analytes in

471

study no salt was added in the further experiments.

472

In this procedure, the equilibrium is achieved in few seconds due to the large contact

473

surface between tiny droplets formed and the sample. Therefore, the mass transfer of

474

the analytes from aqueous matrix to the extraction solvent was quickly realized in less

475

than 1 min. In short, DLLME can be regarded as a time-independent method.

ip t

476 3.1.2. Optimization of the Dderivatization conditions

478

To shorten the sample preparation process, the recent application of microwave

479

radiation offers a good way to provide high acceleration rates in a short time for

480

derivatization reactions (Amaral et al. 2013). In this work the usefulness of microwave-

481

assisted for the derivatization of UV-filters and BPA was compared to that of

482

conventional reaction process. The effects of irradiation time and power level of

483

microwave irradiation were investigated. Initially, the results obtained using different

484

silylation reagents (BSTFA with 1% TMCS and MTBSTFA) were also compared to

485

select the most appropriate derivatization reagent. Thus, 50 µL of a mix standard

486

solution of 13 UV-filters and BPA, at 100 µg/L each, was previously evaporated to

487

dryness and further derivatized by adding 50 µL of the above mentioned silylating

488

reagents. All the reactions were performed for 30 min at 75°C, an optimal set of

489

conditions established in a previous study about derivatization with conventional

490

heating (Zhang and Lee 2012). After cooling, the derivatized solutions were directly

491

injected into the GC-MS. Notwithstanding, the good response obtained with MTBSTFA

492

for the most of thesome compounds, IMC, 4-MBC, EPABA and EHMC the BSTFA with

493

1% TMCS provided a significantly high intensity chromatographic response for

494

compounds than contain a labile H (OH) such as BP3, BP1, DHMB, BENZO and BPA,

495

provably due to its high reactivity. Similar results were reported in the literature by

496

Zhang and Lee (2012) and Kotnik et al. (2014). Some compounds, namely IMC, 4-

497

MBC, EPABA, EHMC, OC and BMDM could be detected without derivatization. The

498

analytical response obtained from the direct injection of this analytes in GC-MS was

499

similar to those obtained after the derivatization step, which proves that are no losses

500

during the derivatization, so securing the use of a single injection per extract. According

501

to the obtained results, BSTFA with 1% TMCS was chosen as derivatizing reagent.

502

To investigate the effect of microwave irradiation time on the derivatization of the

503

compounds under study, four irradiation times (2, 3, 5 and 7 min) were selected for

504

microwave-assisted derivatization at 600 W irradiation power. The efficiency of the

505

microwave method was directly compared with derivatization by means of conventional

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Page 16 of 36

heating (30 min at 75°C). In all the experiments a mix solution of 13 UV-filters and

507

BPA, at 100 µg/L, was dried under a nitrogen stream, and derivatized with 50 µL of

508

BSTFA with 1% TMCS. As shown in Figure 4a3a, microwave irradiation during 2-5 min

509

resulted nearly always in a response peak area close or higher to that obtained by the

510

conventional heating, while a longer irradiation time (7 min) have shown to decrease

511

peak areas for most of the analytes. For most of the analytes a 5 min irradiation

512

showed the best results. In Figure 4b 3b results from an experiment consisting in 5

513

min exposure with different microwave power levels can be observed. Irradiation at 600

514

W gave consistently larger peak areas than lower power levels (240 and 400 W). So, to

515

insure the maximum efficiency of the derivatization procedure to all analytes under

516

study, a 5 min exposure at a microwave irradiation power 600 W was selected.

us

517

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506

518 Method performance

520

The linearity of the method was several times tested (over the study) using matrix-

521

matched calibration solutions (blank water samples solutions spiked with analyte

522

standards,)

523

concentrations ranged from 10 to 5000 ng/L from the analytes in study (Table 24). The

524

results obtained demonstrated a good linearity within the tested interval, with mean

525

correlation coefficients (r) higher than 0.9970 for all analytes.

