Differential chemical profiling to identify ozonation by-products of estrone-sulfate and first characterization of estrogenicity in generated drinking water

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 7 9 1 e3 8 0 2

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Differential chemical profiling to identify ozonation by-products of estrone-sulfate and first characterization of estrogenicity in generated drinking water Marc Bourgin a, Gae¨l Gervais a, Emmanuelle Bichon a,*, Jean-Philippe Antignac a,b, Fabrice Monteau a, Gae¨la Leroy c, Lauriane Barritaud c, Mathilde Chachignon c, Vale´rie Ingrand c, Pascal Roche d, Bruno Le Bizec a a

LUNAM Universite´, ONIRIS, Laboratoire d’Etude des Re´sidus et Contaminants dans les Aliments (LABERCA), 707, 44307 Nantes, France b INRA, Nantes, France c Veolia Environnement Recherche et Innovation, Saint Maurice, France d Veolia Environnement Recherche et Innovation, Maisons-Laffitte, France

article info

abstract

Article history:

For a few years, the concern of water treatment companies is not only focused on the

Received 22 October 2012

removal of target micropollutants but has been extended to the investigation of potential

Received in revised form

biologically active by-products generated during the treatment processes. Therefore, some

18 March 2013

methods dedicated to the detection and structural characterization of such by-products

Accepted 22 March 2013

have emerged. However, most of these studies are usually carried out under simplified

Available online 18 April 2013

conditions (e.g. high concentration levels of micropollutants, drastic treatment conditions, use of deionized or ultrapure water) and somewhat unrealistic conditions compared to that

Keywords:

implemented in water treatment plants. In the present study, a real field water sample was

Chemical food safety

fortified at the part-per-billion level (50 mg L
1) with estrone-3-sulfate (E1-3S) before being

Ozonation

ozonated (at 1 mg L
1) for 10 min. In a first step, targeted measurements evidenced a

Water

degradation of the parent compound (>80%) in 10 min. Secondly, a non-targeted chemical

Estrone-3-sulfate

profiling approach derived from metabolomic profiling studies allowed to reveal 11 ozon-

By-products

ation by-products, among which 4 were found predominant. The estrogenic activity of

High-resolution mass spectrometry

these water samples spiked with E1-3S before and after treatment was assessed by the ERCALUX assay and was found to decrease significantly after 10 min of ozonation. Therefore, this innovative methodological strategy demonstrated its suitability and relevancy for revealing unknown compounds generated from water treatment, and permitted to generate new results regarding specifically the impact of ozonation on estrone-3-sulfate. These results confirm that ozonation is effective at removing E1-3S in drinking water and indicate that the by-products generated have significantly lower estrogenic activity. ª 2013 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ33 2 40 68 78 80. E-mail address: [email protected] (E. Bichon). 0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2013.03.050

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1.

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 7 9 1 e3 8 0 2

Introduction

In recent years, there has been an increasing concern worldwide about the contamination of waters by endocrine disrupting compounds (EDCs) (Richardson and Ternes, 2011). Indeed, these compounds are suspected to have deleterious impact on animal and human health, especially regarding the reproduction and development functions (Colborn et al., 1993; Jones and Hajek, 1995; Pickering and Sumpter, 2003). A wide range of compounds has been reported as EDCs and includes (i) natural compounds like steroid hormones and (ii) anthropogenic molecules like synthetic hormones and various chemicals (pesticides, plasticizers, flame retardants, etc.). Estrone-sulfate (E1-3S) (Table 1) mainly originates from the metabolic conversion of active estrogens, estradiol and estrone, which are mainly excreted by urine as sulfated estrone conjugates (Ying et al., 2002). This compound is also the major constituent of a commercial hormone replacement therapy, namely Premarin, which is commonly used in postmenopausal women. Estrone-sulfate has been detected in many aquatic matrices like river waters (detected up to 10 ng L
1) (Isobe et al., 2003; Rodriguez-Mozaz et al., 2004) and wastewaters (detected up to 160 ng L
1) (D’Ascenzo et al., 2003; Kumar et al., 2009; Pedrouzo et al., 2009). Unlike glucuronide conjugates, sulfate forms of estrone appeared to be relatively persistent against sewage treatment (D’Ascenzo et al., 2003; Desbrow et al., 1998; Ternes et al., 1999a) and were detected in wastewater treatment plant effluents (at concentrations up to 35 ng L
1) (Koh et al., 2007; Pedrouzo et al., 2009). The deconjugation of sulfated form may subsequently occur in the environment (Andersen et al., 2003; Baronti et al., 2000; Desbrow et al., 1998; Ternes et al., 1999b) and may release the active free compound, estrone. Thus, as a potential endocrine disruptor compound, estrone-sulfate is considered as an emerging contaminant to be eliminated in the water treatment plants. Ozone-based processes have been demonstrated to be efficient in drinking water treatment for disinfection, discoloration, taste and odor control, biodegradability increase and oxidation of many organic contaminants. Among them, the most studied are pesticides, pharmaceuticals and personal

care products (Esplugas et al., 2007; Ikehata et al., 2008; Snyder, 2008) and natural and synthetic hormones (Esplugas et al., 2007; Ning et al., 2007; Snyder, 2008). However, to our knowledge, no study has been specifically dedicated to the ozonation of estrone-sulfate so far. Simultaneously to the degradation of emerging contaminants, by-products formation during water treatment has to be also monitored. The analysis of a mixture of known and unknown compounds at low concentrations is usually performed by advanced chromatography coupled to mass spectrometry (Bester, 2009; Celiz et al., 2009; Richardson, 2002; Zwiener and Frimmel, 2004). High-resolution mass spectrometry appears particularly relevant for such analyses of compounds in complex matrices, especially due to its high mass accuracy (<2 ppm) and high sensitivity (femtogram range). Consequently, the interest toward liquid chromatography e high resolution mass spectrometry (LC-HRMS) has been growing and it has been applied for the trace analysis of organic micropollutants in environmental samples, including transformation products detected by non-targeted screening (Krauss et al., 2010). A non-targeted screening consists in the differential global profiling of two different groups of samples, called hereafter ‘treated’ and ‘control’, and subsequent detection of mass spectrometric signals (ions characterized by their mass-tocharge ratios and retention times), presenting significant different abundances between both groups of samples to compare. These discriminant ions were assumed to be likely disinfection by-products. It has been notably implemented in the field of metabolomics to reveal relevant metabolites or biomarkers of interest (Courant et al., 2009; Ruan et al., 2008) for predictive diagnostic or toxicological purposes. However, non-targeted screening has been recently demonstrated to be relevant for the identification of disinfection products in conditions as close as possible to conditions applied in water treatment plants. Thus, Helbling et al. (2010) demonstrated the relevancy of non-targeted screening for the identification of biotransformation products from micropollutants. Recently, Gervais et al. (2011) developed an innovative approach for revealing transformation products generated during drinking water treatments and applied this approach for the chlorination of ethinylestradiol. Though chlorination

