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RESEARCH ARTICLE

Determination of phenols and pharmaceuticals in municipalwastewaters from Polish treatment plants by ultrasound-assistedemulsification–microextraction followed by GC–MS

Urszula Kotowska & Justyna Kapelewska & Joanna Sturgulewska

Received: 23 March 2013 /Accepted: 3 June 2013 /Published online: 2 July 2013# The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract A method combining ultrasound-assisted emulsi-fication–microextraction (USAEME) with gas chromatogra-phy–mass spectrometry (GC–MS) was developed for simul-taneous determination of four acidic pharmaceuticals, ibu-profen, naproxen, ketoprofen, and diclofenac, as well as fourphenols, 4-octylphenol, 4-n-nonylphenol, bisphenol A, andtriclosan in municipal wastewaters. Conditions of extractionand simultaneous derivatization were optimized with respectto such aspects as type and volume of extraction solvent,volume of derivatization reagent, kind and amount of buff-ering salt, location of the test tube in the ultrasonic bath, andextraction time. The average correlation coefficient of thecalibration curves was 0.9946. The LOD/(LOQ) values ininfluent and effluent wastewater were in the range of 0.002–0.121/(0.005–0.403) μg L−1 and 0.002–0.828/(0.006–2.758)μg L−1, respectively. Quantitative recoveries (≥94 %) andsatisfactory precision (average RSD 8.2 %) were obtained.The optimized USAEME/GC–MS method was applied fordetermination of the considered pharmaceuticals and phe-nols in influents and treated effluents from nine Polish mu-nicipal wastewater treatment plants. The average concentra-tion of acidic pharmaceuticals in influent and effluent waste-water were in the range of 0.06–551.96 μg L−1 and 0.01–22.61 μg L−1, respectively, while for phenols were in therange of 0.03–102.54 μg L−1 and 0.02–10.84 μg L−1, re-spectively. The removal efficiencies of the target compoundsduring purification process were between 84 and 99 %.

Keywords Ultrasound-assisted emulsification–microextraction . Gas chromatography–mass spectrometry .

Acidic pharmaceuticals . Phenols . Influents . Effluents .

Municipal wastewater treatment plant

Introduction

Widespread accessibility and ever-increasing use of pharma-ceuticals as well as cosmetics and cleaning agents haveresulted in the appearance and increase in the concentrationof a great number of new chemical compounds in sewageproduced by humans. Under the current legal regulations, itis not obligatory to determine the content of pharmaceuticalsor other organic trace contaminants in wastewater. Nor is thepresence of these compounds in the wastewater taken intoaccount in the planning and management of the sewagetreatment process. As a result, different types of compounds,including pharmaceuticals and endocrine-disrupting com-pounds, penetrate into the environment posing a serious risknot only to aquatic organisms but also to humans. For thatreason, it is essential to analyze the content of these com-pounds in wastewater.

Ibuprofen, naproxen, ketoprofen, and diclofenac are non-steroidal anti-inflammatory drugs (NSAIDs). They havebeen widely used not only to treat various inflammatorydisorders and for pain relief but also for their antipyreticeffect. Nearly all of these substances are available withouta doctor’s prescription at lower doses. Publications regardingNSAIDs in the environment confirm their toxicity to manyanimal species (Oaks et al. 2004). Substances such as 4-octylphenol, 4-n-nonylphenol, bisphenol A, and triclosanare included into the group of endocrine-disrupting com-pounds (Esplugas et al. 2007). 4-Octylphenol and 4-n-nonylphenol are used for the production of elasticizers,technical-grade abstergents, pesticide emulsifiers, as wellas trimming in spinning and weaving. Bisphenol A is a rawmaterial for producing epoxide resins, polycarbonates, andpolystyrene resins, which has been used to stabilize phenol

Responsible editor: Philippe Garrigues

U. Kotowska (*) : J. Kapelewska : J. SturgulewskaInstitute of Chemistry, University of Bialystok, ul. Hurtowa 1,15-399 Bialystok, Polande-mail: [email protected]

Environ Sci Pollut Res (2014) 21:660–673DOI 10.1007/s11356-013-1904-6

formaldehyde. Triclosan is used as an ingredient in personalhygiene and household products such as soaps, toothpaste,mouthwash, deodorants, detergents, and disinfecting lotions.

Determining pharmaceuticals and endocrine-disruptingcompounds in wastewater and other environmental samplesis a very difficult task due to low concentrations and complexmatrix of the substances. The highest concentrations of theanalyzed compounds reaching several dozens of microgramsper liter have been recorded for wastewater entering treat-ment plants (Gómez et al. 2007b; Ramos Payán et al. 2010;Regueiro et al. 2009). In effluents, the concentration ofparticular compounds has been found to be from a few tohundreds of times lower than in the influent wastewater(Gómez et al. 2007b; Yiantzi et al. 2010). Analysis of thecompounds in rivers showed the concentrations of the con-taminants reaching several hundred nanograms per liter, inlake waters up to tens of nanograms per liter, and in the seaup to several nanograms per liter (Beck et al. 2005; Ozcanet al. 2010; Regueiro et al. 2009).

