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Degradation of 17β-estradiol in aqueous solution byozonation in the presence of manganese(II) and oxalicacidLiying Jiang a , Lu Zhang a , Jianmeng Chen a & Hong Ji aa College of Biological and Environmental Engineering, Zhejiang University of Technology,Hangzhou, 310032, ChinaAccepted author version posted online: 01 May 2012.Version of record first published: 25May 2012.

To cite this article: Liying Jiang , Lu Zhang , Jianmeng Chen & Hong Ji (2013): Degradation of 17β-estradiol in aqueoussolution by ozonation in the presence of manganese(II) and oxalic acid, Environmental Technology, 34:1, 131-138

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Environmental TechnologyVol. 34, No. 1, January 2013, 131–138

Degradation of 17β-estradiol in aqueous solution by ozonation in the presenceof manganese(II) and oxalic acid

Liying Jiang∗, Lu Zhang, Jianmeng Chen and Hong Ji

College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China

(Received 6 July 2011; final version received 25 April 2012 )

Natural estrogens, such as 17β-estradiol (E2), are the main substances responsible for estrogenic activity found in domesticsewage. In the work described herein, the degradation of E2 has been investigated by single ozonation and catalytic ozonationin the presence of manganese ion (Mn2+) and oxalic acid. The presence of Mn2+ and oxalic acid in the ozonation processessignificantly improved the E2 degradation and, hence, the reduction of estrogenic activity in aqueous solution. The addition ofMn2+ and oxalic acid produced many more hydroxyl radicals in the catalytic ozonation system than in the single ozonationsystem. Oxidation products formed during ozonation of E2 have been identified by means of gas chromatography–massspectrometry (GC-MS), on the basis of which a possible reaction pathway for E2 degradation by ozonation is proposed.E2 was first oxidized to hydroxyl-semiquinone isomers, and these were subsequently degraded to low molecular weightcompounds such as oxalic acid and malonic acid. The latter were easily oxidized by ozone to form carbon dioxide (CO2).The results demonstrate that the ozonation–Mn2+–oxalic acid system may serve as a powerful tool for removing E2, and theaddition of Mn2+ and oxalic acid is favourable for the complete removal of estrogenic activity induced by steroid estrogensin aqueous solution.

Keywords: catalytic ozonation; 17β-estradiol (E2); hydroxyl radicals; manganese ion; degradation pathway

Introduction

Estrogen hormones in the environment have receivedincreased attention because low concentrations (e.g. a fewng L−1) of such hormones may have adverse effects onthe normal activity of the endocrine system in both ani-mals and humans [1,2]. Published studies have shownthat these compounds can interfere with reproductive sys-tems by producing an unnatural response in the endocrinesystem [3–5]. In particular, natural estrogens such asestrone (E1) and 17β-estradiol (E2), as well as syn-thetic estrogens such as 17α-ethynylestradiol (EE2) andlevonorgestrel (LNG), have been found in the effluentof wastewater treatment plants at concentrations in theng L−1 range owing to incomplete removal during thewastewater treatment process, and are considered to bethe major compounds responsible for observed endocrine-disrupting effects in wild fish. E2, excreted from animalsand human beings, has been identified as having the high-est endocrine-disrupting activity [6,7]. High levels of E2of 150 ng L−1 in raw domestic wastewater and 64 ng L−1

in domestic wastewater treatment plant effluent have beenreported [8,9]. A recent study demonstrated that exposure toE2 (>16 ng L−1) affected the reproduction of male marinefish [10]. Furthermore, E2 resists degradation during thetypical sewage treatment operation and is released into

∗Corresponding author. Email: jiangly@zjut.edu.cn

surface waters [11]. Therefore, it is very important andnecessary to develop innovative treatment systems to dealwith estrogens, especially E2.

