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International Journal of Hygiene and Environmental Health 217 (2014) 506–514

Contents lists available at ScienceDirect

International Journal of Hygiene andEnvironmental Health

jou rn al hom ep age: www.elsev ier .com/ locate / i jheh

oxicity of the micropollutants Bisphenol A, Ciprofloxacin, Metoprololnd Sulfamethoxazole in water samples before and afterhe oxidative treatment

essica Richarda,b,∗, Andrea Boergersc, Claudia vom Eyserc, Kai Besterd, Jochen Tuerkb,c

IWW Rheinisch-Westfälisches Institut für Wasserforschung gGmbH, Mülheim, GermanyCentre for Water and Environmental Research (ZWU), University of Duisburg-Essen, GermanyInstitut für Energie- und Umwelttechnik e.V., IUTA (Institute of Energy and Environmental Technology), Duisburg, GermanyDepartment of Environmental Science, Aarhus University, Roskilde, Denmark

r t i c l e i n f o

rticle history:eceived 17 September 2012eceived in revised form4 September 2013ccepted 29 September 2013

eywords:dvanced oxidation processesaste water treatment

zoneV/H2O2

icropollutants

a b s t r a c t

The amount of organic micropollutants detected in surface waters increases steadily. Common wastewater treatment plants are not built to remove these substances. Thus there is a need for new tech-nologies. A promising technology is the use of advanced oxidation processes through which organicmicropollutants can be removed from waste water. However, the formation of oxidation by-products islikely and needs to be investigated since the by-products not only differ from their parent compounds inregard to their chemical and physical properties but they can also differ in toxicity. Therefore this studywas designed to combine chemical and toxicological analyses of the advanced oxidation (O3 [5 mg/L] orUV/H2O2 [Hg-LP lamp; 15 W; 1 g/L H2O2]) of waste water treatment plant effluents and pure water. Efflu-ent samples from conventional activated sludge waste water treatment (mechanical treatment, activatedsludge basin, and primary as well as secondary treatment steps) and high-purity deionized water (purewater) were spiked with Bisphenol A, Ciprofloxacin, Metoprolol or Sulfamethoxazole and treated with

n vitro toxicityntibiotics-Blockerisphenol A

O3 or UV/H2O2. For the toxicological analyses mammalian cells (CHO-9, T47D) were exposed to the watersamples for 24 h and were tested for cytotoxicity (MTT Test), genotoxicity (Alkaline Comet Assay) andestrogenicity (ER Calux®). The results indicate that the oxidative treatment (O3 or UV/H2O2) of Bisphe-nol A, Metoprolol, Sulfamethoxazole or Ciprofloxacin in waste water did not result in toxic oxidationby-products, whereas the UV/H2O2 treatment of Bisphenol A and Ciprofloxacin in pure water resulted inby-products with cytotoxic but no estrogenic effects after 60 min.

ntroduction

Increasing numbers of organic micropollutants like pharma-euticals and personal care products are detected in surfaceaters (Fick et al., 2009; Hirsch et al., 1999; Tambosi et al., 2010;ieno et al., 2007). These micropollutants are not removed by theonventional activated sludge treatment and are thus reachingurface waters where they are found at concentration levels of upo several �g/L (Bergmann et al., 2011; Heberer et al., 2002; Kornert al., 2000; Maurer et al., 2007; Reungoat et al., 2010; Tambosi

t al., 2010). The occurrence of chemical substances in surfaceaters has been linked to biological effects, such as estrogenic,utagenic or genotoxic effects, in aquatic organisms (Desbrow

∗ Corresponding author at: IWW Rheinisch-Westfälisches Institut für Wasser-orschung gGmbH, Moritzstraße 26, 45476 Mülheim an der Ruhr, Germany.el.: +49 208 40303 362.

E-mail address: j.richard@iww-online.de (J. Richard).

438-4639/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.ijheh.2013.09.007

© 2013 Elsevier GmbH. All rights reserved.

et al., 1998; Korner et al., 2001; Liney et al., 2006; Schrank et al.,2009; Tambosi et al., 2010). This in fact is in contrast to theWater Framework Directive which requires a “good chemical andbiological status” of all water bodies until 2015 (EU WFD, 2000).Therefore it is necessary to apply additional treatment technolo-gies to remove remaining micropollutants from WWTP effluents.Advanced oxidation processes (AOPs) have been described as auseful tool for the removal of micropollutants from waste water(Andreozzi et al., 1999; Ikehata et al., 2008; Misik et al., 2011;Tuerk et al., 2010) and in the last years the first WWTP wereupgraded with a full scale ozonation in Germany and Switzerland(Hollender et al., 2009; MKUNLV, 2011). Various AOP methods aredescribed and the treatment with O3 and UV/H2O2 are the twomost effective techniques also in regard to removing toxic effectsof micropollutants (Ikehata et al., 2008; Ormad et al., 2008; Stalter

et al., 2011; Ternes et al., 2003; Tuerk et al., 2010).

