Chemosphere 111 (2014) 13–17

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The impact of chromophoric dissolved organic matteron the photodegradation of 17a-ethinylestradiol (EE2)in natural waters

http://dx.doi.org/10.1016/j.chemosphere.2014.03.0620045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +48 585236842; fax: +48 585236678.E-mail address: ocewg@ug.edu.pl (W. Grzybowski).

Waldemar Grzybowski ⇑, Jerzy SzydłowskiGdansk University, Institute of Oceanography, Al. Pilsudskiego 46, 81-378 Gdynia, Poland

h i g h l i g h t s

� Photodegradation of 17a-ethinylestradiol (EE2) was studied natural water samples.� Photodegradation was enhanced in presence of chromophoric dissolved organic matter.� Half-life times in river and sea water were about one and two days, respectively.

a r t i c l e i n f o

Article history:Received 25 November 2013Received in revised form 10 March 2014Accepted 15 March 2014

Handling Editor: Klaus Kümmerer

Keywords:17a-EthinylestradiolPhotodegradationSunlightChromophoric dissolved organic matter

a b s t r a c t

17a-Ethinylestradiol (EE2), the potent estrogen which forms the basic constituent of the contraceptivepill, can undergo degradation in natural waters by sunlight and via secondary reactions initiated byphoto-excited dissolved organic matter. The current paper presents the findings of an investigation intothe irradiation process of EE2 when dissolved in natural waters. This investigation was carried out undersimulated sunlight in samples of sea, river and distilled water at a 17a-ethinylestradiol concentration of300 ng L�1.

Several notes of significance may be made on the basis of these results. Firstly, an enhancement of thedegradation, observed in the presence of co-absorbing dissolved organic matter, was shown to beproportional to the absorbance of the sample. Secondly, the kinetics of the process obtained during thisinvestigation were within the range of previously reported findings, despite the fact that significantlyhigher concentrations of EE2 were used in earlier studies. Finally, the environmental half-life times for17a-ethynyloestradiol, calculated from the results of the experiments, were found to be one and twodays in the top layer of river and sea water respectively.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

17a-Ethinylestradiol (EE2), the basic constituent of the contra-ceptive pill, is released into surface waters from communal sewagetreatment plants and, despite occurring at very low concentrations(in the range of ng L�1), can exert noticeable effects on aquatic bio-ta (Sumpter and Johnson, 2005; Kidd et al., 2007). It is not onlymore stable in the natural environment than natural estrogens(Ying and Kookana, 2003; Robinson and Hellou, 2009) but also pos-sesses the greatest estrogenic potency (Thorpe et al., 2003; van denBelt et al., 2004).

The light absorption band of EE2 partly overlaps with the solarspectrum and this xenobiotic may therefore be subject to

degradation via direct/primary photoreactions. Indeed, there arestudies showing that radiation representative to that of sunlight(wavelength > 280 nm) decreases the concentration of 17a-ethyn-yloestradiol in solutions (Table 2). Aside from direct photolysis, asignificant role in the removal of EE2 is played by indirect(sensitized) reactions initiated by other light absorbers. Reportsof experiments with natural water samples (Table 2) clearly showenhancement of EE2 photodegradation as a result of secondaryreactions initiated by photo-excited chromophoric dissolved or-ganic matter (CDOM), the main component in the pool of dissolvednatural organic substances. This material, referred to in the past ashumic substances, is the most important sunlight absorber in nat-ural waters (Lean, 1998). Its significance in aquatic photochemistryhas been proved in numerous studies (see Mopper and Kieber,2002 and references therein).

Table 1Physico-chemical characteristics of irradiated waters.

pH Salinity (practical salinity units) DOC (mg L�1) Fetot (lM) NO3� + NO2

� (lM)

Baltic Sea 8.1 6.9 4.9 ± 0.2 b.da b.db

Vistula River 7.9 0.2 10.8 ± 0.3 0.18 ± 0.05 6.2 ± 1.2

a Below detection limit (0.02 lM).b Below detection limit (0.8 lM).

Table 2Degradation rate constant (k) and half-life for EE2 photodegradation under different experimental conditions.

