Evaluation of the presence of endocrine-disrupting compounds in dissolved and solid wastewater treatment plant samples of Gran Canaria island (Spain)

Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 790570, 15 pages http://dx.doi.org/10.1155/2013/790570 Research Article Evaluation of the Presence of Endocrine-Disrupting Compounds in Dissolved and Solid Wastewater Treatment Plant Samples of Gran Canaria Island (Spain) T. Vega-Morales, Z. Sosa-Ferrera, and J. J. Santana-Rodríguez Departamento de Qu´ ımica, Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Spain Correspondence should be addressed to J. J. Santana-Rodr´ ıguez; [email protected] Received 13 May 2013; Revised 29 July 2013; Accepted 31 July 2013 Academic Editor: Koichiro Wada Copyright © 2013 T. Vega-Morales et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Liquid and solid samples from two wastewater treatment plants (WWTPs) on Gran Canaria Island (Spain) have been tested for the presence of compounds with endocrine-disrupting properties. The selected degradation stages were sampled bimonthly from each WWTP over the 12-month period from July 2010 to July 2011. The analytical methods used for the determination of the endocrine-disrupting compounds (EDCs) were based on on-line solid phase extraction, microwave-assisted extraction (MAE), and ultrasonic-assisted extraction (UAE) coupled to UHPLC-MS/MS. All of the hyphenated methodologies employed in this work showed good recoveries (72–104%) and sensitivities, with LODs lower than 7.0 ng L−1 and 6.3 ng g−1 for the dissolved and solid fractions, respectively. We have also evaluated the estrogenicity of the samples in terms of their estradiol equivalent concentrations (EEQs). The chemical analysis of the selected EDCs revealed fairly low concentrations for both natural and synthetic oestrogens, alkylphenolic compounds, and bisphenol-A in each of the dissolved, particulate, and sludge samples (ng L−1 or ng g−1). However, the estimated estrogenic activity indicated that the majority of samples could represent an important environmental risk, clearly surpassing the threshold to exert deleterious consequences on living beings. 1. Introduction The current concern about endocrine-disrupting compounds (EDCs) is based on scientific facts that collectively indicate that EDCs potential could induce unhealthy changes in humansandwildlifespeciesevenatlow,ngL −1,concentration levels [1–3]. Among EDCs, those that mimic endogenous oestrogens are particularly important because of their central role in reproductive functions [4]. This type of endocrine disrupter, referred herein as estrogenic endocrine disrupting compounds (e-EDCs), interacts with the human oestrogen receptor (hER𝛼), which has larger and more flexible binding sites than what natural oestrogen (17𝛽-estradiol) requires and is therefore a more vulnerable target for a broad range of interferents with high structural diversity [4]. Moreover, because estrogenic receptors are quite similar between different vertebrates, e-EDCs may affect the endocrine functions of many animal species within theecosystem.Theissueofwhetherhumansandwildlife suffer deleterious consequences resulting from the exposure to these chemicals has been extensively discussed in many reviews (e.g., [5,6]). There is evidence that prolonged exposure to these substances, even at trace concentrations, could be a causal factor in breast cancer [6] and testicular germ cell cancer [7]. e-EDCs have also been correlated to increased rates of hypospadias and cryptorchidism [8]aswellasthedecreasing sperm count observed in many countries [9]. The effects of these substances on wildlife species have also been shown in numerous scientific papers [10–12]with similar expectations. In short, several alterations have been reported in a wide range of animal species, such as decreased fertilityinbirds,fish,andmammals;demasculinisationand feminisation of fish, shellfish, gastropods, birds, and mammals; reduced efficacy of the incubation processes in fish, birds, and turtles; immune system disorders in birds and mammals; and alterations in the thyroid of birds and fish [13]. e-EDCs can enter the environment from a variety of sources. However, the vast majority of them are generally focused in localised “hot spots” of chemical discharges, 2BioMed Research International such as wastewater treatment plants (WWTPs) effluents, agricultural runoff, and landfills, which create a continuous input of these pollutants into