Despite the benefits of AOP a few studies were able to show thatAOPs like ozonation or photo-Fenton reactions result in the forma-tion of toxic by-products (Dirany et al., 2011; Garcia-Käufer et al.,

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J. Richard et al. / International Journal of Hygi

012; Larcher et al., 2012; Li et al., 2013; Schrank et al., 2009; Staltert al., 2010) which can be overcome by the use of e.g. sand filtrationr activated carbon after the oxidative treatment (Abegglen, 2009;talter et al., 2010, 2011).

The compounds tested in this study were Metoprolol (�-drenergic receptor antagonist), Sulfamethoxazole, Ciprofloxacinantibiotics), and Bisphenol A which is used in polymeric resins.hese compounds have been chosen as representatives for thelasses of Sulfonamide antibiotics, �-blocker and Fluoroquinolonesnd because of their constant occurrence and their reported con-entrations in the environment up to several �g/L (Giger et al.,003; MKUNLV, 2011; vom Eyser et al., 2013). In addition, Bisphe-ol A is suspected to have an influence on human health andherefore has been added to the Community Rolling Action Plan byhe ECHA in 2012 (ECHA, 2012). Currently there are no environmen-al quality standards (EQS) described in any guideline, however,he Oekotoxzentrum proposed acute and chronic EQS for Meto-rolol (acute: 76 �g/L; chronic 64 �g/l), Sulfamethoxazole (acute:.7 �g/L; chronic 0.6 �g/l) and Bisphenol A (acute: not proposed;hronic 1.5 �g/l) (EAWAG, 2013).

The annual prescriptions of Ciprofloxacin add up to 33,000 kgeading to a widespread distribution of this compound in surface

aters (Bergmann et al., 2011; Schwabe, 2010). The degradationechanisms and pathways of Ciprofloxacin are well described (deitte, 2012). Ciprofloxacin is still of particular concern since geno-

oxic and mutagenic effects have been reported (Hartmann et al.,999; Hu et al., 2007; Kümmerer et al., 2000; Yim et al., 2011). Theeneral toxicity of Ciprofloxacin has been investigated by Gürbayt al. (2005) resulting in an IC50 value (half maximal inhibitory con-entration) of 100 mg/L. Bacterial tests with Vibrio fischeri revealedn EC50 value (half maximal effective concentration) greater than.9 mg/L (Hernando et al., 2007).

Several transformation products such as nitro-aromatic com-ounds or sulfanimalide, an active sulphonamide, for Sulfametho-azole (SMX) are described in the literature (Abellan et al., 2008;am and Marbury, 2005; Yargeau et al., 2008). Studies on the tox-city of Sulfamethoxazole showed, that the EC50 values vary (�g/Lo mg/L) between the test and organism used (Baran et al., 2006;irany et al., 2011; Escher et al., 2005; MKUNLV, 2011; Wammert al., 2006). Yargeau et al. (2008) were also able to show that aixture of several transformation products of SMX had an effect

n human hepatocellular carcinoma cells (Yargeau et al., 2008).Metoprolol is the most frequently prescribed �-blocker in

ermany (Bergmann et al., 2011). Benner and Ternes (2009) wereble to show the formation of transformation products (aldehydeoieties) after ozonation of Metoprolol. EC50 values of 31 mg/L

van den Brandhoff and Montforts, 2010), 63.9 mg/L (Huggett et al.,002), or 200 mg/L (Hernando et al., 2004), as well as a low-st observed effect concentration of 12 mg/L in Daphnia magnaDzialowski et al., 2006) have been published. Cheong et al. (2008)eported an IC50 value of 2.74 g/L for Metoprolol in human ocularell lines.