EE2 concentration (solute) k (h�1)/half-life time Irradiation device; optical path length (l) Source

300 ng L�1

(Distilled water) 0.04 ± 0.02/17 h See text This study(Baltic Sea) 0.06 ± 0.02/12 h(Vistula River) 0.11 ± 0.03/6 h0.6 mg L�1

(Lake water) 0.03/23 h Sunlight, summer sunny day 41�N; l = 1.4 cm Zuo et al. (2013)10 lg L�1

(Distilled water) 0.08 ± 0.02/7 h Solar simulator k > 300 nm; l = 1 cm Whidbey et al. (2012)(10 mg L�1 Solution of Swuanee River fulvic acid) 0.13 ± 0.03/5 h500 ng L�1

(Distilled water) 0.013 ± 0.006/53 h Fluorescent lamp UVB = 133 lW cm�2 Atkinson et al. (2011)(Ottawa River) 0.001 ± 0.004/693 h(Lake Cromwell) 0.021 ± 0.014/33 h(Raisin River) 0.010 ± 0.011/69 h1 mg L�1

(Distilled water) �0.09/7 h Fluorescent lamp UVA = 115.6 Wm�2; k > 300 nm; l = 25.5 cm Puma et al. (2010)10–40 mg L�1

(Distilled water) 0.61/1 h Sunlight simulator (300–800 nm); l � 2 cm Matamoros et al. (2009)(Ebre river) 0.69/1 h(Besos River) 0.73/1 h(Mediterranean Sea) 0.62/1 h(Besos River) 0.007/99 h Sunlight, May, 41�N; l � 2 cm0.150 mg L�1

(Distilled water) �0.12/6 h Fluorescent lamp (k > 290 nm); l = 6.75 cm Mazellier et al. (2008)8 mg L�1

(Estuarine seawater) �0.05/14 h Sunlight 41�N; l = 1.4 cm Zuo et al. (2006)1–2 lg L�1

(Milli-Q water) 0.02 ± 0.002/28 h Sunlight simulator (290–700 nm) 765 W m�2; l = 1.5 cm Lin and Reinhard (2005)(Santa Ana River) 0.30 ± 0.015/2 h100 lg L�1

(Thames River) 0.0055/126 h Solar light simulator Jürgens et al. (2002)

14 W. Grzybowski, J. Szydłowski / Chemosphere 111 (2014) 13–17

In the majority of laboratory experiments EE2 concentrationsexceeded ‘‘natural’’ levels by three to six orders of magnitude.Those results can still be reasonably extrapolated to natural aqua-tic systems, however, provided that the irradiated water samplecontains low amounts of other light absorbing species (as is thecase, for example, with ocean water), thus showing EE2 photodeg-radation to be driven mainly by the primary reactions. However, insamples of high concentration of natural organic matter the reac-tions sensitized by CDOM have to be taken into account. As alreadymentioned, much of the research has been conducted with EE2concentrations comparably high to that of natural organic matter.The question arises therefore whether the relative share of indirectreactions could have been underestimated in those studies. This is-sue was addressed within the current study which purpose was toexamine the photodegradation of EE2 in solutions where itsconcentration was several orders of magnitude lower than thatof natural organic matter.

2. Materials and methods

River and sea water samples were taken from the Vistula Riverat Tczew (Poland) and from Gdansk Bay (Baltic Sea) respectively.The samples were aged at room temperature for several months.The physico-chemical parameters were determined for water

samples filtered through �0.8 lm Whatman GFF filters. The con-centration of dissolved organic carbon was measured using a Shi-madzu TOC-5000 analyser. The total iron concentration wasdetermined by a colorimetric method (Collins et al., 1959) using100 mm long glass cuvette. Prior to performing the analysis thesamples were UV irradiated in order to release organically boundiron (for details see Kononets et al., 2002). The sum of nitrateand nitrite was measured colorimetrically as described by Grashoffet al. (1983). The changes in oxygen concentration in water weremonitored using a dissolved oxygen meter.

Directly before photochemical experiment, the filtered samplesof river, sea and Millli-Q water were shaken vigorously for 5 min inglass jars then enriched with 17a-ethinylestradiol (Merck) to con-centration of 300 ng mg L�1 and poured into quartz glass tubes(1 cm inner diameter). The tubes contained small air-filled head-spaces. The samples were irradiated in triplicates, with the tubestightly sealed with ground glass stoppers to prevent evaporationand dark control samples additionally covered with aluminum foil.The tubes were placed horizontally in a shallow water bath, thetemperature of which was kept below 30 �C using tap watercooling, and irradiation was then performed using a solar simula-tor (SOL 500, Dr. Hönle, Germany) equipped with a H1 filter.Sub-samples of 0.5 ml were withdrawn during the irradiationprocess. Radiation intensity was measured with UV-B SKU 430(280–315 nm) and UV-A SKU 420 (315–380 nm) broadband

irra

dian

ce [

W m

-2]

abso

rban

ce

wavelength [nm]

300 ng/l EE2

river water

sea water

irradiance

Fig. 1. Emission spectrum of the light source and absorbance of 0.01 m deep layers of Vistula River and Baltic Sea water (absorbance of EE2 was multiplied by 1000 to fit thescale).