The technique most extensively used for the isolation oftarget compounds from environmental samples is solid-phase extraction (SPE) (Ballesteros et al. 2006; Gatidouet al. 2007; Gómez et al. 2007a, b; Gracia-Lor et al. 2010;Liu et al. 2004; Yu and Wu 2013). In the case of acidicpharmaceuticals, other techniques have also been applied,such as liquid–liquid microextraction (LLME), hollow fiberliquid-phase microextraction (HF-LPME), and microwave-assisted extraction (MAE) (Antonić and Heath 2006;Quintana et al. 2004; Ramos Payán et al. 2010). To isolatetarget phenols, apart from SPE and solid-phase micro-extraction (SPME), stir bar sorptive extraction (SBSE) andLLME and its variants, dispersive liquid–liquid microextrac-tion (DLLME) and vortex-assisted liquid–liquid micro-extraction (VALLME), are also applied (Kawaguchi et al.2008; Regueiro et al. 2009; Yiantzi et al. 2010; Zgoła-Grześkowiak 2010). The final stage of the determination ofpharmaceuticals and phenols in water samples requires ex-tremely sensitive and selective techniques. Currently, for thispurpose primarily chromatographic methods are used, i.e.,gas chromatography (GC), high-performance liquid chroma-tography (HPLC), and ultrahigh-pressure liquid chromatog-raphy (UHPLC). The detector typically employed in con-junction with chromatographic techniques is mass spectrom-eter (MS). Another detector applied in gas chromatographyassays is flame ionization detector (FID), while in HPLC thediode array detector (DAD), fluorescence detector (FLD),and ultraviolet detector (UV) are used.

Ultrasound-assisted emulsification–microextraction report-ed by Regueiro et al. (2008) is one of the LLME modes usedin addition to the techniques such as DLLME, VALLME, HF-LPME, and single-drop microextraction (SDME). Liquid-phase microextraction techniques are now widely employeddue to a great number of advantages that they offer, such as

low consumption of organic solvents, simplicity of experi-ment, high extraction efficiency, and low costs. In USAEME,a small amount of organic solvent of the order of microliters isintroduced into water sample and the system is treated withultrasound. The organic solvent dissipates into microdroplets,thus creating a considerably large contact area of both theaqueous and organic phases. The use of ultrasound facilitatesthe emulsification process and speeds up the determination ofequilibrium between the two phases. Immediately after theextraction, centrifugation is applied for phase separation andorganic extract is analyzed. The use of ultrasound makes itpossible to bypass the disadvantages connected with othermicroextraction techniques, such as a relatively long extrac-tion time in VALLME, HF-LPME, and SDME as well as thenecessity of using relatively large amounts of disperser sol-vent in DLLME. Owing to its many advantages, USAEMEhas gained a considerable significance since its first use 5 yearsago. The technique has been used, among others, to determinepolycyclic aromatic hydrocarbons (PAH), pesticides, poly-chlorinated biphenyls (PCBs), polybrominated diphenylethers(PBDE), haloanisoles, bisphenols, chlorinated phenoxyaceticacids as well as inorganic selenium (Fontana et al. 2009;Fontana and Altamirano 2010; Najafi et al. 2012; Ozcanet al. 2009a, b, 2010; Pizarro et al. 2012a; Viñas et al. 2012;Zhang and Lee 2012). The determination process has beenmainly conducted in environmental waters (Fontana et al.2009; Fontana and Altamirano 2010; Najafi et al. 2012;Ozcan et al. 2009a, b, 2010; Zhang and Lee 2012) and alsoin wine (Pizarro et al. 2012b), paper, toys, and baby products(Viñas et al. 2012). USAEME procedures generally make useof extraction solvents denser than water, e.g., chloroform(Fontana et al. 2009; Fontana and Altamirano 2010; Ozcanet al. 2009a). Utilization of solvents of lower densities thanwater requires special dishes or additional procedural steps toenable the separation of the solvent from water samples andconsequently may lead to the deterioration of reproducibilityand accuracy of final results (Pizarro et al. 2012b; Zhang andLee 2012). The solidification of a floating drop of a low-densitysolvent requires employing a larger volume of extracting me-dium in comparison with a traditional USAEME procedure(Pizarro et al. 2012a).

In this study, a simple and sensitive analytical procedurefor simultaneous determination of acidic pharmaceuticalsand phenols at low concentrations has been optimized.USAEME has been used for the isolation whereas GC–MSin the selected ion monitoring (SIM) mode has been appliedfor the separation and determination of analytes. The effectsof various extraction and derivatization parameters, i.e., thetype of organic solvent and solvent volume, extraction time,location of test tube in ultrasonic bath, derivatization reagentvolume, and kind and amount of buffering salt, were inves-tigated. The developed USAEME/GC–MS method wasemployed to determine target compounds in influents and

Environ Sci Pollut Res (2014) 21:660–673 661

treated effluents from wastewater treatment plants of ninecities in Poland.