Bisphenol A (BPA) is a suspected anthropogenic endocrine dis-uptor. Although it is less estrogenic toward aquatic organismshan natural hormones, BPA has been reported to be able tonduce feminization phenomena in various species of animals atigh concentrations (Metcalfe et al., 2001). The oxidation of BPAesulted in the formation of several by-products of which some con-ained additional hydroxyl-groups. Other products were formed by

ring opening of the original compound (Deborde et al., 2008;ee et al., 2004). Its toxicity has been studied in regard to itsndocrine disrupting effects. EC50 values range from 10 �g/L to

53 �g/L depending on the test system and the exposure time (Wut al., 2011; Zhang et al., 2010). Golub et al. (2010) reviewed thevailable literature on Bisphenol A concerning its toxicity and con-luded that Bisphenol A mainly affects the offspring viability, sex

d Environmental Health 217 (2014) 506–514 507

differentiation, immune hypersensitivity and gender differentiatedmorphology thus affecting the endocrine system when prenatallyexposed (Golub et al., 2010). However, Umar et al. (2012) con-clude that knowledge about the environmental fate of BPA is mainlybased on chemical analyses and not biological effects. Studies of thetoxicological effects of the isolated transformation products shouldtherefore be conducted (Umar et al., 2012).

Despite all these results the risk assessment of micropollutantsis currently mainly based on the chemical identification of the sin-gle compounds, but it is not considering possible biological effectsof mixtures in complex matrices (Escher and Leusch, 2011).

The objective of this study was to investigate micropollutants(Bisphenol A, Ciprofloxacin, Metoprolol and Sulfamethoxazole)and their oxidation by-products in waste water treatment planteffluents and pure water by chemical and toxicological in vitro anal-yses using concentrations comparable to those detected in surfacewaters.

Materials and methods

Chemicals

High-purity deionized water (pure water) was produced byan Elix 10 – Milli-Q Plus water purification system (Millipore,Eschborn, Germany). All chemicals (if not otherwise stated) and theenzyme catalase were purchased from Sigma–Aldrich (Taufkirchen,Germany) in highest quality (≥98%). 10 mg of the respective refer-ence compound was dissolved in 20 mL water:acetonitrile (50:50,v/v) to prepare a stock solution with a concentration of 0.5 g/L. Thecalibration standards were dissolved in deionized water.

The used effluents of the WWTP Duisburg – Vierlinden stillcontained organic micropollutants. The concentrations of the foursubstances were in the range of 50 ng/L for Bisphenol A, 100 ng/Lfor Ciprofloxacin, 600 ng/L for Metoprolol and 700 ng/L for Sulfa-methoxazole. The DOC (dissolved organic carbon) was between 6and 8 mg/L and the pH ranged from 7 to 8.5. The used methods forquantification are described in the section “chemical analysis”.

AOP equipment

The different oxidation experiments were carried out in labora-tory scale using a 15 W Hg-LP UV lamp (Heraeus Noblelight, Hanau,D) or a COM-CD-HF 2 ozone generator (Anseros, Tübingen, D).

UV oxidation was investigated with and without the addi-tion of hydrogen peroxide. The laboratory scale plant (volumeof 1 L) was filled with spiked and unspiked samples (pure waterand WWTP effluent). All experiments were carried out at a con-stant temperature (Lauda-Thermostat, Lauda, Königshofen, D). Thewater was pumped through the laboratory scale plant by a flex-ible tube pump (Multifix constant M 838, Alfred Schwinherr KG,Schwäbisch-Gmünd, D). Time controlled sampling was performed.

For ozonation experiments ozone water was produced by con-veying ozone gas through ice-bath-cooled pure water. The ozonewas produced using technical oxygen and an ozone generator. Theexperiments were performed in batch-mode by adding high con-centrated ozone water to spiked and original water samples (purewater and waste water treatment plant effluent). The concentra-tion of the ozone in the water was determined by measuring theabsorption using an UV VIS spectrometer (SPECORD® PC 200, Ana-lytik Jena AG, Jena, D) at 260 nm with a molar absorption coefficientof 3300 [mol−1 cm−1].

Chemical analysis

Samples were analyzed with an LC 20 HPLC system (Shimadzu,Duisburg, D), which was coupled to a 3200 Q Trap system (AB

508 J. Richard et al. / International Journal of Hygiene an

Table 1Precursor ion (Q1), quantifier, and qualifier ions (Q3) of the HPLC–MS/MS quantifi-cation measurements.

Compound m/z (Q1)Precursor m/z (Q3)Quantifier m/z (Q3)Qualifier

Ciprofloxacin 332.2 231.0 314.3Metoprolol 268.0 72.1 56.1Sulfamethoxazole 254.1 156.2 92.1

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exposure medium was prepared directly before usage as describedbelow. For the wells containing the 17�-estradiol standard series,

ciex, Darmstadt, D). Data evaluation was done with AnalystTM

.5 (AB Sciex, Darmstadt, D). The separation was performed on a50 mm × 2 mm Synergi 4u Polar RP column (Phenomenex, Aschaf-enburg, D) with a water–acetonitrile gradient of 0.1% formic acidn water (v/v) (mobile phase A) and 0.1% formic acid (v/v) in purecetonitrile (mobile phase B) with a flow rate of 0.3 mL/min at 30 ◦C.