W. Grzybowski, J. Szydłowski / Chemosphere 111 (2014) 13–17 15

sensors produced by Skye Instruments. The readouts of the UVBand UVA sensors at the tubes surface were 2.0 W m�2 and31.2 W m�2 respectively. The sensors, when exposed to sunlightradiation on a sunny afternoon in late June in Gdynia (54 �N),showed corresponding values of 2.3 W m�2 and 26.8 W m�2.

The absorption spectra of the natural water samples were mea-sured with a Perkin Elmer Lambda 3b dual-beam spectrophotom-eter, using 100 mm long quartz cuvettes with Milli-Q water asreference. The absorbance values of EE2 solution (Fig. 1) were cal-culated using data from the measurements of 3 mg L�1 EE2 solu-tion. 10% methanol solution served as a reference in this case.

Analysis of 17a-ethinylestradiol concentration changes wascarried out using commercial EE2 enzyme-linked immunosorbentassay (ELISA) kits in accordance with the instructions of the man-ufacturer (Tokiwa Chemical Industries, Japan). This method isbased on the competition between a labelled standard EE2 andthe EE2 in the sample, in terms of binding to a specific monoclonalantibody. The amount of labelled EE2 bound to the antibody(determined by the addition of a colouring reagent) is inverselyproportional to the amount of EE2 in the sample. The assay is cal-ibrated using a standard solution of EE2 supplied with the kit. Themethod has been verified against instrumental techniques byHuang and Sedlak (2001) and has been applied in other environ-mental research (Hintemann et al., 2006; Shved et al., 2008; Fergu-son et al., 2013).

3. Results and discussion

Selected physico-chemical characteristics of water serving asEE2 solute were determined in the aged samples of Baltic Seaand Vistula River water after filtering through Whatman GFF filters(Table 1).

In addition, the filtered samples have been checked for bacterialactivity by measuring biochemical oxygen demand after 24 h longincubation in the dark at room temperature. There were no mea-surable changes in dissolved oxygen concentration during incuba-tion period (0.3 mg L�1: the highest standard deviation of dissolvedoxygen measurement was used as a conservative measure ofprecision).

Fig. 1 shows the optical properties of the irradiated samples andthe emission spectrum of the sunlight simulator.

As seen in Fig. 1, the absorbance of the samples at wavelengthslonger than 290 nm was below 0.1. This means that irradiatedsamples were optically thin and that absorbing species (i.e. EE2and CDOM) did not overshadow each other. Irradiation resultedin a decrease in CDOM absorption along the whole spectrum.Absorbance values at 300 nm decreased 20% in Baltic Sea waterand 25% in Vistula River water.

The EE2 concentration in the non-enriched water samples wasnot statistically different from the zero standard provided by theELISA kit manufacturer. An initial EE2 concentration of 300 ng L�1

therefore permitted monitoring without the need for pre-concen-tration and, in the enriched samples kept in darkness during irra-diation, no measurable decrease was observed. However, incontrast to the distilled water solutions, initial analysis of theenriched natural water samples showed lower than nominal con-centrations of EE2. The apparent decrease ranged up to 30 ng L�1

(see Fig. 2).In spite of the very low absorbance of the distilled water solu-

tion, a significant decrease in EE2 concentration was noted after8 h of irradiation. The direct photodegradation of EE2 by UV repre-sentative of sunlight (wavelength longer than 280 nm) had alreadybeen observed in previous studies (Table 2) but at significantlyhigher EE2 concentrations.

The EE2 concentrations decreased at the highest rate in theVistula River water solution. The photodegradation rates were inagreement with the optical properties and iron content of theirradiated solutions in that the higher the absorbance/iron concen-tration, the greater the effects of irradiation. The enhanced photo-degradation may be attributed to secondary reactions initiated(sensitized) by CDOM present in the natural water samples (Mop-per and Kieber, 2002). Since most of the iron in natural waters isorganically bound (Morel and Hering, 1993), CDOM photochemis-try cannot be straightforwardly separated from iron-inducedphotochemistry (Boule et al., 1999). The efficiency of CDOM in17a-ethynyloestradiol degradation has been assessed by dividingthe concentration change by the absorbed energy dose (the latterwas calculated using the absorbance spectra of the samples andthe emission spectrum of the radiation source). The calculationsmade for an irradiation period of 8 h show that sea water CDOMis approximately two times more efficient.