the environment [14]. Despite this fact, the relatively high lipophilicity and persistence of e-EDCs enable their bioaccumulation and biomagnification within the environment, and therefore, a more globalised phenomenon of endocrine disruption is possible. The primary objective of the present work was to develop a comprehensive evaluation of the endocrine-disrupting activity in different fractions of wastewater samples (dissolved phase, particulate phase, and sludge) taken from two different wastewater treatment plants (WWTPs) on the island of Gran Canaria (Spain). The chemicals analysed in this work included both natural and synthetic steroids as well as nonsteroidal compounds (Table 1). Regardless of their nature, all of the selected compounds share the ability to mimic endogenous estradiol and have been frequently found in both WWTP effluents and surface waters. To achieve our objective, we evaluated the estrogenicity of the samples in terms of their estradiol equivalent concentrations (EEQs) [15,16]. Because the effects of estrogenic activity in eEDCs have proved to be additive, the EEQ can be defined as the sum of the concentrations for each individual eEDC after normalising by an estradiol equivalency factor (EEF) [17]. These EEFs are defined as the quotient of half maximal effective concentrations of estradiol and other EDC (EC50E2/EC50EDC) and are set to 1 for estradiol (E2) [18]. To estimate the concentration of each e-EDC, we employed a variety of extraction/purification procedures that have been previously published by our research group [19,28, 29]. These methods include microwave-assisted extraction (MAE) for sludge samples, ultrasonic-assisted extraction (UAE) for particulate matter, and on-line solid Phase Extraction (SPE) for the dissolved fraction. All of these extraction techniquesweresubsequentlycoupledtoanUHPLC-MS/MS instrument for the identification and quantification of each compound. In turn, the calculations of the EEFs were carried out by averaging the most recent values reported in the scientific literature. Given the wide range of EEF values observed even amongst the same e-EDC, we considered several biological, based assays (BBAs), such as the oestrogen receptor (ER) binding assay, the yeast oestrogen screen (YES), and the oestrogen responsive chemically activated luciferase expression (ER-CALUX), to obtain significant EEFs for each analyte being studied. This approach has been successfully employed before in other publications [17,30]. To the best of our knowledge, this is the first attempt to estimate the potential estrogenic risks in this region and one of the few reports [17,21,31] that take into consideration the different fractions of WWTPs samples that are released into the environment. 2. Method and Materials 2.1. Chemicals. The natural oestrogens 17𝛽-estradiol (E2), estrone (E1), and Estriol (E3); synthetic steroids 17𝛼ethinylestradiol (EE) and diethylstilbestrol (DES); and nonsteroidal compounds bisphenol-A (BPA), nonylphenol (NP), and octylphenol (OP) were purchased from Sigma Aldrich in greater than 98% purity (Madrid, Spain). Stock solutions were prepared at 1000 𝜇gmL −1 by dissolving each standard in methanol and storing in glass-stoppered bottles at −18∘C. Short ethoxylated chains (AP1-2EOs) were directly acquired as stock solutions (10 𝜇gmL −1 in 1 mL) in acetone andstoredat−18∘C. Long-chained APnEOs (𝑛≥3) were only available in technical mixtures. Igepal CO210, CO520, and CO720 contained a range of NPnEO oligomers with 3–12 ethoxy units (EO), whereas Igepal CA210, CA520 and CA720 contained the same EO range of OPnEO oligomers. Stock solutions (1000 𝜇gmL −1)oflong-chainalkylphenolic ethoxylated surfactants were also prepared by dissolving the appropriate quantities. These solutions were used to calculate the concentrations of the analysed samples; however, considering their low to nonexistent estrogenic potential, we have not included them in the EEQ calculations. 