The quantification of Ciprofloxacin, Metoprolol and Sulfame-hoxazole was carried out in multiple reaction monitoring (MRM)

ode utilizing positive electrospray ionization. The calibrationas weighted 1/x with a linear regression. The limits of detectionere 10 ng/L for Ciprofloxacin, Metoprolol and Sulfamethoxazole.

n Table 1, the precursor, quantifier and qualifier ions of thePLC–MS/MS measurements are given.

Quantification of Bisphenol A was done by a Trace GC Ultra cou-led to a DSQ Mass spectrometer (Thermo-Scientific, Dreieich, D).ingle ion mode measurements of m/z = 357 and m/z = 372 weresed for quantification with a limit of detection of 1 ng/L. Separa-ion was carried out with a Restek Rxi – 5Sil-MS (30 m × 0.25 mm,.25 �m, Restek, Bellefonte, PA, USA).

Transformation products were investigated by a non-targetPLC–MS method. The samples were measured using posi-

ive (ESI(+)) and negative (ESI(−)) ionization. The gradient wasncreased linearly from 5% to 100% methanol within 20 min. The

easurement of all substances and their transformation productsas performed by an information dependent acquisition (IDA)

xperiment. The MS scan range was set to m/z = 50–500 while theinear ion trap (EMS) was adjusted to a peak intensity of ≥1000ounts. Rolling collision energy was turned on, former target ionsere excluded for 2 s and the mass tolerance was set to 250 mmu.

sotopes within a 4.9 Da window were excluded. Enhanced prod-ct ion (EPI) scans were received for the two most intense masses.arameters for EMS and EPI scans were: curtain gas 30 V, ion sourceemperature 550 ◦C, ion source gases 30 and 30 psi, ion transferoltage 5000 V, declustering potential 20 V, entrance potential 10 Vnd collision energy 10 eV.

oxicological methods

ell cultureCHO-cells (Chinese Hamster ovary; ECACC, Salisbury, UK) were

rown in HAM’s F12 medium supplemented with 10% FCS (fetal calferum), 0.5% Gentamycin and 0.5% l-glutamine at 37 ◦C and 5% CO2onditions. These cells were used for the MTT Test and the Alkalineomet Assay.

T47D cells (human ductal breast epithelial tumor cells; BDS,msterdam, Netherlands) were grown in DMEM F12 (Dulbecco’sodified Eagle medium) supplemented with 7.5% FCS, 0.5% Gen-

amycin and 1% l-glutamine at 37 ◦C and 5% CO2 conditions. Duringhe assay DMEM F12 without phenol red (exposure medium) sup-lemented with 4.5% stripped FCS (BDS, Amsterdam, Netherlands)nd 1% NEAA (non-essential amino acids) was used. T47D cells weresed for the ER Calux.

All media were purchased from Gibco (Karlsruhe, D) and sup-lements from c.c.pro (Oberdorla, D).

d Environmental Health 217 (2014) 506–514

CytotoxicityTo detect cytotoxic effects the MTT-(3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide) test was performed(Mosmann, 1983). 200 �L HAM’s F12 medium containing 100,000CHO cells were added to the wells of a 96-well plate and incu-bated overnight. The cells were then exposed to the spiked wastewater or pure water samples (ratio of 1:10 in fresh medium), thenegative control (HAM’s F12 medium containing 10% pure water)and 50 �M MMA III as (monomethylarsonous acid) the positivecontrol for 24 h. After exposure the medium was removed, 100 �Lfresh medium and 10 �L MTT solution (5 mg MTT dissolved in1 mL PBS) were added and the cells further incubated at 37 ◦Cand 5% CO2 for 2 h. Before measurement the staining solutionwas removed and the cells were lysed using 100 �L lysis solu-tion (99.4 mL dimethylsulfoxide; 0.6 mL acetic acid; 10 g sodiumdodecyl sulfate). The absorption was then measured at 595 nm.Each sample was tested three times and the viability and standarddeviation were calculated.