Table 1 e Chemical and physical properties of estrone-sulfate. Estrone-3-sulfate (E1-3S)

Structural formula

CAS #: 438-67-5 12

1

O

Molecular weight: 350.42928 g mol
1




Precursor ion [MeH] : 349.11152 amu

HO

S O

10

2

A O

14

B

4

8 7

6

17

D

C

9

5

3

O

13

11

Molecular formula: C18H22O5S

CH3

15

16

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 7 9 1 e3 8 0 2

of this compound was already widely investigated in ultrapure water, this technique was successful to determine 8 products in treatment plant conditions. Among them, six were reported for the first time. The present study reports the results obtained during a similar investigation focused on the impact of ozonation to a drinking water spiked with estrone-sulfate. The aims of this study were as follows: (i) to produce water samples and control the ozonation efficiency; (ii) to reveal ozonation by-products of estrone-sulfate by using an approach derived from the metabolomic studies, consisting in a differential non-targeted profiling strategy based on liquid chromatography coupled to high resolution mass spectrometry (LCHRMS); (iii) to propose a chemical structure for the revealed treatment by-products by functional group investigation and (HR)MSn fragment interpretation and (iv) to assess the potential estrogenic activity of these by-products generated by the ozonation treatment using ER-CALUX bioassay.

2.

Material and methods

2.1.

Reagents and chemicals

Acetonitrile, dichloromethane, ethanol and methanol were HPLC grade and purchased from Carlo Erba Reactif (Val de Reuil, France). Ammonium acetate was from Merck (Darmstadt, Germany). Ultrapure water (UPW) was produced with a Thermo Scientific Brandstread Nanopure system (Thermo Fischer Scientific, Waltham, MA, USA). Deuterium oxide (99.9 atom % deuterium) was purchased from Aldrich (Saint Louis, MO, USA). Standards of estrone-3-sulfate, 6-keto-estrone, 11-ketoestrone, 11-hydroxy-estrone and 16-hydroxy-estrone (purity>98%) were purchased from Steraloids (Newport, RI, USA). Individual stock solutions of estrone derivatives at 1 mg mL
1 were prepared for the structural characterization of ozonation by-products as follows: 5 mg of compounds were dissolved in 5 mL of ethanol. Working solutions (100 ng mL
1) were prepared by diluting 1 mL of stock solution in 9 mL of ethanol. Fifty five milligrams of sulfur trioxide pyridine complex (Merck, Hohenbrunn, Germany) were dissolved in 10 mL of pyridine. This solution was implemented to conjugate the estrone derivatives to their sulfate forms.

2.2. Water samples preparation and ozonation treatment The real field water used for the ozonation experiments was collected from a Drinking Water Treatment Plant (DWTP), preozonated then filtered through granular activated carbon. This water was considered as initially free of E1-3S, no trace of this compound being detected on the basis of targeted LC-MS/ MS measurement at the limit of quantification of 1 ng L
1. Solutions to ozonate were prepared as follows: a first stock solution of estrone-sulfate (100 mg L
1) was prepared by dissolution of 10 mg of estrone-sulfate in 100 mL of methanol. A second stock solution (1 mg L
1) was obtained by introducing 1 mL of the first stock solution in a 100 mL volumetric

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flask. Methanol was evaporated under a gentle stream of nitrogen and estrone-sulfate was redissolved in ultrapure water by placing the flask in an ultrasonic bath. Working solutions (100 ng L
1 e 10 mg L
1) were prepared after adequate dilution of the latter stock solution in drinking water. Before being fortified, the collected water sample was characterized by pH, Total Organic Carbon (TOC) and alkalinity values of 7.8, 1.83 mg L
1 and 4.4 meq L
1, respectively. Ozonation experiments were carried out according to the “Ozotest” method developed by Roche and co-workers (Roche et al., 1994) to evaluate the effects of ozonation and enable the calculation of ozone consumption and ozonation efficiency. Ozone was produced in situ by a laboratory ozone generator (BMT 802X, BMT Messtechnik, Stahnsdorf) fed with pure oxygen (Air Liquide, N48 oxygen quality). Ozone gas concentration and ozone flow rate were adjusted to 100 g m
3 and 40 L h
1, respectively. A 1.4 L sealed glass reactor was filled with one liter of water sample (spiked or not) overfilled with an extra volume of water sample. The same extra volume of gaseous ozone, ca. 10e155 mL corresponding to 1e15.5 mg, was drawn with a glass syringe from the outlet line of the ozone generator and was injected in the reactor via a septum. The excess of water was discarded through the bottom tap with the reactor over pressure in order to obtain exactly 1 L of sample to treat at atmospheric pressure and initial ozone concentration of 1e15.5 mg L
1. The reactor was then placed on an automatic agitator for 10 min. After the ozone contact, the sample was driven out by nitrogen pressure (inert gas, N50 quality) through the bottom tap. Before complete evacuation of the water sample, a small amount of water was kept in the reactor to create a water interface able to trap the gas phase in the flask. On the one hand, dissolved ozone concentration in water was determined using the indigo method as follows (Bader and Hoigne´, 1981): dissolved ozone in a 1-mL aliquot was titrated by a solution of potassium indigo trisulfonate. UV absorbances measured for the residual ozone determination were determined with a DR 4000 Hach spectrophotometer. On the other hand, residual ozone in the gas phase, trapped in the reactor, was also determined, by iodometry (IOA, 1987) as follows. Forty milliliters of a potassium iodide solution at 200 mg L
1 was introduced in the reactor with a syringe via the septum shortly after withdrawal of the aqueous phase. Glass reactor was vigorously shaken during 2 min to perform the reaction between potassium iodide and residual ozone in the gas phase. The mixture was then collected in a beaker and sulphuric acid (pH 1e2) was added. The titration of formed iodine was then performed with a NaS2O3 standard solution at the concentration of 0.01 mol L
1. Reaction in the ozonated water samples was quenched by stripping ozone with nitrogen bubbling for 10 min. Water samples were finally kept at
20  C until analysis.