526

Recovery Extraction efficiency and intra-day repeatability were determined on blank

527

water samples spiked with BPA and UV-filters at three concentration levels, being each

528

test performed six times. Table 4 2 shows average of recoveries extraction efficiency

529

and intra-day repeatability expressed as relative standard deviation (RSD). Mean

530

recoveries extraction efficiencye for the lowest level (50 ng/L) ranged from 55 to 102%,

531

from 58 to 91% for the middle level (1000 ng/L) and from 68 to 105% when the highest

532

level (2500 ng/L) was evaluated. Lowest recoveries extraction efficiency were observed

533

for THB and BP1, which can be explained by their lower lipophilicity (lower Log Kow) so

534

lower affinity for tetrachloroethylene used as extractor solvent. RSD values of the intra-

535

day repeatability ranged from 2% to 22%; as expected, higher RSD values were

536

obtained when the lowest level (50 ng/L) was evaluated. Inter-day repeatability was

537

evaluated at 1000 µg/L (Table 42). Results obtained were similar or just slightly higher

538

than those obtained from intra-day repeatability, ranging from 2% to 18%. The results

539

reported provide evidence that the optimized method achieves for nearly all analytes

540

acceptable recoveries levels of extraction efficiency (between 70 and 120%) and

541

repeatability (RSD≤20%), in line with criteria sets by EU guidelines (EC, 2005).

as

described

in

the

Experimental

section.

Standard

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Page 17 of 36

The detection limits of the method were determined by successive analyses of

543

chromatographic sample extracts with decreasing amounts of the compounds until a

544

3:1 signal-to-noise ratio was reached. The lowest assigned value obtained was 2 ng/L

545

for HS, BP3, and EHMC (see Table 24). Similar values were obtained by Poiger et al.

546

2004 and Magi et al. 2013. The method quantification limits were established as the

547

lowest concentration assayed able to be quantified with acceptable accuracy (70-110%

548

of recoveryextraction efficiency) and precision (RSD of ≤20%) (EC, 2005), which were

549

the lowest calibration level of the calibration curve.

ip t

542

550 Presence of UV- filters and BPA in Portuguese WWTPs

552

The presence of BPA and seven out of thirteen UV-filters assayed was noticed in

553

influent/effluent waters collected from the 15 Portuguese WWTPs surveyed in this

554

work, as can be seen in Table 53. Overall, data reveal a pronounced seasonal

555

variation with influent loads higher in the warmer season (July 2013) than in the colder

556

one (March 2013), reflecting an increased use of sunscreen products in summer.

557

Furthermore, levels found in influent wastewaters were far above the corresponding

558

levels observed in effluent waters indicating significant elimination in the WWTPs.

559

The most prevalent compound was BP3 which was detected in 79 % of the 90 samples

560

analysed (71 samples), although at levels below LOQ in 13 effluent samples. This

561

finding was not surprising taking into account the high water solubility of the compound,

562

and the fact that BP3 be used not only in sunscreens and cosmetics but also in plastics

563

like light stabilizers (Krause et al., 2012). Levels found ranged from n.d. to 323.3 ng/L

564

in influent waters, and from n.d. to 68.2 ng/L in effluent waters, with average levels (not

565

considering n.d. and
566

are somewhat similar to the results reported by Tsui et al. (2014) that ranged from 111

567

ng/L (WWTPs effluent) to 284 ng/L (WWTPs influent). The marked seasonal variation

568

above referred could be clearly seen in BP3 levels. In influent waters mean values of

569

34.1, 58.2, and 181.2 ng/L were obtained for March, May, and July collections,

570

respectively, while mean values of 18.1, 19.8, and 49.3 ng/L, respectively, were

571

obtained for effluent waters.

572

The second most prevalent UV-filter was EHMC, although at a marked distance of

573

BP3. It was detected in 12 out of 45 influent water samples (27 %) and in 9 out of 45

574

effluent water samples (20%). EHMC levels ranged from n.d. to 689.5 ng/L in influent

575

waters, and from n.d. to 483.4 ng/L in effluent waters, with average levels (not

576

considering n.d. and
577

highs mean values are strongly influenced by the levels found in the July collection of

578

WWTP of Beirolas: 689.5 and 483.4 ng/ in influent and effluent waters, respectively.

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Page 18 of 36

Figure 5 4 represents a total ion chromatogram of Beirolas influent and the selective

580

ion monitoring chromatogram of EHMC.

581

Anyway, the levels observed were slightly lower than those reported by Balmer et al.

582

(2005) for Swiss WWTPs. The seasonal variation was so stark, that 20 out of 23

583

positive samples were collected in July.