GenotoxicityThe Alkaline Comet Assay was used to detect genotoxic effects

as previously described by Ostling and Johanson (1984) and latermodified by Singh et al. (1988). 100,000 CHO cells were seededin each well of a 24-well plate and incubated overnight beforethe cells were exposed to the spiked waste water and pure watersamples (ratio of 1:10 in fresh medium) and the negative control(HAM’s F12 medium containing 10% pure water) for 24 h. The cellswere also exposed against 0.1 mg/mL N-ethyl-N-nitrosourea (ENU)for 30 min, which was used as a positive control. After exposurethe medium was removed, the cells were trypsinated and resus-pended in PBS. 20 �L of cell suspension containing 8000 cells weremixed with 45 �L low melting point agarose (Invitrogen, Ltd., Pais-ley, Scotland, UK) and added to a chamber sticking to a gelbond film.After solidification of the agarose the cells were lysed overnightby incubation in lysis solution (10 mL Tris-solution (1 M), 146.1 gNaCl, 100 mL EDTA (1 M) und 10 g N-laurylsarcosine sodium saltwere filled up to 1 L with Millipore water) at 4 ◦C. Before elec-trophoresis the gels containing the lysed cells were incubated inelectrophoresis solution (4 ◦C) for 20 min. Then 25 V/300 mA cur-rent were applied for another 20 min. The gels were then incubatedin neutralization solution for 30 min and transferred to ethanolabsolute and further incubated for 2 h before they were left to dry.The analysis was performed using a CCD camera attached to a flu-orescence microscope (Leica DMBL, Leitz GmbH & Co KG, Wetzlar,D) and the Comet Assay 4 Software (Perceptive Instruments, Suf-folk, UK). The extent of damaged DNA is displayed as the relativeOlive Tail Moment (OTM) which was calculated for each samplein relation to the corresponding negative control. The significanceof the differences between the results from the water samplesand the negative controls was calculated using the non-parametricMann–Whitney U test and is displayed as asterisks (highly signifi-cant increase in DNA damage: ***p < 0.001).

EstrogenicityThe ER Calux (BioDetection Systems (BDS), Amsterdam,

Netherlands) was performed to test the spiked pure water sam-ples for estrogenic effects. T47D cells were seeded into a 96-wellplate and incubated over night. The next day the medium wasremoved, fresh medium added and the cells were incubated foranother 24 h, before they were exposed to the standard seriesof 17�-ethinylestradiol with final concentrations between 0 and30 pM/100 �L and the water samples (ratio of 1:10) for 24 h. The

medium (DMEM F12 without phenol red) and pure water weremixed at a 1:10 ratio. The exposure medium for the wells

ene and Environmental Health 217 (2014) 506–514 509

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Table 2Final concentrations of the four micropollutants used during toxicity testing of purewater and waste water treatment plant effluents after exposure. Oxidation was per-formed with c (O3) = 5 mg/L and UV/H2O2 (Hg-LP lamp, P = 15 W and c (H2O2) = 1 g/L).

Micropollutant (mg/L) Ozonation UV/H2O2

Bisphenol A 1.4 0.1Ciprofloxacin 1.4 0.1

J. Richard et al. / International Journal of Hygi

ontaining the water samples, medium and DMSO (dimethyl sulf-xide) were mixed at a 1:1000 ratio. The T47D cells were thenxposed to the water samples at a 1:10 ratio of medium and sample.fter the 24 h exposure the medium was removed and 50 �L lysisolution was added to each well and the plate was incubated for5 min at room temperature. Successively, 30 �L of the supernatantas transferred to a white 96-well plate. Immediately before mea-

urement 30 �L GlowMix were added and the luminescence wasetermined using a Tecan GENios plate reader (Tecan; Crailsheim,). Statistical analysis and the calculation of the estradiol equiva-

ents (ER Calux EEQ) was performed using the provided excel sheetrom BDS.

Each sample was tested independently at least three times usinghree replicates per test in the MTT Test, the Alkaline Comet Assaynd the ER Calux.

utagenicityTo detect mutagenic effects of the tested pure water samples,

he Ames MPFTM 98/100 Aqua Test (Xenometrix, Ch) was usedith a minor modification. Instead of testing a serial dilution with

ix concentrations for the exposure, six single samples with onlyne concentration were tested to use the highest possible concen-ration of the analyzed substance. The remaining steps were doneccording to the manual. In short: the two bacterial strains TA98nd TA100 were grown for 16 h in a shaking water bath at 37 ◦Cnd were then exposed to pure water samples containing 0.1 mg/Liprofloxacin before and after UV/H2O2 treatment for 90 min in a4-well plate. After the exposure time indicator medium was addedo each well and each sample was transferred to 48 wells of a 384-ell plate and further incubated for 48 h according to the provider’sanual. Then the number of revertants was determined by count-

ng the number of wells where a color change had occurred. Allxperiments were repeated independently at least 3 times usingne replicate.

ample treatment and exposure of cells

The selected four substances were added to water samples,ither pure water (high-purity deionized water) or WWTP efflu-nt, and these samples were then oxidized using ozone (5 mg/L)r UV/H2O2 (Hg-LP, 15 W, c (H2O2) = 1 g/L). After UV/H2O2 treat-ent 0.25 mL of catalase (c = 0.4 kU/mL) were added to degrade

emaining peroxides. The use of pure water as well as WWTP efflu-nt allowed the identification of oxidation by-products as well ashe analyses of matrix effects.