2.3.

SPE-LC-MS/MS targeted analysis of estrone-sulfate

The targeted analysis of E1-3S in water samples was carried out as follows. One hundred milliliters of defrosted sample were passed through an Oasis HLB cartridge (200 mg, 6 mL), previously conditioned with 5 mL of MeOH and 5 mL of ultrapure water. After drying the cartridge, analytes were eluted

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with 10 mL of methanol-acetonitrile (95:5, v/v) and evaporated to dryness with a gentle nitrogen stream. Extracts were finally dissolved in methanol-ultrapure water (1:1, v/v) for injection. Recovery experiments of E1-3S in water samples fortified at 7 different levels (from 1 to 100 ng L
1) in the same conditions showed recoveries ranging from 60% to 70% (data not shown). Thirty-five microliters of extract were injected on a surveyor liquid chromatography system coupled to a Quantum triple-stage quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) operating in negative ionization mode. The chromatographic separation was carried out on a Thermo Fisher Scientific Hypersil BDS column (2.1  100 mm; 3 mm particle size) at 0.2 mL min
1 under the following gradient elution conditions with (A): methanol and (B): ammonium acetate (25 mM) in water: t ¼ 0 min, AeB (5:95, v/v); t ¼ 10 min, AeB (90:10, v/v); t ¼ 14.5 min, AeB (90:10, v/v); t ¼ 15 min, AeB (5:95, v/v); t ¼ 22 min, AeB (5:95, v/v). Estronesulfate was monitored in the Multiple Reaction Monitoring (MRM) acquisition mode using two distinct diagnostic signals (349.1 > 269.1, 349.1 > 145.1).

2.4.

Untargeted analysis of ozonation by-products

2.4.1.

Sample preparation and HPLC-HRMS analysis

Aliquots (500 mL) were first defrosted and then loaded on a previously conditioned solid-phase extraction (SPE) cartridges (Oasis HLB, 6 mL, 500 mg). After air-drying, analytes were eluted from cartridges with three successive 2 mL volumes of methanol, acetonitrile and dichloromethane. Collected extracts were evaporated to dryness under a gentle stream of nitrogen and reconstituted in 200 mL of a methanol-ultrapure water mixture (1:1; v/v). Final extracts were injected on an Agilent Technologies HP 1200 series liquid chromatography device equipped with a Phenomenex (Torrance, CA, USA) Gemini C18 column (50 mm  2 mm I.D., 3 mm particle size). To separate the analytes, a chromatographic gradient with acetonitrile (solvent A) and 0.1% acetic acid in water (solvent B) was applied at 0.3 mL min
1 as follows: t ¼ 0 min, AeB (1:99, v/v); t ¼ 1 min, AeB (1:99, v/v); t ¼ 15 min, AeB (100:0, v/v); t ¼ 17 min, AeB (100:0, v/v); t ¼ 18 min, AeB (1:99, v/v); t ¼ 25 min, AeB (1:99, v/ v). These HPLC conditions were later called conditions #1. MS data were acquired thanks to a Thermo Scientific (Waltham, MA, USA) LTQ-OrbitrapTM high-resolution hybrid mass spectrometer, incorporating a linear ion trap LTQ and a Fourier transform mass analyzer Orbitrap. MS data were acquired using negative electrospray ionization in full scan mode (m/z 70e800 amu) at 30,000 resolution. XCalibur v2.0.7 (Thermo Fisher Scientific, Waltham, MA) software was used for chromatogram analysis and interpretation.

2.4.2. Detection of ozonation by-products by a metabolomiclike profiling The applied global differential profiling strategy was previously described elsewhere and was based on a comprehensive analytical workflow including appropriate data processing and analyzing tools using the XCMS data processing software (Smith et al., 2006). Briefly, initially acquired LC-HRMS data files (proprietary RAW format) were converted in an exchangeable format (open

source NetCDF format) and were divided into two groups, namely ‘Control’ versus ‘Treated’ water samples. ‘Treated’ samples corresponded to water spiked with E1-3S and then treated by ozonation. ‘Control’ samples corresponded either to (i) non-spiked and non-ozonated samples, (ii) spiked and non-ozonated samples or (iii) non-spiked and ozonated samples. Global LC-HRMS profiles generated from the two groups of samples (‘Treated’ and ‘Control’ samples) were then compared with the XCMS freeware. As a result from the data processing step, a list of detected ions (characterized by their m/z and retention time values) presenting a statistically significant difference of abundance between both groups was generated. Various criteria were then employed to highlight potential compounds of interest, including statistical (fold change >2, p-value <0.05) or analytical (visualization of the corresponding extracted ion chromatograms) criteria. An accuracy error threshold of 5 amu was set as a limit to the calculation of possible elemental compositions. Additional conditions taken into consideration for calculating elemental compositions included the upper limits on the number of carbon, hydrogen, oxygen, nitrogen, sulfur and halogen atoms (set at 20, 30, 10, 5, 3 and 5 respectively), the nitrogen rule and the range of double-bond equivalent.