Experimental

Reagents and solvents

Ibuprofen (IBP), naproxen (NAP), ketoprofen (KET), diclo-fenac (DIC), 4-octylphenol (OP), 4-n-nonylphenol (NP), bis-phenol A (BPA), pyrene (PYR), 1,2-dichlorobenzene (1,2-DCB), and decane (DEC) were obtained from Sigma-Aldrich (Germany). Triclosan (TRC) was obtained fromFluka (Italy). Carbon tetrachloride (CTC) was purchased fromMerck (Germany). Chloroform (CHL), toluene (TOL), aceticanhydride, sodium hydrogen phosphate (V), and methanolwere obtained from POCH (Poland). Series of C10–C22 n-alkanes used to determine retention indices of target com-pounds were purchased from Fluka (Switzerland).

Stock solutions containing all analytes (at 1 mg mL−1 ofeach) were prepared in methanol and stored at −18 °C for notlonger than 1 month. Working solutions were prepared bydiluting the stock standard solution in methanol and stored at−18 °C for not longer than 2 weeks. Deionized water wasfrom purification system (Milli-Q RG, Millipore, USA) andwas stored in a glass bottle.

Wastewater samples

The wastewater samples were obtained from wastewater treat-ment plants (WWTPs) in nine cities in Poland. In these plants,the treatment process includes both mechanical purificationand biological purification with the use of activated sludge.Figure 1 shows the locations and average daily capacity Qd

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of the mentioned wastewater treatment plants. Average dailysamples of influents and treated effluents were collected inglass bottles and transported to the laboratory. Upon arrival, thesamples were filtered through a membrane filter with 0.45 μmpore size and acidified with concentrated hydrochloric acid topH=2. Later, the wastewater samples were stored at −18 °C.

The procedure of ultrasound-assisted emulsification–microextraction (USAEME) with in situ derivatization

For the simultaneous USAEME and derivatization, aliquotsof 5-mL water samples were placed in 10-mL glass centri-fuge tubes containing previously weighed 0.1 g of sodiumhydrogen phosphate. The extraction solvent (carbon tetra-chloride, 40 μL) containing pyrene (5 μg L−1) as an internalstandard and the derivatization reagent (acetic anhydride,300 μL) were added to the water sample and mixed.Immediately after, the tube was immersed in an ultrasonic

Unitra Unima (Poland) water bath in such a way that thelevels of both liquids (i.e., the bath and the sample) wereequal. Extractions were performed at 42 kHz of ultrasoundfrequency and 230 W of power for the duration of 5 min atroom temperature. Emulsions were disrupted by centrifuga-tion at 4,000 rpm/min for 5 min in an MPW-250 MedInstruments (Poland) laboratory centrifuge. In effect, theorganic phase settled at the bottom of the conical tube.After centrifugation, carbon tetrachloride was removed usinga 100-μL Hamilton syringe (USA) and transferred into a150-μL microvial with integrated insert. GC–MS analysiswas then performed as described in “GC–MS conditions”.Consecutive steps taken to isolate the target compounds areshown in Fig. 2.

GC–MS conditions

Analysis was performed with a HP 6890 gas chromatographwith a mass spectrometric detector MSD5973 and HP 7673autosampler (Agilent Technologies, USA). This device wasequipped with HP-5MS (5% phenylmethylsiloxane) size 30mlength×0.25 mm, i.e., coated with 0.25 μm film thickness andsplit/splitless injector. The injector worked in splitless mode.Helium of purity 99.999 % was used as carrier gas at a flowrate of 1 mL min−1. The injector temperature was 250 °C. Theoven temperature was programmed from 150 °C, increased at5 °C/min, to 250 °C. The total run time was 20 min.

The MS detector worked in selected ion monitoringmode. The electron impact source temperature was 230 °Cwith electron energy of 70 eV. The quadrupole temperaturewas 150 °C, and the GC interface temperature was 280 °C.The retention times of target compounds are shown inTable 1 together with the quantification and identificationof ions. The mass spectra of acetylated derivatives of thetarget compounds are presented in Fig. 3. The registeredmass spectra of acetylated IBP, NAP, KET, DIC, TRC, andBPA are compatible with those in literature (NIST ChemistryWebBook 2011). As for the mass spectra of OP and NPacetylated derivatives, the perusal of relevant literatureproved that they have not been published until now.Because of the absence of these data in the NIST MS data-base, computer software wrongly indicated “octylphenol” asa result of determination of octylphenol acetylated deriva-tive, and likewise, “nonylphenol” as a result of determinationof nonylphenol acetylated derivative. The presence of peakscorresponding to the masses 42 units greater (i.e., 248, 262)than molar masses of octylphenol and nonylphenol in themass spectra was proof that these compounds existed in theforms of the acetylated derivatives. Another evidenceconfirming the effectiveness of the conducted derivatizationis the increase of retention times at which the peaks corre-sponding to octylphenol and nonylphenol were registeredafter the acetylation process.