In recent years, ozonation has been considered to bea promising technique owing to its high efficiency inremoving these estrogen compounds [12,13]. Bila et al.reported that high E2 removal efficiencies (>99%) wereachieved with low ozone dosages, and that estrogenicactivity removal increased with ozone dosage but wasnot completely removed at pH 7 or 11 [14]. Irmak et al.showed that the time needed for complete conversion of0.1 mmol of E2 was 55 min for an applied O3 dose of15.78 × 10−3 mmol min−1, and the intermediate productsformed were determined [15]. A disadvantage of usingozonation alone for treating wastewater is that a largeamount of energy is required for ozone generation. More-over, full mineralization of estrogen is not achievable atthe O3 doses commonly used in water treatment, and con-sequently biological activity is not completely removedsince a variety of transformation products may be generated.Hence, it is important not only to identify the major initialtransformation products, but also to assess their estrogenicactivity relative to that of the parent compound.

In the last two decades, metal-catalytic homogeneousozonation, combining a transition metal with ozone,has attracted considerable interest because of the need

ISSN 0959-3330 print/ISSN 1479-487X online© 2013 Taylor & Francishttp://dx.doi.org/10.1080/09593330.2012.689358http://www.tandfonline.com

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to improve ozonation efficiency and optimize economicefficiency. Several metal-catalytic ozonation systems, suchas O3–Mn(II), O3–Fe(II), O3–Zn(II), O3–Co(II) and O3–Ni(II), have been investigated [16–20]. The degrada-tion efficiency of organics in catalytic ozonation systemsexceeds that achieved by ozonation alone. Manganese saltshave been used as catalysts for ozonation in numerousstudies [21–24]. Ni et al. studied the ozonation of 2-chlorophenol solution in the presence of various kinds ofmetallic ions, including Pb2+, Cu2+, Zn2+, Fe2+, Ti2+ andMn2+ [25]. It was found that the reaction rate increased in allcases, and the best result was obtained by using Mn2+. Maand Graham reported that the oxidation of atrazine by ozonewas greatly enhanced in the presence of a small amount ofMn(II) [26]. Xiao et al. demonstrated that a trace amount ofMn2+ accelerated the mineralization of 2,4-dichlorophenolby ozone. Furthermore, several studies have indicated thatmetal ions such as Mn(II) or Mn(IV) form complexes withsome organic compounds (e.g. oxalic acid), thereby increas-ing the rate of ozonation [27]. Xiao et al. reported that theaddition of Mn2+ coupled with oxalic acid accelerated thedegradation of 2,4-dinitrotoluene (DNT) [28].

In the present study, E2 has been selected as a targetcompound, and its degradation efficiency and mechanismsin different ozonation systems (including ozonation alone,manganese-catalysed ozonation coupled with oxalic acid)have been investigated. The objectives of this study were:(i) to compare the degradation efficiency and estrogenicactivity removal in several ozonation systems; (ii) to investi-gate the generation of hydroxyl radicals in several ozonationsystems; (iii) to propose possible reaction mechanismsfor E2 ozonation based on the by-products identified byGC-MS.

Materials and methodsChemical reagents17β-Estradiol (Sigma-Aldrich, >99% pure) was used asa model compound for catalytic ozonation. A stock solu-tion of E2 at a concentration of 0.0368 mm was preparedby diluting an initial solution of 3.68 mm (1000 mg L−1)in methanol, and was stored at 4 ◦C. Ozone was pro-duced with an ozone generator (CHYF-3A, rated flow50 mg min−1, Hangzhou Rongxin Electronic EquipmentCo., Ltd, China), and stock solutions thereof were pro-duced by sparging ozone-containing oxygen through Milli-Q water that was cooled in an ice bath. A 50 mg L−1

manganese stock solution was prepared by dissolvingMnSO4 · H2O in deionized water. An internal standard,17β-estradiol-d4, purchased from Cambridge Isotope Lab-oratories (USA), was used to quantify the target compounds.Bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing1% trimethylchlorosilane (TMCS) was purchased fromSigma-Aldrich. All the organic solvents used, as well as 4-hydroxybenzoic acid, oxalic acid and sodium thiosulphate,

were obtained from a local chemical company and usedwithout further purification. All glassware was soaked in10% HNO3, then rinsed with tap water and deionizedwater.