2.4.3. Isolation and structural identification of ozonation by-products Twenty microliters of the SPE extract, reconstituted in methanol-ultrapure water (1:1, v/v), were injected on a Transcend HPLC pump with automatic autosampler (Thermo Scientific, Whaltman, MA, USA) equipped with a Nacalai (Kyoto, Japan) Cosmosil p-Naphtyl column (250 mm  4.6 mm I.D., 5 mm particle size). The mobile phase was a mixture of acetonitrile (solvent A) and 0.1% acetic acid in ultrapure water (solvent B) applied at a flow rate of 1.0 mL min
1. These chromatographic conditions were later called HPLC conditions #2. One quarter of the flow was introduced into a Thermo Fisher (Whaltman, MA, USA) Exactive high-resolution Fourier transform mass spectrometer while the rest was collected into glass vials with a collection time of 30 s per fraction. The sample was injected 5 more times and equivalent fractions were gathered as a function of their collection time. Recombined fractions were evaporated to dryness and finally reconstituted in 150 mL of MeOH-H2O (1:1, v/v). Fragmentation experiments were conducted in order to identify the generated treatment by-products (TBPs). Collected fractions were introduced into the atmospheric pressure interface by infusion at the flow rate of 5 mL min
1 with a syringe pump. Using the LTQ-OrbitrapTM system, precursor ions were selected in the linear trap and fragmented with an activation Q of 0.250 and a relative collision energy of 40%. Product ions were scanned from the lowest possible m/z value (typically 1/3 of the precursor ion m/z) to the m/z of the precursor ion. Analogues of estrone-3-sulfate, i.e. 11-hydroxy-, 16hydroxy-, 6-keto- and 11-keto-estrone-3-sulfate, were synthesized in our lab as follows: 200 mL of a working standard solution were placed in a vial and evaporated to dryness. Extracts were reconstituted in 200 mL of acetonitrile and 200 mL of sulfur trioxide solution. Solutions were kept sealed overnight at 45  C. Mixtures were evaporated to dryness and finally

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dissolved in 200 mL of ultrapure water-methanol (1:1, v/v) before being injected in the HPLC system.

2.4.4.

Characterization of estrogenic potency by ERa-CALUX

Bioassays were performed with human bone cell lines (U2-OS), incorporating the firefly luciferase gene coupled to responsive elements as a gene reporter for the presence of estrogens. Cells were plated in 96-well plates (6000 cells/well) with phenol red-free DF medium (100 ml) supplemented with 7.5% dextran-coated charcoal-stripped FCS. Following 2-day incubation, medium was removed and replaced with fresh assay medium and cells incubated with solution to be tested for 24 h. Samples were prepared by extracting 250 mL of ozonated (1 mg O3 L
1) or non-ozonated water with preconditioned SPE cartridges (Oasis HLB, 6 mL, 500 mg). After drying cartridges, analytes were eluted with ethyl acetate (2  5 mL). Combined extracts were evaporated and samples reconstituted in DMSO (40 mL). Extracts were finally diluted 1000 times before being applied to cells. Exposed cells, express not only proteins that are under normal circumstances associated to responsive elements but also luciferase. After addition of the substrate luciferine, light is emitted and detected using a luminometer (Lucy2; Anthos Labtec Instruments, Austria). The amount of emitted light (at 562 nm) is proportional to the amount of ligand-specific receptor binding, which is benchmarked against the relevant reference compound (17b-estradiol). In our case, samples were simply assayed at a single dilution level.

5  103 and 100 ng L
1). At E1-3S initial concentrations fixed at 10  106 ng L
1, the degradation of parent compound increased with ozone dose. Indeed, the degradation efficiency was only 35% for 1 mg O3 L
1 and was almost complete (>92%) for an ozonation rate superior to 5 mg L
1. At much lower E1-3S initial concentrations (100
50  103 ng L
1), E1-3S removal was generally well advanced for an ozone dose of 1 mg L
1 within 10 min, as its value was 80% at the initial concentration of 50  103 ng L
1 and it was almost complete (>99%) at the initial concentration of 100 ng L
1. In these treatment conditions, a residual concentration of ozone was measured in both aqueous and gas phases. Ozone consumption and residuals in both phases (gas and liquid) varied as a function of ozone dose. According to ozone demand and Henry’s law, transfer efficiency, defined as the ratio (added ozone e residual ozone in the gas phase)/added ozone, varied very slightly from 50% to 61% for increasing E1-3S initial concentration. Following this evidence of the removal of E1-3S by ozonation, the detection and identification of by-products needed to be investigated. Solutions initially spiked at 50✕103 ng L
1 and ozonated at 1 mg L
1 were implemented. The slight difference in ozone transfer efficiency between 100 ng L
1 and 50  103 ng L
1 of E1-3S let us suppose that ozonation by-products would be likely the same. However, an initial E1-3S concentration of 50  103 ng L
1 is more convenient to detect a maximum of compounds.

3.2.

3.

Results and discussion

3.1. Impact of ozonation in terms of estrone-3-sulfate degradation Real drinking water samples spiked at different amounts of E13S (from 100 ng L
1e10 mg L
1) were subjected to an ozonation process. Treatment conditions, including contact time (10 min) and ozone concentration (1e15.5 mg L
1), were set to be as close as possible to real conditions used in drinking water treatment plants (i.e. not buffered, presence of organic and inorganic matter, etc.). The experimental parameters characterizing this ozonation process are summarized in Table 2. This table presents the E1-3S removal rate as a function of the ozone concentration (from 1 to 15.5 mg L
1) and E1-3S initial concentrations (from high concentrations, i.e. 10  106 ng L
1, to environmental concentrations, i.e. 50  103,

Detection of the E1-3S ozonation by-products

After the acquisition of global LC-HRMS profiles for all samples of interest, a preliminary visual comparison of the global chromatographic traces (TIC) obtained in Full Scan mode between ‘Control’ and ‘Treated’ samples was undertaken. Fig. 1a, corresponding to the chromatogram of a spiked but non-ozonated water sample, shows the presence of different peaks, among which the diagnostic signal of E1-3S (Rt ¼ 9.2 min) and these of components naturally occurring in the matrix (Rt ¼ 9.4e15.8 min). Regarding the chromatogram of the non-spiked and ozonated water samples (Fig. 1b), only the peaks associated to the naturally occurring organics and their ozonation by-products (Rt ¼ 9.4e15.8 min) were present. The analysis of a spiked and ozonated sample led to the chromatogram presented on the Fig. 1c, showing the presence of various peaks. Among these, those corresponding to residual E1-3S, naturally occurring organics, and their ozonation by-products, were still present. However, 4 other peaks at lower