662 Environ Sci Pollut Res (2014) 21:660–673

Determination of retention indices

To determine the retention indices of analytes, the mixture ofC10–C22 n-alkanes was analyzed in the same GC–MS con-ditions as target compounds. The values of retention timeswere used to calculate linear retention indices from Eq. (1):

RI ¼ 100 tx−tnð Þ= tnþ1−tnð Þ þ 100n ð1Þwhere tx, tn, and tn+1 are the retention times of compound x,and n-alkanes with the number of carbon atoms in the mol-ecule n and n+1, respectively (tn<tx<tn + 1) (van Den Dool

and Kratz 1963). The retention indices of target compoundsare shown in Table 1.

Results and discussion

Optimization of extraction and derivatization procedure

All tests during the optimization process had been carried outusing distilled water containing 100 μg L−1 of each targetcompound.

Fig. 2 Schematic diagram of isolation of acidic pharmaceuticals and phenols by USAEME with in situ derivatization

Fig. 1 Location and average daily capacity Qdð of the studied wastewater treatment plants


Organic solvent selection

Properties of extraction solvent have crucial impact on theefficiency of the liquid–liquid extraction process. The organicsolvent must have low water solubility and high affinity withthe isolated compounds. In the USAEME technique, the choiceof the solvent is also dictated by the possibility of forming

emulsion during the extraction procedure. Accordingly, fivedifferent solvents, 1,2-dichlorobenzene, carbon tetrachloride,chloroform, n-decane, and toluene, were examined in the pre-liminary experiments. In their course, 100 μL of solvent wasadded to 5-mL aliquots of the target compounds solution, andthe samples were placed in an ultrasonic bath for 5 min.Emulsification was observed in all the cases. After

Table 1 The chemical abstract service (CAS) registry numbers, mo-lecular weights (MW), octanol–water partition coefficients (pKow),dissociation constants (pKa), chemical structure of target compounds,

and retention time (tR), retention index (RI), and quantification andidentification ions (m/z) of acetylated derivatives of target compounds

Compound Target compounds Acetylated derivatives of target compounds

Structure CAS number MW pKa pKow tR (min) RIQuantification and

identification ions (m/z)f

IBP

CH3

OH

OCH3

H3C

15687-27-1 206 4.91a 3.50a 4.91 1550 ± 1 161, 177, 220

OP

OH

H3C 949-13-3 204 10.39b 4.12d 9.57 1874 ± 1 107,206, 248

NP

OH

H3C

104-40-5 218 n.a.e 4.48d 11.28 1977 ± 1 107,220, 262

NAP

CH3

OH

O

OH3C

22204-53-1 230 4.15a 3.18a 12.28 2035 ± 1 170,185, 244

KET CH3

OOHO

22071-15-4 254 3.89a 3.12a 14.30 2161 ± 1 105,209, 268

DICCl

Cl

NH

OH

O15307-86-5 296 4.15a 4.40a 14.80 2182 ± 1 214, 242, 309

TRC

Cl

O

Cl

Cl

OH

3380-34-5 289.5 4.5c 4.53a 1.95 2192 ± 1 218,288, 290

BPA

CH3

CH3

OHHO 80-05-7 228 10.3a 3.32a 17.92 2375 ± 1 213, 228, 270

a LOGKOW—Sangster Research Laboratories 2013b BŁędzka et al. 2010c Van Den Dool and Kratz 1963d Ahel and Giger 1993e Not availablef Bolded ions selected for monitoring


centrifugation, the extraction solvents with density higher thanwater (1,2-dichlorobenzene, carbon tetrachloride, and

chloroform) were removed with a syringe from the conicalbottom of the test tube. n-Decane and toluene, which have

Fig. 3 Mass spectra of acetylated derivatives of the target compounds


lower density than water, were collected from the bottom of thetube after the removal of water. In the cases of DIC, KET, OP,NP, TRC, and BPA, the largest peak areas were obtained usingchloroform, whereas in the case of IBP and NAP, the largestpeak areas were obtained using carbon tetrachloride as theextraction solvent. During these experiments, it was noted thatintroducing chloroform into the chromatographic system re-sults in considerable elevation of the baseline on the chromato-grams, and at the same time reduces repeatability of IBP, NAP,and KET peak areas. That is why eventually carbon tetrachlo-ride was selected as the optimum extraction solvent for furtherexperiments.

Effect of solvent volume

The amount of solvent used in extraction has a directimpact on the condensation of the analyte: the lower thevolume of organic phase, the greater the concentration ofthe analyte, which effects in lowering the limit of quanti-tation (LOQ). What is more, using the smallest possibleamounts of toxic solvents conforms with the guidelines of“green chemistry”.