Ozonation procedureThe catalytic ozonation experiments were carried out in500 mL vials at room temperature (25 ◦C). The aqueoussolution consisted of Milli-Q purified water spiked with10 μM E2 and adjusted to pH 7 with 10% HCl or NaOH.Manganese(II) and oxalic acid were introduced, and thereaction was initiated by ozone solution added under vigor-ous stirring. In this study, two different ozone concentrations(15 and 40 μM), coupled with Mn2+ and oxalic acid, wereselected. Samples (5 mL) of the reaction mixture were takenafter the desired time intervals and immediately quenchedwith sodium thiosulphate. Samples were stored at 4 ◦C andwere analysed within 24 h.

Chemical analysisDetermination of E2 and intermediates17β-Estradiol and intermediates in aqueous solution wereextracted with dichloromethane, derivatized, and then anal-ysed by GC-MS [29,30]. Briefly, the extracted liquid sam-ples were spiked with 17β-estradiol-d4 and then derivatizedwith pyridine and BSTFA. The derivatized samples wereinjected into a Hewlett-Packard (HP) 6890 gas chromato-graph equipped with an HP 5-MS column (30 m × 0.25 mmi.d.; 0.25 μm film thickness) and an HP 5975 mass-selectivedetector system. Helium carrier gas was maintained at aconstant flow rate of 1.0 mL min−1. The MS was operatedin full-scan or selected-ion monitoring mode with positiveionization by electron impact. The inlet and MS transfer linetemperatures were maintained at 280 ◦C, and the ion sourcetemperature was 250 ◦C. Each sample (2 μL) was injectedin splitless mode. To separate the compounds, the followingGC column temperature programme was used: 100 ◦C for1 min, increased to 200 ◦C by a ramp of 10 ◦C min−1 and to300 ◦C by a ramp of 3 ◦ C min−1, and maintenance at 300 ◦Cfor 10 min.

Analysis of dissolved ozone, Mn2+ and hydroxyl radicalsDissolved ozone in the aqueous phase was determinedby the indigo method [31]. The concentration of Mn2+ions was determined using an inductively coupled plasmamass spectrophotometer (Agilent 7500a). Hydroxyl rad-icals react with 4-hydroxybenzoate (HDB) to produce3,4-dihydroxybenzoate (DHDB), and the concentration ofthe latter was analysed by HPLC (Agilent Technologies1200 Series) equipped with an Eclips XDB-C18 reversed-phase column (5 μm, 4.6 × 150 mm) and a UV detector(λ = 262 nm) [32]. The mobile phase buffer was a mixtureof methanol and phosphoric acid (1 mL L−1) (50:50, v/v).

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The column was operated isocratically with a flow rate of0.8 mL min−1, and was maintained at 30 ◦C.

Evaluation of estrogenic activitiesA two-hybrid yeast (Y187), containing estrogen receptor-α(ERα) and co-activator sequences, was constructed and wasprovided by the Research Centre for Eco-EnvironmentalScience, China. Yeast cell culture and yeast assay wereconducted as described by Gaido et al. [33] and Li et al.[34] with some modifications. The samples were seriallydiluted in dimethyl sulphoxide (DMSO). Aliquots (5 μL)from serial dilutions of test samples were combined with995 μL of medium containing 5 × 103 yeast cells mL−1,resulting in a test culture. Aliquots (200 μL) of the testcultures were transferred to a 96-well plate and incu-bated at 30 ◦C with vigorous orbital shaking (800 rpm)for 2 h. The cell density of the culture was then mea-sured at 600 nm. Portions (50 μL) of the test culture weretransferred to a new 96-well plate, 120 μL of Z-buffer(16.1 g L−1 Na2HPO4 · 7H2O, 5.5 g L−1 NaH2PO4 · H2O,0.75 g L−1 KCl, 0.246 g L−1 MgSO4 · 7H2O) and 20 μL ofchloroform were added, and the assay solutions were care-fully mixed (vortexing for 1.5 min). The enzyme reactionwas initiated by the addition of 40 μL of o-nitrophenyl-β-D-galactopyranoside (13.3 mmol L−1, dissolved in Z-buffer) and incubated at 30 ◦C for 60 min. The reactions