Table 2 e Effect of ozone on estrone-sulfate removal (%) and related operational parameters. E1-3S initial concentration (ng L
1)
1

Ozone dose (mg L ) E1-3S removal (%) Residual O3 in water (mg L
1) Residual O3 in gas(mg L
1) Consumed O3 (mg L
1) Transferred O3 (mg L
1) Transfer efficiency (%)

10  106

10  106

10  106

10  106

10  106

50  103

5  103

100

15.5

9.5

5

2

1

1

1

1

>99 0.35 1.71 13.44 13.79 89

>99 0.00 0.41 9.09 9.09 96

92 0.01 0.02 4.97 4.98 100

70 0.01 0.00 1.99 2.00 100

35 0.02 0.00 0.98 1.00 100

80 0.44 0.39 0.16 0.61 61

91 0.22 0.45 0.33 0.55 55

>99 0.21 0.50 0.28 0.50 50

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Fig. 1 e Total ion currents (TICs) obtained for (a) a E1-3S-spiked and non-ozonated sample, (b) a non-spiked and ozonated sample and (c) a spiked and ozonated sample.

retention times (Rt ¼ 7.5e8.5 min) could correspond to E1-3S ozonation by-products. Consequently, the preliminary visual interpretation of some relevant extracted ion chromatograms (EICs) enabled to determine 4 predominant ozonation byproducts. However, our metabolomic-like approach, based on a comprehensive data processing, was subsequently implemented to potentially reveal more ozonation by-products. This strategy (comprehensive deconvolution of the acquired profiles using XCMS software and dedicated algorithm and script) ensured the extraction of a total of 576 ions of potential interest from the generated global chemical profiles. After the examination of the corresponding extracted ion chromatograms, 86 ions presenting significant difference between ‘Treated’ and ‘Control’ groups were selected as more particularly relevant. After grouping MS signals possibly assigned to identical compounds (information redundancy commonly observed in MS due to the presence of isotope contributions, adducts and/or in-source fragment products.), the list of these ions was finally reduced to 11 potential compounds of interest. The list of the 11 putative by-products highlighted by this approach is presented in Table 3. Four ozonation by-products, namely TBP4, TBP7, TBP10 and TBP11, corresponded to the 4 compounds previously observed on the TICs (Fig. 1c). All the retention times of these 11 compounds were lower than this of E1-3S, confirming the higher polarity of the ozonation by-

products, in accordance with the basic chemical principles of ozonation reactions (Bailey, 1982). The mass-to-charge ratios of proposed raw chemical formulae (Table 3) were close to observed m/z ratios with accuracies within 1.3 amu. Proposed raw chemical formulae were similar to that of estrone-sulfate as the number of carbon and sulfur atoms were the same as the number of these atoms in the E1-3S raw chemical formulae; only the number of oxygen and hydrogen atoms increased. Consequently, proposed ozonation by-products appeared to be oxidized forms of estrone-sulfate. Moreover, the abundances of the products were estimated on the basis of the ratio between the associated peak and the peak of estrone-sulfate before ozonation, and hypothesizing that all the studied compounds presented identical response factors. Indeed, as ionization certainly occurred on the sulfate group, responses factors may be considered similar for the parent compound and its ozonation by-products. Table 3 confirms the presence of 4 predominant by-products after 10 min of treatment, namely TBP4, TBP7, TBP10 and TBP11, with a respective abundance of 11, 8, 17 and 11%, compared to estrone-sulfate before treatment. The abundance of other byproducts was very weak (<1%). Moreover, the sum of peak areas for all the compounds detected after treatment (ozonation by-products and residual estrone-sulfate) was lower (67%) than the initial area of estrone-sulfate, before treatment.

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Table 3 e List of highlighted ozonation by-products of estrone-sulfate with their respective retention time, proposed raw formula, ring and double bond equivalents (RDBE), mass accuracy and abundance after contact time. Compound Rt (min) TBP1 TBP2 TBP3 TBP4 TBP5 TBP6 TBP7 TBP8 TBP9 TBP10 TBP11 E1-S

6.71 6.78 6.92 7.55 7.57 7.74 7.80 8.00 8.13 8.26 8.50 9.25

Observed Raw formula RDBE Theoretical Mass accuracy Relative abundance monoisotopic mass hypothesis monoisotopic mass (D amu) after treatmenta 381.1007 393.0645 379.0852 365.1060 395.0801 397.0958 379.0854 399.1116 377.0698 365.1057 363.0903 349.1121

C18H22SO7 C18H18SO8 C18H20SO7 C18H22SO6 C18H20SO8 C18H22SO8 C18H20SO7 C18H24SO8 C18H18SO7 C18H22SO6 C18H20SO6 C18H22SO5

8 10 9 8 9 8 9 7 10 8 9 8

381.1019 393.0654 379.0862 365.1070 395.0811 397.0968 379.0862 399.1125 377.0705 365.1070 363.0913 349.1108

1.2 1.0 1.0 1.0 1.0 1.0 0.8 0.9 0.7 1.3 1.0 1.2

<1% <1% <1% 11% <1% <1% 8% <1% <1% 17% 11% 20%

a calculated as the mean ratio (n ¼ 8) of the area associated peak to the mean area (n ¼ 4) of estrone-sulfate at initial conditions (by supposing that response coefficients were equal for all compounds).

This may be explained by two reasons: (i) the hypothesis of similar response factors is inadequate or (ii) the existence of other compounds generated during the ozonation treatment but not detectable using the present analytical conditions.