In order to obtain the highest extraction efficiency of theUSAEME procedure, the volume of extraction solvent hadto be optimized. For this purpose, different volumes ofcarbon tetrachloride in the range of 40–100 μL were ex-amined (Fig. 4). The analysis showed that reduction ofsolvent volume is accompanied by growth of peak areasof the analyzed compounds. The amount of solvent recov-ered after the extraction process conducted with the use of40 μL of carbon tetrachloride ranged between 25 and

30 μL, and the used volume was the smallest whichallowed the introduction of the sample into chromatographwith autosampler. All in all, the volume of carbon tetra-chloride equal to 40 μL was chosen as the optimal volumefor further investigations.

Effect of manual shaking of sample

Manual shaking was used to ensure that the extractionsolvent and aqueous sample were adequately mixed beforethe ultrasound treatment. Manual shaking was conductedfor 10 s after injection of extraction solvent and derivati-zation agent but before the sonication stage. It was ob-served that the extraction efficiency and reproducibilityimproved when this step of the procedure was added.Thus, manual shaking was used in subsequent optimizationexperiments.

Effect of the test-tube material

Furthermore, the impact of extraction vessel material on theefficiency of the reaction was also investigated. For thispurpose, the extraction procedure was conducted in bothglass and polypropylene test tubes. In the course of theinvestigation, it was observed that the greater efficiency ofthe process with all the compounds was achieved when usingglass tubes. It can be speculated that the analyzed com-pounds are subject to adsorption on polypropylene surface,which leads to a fall in efficiency of the extractions proceed-ing in polypropylene test tubes. Therefore, glass test tubeswere selected for further experiments.

Fig. 4 The influence of solventvolume on extraction efficiencyof target pharmaceuticals andphenols (n=3)


Effect of location of the test tube in the ultrasonic bath

In conducting the optimization process, it was noticed thatthe ultrasonic bath was not homogeneous and two sources ofultrasound waves could be observed. According this, weplaced the test tubes in the left source and in the right sourceof the ultrasound. Holding the test tube in the right sourcegave a higher extraction efficiency. Therefore, placing of testtubes in the right source was selected for further optimizationexperiments.

Effect of kind and amount of buffering salt

In the in situ derivatization technique with the use of aceticanhydride, it is necessary to use an addition of buffer salt.This is because stable alkaline reaction environment has tobe ensured in order to prevent the reaction equilibrium shifttowards the substrates and to ensure greater efficiency of theprocess. In acylation processes conducted with the use ofacetic anhydride, sodium hydrogen carbonate is the mostfrequently used buffer salt. But in this particular case whensodium hydrogen carbonate was used, carbon dioxide bub-bles interfering with the collection of the organic phaseappeared. They were produced as a consequence of decom-position of carbonic acid generated by carbonates in thepresence of the acetic acid formed from the anhydridehydrolysis.

For this reason, in the described experiments sodiumhydrogen phosphate was used. To study the effects of thevarious amounts of added sodium hydrogen phosphate onthe derivatization process, experiments were performed withdifferent quantities of this salt (0.1–0.4 g) per 5 mL ofwastewater sample. Figure 5 illustrates the influence ofadded quantity of sodium hydrogen phosphate on extraction

efficiency of the target compounds. It can be noticed that theresults obtained with different concentrations of the salt werevery similar; hence, the amount of 0.1 g was used in subse-quent experiments.

Effect of derivatization reagent volume

The influence of the volume of acetic anhydride on therelative peak area was studied in the range of 200–350 μL (Fig. 6). The results indicated that the volumeof acetic anhydride equal to 300 μL should be chosen asoptimal.

Effect of the extraction time

The time of isolation is of great significance in all extractionprocedures. In USAEME, extraction time is defined as thetime between injection of extraction solvent and the end ofthe sonication stage (Ma et al. 2009). To determine theinfluence of the extraction time on isolation efficiency, theparameter was examined in the range of 5–15 min. Thehighest values of IBP, NAP, and KET peak areas wereachieved during the extraction which lasted 15 min, but thedifferences between individual results obtained with differ-ent times were not statistically significant. In the cases of OP,NP, DIC, TRC, and BPA, equally high levels of extractionefficiency were achieved when the process continued for 5and 10 min, while the results obtained after 15 min wereconsiderably lower. Presumably, under the prolonged influ-ence of ultrasound, the equilibrium of derivatization shiftstowards the substrates, resulting in the decrease of efficiencyof the concurrently proceeding extraction and derivatizationprocesses. Keeping that in mind, 5-min extraction time waschosen as the most suitable for further studies.