were terminated by the addition of 100 μL of Na2CO3(1 mol L−1). After centrifugation at 12,000 g for 15 min,200 μL of the supernatant was transferred to a new 96-wellplate, and the absorbance at 420 nm was measured. The β-galactosidase activity was calculated according to reportsby Ma et al. [35].

Results and discussionE2 degradation in different ozonation systemsExperiments were performed on different ozonation sys-tems to investigate the degradation efficiency of E2, includ-ing ozonation alone, ozonation–Mn2+, and ozonation–Mn2+–oxalic acid. Lee and von Gunten reported that 40 μMof ozone was depleted in less than 2 min, indicating lowstability in solution [36]. Hence, a short reaction time wasselected in these experiments.

Figure 1 presents the results of E2 degradation in thedifferent ozonation systems. When either 15 or 40 μMozone was added, the concentration of E2 decreasedwith increasing reaction time in each of the processes,that is, ozonation alone, ozonation–Mn2+ and ozonation–Mn2+–oxalic acid. The efficiency of E2 degradation washigher in the presence of Mn2+ or Mn2+–oxalic acidcompared with the case of ozonation alone, and the high-est E2 degradation efficiency was observed for ozonationwith the simultaneous addition of Mn2+ and oxalic acid.

0.0

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+Mn2+

+OA

(b)

Time (min)

+Mn2+

+Mn2+

+OA

O3

O3

O3

O3

O3

O3

Figure 1. E2 degradation in several ozonation systems: (a) O3: 15 μM O3 alone; O3 + Mn2+: 15 μM O3 coupled with 75 μM Mn2+;O3 + Mn2+ + OA: ozonation catalysed by 15 μM O3 coupled with 75 μM Mn2+ in the presence of 230 μM oxalic acid. (b) O3: 40 μM O3alone; O3 + Mn2+: 40 μM O3 coupled with 200 μM Mn2+; O3 + Mn2+ + OA: ozonation catalysed by 40 μM O3 coupled with 200 μMMn2+ in the presence of 611 μM oxalic acid. Conditions: T = 25 ◦C, initial pH 7.0, initial E2 concentration of 10 μM.

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alac

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(b):O3=40 M

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2+O3

Figure 2. Reduction of estrogenic activity after 5 min treatment by several ozonation systems: (a) O3: 15 μM O3 alone; O3 + Mn2+:15 μM O3 coupled with 75 μM Mn2+; O3 + Mn2+ + OA: ozonation catalysed by 15 μM O3 coupled with 75 μM Mn2+ in the presenceof 230 μM oxalic acid. (b) O3: 40 μM O3 alone; O3 + Mn2+: 40 μM O3 coupled with 200 μM Mn2+; O3 + Mn2+ + OA: ozonationcatalysed by 40 μM O3 coupled with 200 μM Mn2+ in presence of 611 μM oxalic acid. Conditions: T = 25 ◦C, initial pH 7.0, initial E2concentration of 10 μM.

More than 90% of the E2 was degraded within 5 min byozonation with the simultaneous addition of Mn2+ andoxalic acid in both the 15 and 40 μM ozonation sys-tems. On the basis of the above results, it is clear thatE2 was consumed very rapidly in the ozonation sys-tems, and that E2 degradation was significantly promotedwhen Mn2+ and oxalic acid co-existed in the ozonationsystem.