Since all of the irradiated solutions were optically thin, it wasassumed that photoreactions during irradiation had adhered to

0

50

100

150

200

250

300

350

distilled water + EE2 (300 ng L-1) sea water + EE2 (300 ng L-1) river water + EE2 (300 ng L-1)

EE

2 co

ncen

trat

ion

[ng

L-1

]

0 h 8 h 16 h

Fig. 2. Photodegradation of 17a-ethynyloestradiol (EE2) dissolved in sea, river and distilled water; mean ± mean standard deviation (n = 3).

16 W. Grzybowski, J. Szydłowski / Chemosphere 111 (2014) 13–17

the law of first order kinetics (Leifer, 1988). For each sample, deg-radation rate constant (k) and half-life (t½) were calculated usingthe following equations:

cðtÞ ¼ cðt0Þe�kt ð1Þ

t1=2 ¼ ln 2=k ð2Þ

Note: C(t0) = EE2 concentration at time zero, C(t) = EE2 concen-tration at time of irradiation.

To compare the obtained results with those presented in litera-ture is difficult due to the fact that different light sources and irra-diation conditions were used in different studies. In addition tothis, a lack of detailed optical characteristics for light sources andirradiated samples does not allow for normalization of the rela-tionship between degradation and the amount of light absorbed.Table 2 presents the photodegradation characteristics obtainedduring this study, together with previously published findings(some values have been calculated from the available figures). Onlydata from experiments in which sunlight or a source of sun-likeradiation was used has been included here.

Despite different concentrations, the degradation kinetics ob-tained for pure EE2 solutions are generally comparable withinthe precision of the methods. The only exception (an order of mag-nitude higher reaction rate constant) was reported for an experi-ment with very high EE2 concentration. In this case, however,calculation of solution absorbance (e280 nm � 2000 M�1 cm�1,c � 0.1 mM, l = 2 cm) suggests that the irradiated sample was notoptically thin and that an assumption of first order kinetics cannotbe applied (Leifer, 1988).

The rates for degradation in solutions containing CDOM ob-tained in this study do not differ significantly from those acquiredin previous studies for solutions of significantly higher EE2 concen-trations. Moreover, the range of reported photodegradation rates isrelatively narrow considering the enormous span of EE2 concen-trations and CDOM levels supposedly tested during these experi-ments. This suggests high and ubiquitous potential of naturalorganics to assist in the sunlight-induced removal of EE2 from nat-ural waters although such a conclusion may only be given tenta-tively due to lack of data on amount/absorbance of co-irradiatednatural organic matter.

For the results obtained in this study to be extrapolated to ac-tual conditions in the aquatic environment, adjustment to theshape of irradiation vessel is required. A sample in a tube receivesradiation from practically all directions due to radiation reflection(e.g. off walls) and, as a result, direct photoreactions in tubes arefaster than in a water column, where radiation is only receivedfrom above. The so-called geometry factor, defined as a ratio of rate

constant in a tube to that in a ‘‘flat’’ layer, was assumed to be 2after Leifer (1988). Thus, the corrected half-life times for BalticSea and Vistula River water are 24 and 12 h respectively.

It should be stressed that the presented data enable predictionsto be made in relation to 17a-ethynyloestradiol degradation onlyat the very top of the water column. Photodegradation in the thick-er, optically obscured layer will be dominated by CDOM-induced,secondary reactions and will be difficult to monitor on account oflight attenuation and mixing in the water column. Moreover, acomprehensive study of this phenomenon would not only requirethe basic optical characteristics of a sample and an irradiationsource, but also discriminating the impact of other non-CDOMphotoreactive components present in the water. Of these, nitrate,iron (Chowdhury et al., 2011) and halide ions (Grebel et al.,2012) have already been proved to be significant factors in thephotodegradation of 17b-estradiol, the natural counterpart of EE2.

4. Conclusions

Since the radiation intensity used during the experiment wasconstant (in contrast to natural conditions), irradiation time wasconverted into a number of ‘‘real’’ days using data published byDiffey (2002) on the daily dose of UVA radiation at a similar lati-tude (55 �N). The daily dose measured in June was up to150 J cm�2, equivalent to 13 h of irradiation time under the lampused during the current study. Thus, 17a-ethinylestradiol concen-tration in the top layer of the Vistula River and the Baltic Sea can behalved in the course of one and two sunny days respectively. Incomparison to biodegradation, in which EE2 half-life time mayrange from 5 (Robinson and Hellou, 2009) to 35 d (Ying and Koo-kana, 2003) and up to even 80 d (Zuo et al., 2013), sunlight-drivendegradation seems to be the most effective natural mode of remov-ing EE2 from the aquatic environment.

Acknowledgment

This study was funded by the Polish Ministry of Science andHigher Education through Grant No. N N306 254939 for theresearch project entitled ‘‘Phototransformation of the syntheticsteroid estrogen in Gdansk Bay water’’.

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