Fig. 5 The influence of saltingout effect on extractionefficiency of target compounds(n=3)


Method validation

Tables 2 and 3 present an overview of the method’s perfor-mance, including linearity, limits of quantification (LOQ)and detection (LOD), repeatability, and recovery for theseanalytes when using deionized water as the sample matrix(Table 2) and influent and effluent wastewater as the samplematrix (Table 3). Calibration curves were obtained by spik-ing the wastewater samples with eight concentration andperforming the extraction. Calibration curves were linearwithin the studied concentration range, with coefficients ofcorrelation greater than 0.987 for all the target compounds.RSD of the determination ranged from 2.0 to 16.8 %,depending on the analyzed compound and the kind of matrix

used. The LOQ, determined as the analyte concentrationcorresponding to a signal/noise ratio of 10, ranged from0.005 to 2.758 μg L−1. The LOD, defined as the concentra-tion that corresponds to three times the standard deviation ofblanks, were between 0.002 and 0.828 μg L−1. Recoverieswere evaluated by spiking matrix before extraction and com-pare the signal obtained with the extract spiked after. Wherethe target compounds were initially present in the wastewatersamples, the areas of peaks registered during blank analysiswere subtracted from the corresponding peak areas of spikedsamples. Recoveries were obtained from influent and effluentwastewater samples at concentrations of 1 μg L−1 in the casesof OP, NP, TRC, BPA, and DIC, and at 10 μg L−1 in the casesof IBP, NAP, and KET; these values are shown in Table 3. In

Table 2 Analytical characteristics of the USAEME/GC–MS method

Analyte Linearity LODa LOQb Repeatability Recovery

Range (μg L−1) R2 (μg L−1) (μg L−1) (RSD%; n=3; 1 μg L−1) (%; n=3; 1 μg L−1)

BPA 0.01–10 0.9967 0.0001 0.0003 8.7 95

TRC 0.01–10 0.9942 0.0005 0.0015 2.7 97

NP 0.05–10 0.9937 0.0003 0.001 5.2 105

OP 0.05–10 0.9936 0.0003 0.001 4.8 104

DIC 0.05–10 0.9982 0.003 0.009 6.9 99

IBP 0.5–10 0.9907 0.005 0.015 7.5c 95c

NAP 0.5–10 0.9968 0.081 0.269 6.5c 97

KET 2–10 0.9933 0.061 0.204 6.8c 94c

a Limit of detectionb Limit of quantificationc 10 μg L−1

Fig. 6 The influence ofderivatization reagent volumeon extraction efficiency of targetcompounds (n=3)


Table 3 Analytical characteris-tics of the USAEME/GC–MSmethod obtained with the use ofinfluent and effluent wastewateras the sample matrix

a Limit of detectionb Limit of quantificationc 10 μg L−1

Analyte Linearity LODa LOQb Repeatability Recovery

Range (μg L−1) R2 (μg L−1) (μg L−1) (RSD%; n=3;1 μg L−1)

(%; n=3; 1 μg L−1)

Influent wastewater

BPA 0.01–10 0.9970 0.002 0.005 16.8 94

TRC 0.01–10 0.9960 0.003 0.009 3.3 97

NP 0.05–10 0.9966 0.008 0.027 5.3 103

OP 0.05–10 0.9975 0.011 0.035 4.9 106

DIC 0.01–10 0.9988 0.003 0.009 10.0 100

IBP 0.5–100 0.9960 0.121 0.403 7.8c 105c

NAP 0.5–100 0.9933 0.073 0.244 2.0c 103c

KET 0.5–100 0.9971 0.059 0.196 13.8c 96c

Effluent wastewater

BPA 0.01–10 0.9977 0.002 0.006 11.1 98

TRC 0.01–10 0.9908 0.003 0.010 2.8 102

NP 0.1–10 0.9938 0.029 0.098 13.5 108

OP 0.1–10 0.9894 0.030 0.099 9.6 103

DIC 0.01–10 0.9941 0.003 0.010 4.3 96

IBP 5–100 0.9937 0.762 2.540 13.1c 101c

NAP 5–100 0.9945 0.354 1.179 7.0c 96c

KET 5–100 0.9873 0.828 2.758 6.1c 97c

Fig. 7 The chromatograms registered during analysis of influent (a) and effluent (b) wastewater samples from WWTP B


the case when influent wastewater was used as the samplematrix, recoveries were between 94 and 106 %. When usingtreated effluents as the sample matrix, the recoveries werebetween 96 and 108 %. The comparison of the performanceof proposed method with methods usually used for the anal-ysis of target compounds in wastewater samples shows manyadvantages of USAEME/GC–MS procedure. The recoveries

obtained in the presented work are better than those deter-mined by SPE/GC–MS and SPE/LC–MSmethods, which areusually involved in such determinations, while repeatability ison similar level. The sensitivity of USAEME/GC–MS meth-od, expressed as LOD values, is better than those obtainedusing SPE technique for phenols, while for pharmaceuticalsit is comparable or slightly worse (Ballesteros et al. 2006;

Table 4 Concentration of the pharmaceuticals and phenols in wastewater samples from Polish wastewater treatment plants (WWTPs) (n=3)

Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range(μg L−1) (μg L−1) (μg L−1) (μg L−1) (μg L−1) (μg L−1) (μg L−1) (μg L−1)

Ibuprofen Naproxen Ketoprofen Diclofenac

WWTPA IW 8.48±1.95 n.d.–13.40 41.78±2.32 n.d.–41.78 8.14±0.52 n.d.–8.14 0.06±0.05 n.d.–0.06