Estrogenic activity reduction in different ozonationsystemsIn order to test whether estrogens could be transformed intonon-estrogenic compounds by the ozonation systems, theestrogenic activities in solution were measured using a yeasttwo-hybrid system. Figure 2 shows the estrogenic activitiesremaining after 10 μM E2 was treated for 5 min in the 15and 40 μM ozonation systems.

It can be seen in Figure 2 that the estrogenicactivities in solution were decreased dramatically inthe respective ozonation systems. When 15 μM O3was added, the estrogenic activity was reduced to 1/8by ozonation alone, to 1/18 by ozonation–Mn2+ andto 1/45 by ozonation–Mn2+–oxalic acid, as expressedas a fraction of the initial activity (Figure 2a). Atan ozone dose of 40 μM, the estrogenic activity wasdecreased to 1/16, 1/36, and 1/160 in the processesof ozonation alone, ozonation–Mn2+ and ozonation–Mn2+–oxalic acid, respectively. This result suggested

that the presence of Mn2+ and oxalic acid couldincrease the degree of mineralization of E2 in ozonationsystems.

•OH production in different ozonation systemsIt is now widely accepted that ozone reacts in aqueoussolution with various organic and inorganic compounds,either by direct selective reactions of molecular ozone orthrough a radical-type reaction involving hydroxyl radicalsgenerated by the decomposition of ozone in water [37].Because of the rapid decomposition of dissolved ozone incatalytic ozonation, it was necessary to determine whethermany more hydroxyl radicals were produced in the presenceof Mn2+ or Mn2+–oxalic acid. The aromatic compound 4-hydroxybenzoic acid was used to trap •OH, and the DHDBproduced was quantified to assess the amount of hydroxylradicals. It is shown in Figure 3 that the concentrationof DHDB increased sharply within 2 min in the respec-tive ozonation systems. Furthermore, compared with thatin the case of ozonation alone, the concentration of DHDBwas increased by 16% and 23% in the processes involvingozonation–Mn2+ and ozonation–Mn2+–oxalic acid, respec-tively, which indicated that the presence of Mn2+ andoxalic acid favoured the additional production of hydroxylradicals. Thus, the greater E2 degradation and reductionof estrogenic activity could be attributed to an increaseof hydroxyl radical concentration produced by Mn2+ andoxalic acid.

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5 10 15 200.0

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DB

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+OA

Figure 3. Concentration of 3,4-dihydroxybenzoate (DHDB) inozonation systems generating hydroxyl radicals. Conditions: O3:15 μM O3 alone, O3 + Mn2+: 15 μM O3 coupled with 75 μMMn2+, O3 + Mn2+ + OA: ozonation catalysed by 15 μM O3 cou-pled with 75 μM Mn2+ in the presence of 230 μM oxalic acid.T = 25 ◦C, initial pH 7.0, initial E2 concentration of 10 μM.

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Figure 4. Concentration of dissolved manganese in ozona-tion systems. Conditions: O3 + Mn2+: 40 μM O3 coupledwith 200 μM Mn2+; O3 + Mn2+ + OA: ozonation catalysed by40 μM O3 coupled with 200 μM Mn2+ in the presence of 611 μMoxalic acid. Conditions: T = 25 ◦C, initial pH 7.0, initial E2concentration of 10 μM.

Analysis of intermediatesMn2+ concentration in solutionAs previously assumed, Mn must exist in ionic form forthe sustainable production of hydroxyl radicals in solu-tion. As shown in Figure 4, the concentration of dissolvedmanganese ions decreased slightly within 2 min, but thenremained constant within 20 min in the ozonation systems,which was consistent with the reduction of E2. This canbe rationalized in terms of the catalytic mechanism pro-posed by Xiao et al. [28]. In the ozonation systems, Mn2+catalysed the decomposition of ozone to generate hydroxylradicals, and at the same time the Mn2+ was oxidized tohigher-valent manganese oxide. Pollutants were degraded

Table 1. Main intermediates identified by GC-MS in singleozonation of E2.