For the exposure experiments the waste water and pure wateramples spiked with either of the four substances were added tohe cells in a ratio of 1:10 into medium. The final concentrations ofhe substances during exposure are listed in Table 2.

esults and discussion

ethod adaptation

The methods used in this study are routine methods for theesting of e.g. pharmaceuticals and other substances. In order toest water samples for the detection of the toxicity of micro-ollutants the applied toxicological methods had to be adaptedo prevent methodological errors. Therefore different amounts ofater have been added to the cell culture medium and were ana-

yzed for cytotoxic and genotoxic effects. In addition the enzymeatalase (0.25 mL; c = 0.4 kU/mL) was added to samples treated with

V/H2O2 to degrade the remaining peroxides to water and oxygenaving no more toxic effects. With a water content of 10% no cyto-oxic (Fig. 1a) or genotoxic (Fig. 1b) effects were detected. Thuspiked waste water and pure water samples had to be transferred

Metoprolol 1.4 0.1Sulfamethoxazole 1.4 0.1

into cell culture medium at a ratio of 1:10 to avoid false positiveeffects resulting from osmotic pressure.

The use of H2O2 during UV treatment led to the formation ofperoxides which were not completely removed after 60 min oftreatment causing a decrease in cell viability and an increase inDNA damage, thus also leading to false positive results. Throughthe addition of the enzyme catalase (0.25 mL; c = 0.4 kU/mL) theremaining peroxides were degraded to water and oxygen havingno more toxic effects (Fig. 1).

Spiking experiments

The experiments with the four tested substances were per-formed with an exposure of the spiked pure water and wastewater treatment plant effluents resulting in the following finalconcentrations of the substances during exposure (Table 2). Theconcentrations were chosen because of the accompanying non-target analysis.

Ciprofloxacin

Ciprofloxacin was tested for its cytotoxic and genotoxic effectsbefore and after ozonation (5 mg/L) or UV/H2O2 treatment.The results displayed in Fig. 2 clearly show that 1.4 mg/L ofCiprofloxacin were neither before nor after ozonation cytotoxic(Fig. 2). However, the UV/H2O2 oxidation of 0.1 mg/L Ciprofloxacinled to the formation of cytotoxic oxidation by-products since beforetreatment no effects were detected and the test for peroxides wasnegative (Fig. 2b). In addition no increase in DNA damage was seenbefore and after ozonation (Fig. 2c) as well as after UV treatment ofthe WWTP effluent containing Ciprofloxacin, while the cytotoxicsample (Fig. 2b #, pure UV/H2O2 treated) was not tested in theAlkaline Comet Assay.

Ciprofloxacin (0.1 mg/L in pure water) has also been tested forits potential to induce mutations using the Ames Test (Salmonellatyphimurium strains TA98 and TA100) before and after 10, 30 or120 min of UV/H2O2. However, Ciprofloxacin did not induce muta-genic activities (a significant increase in the number of revertants)in the Ames Test in any of the samples.

Bisphenol A

Bisphenol A was tested at a concentration of 1.4 mg/L (ozona-tion) and 0.1 mg/L (UV/H2O2), in pure water and WWTP effluent.The ozonation with 5 mg/L ozone did not lead to the formation ofcytotoxic or genotoxic oxidation by-products (Fig. 3a and c). How-ever, the UV/H2O2 treatment of Bisphenol A in pure water revealedcytotoxic by-products after 60 min of reaction time (Fig. 3b; pureUV/H2O2 treated; dark gray bar marked with ***). This sample wasadditionally tested for remaining peroxides but none were detectedand thus the toxicity can be related to formed by-products dur-

ing UV/H2O2 treatment. In comparison the WWTP effluent sampledid not show cytotoxic effects possibly due to competing reactionsof Bisphenol A with the water matrix. Except the one cytotoxicsample (marked with # in Fig. 3d), all others were tested for

510 J. Richard et al. / International Journal of Hygiene and Environmental Health 217 (2014) 506–514

F UV trt reatm

gi

Brps

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ig. 1. Method adaptation for the use of pure water as a matrix and H2O2 duringesting of pure water with (dark gray bars) and without (light gray bars) UV/H2O2 t

enotoxicity but none of these samples showed significant changesn DNA damage.