(NS=3) EW n.d. n.d. n.d. n.d. n.d. n.d. 0.011±0.007 n.d.–0.011

WWTP B IW 30.46±3.65 n.d.–65.28 27.79±5.02 n.d.–117.23 9.46±0.96 n.d.–28.65 1.07±0.31 n.d.–3.32

(NS=20) EW 22.61±7.93 n.d.–41.07 n.d. n.d. n.d. n.d. 0.09±0.02 n.d.–0.16

WWTP C IW 31.25±4.37 n.d.–74.20 4.89±1.37 n.d.–12.03 21.61±2.17 n.d.–21.61 1.21±0.06 n.d.–1.41

(NS=9) EW 9.96±1.78 n.d.–18.30 7.76±1.37 n.d.–7.76 n.d. n.d. 0.07±0.07 n.d.–0.07

WWTP D IW 10.38±1.50 n.d.–18.42 4.01±2.34 n.d.–7.31 0.95±0.52 n.d.–0.95 0.65±0.05 n.d.–1.09

(NS=6) EW n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

WWTP E IW n.d. 0.78±0.19 n.q. n.d.

(NS=1) EW n.d. n.d. n.d. n.d.

WWTP F IW 3.41±0.85 n.d. n.d. n.q.

(NS=1) EW n.d. n.d. n.d. n.d.

WWTP G IW 10.10±0.75 2.50±1.12 n.d. n.q.

(NS=1) EW n.d. n.d. n.d. n.q.

WWTP H IW 18.54±3.18 2.03±0.09 n.q. n.q.

(NS=1) EW n.d. n.d. n.d. n.q.

WWTP I IW 19.01±0.16 551.96±104.35 233.63±44.58 3.54±0.65

(NS=1) EW n.d. n.d. n.d. 2.56±0.50

Octylphenol Nonylphenol Triclosan Bisphenol A

WWTPA IW 0.44±0.06 n.d.–0.44 1.52±1.23 1.01–2.21 n.d. n.d. 0.69±0.12 n.d.–0.69

(NS=3) EW 0.11±0.01 n.d.–0.11 0.88±0.16 n.d.–0.88 n.d. n.d. n.d. n.d.

WWTP B IW 1.90±0.21 n.d.–5.71 4.49±0.77 n.d.–16.92 1.33±0.15 n.d.–6.85 0.97±0.30 n.d.–2.88

(NS=20) EW 0.77±0.13 n.d.–4.02 0.80±0.11 n.d.–2.04 0.54±0.15 n.d.–0.82 0.61±0.10 n.d.–1.20

WWTP C IW 1.07±0.14 n.d.–0.36 10.60±1.93 n.d.–23.60 0.51±0.05 n.d.–1.99 0.21±0.02 n.d.–0.39

(NS=9) EW 0.14±0.02 n.d.–0.14 0.23±0.03 n.d.–0.23 0.06±0.01 n.d.–0.10 n.d. n.d.

WWTP D IW 0.33±0.13 n.d.–0.54 2.11±1.01 n.d.–3.25 0.19±0.01 n.d.–0.19 0.99±0.10 n.d.–2.20

(NS=6) EW n.d. n.d. 0.19±0.02 n.d.–0.19 0.91±0.21 n.d.–0.91 1.45±0.15 n.d.–2.10

WWTP E IW 0.84±0.19 0.45±0.14 0.87±0.15 0.54±0.35

(NS=1) EW 2.28±0.08 n.d. 0.84±0.07 10.84±0.37

WWTP F IW 2.35±0.35 9.59±0.60 0.19±0.08 2.41±0.88

(NS=1) EW 1.60±0.87 n.d. n.q. 1.75±1.26

WWTP G IW 1.64±0.27 8.58±1.05 0.47±0.14 1.22±0.23

(NS=1) EW n.q. n.d. 0.10±0.05 n.q.

WWTP H IW 1.06±0.21 n.d. 0.26±0.05 0.53±0.78

(NS=1) EW n.q. n.d. 0.02±0.13 n.q.

WWTP I IW n.d. 102.54±19.08 n.d. 0.03±0.01

(NS=1) EW n.d. n.d. n.d. 1.41±0.24

SD standard deviation, NS number of samples, IW influent wastewater, EW effluent wastewater, n.d. not detected, n.q. not quantified


Gatidou et al. 2007; Gómez et al. 2007a, b; Gracia-Lor et al.2010; Yu and Wu 2013).

Occurrence of target phenols and pharmaceuticalsin municipal wastewaters from Polish WWTPs

The developed USAEME/GC–MS method was applied fordetermination of ibuprofen, naproxen, ketoprofen, diclofenac,octylphenol, nonylphenol, triclosan, and bisphenol A in influ-ents and effluents from municipal wastewater treatment plantslocated in nine cities of Poland (see Fig. 1). The chromatogramsof influent (a) and effluent wastewater samples (b) fromWWTP B are shown in Fig. 7. The occurrence of targetcompounds and levels of contamination in the analyzed waste-water samples are summarized in Table 4.