3.3. Fractionation and structural elucidation of ozonation by-products Preparative chromatography was first performed and ensured the isolation of the ozonation by-products. Isolated by-products were then injected into atmospheric pressure interface in negative electrospray mode by infusion. It appeared that only the 4 predominant compounds, namely TBP4, TBP7, TBP10 and TBP11, were collected in a sufficient amount to determine their structures. A first approach implemented for structural characterization is based on hydrogen/deuterium (H/D) exchange method: mass spectra of purified by-products, dissolved either in ultrapure water or in deuterium oxide, were generated and the difference in molecular weight was measured to determine the number of exchangeable hydrogen atoms in the molecule. A second approach for the structural assignment was based on the interpretation of fragmentation pathways and the comparison with fragmentation patterns of commercial standards, if available. This technique was usually supported by multiple-stage MSn mass spectrometry using collisioninduced dissociation. High mass accuracy measurements enabled the determination of the molecular formula of precursor and fragment ions, as presented in Table 4. The MS2 spectrum of estrone-sulfate showed a unique fragment ion at m/z 269.1533, probably corresponding to the desulfated ion. The MS3 spectrum showed the presence of different fragment ions ranging from m/z 145.0652 to m/z 253.1228. Elemental compositions were proposed for these fragments ions, according to the formula predictor software, and presented in Table 4. A fragmentation pathway was proposed in Fig. 2 and suggested the successive opening of ring D and ring C, as proposed by Bourcier and her co-workers (Bourcier et al., 2010). The detailed mass spectrometric analysis of the fragmentation pattern of estrone-sulfate provides a

basis to determine structural assignment for the ozonation by-products. To our knowledge, no study has been investigated sulfateconjugated phenolic compounds yet. Sulfate group is an electron-withdrawing group so its presence on the aromatic group may hinder the reaction usually observed on phenolic compounds, especially the formation of hydroxylated compounds at the ortho-position by electrophilic substitution. Moreover, due to the substantial deactivation of the conjugated phenol ring toward electrophilic reaction, ozonation reaction was assumed to be likely dominated by attack of hydroxyl radicals HOC. Hydroxyl radicals are more oxidative and less selective than molecular ozone and its reaction rate constants are about 4e5 orders of magnitude greater than rate constants of ozone for the same micropollutant (Dodd et al., 2006; Huber et al., 2003). Accordingly, hydroxyl radicals were likely crucial active species during the generation of ozonation by-products and may be able to oxidize many sites on estronesulfate molecule.

3.3.1.

Compound TBP11

Compound TBP11 was eluted at the retention time of 8.50 min with HPLC conditions #1 and showed a deprotonated molecular ion at m/z 363.0903, i.e. 14 amu higher than that of estrone-sulfate, probably corresponding to the raw chemical formula of C18H20SO6. Moreover, no change in m/z was observed during the hydrogen/deuterium exchange experiment, suggesting that TBP11 could be a carbonylated form of estrone-sulfate. By-product TBP11 and both custom-made compounds at m/z 363.0909, i.e. 6-keto-estrone-sulfate and 11-keto-estrone-sulfate, were injected individually in HPLC conditions #2 to determine their experimental HPLC retention times (Table 4). They were measured at 25.62 min, 25.67 min and 24.19 min for TBP11, 6-keto-estrone-sulfate and 11-ketoestrone-sulfate, respectively, indicating that TBP11 may be considered as 6-keto-estrone-sulfate. The MS2 spectrum of TBP11 and 6-keto-estrone-sulfate were thus compared and both showed only one fragment ions at m/z 283.1340, probably associated to a loss of SO3. No [MHeH2SO4]- ion (by the step-wise loss of SO3 then H2O, i.e.

3798

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 7 9 1 e3 8 0 2

Table 4 e List of the main ozonation by-products of E1-3S revealed by the metabolomic-like approach and synthesized analogues of E1-3S characterized by their retention times in two specific HPLC system, their fragmentation pattern and their proficiency for H/D exchange. (*) indicates the precursor selected for further CID fragmentation. Compound

Retention time with HPLC conditions #1 (min)

MS2

MS

MS3

m/z

Assignment

m/z

Assignment

m/z

Assignment

239.1439 225.0921 223.1128 209.0972 195.0815 171.0814 265.0868 253.0869 237.0920 225.0920 185.0607 171.0450 239.1076 225.1282 209.0971 195.0815 171.0815 267.1021 173.0604 159.0449 253.1228 225.1280 225.0915 171.0811 159.0806 145.0652 269.1170 241.0856 227.1064 187.0754 175.0755 161.0599 269.1167 241.0855 187.0754 175.0755 161.0599 251.1071 239.1072 225.1280 209.0966 171.0811 213.1282 171.0812 159.0812 145.0656 267.1013 173.0599 159.0443 255.1014 239.1066 227.1067 185.0963 171.0807 145.0652

C17H19O
C15H13O
2 C16H15O
C15H13O
C14H11O
C12H11O
C17H13O
3 C16H13O
3 C16H13O
2 C15H13O
2 C12H9O
2 C11H7O
2 C16H15O
2 C16H17O
C15H13O
C14H11O
C12H11O
C17H15O
3 C11H9O
2 C10H7O
2 C17H17O
2 C16H17O
C15H13O
2 C12H11O
C11H11O
C10H9O
C17H17O
3 C15H13O
3 C15H15O
2 C12H11O
2 C11H11O
2 C10H9O
2 C17H17O
3 C15H13O
3 C12H11O
2 C11H11O
2
C10H9O2 C17H15O
2 C16H15O
2 C16H17O
C15H13O
C12H11O
C15H17O
C12H11O
C11H11O
C10H9O
C17H15O
3 C11H9O
2 C10H7O
2 C16H15O
3 C16H15O
2 C15H15O
2 C13H13O
C12H11O
C10H9O


TBP4

7.55

365.1060*

C18H21SO
6

285.1484 267.1379*

C18 H21 O
3 C18 H19 O
2

TBP7

7.80

379.0854*

C18H19SO
7

299.1279 281.1175*

C18 H19 O
4 C18 H17 O
3

TBP10

8.26

365.1057*

C18H21SO
6

285.1483 267.1378*

C18 H21 O
3 C18 H19 O
2

TBP11

8.50

363.0903*

C18H19SO
6

283.1326*

C18 H19 O
3

E1-3S

9.25

349.1121*

C18H21SO
5

269.1533*

C18 H21 O
2

2-hydroxy-estrone3-sulfate

n.a.

365.1070*

C18H21SO
6

285.1483*

C18 H21 O
3

4-hydroxy-estrone3-sulfate

n.a.