In addition to the cytotoxicity and genotoxicity evaluation the

isphenol A samples were also tested for estrogenic activity. Theesults of the ER Calux showed that the untreated Bisphenol A sam-le resulted in estrogenicity whereas the UV/H2O2 treated sampleshowed no more estrogenic effects (Table 3).

ig. 2. Cytotoxicity (a, b) and genotoxicity (c, d) testing of pure water (pure) or WWTP effl3; Ciprofloxacin = 1.4 mg/L) or UV/H2O2 treatment (UV/H2O2; 15 W Hg-LP; 1 g/L H2O2; C

**p < 0.001.

eatment: (a) cytotoxicity (MTT Test) and (b) genotoxicity (Alkaline Comet Assay)ent (***p < 0.001, significantly different to negative control).

Sulfamethoxazole

The results of the toxicity tests (MTT Test and Alkaline Comet

Assay) with 1.4 mg/L (Ozonation; 5 mg/L O3) or 0.1 mg/L (UV/H2O2;15 W Hg-LP; 1 g/L H2O2) demonstrated that neither Sulfametho-xazole itself nor the samples after oxidation led to the detectionof cytotoxic (Fig. 4a) or genotoxic (Fig. 4b) effects. There were no

uent (WWTP) before (light gray bars) and after (dark gray bars) ozonation (5 mg/Liprofloxacin = 0.1 mg/L). # not tested for genotoxicity because of cytotoxic effects.

J. Richard et al. / International Journal of Hygiene and Environmental Health 217 (2014) 506–514 511

F TP efflO 2O2; B*

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ig. 3. Cytotoxicity (a, b) and genotoxicity (c, d) testing of pure water (pure) or WW3; Bisphenol A = 1.4 mg/L) or UV/H2O2 treatment (UV/H2O2; 15 W Hg-LP; 1 g/L H

**p < 0.001.

ifferences between the untreated samples or the two oxidationethods and the negative controls. Thus no toxic oxidation by-

roducts have been formed.

etoprolol

The results for Metoprolol are comparable to the results for thexidation experiments with Sulfamethoxazole. They showed noytotoxic effects after ozone or UV/H2O2 treatment of both water

amples (pure water and WWTP effluent) containing Metopro-ol with maximum concentrations of 1.4 mg/L The Alkaline Cometssay also did not show a significant increase in DNA damage forny of the tested samples (data not shown). No difference was

able 3strogenicity of pure water containing 0.1 mg/L Bisphenol A before and afterV/H2O2 treatment. Estrogenicity is displayed as ER Calux EEQ (ER Calux estradiolquivalents). LOD = 0.83 ng/L.

Sample (0.1 mg/L Bisphenol A) ER Calux EEQ (ng/L)

Neg. control <0.83Before treatment 5.72After 10 min treatment <0.83After 60 min treatment <0.83

uent (WWTP) before (light gray bars) and after (dark gray bars) ozonation (5 mg/Lisphenol A = 0.1 mg/L). # not tested for genotoxicity because of cytotoxic effects.

observed between pure water and waste water or the respectivewater sample after the two AOPs.

Chemical analyses

In addition to the toxicological tests, the chemical analysisshowed that oxidation by-products were formed during the oxida-tive treatment of 17 mg/L Ciprofloxacin with UV/H2O2 (Fig. 5).The degradation at the piperazinyl substituent is initiated by theaddition of oxygen (m/z = 348, Oxociprofloxacin). This mechanismis well known and published by Calza et al. (2008) and others(de Witte et al., 2009; Tiefenbacher et al., 1994). The oxidativeattack facilitates a ring opening reaction of the heterocycle result-ing in m/z = 308 (de Witte et al., 2009). A further degradation mightoccur by an opening of the benzene ring and an abstraction of thecyclopropyl group (m/z = 228). Defluorinated Ciprofloxacin resultsfrom an oxidative attack after a destabilization of the C-F bond(m/z = 378). Further degradation could occur by a complete removal

of the cyclopropyl substituent (m/z = 290) following a splitting-upof the molecule (m/z = 115). Although these by-products have beendetected no toxicity has been shown for Ciprofloxacin itself and thewater sample after 60 min of oxidative treatment.

512 J. Richard et al. / International Journal of Hygiene and Environmental Health 217 (2014) 506–514

Fig. 4. Cytotoxicity (a, b) and genotoxicity (c, d) testing of pure water (pure) or WWTP effluent (WWTP) before (light gray bars) and after (dark gray bars) ozonation (5 mg/LO3; Sulfamethoxazole = 1.4 mg/L) or UV/H2O2 treatment (UV/H2O2; 15 W Hg-LP; 1 g/L H2O2; Sulfamethoxazole = 0.1 mg/L). ***p < 0.001.