All studied compounds were found in the influents; in theeffluents, all compounds besides KET were registered. Theconcentrations of determined pollutants in influent wastewa-ters varied from values below LOD to several hundreds ofmicrograms per liter, while in treated effluents were betweenLOD and several dozens of micrograms per liter. The targetpharmaceuticals were found with frequency of 56 % for IBP,44 % for NAP, 30 % for DIC, and 21 % for KET in influentwastewater and 14 % for DIC, 12 % for IBP, and 2 % forNAP in effluent wastewater. The highest average valueswere found for NAP (552 μg L−1) and KET (234 μg L−1)in WWTP I. In 32 cases out of 172 (4 pharmaceuticals×43samples), measured concentration was above 10 μg L−1 ininfluent wastewater. It happened 21 times for IBP, seventimes for NAP, and four times for KET. In seven cases outof 172, measured concentration was above 1 μg L−1 ineffluent wastewater. It happened five times for IBP andNAP and one time for DIC. The target phenols were foundwith frequency of 65 % for NP, 47 % for OP and BPA, and44 % for TRC in influent wastewater and 26 % for TRC,23 % for OP, 19 % for BPA, and 14 % for NP in effluentwastewater. The highest values were found for NP(24 μg L−1) and OP (5.7 μg L−1) in influent wastewater. Infour cases out of 172 (4 phenols×43 samples), measuredconcentration was above 10 μg L−1 in influent wastewater. Itwas registered only for NP. In 10 cases out of 172, measuredconcentration was above 1 μg L−1 in effluent wastewater. Ithappened six times for BPA, three times for OP, and one timefor NP.

The concentrations of target phenols and pharmaceuticalswere similar in samples collected from all the wastewatertreatment plants except of WWTPI, where the registeredconcentrations were much greater than in other WWTPs.The WWTPI is a small municipal wastewater treatmentplant; the influents are transported into this plant from house-holds mostly by special waste-removal trucks.

In most cases, concentrations of the target compoundsregistered in effluents were much lower than those found in

influent wastewaters. Nevertheless, the concentrations of OPand BPA registered in WWTPE were higher in effluents thaninfluents. This phenomenon might be explained by the factthat the samples of both influent and effluent wastewaterswere taken at the same time, meaning that they did notoriginate from the same portion of wastewater. Analogoussituation was previously reported during determination ofnaproxen, diclofenac, and ibuprofen in municipal wastewa-ters (Hernando et al. 2006). In order to determine the effec-tiveness of the wastewater purification, collection of theinfluent and effluent samples from the same wastewaterportion was done four times in WWTPB. It was achievedby collecting the effluents 24 h after influent taking. Table 5shows the average concentrations of target pharmaceuticalsand phenols determined in the influents and effluents fromthe same wastewater portion and the removal efficiencieswere calculated. The obtained values of removal efficiencyrates were very high and exceed 90 % for all compoundsexcept DIC.

Conclusions

In the present study, a new analytical methodology based onultrasound-assisted emulsification–microextraction followedby GC–MS determination has been proposed for the determi-nation of acidic pharmaceuticals and phenols. In situ deriva-tization with acetic anhydride, conducted simultaneously withextraction, was successful under the optimized conditions.Scrutiny of the available literature sources shows that thepresent work is first to describe acylation with the useof acetic anhydride applied for determination of acidicpharmaceuticals and OP, NP, and BPA in water samples.

The proposed USAEME technique with in situ derivatiza-tion step offers several advantages in terms of simplicity, lowcost, minimal solvent consumption, and very short time of

Table 5 The average concentrations of target compounds determinedin the influents and effluents from the same wastewater portion and theremoval efficiencies

Analyte Average concentration (μg L−1) Removal efficiency

Influent Effluent

IBP 43.95 1.48 91±15 %

NAP 6.46 0.14 98±5 %

KET 12.07 0.21 98±5 %

DIC 0.38 0.06 84±5 %

OP 0.39 0.02 96±1 %

NP 2.67 0.01 98±3 %

TRC 0.92 0.28 96±6 %

BPA 1.26 0.01 99±1 %


sample preparation. The method has been successfully used todetermine ibuprofen, naproxen, ketoprofen, diclofenac, octyl-phenol, nonylphenol, triclosan, and bisphenol A in influentand effluent municipal wastewater samples obtained fromnine wastewater treatment plants located in Poland. All stud-ied pharmaceuticals and phenols were found in the influentsfrom Polish WWTPs; in the effluents, all compounds besidesKET were registered. The concentrations of pollutants ininfluent wastewaters varied from values below LOD to severalhundreds of micrograms per liter, while in treated effluentsconcentrations were between LOD and several dozens ofmicrograms per liter. Average elimination rates of pharmaceu-ticals and phenols during purification process carried in mu-nicipal WWTP are 93 % and 97 %, respectively.

Open Access This article is distributed under the terms of the CreativeCommons Attribution License which permits any use, distribution, andreproduction in any medium, provided the original author(s) and thesource are credited.

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