584

EPABA was found in 20 out of 45 influent water samples (44.5 %) with levels ranging

585

from 12.2 to 418 ng/l. The mean value in the positive samples was 88.1 ng/L, while the

586

mean value of the positive samples collected in July (5 samples) was significantly high:

587

231.5 ng/L. In effluent waters the absence of the compound was almost complete,

588

being detected in only 5 samples at levels
589

The number of positive (>LOQ) samples of OC was only 16 (12 influent waters and 4

590

effluent samples) even though it should be noted the high values found. OC levels in

591

these samples ranged from 19.6 to 785.5 ng/L in influent waters with a mean of 388.3

592

ng/L, and from 124.6 to 353.5 ng/L with a mean of 247.5 ng/L.

593

The other 3 UV-filters studied, BMDM, 4-MBC, and BP1, were found in a few number

594

of samples (11, 4, and 3, respectively), predominantly in influent water samples. It is

595

worth noting; however, that the two higher values observed in this study was from

596

BMDM, 2935 and 1247.5 ng/L, in two influent water samples collected in July in two

597

different WWTPs.

598

This high levels of BMDM could be explained by the extensively used in sunscreen

599

formulations (UVA filter), therefore it is not surprising that the highest levels were found

600

in WWTPs located near coastal bathing areas. BMDM is usually employed together

601

with OC to prevent its quick degradation. In this study it can be noted that 3 out of the 5

602

higher levels of BMDM (July collection of influent waters from WWTPs 4, 11, and 14)

603

are matched with the 3 higher levels of OC. Interestingly, two other important levels of

604

OC were also found in WWTPs serving coastal bathing areas (WWTP 6 and 15)

605

although in this case not followed by high levels of BMDM.

606

The above observation is not confined to BMDM and OC. Overall the highest total

607

levels of UV-filters were noticed from influent waters collected in July from WWTPs

608

located near bathing areas and near the most densely populated regions. The highest

609

total values (i.e. sum of the levels of all compounds studied) were verified in WWTP 11,

610

located in Lisbon, which is simultaneously a densely populated area and a bathing area

611

– 4507,1 and 1234.6 ng/L in influent and effluent waters, respectively – WWTP 14,

612

located in Algarve, the more important bathing area of Portugal, densely populated in

613

Summer – 3464.7 and 1066.3 ng/L, respectively – and WWTP 4, located near Viana do

614

Castelo, another important bathing region in the North of Portugal – 1417.9 and 301.5,

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Page 19 of 36

respectively. These findings are in according with literature references from other

616

countries (Tsui et al..2014; Sánchez Rodríguez et al., 2015).

617

BPA, the only non UV-filter compound studied, was detected in only 4 out of 15

618

WWTPs, two of them the WTTP 11 and 14, corresponding to those working with most

619

contaminated waters, as above referred, being the other two characterized by the low

620

level of UV-filters observed, WWTP 5 located in a predominantly industrial area (Vale

621

do Ave), and WWTP 12 situated in the farming region of Alentejo. BPA levels are of

622

concerning, ranging from 300.5 to 1274 ng/L (mean level: 574.4 ng/L), in influent

623

waters, and from 61 to 874 ng/L (mean level: 348.2 ng/L), in effluent waters. The

624

presence of BPA at levels higher than 1 µg/L were previously reported by Mohapatra et

625

al. (2011) in WWTPs from Canada.

626

As already reported the presence of BPA and UV filters in effluent wastewaters were in

627

general sharply lower than that observed in influents waters

628

removal in the WWTPs. Data in Table 35 suggest elimination rates in the range of

629

>99% for BP1, 37->99% for BMDM, EPABA, and 4-MBC, 30->99% for OC and EHMC,

630

10-86% for BP3, and 12-72% for BPA, with the caveat that data were generated from

631

influent and effluent sampled on the same day and thus not exactly from the same

632

package of wastewater. On other hand, it is not clear from our data whether all of these

633

compounds are actually degraded or just removed from wastewater by sorption to

634

sewage sludge for example. Additionally, it was not possible to obtain any correlation

635

between the data and the points of sampling due to the high variation on the type of

636

treatments and process involved in the different WWTPs. Notwithstanding, removal

637

efficiencies obtained were in agreement with those reported in literature by Balmer et

638

al. (2005) - 18 to 99% for 4-MBC, BP3, OC and EHMC in WWTPs equipped with

639

mechanical, biological, and chemical treatment and sand filtration - and by Kupper et

640

al. (2006) which reported removal rates of 92-99% for a set of 4 UV-filters including OC

641

and 4-MBC in a WWTP equipped with conventional activated sludge treatment. Lower

642

removal efficiencies has been reported in a study performed in a Chinese WWTP; 28–

643

31% for BP3, 40–43% for EHMC, and 36–38% for OC (Li et al., 2007).