Fig. 5. Emerging transformation products of Ciprofloxacin during UV/H2O2 treatment in pure water. The initial concentration was 17 mg/L and the added concentration ofhydrogen peroxide was 0.3 g/L. Non-target analysis was done using HPLC–MS (ESI+). m/z = mass to charge ratio. In the second graph a part of the main picture is enlarged(top right corner).

ene an

ecepCroabcptfBTtreTfDhpiwtoidm

iffiBZp2

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retabaBbwtombrTStdtfii

J. Richard et al. / International Journal of Hygi

To investigate the general process of by-product formation,xperiments with a pure water matrix were carried out and bothhemical and toxicological analyses were performed. The samexperiments were then done with spiked waste water treatmentlant effluent samples. The above described cytotoxic effects ofiprofloxacin and Bisphenol A in pure water after UV oxidationesult from formed oxidation by-products since no remaining per-xides were detected. The toxic effect occurs only in pure waternd not in WWTP effluent containing Ciprofloxacin which mighte due to matrix effects. WWTP effluents contain a complex matrixomposition, e.g. organic substances, while the pure water sam-les only contain the tested substance. Therefore it is possiblehat the UV/H2O2 treatment of the pure water sample led to theormation of a toxic oxidation by-product since Ciprofloxacin orisphenol A are the only substances to be oxidized in this test.he same treatment of a WWTP effluent sample did not leado toxic effects, which could be the consequence of competingeactions or different reaction pathways (Sein et al., 2009). Theffluent still contained organic material in various compositions.his could lead to competing reactions during oxidation and theormation of different transformation products might occur as well.ifferent degradation patterns depending on the water matrixave already been shown by Zwiener and Frimmerl (2000). Theyroved, that Ibuprofen and Clofibric acid are faster degradable

n distilled water than in surface water. The same observationsere published by Trovo et al. (2009) for the degradation pat-

erns of Sulfamethoxazole. They also showed that the oxidationf Sulfamethoxazole by photo-Fenton reactions only results in anncrease in toxicity in seawater but not in distilled water. Thusifferent oxidation by-products might be found depending on theatrix used.The chemical analysis of the oxidized water samples contain-

ng Ciprofloxacin, Metoprolol and Sulfamethoxazole revealed theormation of several oxidation by-products which confirmed thendings of other groups (Abellan et al., 2008; An et al., 2010;enner and Ternes, 2009; Calza et al., 2008; Trovo et al., 2009;hou and Moore, 1994). In addition further unknown oxidation by-roducts have also been identified by own studies (vom Eyser et al.,013).

onclusions

Advanced oxidation processes seem to be a useful method toemove or pre-oxidize remaining organic micropollutants from theffluents of waste water treatment plants. Although the forma-ion of oxidation by-products is likely, the oxidation of Metoprololnd Sulfamethoxazole did not lead to the formation of toxicy-products comparing treated and untreated samples as wells pure water and WWTP effluents. In contrast, the tests withisphenol A and Ciprofloxacin showed the formation of toxicy-products after 60 min of UV/H2O2 treatment in pure waterhereas the ozonation and the tests in WWTP effluent led to no

oxic responses. These results in general demonstrate that both,zonation and UV/H2O2 oxidation of water containing differenticropollutants lead to the formation of a variety of oxidation

y-products with no (Metoprolol, Sulfamethoxazole) or cytotoxicesponses (Bisphenol A, Ciprofloxacin) depending on the matrix.his indicates that AOP are sufficient in removing Metoprolol,ulfamethoxazole and Ciprofloxacin from WWTP effluents at theested concentrations without the formation of toxic by-products,epending on the type of waste water. Therefore the oxida-

ion of spiked pure water samples might give an idea aboutormed oxidation by-products and their biological effects but real-stic conditions with WWTP effluents still need to be furthernvestigated.

d Environmental Health 217 (2014) 506–514 513

Acknowledgements

The authors would like to thank for financial support from theGerman Federal Ministry of Economics and Technology within theagenda for the promotion of industrial cooperative research anddevelopment (IGF) based on a decision of the German Bundestag.The access was opened by member organization environmen-tal technology and organized by the AiF, Arbeitsgemeinschaftindustrieller Forschungsvereinigungen, Cologne (IGF-Project No.15862N). The authors appreciate the cooperation and promotion ofthe local water board Wirtschaftsbetriebe Duisburg which partici-pated in this project. The authors would also like to thank MelanieGerhards for her technical assistance during the experiments.

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