365.1077*

C18H21SO
6

285.1483*

C18 H21 O
3

11-hydroxy-estrone3-sulfate

n.a.

365.1067*

C18H21SO
6

285.1494 267.1387*

C18 H21 O
3 C18 H19 O
2

16-hydroxy-estrone3-sulfate

n.a.

365.1067*

C18H21SO
6

285.1493*

C18 H21 O
3

6-keto-estrone3-sulfate

n.a.

363.0909*

C18H19SO
6

283.1340*

C18 H19 O
3

11-keto-estrone3-sulfate

n.a.

363.0909*

C18H19SO
6

283.1339*

C18 H19 O
3

H/D exchange

Retention time with HPLC conditions #2 (min)

Yes (þ1)

17.46

Yes (þ1)

21.06

Yes (þ1)

22.14

No

25.62

No

27.98

Yes (þ1)

26.83

Yes (þ1)

27.85

Yes (þ1)

22.25

Yes (þ1)

21.17

No

25.67

No

24.19

3799

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 7 9 1 e3 8 0 2

O

O

O

O

m/z 253.1228

m/z 225.0915

O

O

O

m/z 269.1533

O

m/z 171.0811

m/z 225.1280

O

O

m/z 145.0652

m/z 159.0806

Fig. 2 e Proposed fragmentation pathway of estrone in ESI(L) mode.

98 amu) was observed during the fragmentation of the precursor ion, confirming that no free hydroxyl group was present in this molecule. The MS3 spectra of both compounds presented many similar peaks. Among them, the most intense were m/z 267.1013, 173.0599 and 159.0443. The presence of a fragment ion at m/z 267.1013, probably corresponding to a raw chemical formula C17H15O3 (predicted 267.1016 amu), can be assigned to be formed after the loss of CH4, while the fragment ion at m/z 159.0443, probably corresponding to C10H7O2 (predicted 159.0441 amu), may be generated by the opening of ring C. On the other hand, the presence of ion at m/z 173.0599,

attributed to C11H9O2 (predicted 173.0603), may be due to the direct opening of ring C. Further MS4 fragmentation experiments on this latter fragment ion showed the formation of fragment ion at m/z 145.0288 (data not shown), probably following the opening and the rearrangement of ring B, as proposed in Fig. 3. Anyway, the proposed fragmentation pathway may suggest that carbonyl group is present on the ring B. In spite of these results, fragmentation experiments of 11-keto-estrone-3-sulfate were carried out. Its MS2 spectrum presented an unique peak at m/z 283.1339, similarly to TBP11

O

O O

O

m/z 159.0443

O O

m/z 267.1013

O

O

O O O

m/z 173.0599

O

m/z 145.0288

m/z 283.1340

Fig. 3 e Proposed MS3 fragmentation pathway of 6-keto-estrone-3-sulfate in ESI(L) mode using MS2 base peak (m/z 283.1340) as precursor ion.

3800

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 7 9 1 e3 8 0 2

O

O O

O

m/z 239.1066 O

O

m/z 171.0807

O O

m/z 255.1014

O

m/z 283.1339

O

O

m/z 185.0963 O

O

m/z 227.1067

m/z 145.0652

Fig. 4 e Proposed MS3 fragmentation pathway of 11-keto-estrone-3-sulfate in ESI(L) mode using MS2 base peak (m/z 283.1339) as precursor ion.

and 6-keto-estrone-sulfate, but the MS3 spectrum of 11-ketoestrone-3-sulfate was found totally different. Indeed, it presented major peaks at m/z 255.1014, 239.1066, 227.1067, 185.0963, 171.0807 and 145.0652. A proposed fragmentation pathway was proposed for 11-keto-estrone-3-sulfate in Fig. 4 and confirmed that the carbonyl group was present on ring C. Finally, the HPLC retention times experimentally determined and the mass spectral data confirmed that TBP11 may not be the custom-made compound 11-keto-estrone-3-sulfate but rather 6-keto-estrone-3-sulfate.

3.3.2.

Compounds TBP4 and TBP10

Compounds TBP4 and TBP10 were eluted in HPLC conditions #1 at the retention time of 7.55 min and 8.26 min, respectively. They showed a deprotonated molecule at about m/z 365.1060, corresponding to a gain of 16 amu compared to estronesulfate and probably assigned to C18H22SO6. Hydrogen/deuterium exchange experiments on these compounds confirmed the presence of one exchangeable hydrogen on this compound, suggesting that TBP4 and TBP10 were probably 2 isomeric compounds of hydroxy-estrone-sulfate. Therefore, collected data for TBP4 and TBP10 were compared with those of synthesized compounds, 2-hydroxy, 4-hydroxy-, 11hydroxy- and 16-hydroxy-estrone-3-sulfate. The MS2 spectra of compounds TBP4 and TBP10 showed two fragment ions at about m/z 285.1484 and 267.1379, respectively corresponding to the loss of SO3 and H2SO4 and indicating that hydroxyl groups may be present on an alicyclic ring rather than on aromatic ring. Indeed, MS2 experiments carried out on 2hydroxy- and 4-hydroxy-estrone-3-sulfate showed the presence of a single main fragment ion at m/z 285.1483. Similarly, in the MS2 spectra of the custom-made compound, 16hydroxy-estrone-3-sulfate, no fragment ion at m/z 267.1379 was observed but a fragment ion at m/z 213.1282, probably originating from the total opening of ring D. Therefore,

according to the retention times and the acquired mass spectral data, compounds TBP4 and TBP10 could not be identified as 2-hydroxy-, 4-hydroxy- and 16-hydroxy-estrone3-sulfate. Consequently, the reactivity of sulfate-conjugated phenolic compounds appeared to be different to that of nonconjugated compounds. Further MS3 experiments were however carried out on all these compounds presenting a mass-to-charge ratio of 365.1070 originating either from the ozonation or from benchscale chemical synthesis. Compounds TBP4 and TBP10 presented similar MS3 spectral data but some differences for ions at m/z 239.1 and 225.1 were revealed thanks to the use of highresolution mass spectrometry. Indeed, fragment ions at m/z 239.1439 and m/z 225.0921 obtained after MS3 fragmentation of TBP4 were respectively attributed to C17H19O and C15H13O2, while fragment ions at m/z 239.1076 and m/z 225.0920 obtained by the MS3 fragmentation of TBP10 were supposed to be C16H15O2 and C16H17O. Consequently, both ozonation byproducts presented different fragmentation pathways. Additionally to their close retention times, TBP10 and 11hydroxy-estrone-3-sulfate presented many similar fragment ions at m/z 239.1076, 225.1282, 209.0971 and 171.0815 in the MS3 spectra (Table 4). Consequently, the assignation of TBP10 to 11-hydroxy-estrone-3-sulfate is plausible but the collected information was not sufficient to confirm that. Concerning TBP4, the huge differences observed in terms of HPLC retention times with synthesized hydroxy-estrone-3sulfate compounds hypothesized that TBP4 can be none of the custom-made compounds. Besides, collected spectral data did not allow proposing a structure for this ozonation by-product.