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showing a marked

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644 645

Conclusions

646

The proposed DLLME-GC/MS method allowed a reliable and fast simultaneous

647

quantification of 13 UV-filters and BPA in WWTPs wastewater samples. Sample

648

preparation method is based in a DLLME procedure using tetrachloroethylene and

649

acetone as extractive and dispersive solvent, respectively, followed by a fast

650

microwave silyl derivatization and further analysis by GC-MS. Use of microwave

651

irradiation as heating method for the silylation of the studied compounds, significantly 19

Page 20 of 36

reduced the derivatization time, being 5 min of irradiation at 600 W found so effective

653

as the conventional heating at 75°C for 30 min.

654

Eighth out of the 14 analytes were positively identified in most of the samples of 15

655

WWTPs, during 3 collection sessions, indicating a wide-spread presence of UV-filters

656

and BPA in water environment from various zones of Portugal. The most prevalent UV-

657

filter was BP3 detected in 71 out of 90 samples analyzed, although the higher level has

658

been observed for BMDM, 2935 ng/L. As expected, waters from bathing areas and

659

densely populated areas showed the highest total levels, with a marked incidence in

660

summer collection. Worrying levels of BPA were observed in samples from 4 out of 15

661

WWTPs used in this survey.

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652

662 Acknowledgments

664

This research was supported by grant from the FCT project UID/QUI/50006/2013 and

665

COMPETE FSE/FEDER. S.C.C. is grateful to “POPH-QREN- Tipologia 4.2, Fundo

666

Social Europeu e Fundo Nacional MCTES”.

an

us

663

667 668 References

670

A. Krause, M. Klit, Blomberg J., Sunscreens: are they beneficial for health? An

671

overview of endocrine disrupting properties of UV-filters,” Inter. J. Andrology, 35

672

(2012) 424–436.

ed

M

669

A. Sánchez Rodríguez, M. Rodrigo Sanz, J.R. Betancort Rodríguez, Occurrence of eight UV filters in beaches of Gran Canaria (Canary Islands). An approach to environmental risk assessment. Chemosphere 131 (2015) 85-90.

676

A. Zenger, H. Schmutz, K. Fent, Simultaneous trace determination of nine organic UV-

677

absorbing compounds (UV filters) in environmental samples. J. Chromatogr. A,

678

1202 (2008) 64-74.

679 680

ce pt

673 674 675

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efficiencies and preliminary risk assessment of multiple classes of organic UV

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determination of UV filters in environmental water samples. Anal. Bioanal. Chem.

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785

profiles of bisphenol analogues in municipal sewage sludge in China. Environ.

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Pollution, 186 (2014) 14-19.

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788

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789

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801

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802

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two Swiss lakes. Chemosphere, 55 (2004) 951-63. W. Li, Y. Ma, C. Guo, W. Hu, K. Liu, Y. Wang, T. Zhu, Occurrence and behavior of four of the most used sunscreen UV filters in a wastewater reclamation plant. Water Res., 41, (2007) 3506-3512.

807 808 809

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810

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811

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812

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cr

ip t

804 805 806

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815

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816

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817

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814

Z. Zhang, N. Ren, T. Kunisue, D. Gao, K. Kannan, Determination of benzotriazole and

819

benzophenone UV filters in sediment and sewage sludge. Environ Sci Technol,

820

45 (2011) 3909–3916.

821

ce pt

822 823 824 825 826

829 830 831

Ac

827 828

ed

818

Figure Captions

Figure 1-Locations of WWTPs in Portugal.

832 833

Figure 12- Comparison of average relative peak area response obtained with different

834

extarctive solvents. 24

Page 25 of 36

835 836

Figure 32- Comparison of average relative peak area response obtained with different

837

dispersive solvents and without dispersive solvent.