Retentiontime (min) Intermediates Chemical structure

7.45 Estradiolsemiquinone

O

OHCH3

14.08 2-Hydroxyl-estradiol

CH3

OH

OH

OH

10.55 Oxalic acid

O

OHO

OH

12.50 Malonic acidO

OH

O

OH

by reaction with hydroxyl radicals, and oxalic acid reducedmanganese oxide to lower-valent Mn2+.

Mn2+ + O3 + H+ → Mn3+ + •OH + O2

2Mn3+ + 2H2O → Mn2+ + MnO2 + 4H+

•OH + organic → intermediates

MnO2 + H2C2O4 + 2H+ → Mn2+ + 2CO2 + 2H2O

Mn2+ + O3 + H+ → Mn3+ + •OH + O2

Identification of intermediates in E2 ozonationTo elucidate the structures of the intermediates, the samplesobtained after 2 min in single ozonation and Mn2+–oxalicacid catalytic ozonation were subjected to GC-MS analysis.In single ozonation, many organic intermediates were pro-duced, including 2-hydroxyl-E2, E2 semiquinone, oxalicacid. and malonic acid, as shown in Table 1. In con-trast, these intermediates were not found in the case ofMn2+–oxalic acid catalytic ozonation.

Proposed reaction pathwayBased on analysis of the intermediates, it is suggested thatE2 underwent ozonation in the case of single ozonation.The frontier densities (FEDs) for the E2 molecule havebeen evaluated by Ohko et al. [38], and it was found that

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OH

HO

OH

HO

HOOH

-H2O

OH

O

OH

OH

O

OH

OH

O

OH

OH

O

HO

O

O

HO

OH O O

HO OH

CO2+H2O

O3/ OH

O3/ OH

1

+

564

8

73

2 14

161317

1218

15910

11

E2 2-hydroxyl-E2 radical

E2 semiquinone radical

E2 semiquinone

Oxalic acid Malonic acid

Figure 5. The possible reaction pathway for E2 oxidation by ozone.

the 2FED2HOMO was higher at the phenol moiety, espe-

cially at the C10 and C3 atoms (the values were 0.508 and0.364, respectively). So, the C10 or C3 atoms should bethe sites at which the first electron is extracted. The reac-tion may be initiated by •OH at C2 and C5 on the basisof FED2

HOMO + FED2LOMO values. Referring to published

work [39], a probable pathway for the ozonation of E2 isproposed in Figure 5.

The first addition of an OH radical occurs at the C2 atom,thus producing the corresponding 2-hydroxyl-E2 radical.The attack of •OH was followed by a dehydration reaction,yielding E2 semiquinone radical as a resonance structure.

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E2 semiquinone could then be produced by direct attackof •OH at the C10 or C2 atom of the resonance structure.Subsequently, further oxidation led to breakdown of thearomatic structures, leading to organic acids and ultimatelyto carbon dioxide.

ConclusionThe catalytic ozonation of E2 in aqueous solution has beenstudied in the presence of Mn2+ and oxalic acid. Under theexperimental conditions, the addition of Mn2+ and oxalicacid significantly promoted E2 degradation and the reduc-tion of estrogenic activity. Furthermore, the presence ofMn2+ and oxalic acid favoured the additional productionof hydroxyl radicals, which accelerated E2 degradation.

The concentrations of Mn2+ and organic intermedi-ates were determined in the respective ozonation systems.Manganese maintained dissolved ion status in the catalyticozonation, and was the main catalyst of E2 ozonation. Someorganic intermediates were produced in the ozonation ofE2, and the main products were semiquinones, which werefurther oxidized to organic acids of lower molecular weight.

AcknowledgementsThis study was sponsored by the National Natural Science Foun-dation of China (Grant No. 21107097) and the Natural ScienceFoundation of Zhejiang Province (Grant No. Y5110118).

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