3.3.3.

Compound TBP7

TBP7, eluting at the retention time of 7.80 min in HPLC conditions #1, was attributed to an elemental composition of C18H20SO7 ([M
H]
ion at m/z 379.0854). Considering the

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 3 7 9 1 e3 8 0 2

results of the H/D exchange method, TBP7 could have only one functional group with exchangeable hydrogen. Consequently, TBP7 could correspond to a carbonylated and hydroxylated form of estrone-sulfate. The MS2 spectrum of TBP7 showed two fragment ions at m/z 299.1279 and 281.1175, respectively formed following the loss of SO3 and H2SO4, indicating the presence of one hydroxyl group on a saturated ring rather than on the aromatic ring. Further MS3 experiments led to the formation of several fragment ions at m/z 265.0868, 253.0869, 237.0920, 225.0920, 185.0607 and 171.0450. The presence of this latter fragment ion, assigned to the chemical formula C11H7O2 (predicted m/z 171.0452), is noteworthy and may imply that the carbonyl function may be present on ring B, probably in position 6 according to the same observation as previously for TBP11. However, information collected from the observed fragment ions did not enable to identify the position of hydroxyl group. Therefore, according to the mechanism of ozonation reactions, including here the likely attack of hydroxyl radicals HOC and the proposed chemical structure of the revealed byproducts, the ozonation of estrone-3-sulfate may first lead to the formation of 2 main hydroxylated analogues TBP4 and TBP10, the latter probably being 11-hydroxy-estrone-3sulfate, and 1 carbonylated analogue TBP11, identified as 6keto-estrone-3-sulfate. Then, further oxidation of one of the hydroxylated compounds or the carbonylated one may generate a carbonylated-hydroxylated analogue of estrone-3sulfate, namely TBP7. Here, it is noteworthy to mention that identified byproducts were also detected in the solution initially concentrated at 100 ng L
1 (data not shown). This information confirmed the hypothesis, presented in paragraph 3.1, that ozonation by-products may be the same in both solutions initially concentrated at 100 and 50  103 ng L
1.

3.4.

Effects of ozonation on the estrogenic activity

ER-a-CALUX was selected to measure estrogenic activity in water samples due to its high performances. Indeed, recently, Leusch and his co-workers compared five in vitro bioassays (yeast estrogen screen, ER-CALUX, MELN, T47D-KBluc and ESCREEN) to determine estrogenic activity in environmental waters (Leusch et al., 2010). ER-CALUX was found to be the most relevant bioassays with high robustness, sensitivity, reproducibility and low limit of quantification. The estrogenic activity, expressed in our study as estradiol equivalent EEQ (i.e. equivalent ng of 17b-estradiol per liter of water), was monitored for 2 collected water samples (initially spiked at 50 mg L
1, after 0 and 10 min of ozonation). Before ozonation, the estrogenic activity of E1-3S solution was measured at 1.4 ng L
1 EEQ (repeated twice, data not shown). At the collection time of 10 min, duplicated measured values of estrogenic activity were either lower than the limit of quantification (0.06 ng L
1 EEQ) or equal to 0.37 ng L
1 EEQ (data not shown), indicating a significant decrease of estrogenic activity after the ozonation treatment. Similarly, the response for a solution not spiked and ozonated for 10 min was inferior to the method limit of detection (0.04 ng L
1 EEQ). According to HPLC analyses, approximately 20% of the initially-dosed E1-3S concentration remained in the E1-3S-spiked drinking water

3801

samples following treatment with ozone. By comparison, ozone-treated samples of E1-3S-spiked drinking water elicited estrogenicity responses of a comparable level (i.e., on the order of 20%, accounting for experimental error) relative to initial levels in untreated controls. This suggests that treatment of E1-3S with ozone leads to generation of by-products with relatively low aggregate estrogenicity compared to the parent compound. Unfortunately, the lack of more precise E13S dose-response calibration data within the present study precludes a definitive conclusion in this regard. Nevertheless, it can be concluded on the basis of the available evidence that ozonation under exposure conditions appropriate to practical application yields substantial elimination of E1-3S0 estrogenicity. In spite of these results, the monitoring of complementary biological activities (acute toxicity, chronic toxicity, etc.) would be necessary to characterize more broadly the potential toxicity associated to the generated by-products.

4.

Conclusions

In this study, ozonation had been demonstrated for the first time to decrease significantly the concentration of E1-3S during drinking water processes. Indeed, E1-3S was degraded to below method detection limit in realistic conditions of ozonation of drinking water treatment plant. In parallel with the removal of E1-3S, some ozonation by-products were generated, then revealed and furthermore identified using an innovative post-acquisition metabolomic-like approach. This untargeted screening method enabled to reveal 11 ozonation by-products not previously reported in the literature, four of them being found predominant. Complementary experiments including H/D exchanges and fragmentation by (HR)MSn were carried out to elucidate their chemical structure. In parallel, ER-Calux bioassays were performed to evaluate estrogenic activities of water samples and showed a significant decrease of biological activity after ozonation treatment. Accordingly, the developed approach combining a comprehensive screening of by-products by a metabolomiclike approach and the monitoring of the estrogenic activity of treated waters, was confirmed as a promising strategy to assess the efficiency of a water treatment on emerging contaminants.

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