838 839

Figure 34a- Comparison of average relative peak area response obtained with

840

different derivatization conditions.

ip t

841 842

Figure 34a- Comparison of average relative peak area response obtained with

843

different microwave power.

cr

844

Figure 54- Total ion chromatogram (TIC) of Beirolas influnte and efluent of July

846

obtained by the optimized DLLME- GC–MS method, together with the individual

847

chromatogram in selected ion monitoring (SIM) mode of BP3 and EHMC.

us

845

848

Ac

ce pt

ed

M

an

849

25

Page 26 of 36

M an

12.00

10.00

ed

8.00

ce pt

6.00

4.00

Ac

Relative peak area

Figure 1

us

cr

i

Figure

2.00

0.00

EHS

HS

IMC

4-MBC

BP3

C6H5Cl

BP1

BPA

DHMB

C8H18O

C2HCl

C2Cl4

EPABA

C2H3Cl3

THB

EHMC

OC

BMDM

DBENZO

C2Cl4:C8H18O (3:1)

Page 27 of 36

9

8

7

AC

WHITOUT DISPERSIVE

ed

6

ce pt

5

4

3

Ac

Relative peak area

MEOH

Figure 2

M an

MeCN

us

cr

i

Figure

2

1

0 EHS

HS

IMC

4-MBC

BP3

BP1

BPA

DHMB

EPABA

THB

EHMC

OC

BMDM DBENZO

Page 28 of 36

i

Figure

cr

9.00

7.00

M an

6.00

5.00

4.00

3.00

1.00

0.00 IMC

4-MBC

BP3

75 °C 30 min

BP1

600 W 2 min

BPA

600 W 3 min

DHMB

EPABA

600 W 5 min

THB

EHMC

OC

BMDM

DBENZO

600 W 7 min

9.000

8.000

7.000

Figure 3 b

6.000

5.000

Relative peak area

HS

ce pt

EHS

ed

2.00

Ac

Relative peak area

Figure 3 a

us

8.00

4.000

3.000

2.000

1.000

0.000

EHS

HS

IMC

4-MBC

BP3 240 W 5 min

BP1

BPA 400 W 5 min

DHMB

EPABA

600 W 5 min

THB

EHMC

OC

Page 29 of 36

BMDM DBENZO

Abundance

TIC: BEIR-Efluent.D\data.ms

M an

1300000 1200000

Ion 178.00 (177.70 to 178.70): BEIRO-Ia.D\data.ms Ion 161.00 (160.70 to 161.70): BEIRO-Ia.D\data.ms Ion 134.00 (133.70 to 134.70): BEIRO-Ia.D\data.ms Ion 290.00 (289.70 to 290.70): BEIRO-Ia.D\data.ms

1100000 1000000 900000

EHM

800000 700000 600000 500000 300000

12.82 12.84 12.86 12.88 12.90 12.92 12.94 12.96 12.98 13.00 13.02 13.04

ed

400000 200000 0

1300000 1200000 1100000 1000000 900000

5.00

7.00

IS1

9.00

11.00

13.00

15.00

17.00

15.00

17.00

19.00

TIC: BEIR-Influent.D\data.ms (*)

Ion 285.00 (284.70 to 285.70): BEIR-IP-1.D\data.ms Ion 223.00 (222.70 to 223.70): BEIR-IP-1.D\data.ms Ion 242.00 (241.70 to 242.70): BEIR-IP-1.D\data.ms Ion 77.00 (76.70 to 77.70): BEIR-IP-1.D\data.ms

Ac

800000

IS3

ce pt

100000

Figure 4

us

cr

i

Figure

700000

BP3

600000 500000 400000 300000

9.3

200000

IS3

9.4

9.5

9.6

9.7

9.8

9.9

EHM

100000 0

IS1 5.00

7.00

9.00

11.00

13.00

19.00 Time (min)

Page 30 of 36

Tables

Table 1- Retention times and MS conditions for the GC-MS analysis of UV-filters and BPA. Quantification ions (m/z) are shown in bold type.

ip t

cr

11.60 12.47 15.90 16.50 17.50

357, 386, 327, 221, 171, 373, 73, 299, 223 165, 277, 77, 145, 431, 73, 343, 105 178, 163, 134, 290, 57 249, 204, 232, 360, 70, 112, 178 135, 310, 161, 77, 253, 108, 367 454, 340, 370, 73, 280, 149, 469

us

11.35 11.47 11.75 11.81 12.00 12.91 16.16 16.77 18.54

an

BPAd (IS3 ) BPB (IS2) DHMB EPABA THB EHMC OC BMDM DBENZO

Time Windows SIM ions m/z 5.20 110, 82, 192, 54, 160 195, 135, 57, 307, 7.60 195, 69, 135, 210 178, 161, 134, 248, 89, 118 254, 128, 211, 183, 155, 55 9.10 285, 242, 77, 223, 105 9.85 343, 73, 164, 105, 271, 357, 372, 374, 339, 117, 207 368, 386, 339, 217, 129 11.00

M

tR 5.37 7.96 8.82 8.93 9.24 9.55 10.47 11.37

Ac

ce pt

ed

Analyte BP10 (IS1) EHS HS IMC 4-MBC BP3 BP1 BPA

Page 31 of 36

ip t cr

(ng/L)

Equation

Correlation coefficient r

50 ng/L spiking level % Extraction Intra-day efficiency %RSD

y=0.0007x+0.6236

0.9992

101

12

10-50000

y=0.001x+0.00782

0.9994

76

15

50-50000

y=0.001x-0.4208

0.9985

95

17

50-50000

y=0.003x-0.1697

0.9999

99

10-50000

y=0.003x-2.1907

0.9971

102

500-50000

y=0.007x-0.0346

0.9992

59

500-500000

y=0.0011x-0.3629

0.9980

76

10-50000

y=0.0039x-3.8194

0.9986

10-50000

y=0.0019x-0.5074

0.9990

100-50000

y=0.0003x-0.2773

100-50000

y=0.0034x-0.1918

10-50000

y=0.0031x+0.0837

50-50000

y=0.0007x+0.3807

50-50000

y=0.0002x-0.2017

Inter-day

2500 ng/L spiking level % Extraction Intra-day efficiency %RSD

%RSD

MQL (ng/L)

MDL ng/L

16

10

6

13

10

2

266

10

50

6

EF

% Extraction efficiency

Intra-day %RSD

EF

266

91

15

239

103

13

271

200

82

13

216

89

10

234

250

89

10

234

101

8

M

10-50000

1000 ng/L spiking level

EF

(1000 ng/L)

261

96

3

253

93

6

245

3

50

6

12

268

93

3

245

105

7

276

3

10

2

16

155

68

2

179

99

7

261

2

500

3

16

200

74

7

195

98

6

258

7

500

15

61

17

161

60

5

158

78

2

205

5

10

6

79

10

208

69

5

182

75

7

197

5

10

6

0.9970

55

22

145

58

10

153

68

14

179

18

100

23

0.9991

76

8

200

61

4

161

101

5

266

4

100

2

0.9981

75

11

197

76

8

200

104

5

274

8

10

3

0.9977

69

9

182

77

10

203

105

8

276

10

50

26

0.9991

62

18

163

62

14

163

76

6

200

14

50

30

ep te

d

10

Ac c

EHS HS IMC 4-MBC BP3 BP1 BPA DHMB EPABA THB EHMC OC BMDM DBENZO

Calibration data

linearity

an

Range of Analytes

us

Table 2- Linearity, average of extraction efficiency (%. n=6), intra and inter-day repeatability (%RSD n=6), enrichment factor (EF= [(%Recover x (Vaq/Vsed))/100]), method quantification limit (MQL) and method detection limit (MDL) using a DLLME and GC–MS analysis.

Page 32 of 36

ip t cr

Ac c

ep te

d

M

an

us

Table 3- Occurrence of UV-filters and BPA (ng/L) in influent and effluent wastewater samples collected in Portugal.

Page 33 of 36

Page 34 of 36

d

ep te

Ac c M

an

cr

us

ip t

Ac ce p

te

d

M

an

us

cr

ip t

Electronic Supplementary Material (online publication only) Click here to download Electronic Supplementary Material (online publication only): Figure-S1.pptx

Page 35 of 36

Ac ce p

te

d

M

an

us

cr

ip t

Electronic Supplementary Material (online publication only) Click here to download Electronic Supplementary Material (online publication only): Tables-1to2 - Supplementary.docx

Page 36 of 36