2.2. Sampling Sites and Collection. Samples of wastewater, particulate matter, and sludge were collected bimonthly for a period of 12 months (July 2010–July 2011) from two different WWTPs located in northeastern Gran Canaria island (Spain). This region contains both the vast majority of the population (hosting more than half a million people) and the largest concentration of the limited industrial activity on the island. Therefore, domestic sewage systems are the primary source of raw wastewater flowing into the two WWTPs. The characteristics of each WWTP under study are described below. WWTP1. WWTP1 is based on a conventional activated sludge (CAS) process. In this plant, there was an important agricultural water input (irrigation of agricultural crops) flowing into the plant despite the predominantly domestic nature of the incoming wastewater. This plant possesses a treatment capacity of 10 L seg−1, or approximately 5,000 equivalent inhabitants. Liquid wastewater samples were taken from the output of the secondary treatment, the clarifier-settled tank effluent, whereas the sludge samples were collected from drying tanks that were exposed to the outdoors. WWTP2. WWTP2 employed a novel biomembrane reactor (BMR) treatment process. WWTP1 and WWTP2 receive very similar raw wastewaters with an important agricultural water input given the similarities between both locations. The treatment capacity of this plant slightly surpassed 7000 equivalent inhabitants. In this case, the liquid wastewater samples were taken after the biomembrane filtration process, whereas the sludge samples were collected from the output of thecentrifugeusedtodewaterthesludge. 2.3. Sample Pretreatment. The liquid wastewater samples were collected in glass stoppered amber bottles (2.5 L) and acidified to a pH of <3topreventthelossofthee-EDC targets via biological degradation and abiotic reactions such as hydrolysis [32]. The samples were then stored at 4∘Cand extracted within 48 h. Prior to this extraction, the wastewater samples were filtered through 0.45 𝜇m membrane filters BioMed Research International 3 Table 1: Physicochemical properties of the compounds under study. Compounds Chemical structure Molecular weight Log 𝐾OW aSolubility (mg L−1)b Diylstilbestrol (DES) HO H3C CH3 OH 268.1 5.64 3.32 17𝛼-Ethinylestradiol (EE) HO CH CH3 OH H H H296.4 4.15 4.8 17𝛽-Estradiol (E2) HO CH3 OH HH H272.4 3.94 13.0 Estriol (E3) HO H H H CH3 OH OH 288.4 2.81 13.0 Estrone (E1) HO HH H CH3 O 270.4 3.43 13.0 Bisphenol A (BPA) HO CH3 OH H3C 228.0 3.32 120.0 Nonylphenol (NP) OH R9 220.0 4.48 1.57 Octylphenol (OP) OH R8 206.0 4.12 12.6 4BioMed Research International Table 1: Continued. Compounds Chemical structure Molecular weight Log 𝐾OW aSolubility (mg L−1)b Nonylphenol monoethoxylate (NP1EO) OH O R9 264.0 3.02 4.17 Nonylphenol diethoxylate (NP2EO) OH OO R9 308.0 3.38 4.21 Octylphenol monoethoxylate (OP1EO) OH O R8 250.0 8.0 4.10 Octylphenol diethoxylate (OP2EO) OH O O R8 294.0 13.2 4.00 aOctanol/water partition coefficients. bSolubility at 20∘C. (Millipore, Bedford, MA, USA). This pore size was used as a threshold to separate the dissolved and particulate phases [33]. Therefore, the 0.45 𝜇m filters with the retained particulate matter (between 0.1 and 0.2 g for all samples) were stored at −18∘Cpriortoanalysis.Sludgesampleswerecollected in glass-stoppered flasks and stored in a freezer at −18∘C. The target compound determination of both the particulate matter and sludge samples was conducted within 48 hours. 2.4. Analytical Methods. In the evaluation of the estrogenicity of the environmental samples, the calculation of the e-EDC concentrations was the first goal achieved. We employed three extraction/preconcentration methods that had been previously published by our research group to accomplish this goal [19,28,29]. All of the methods’s hyphenated methodologies employed in this work showed the sensitivity and selectivity needed to determine this class of compounds in wastewater matrices (Table 2). The recoveries for both liquid and solid samples were between 72–104%, whereas the limits of detection ranged from 0.3 to 2.1 ng L−1 in liquid samples, and from 0.1 to 1.9 ng g−1 in solid samples, including both particulate and sludge materials. In addition, the triple quadrupole mass spectrometry detection system employed in this work offered the selectivity criteria (e.g., retention times, parent and product ions, and ion ratios) needed to unequivocally determine the selected compounds. In order to take into consideration the matrix effects, a well-known phenomenon which usually impair the proper ionization of the analytes in complex matrices when using ESI interfaces, we have employed in each methodology a matrix-matched calibration for the quantification of the analytes. This approach allowed us, by the one hand, to not underestimate the results due to the ion suppression observed in the electrospray interface, and on the other, to prevent the use of isotopic labeled internal standards for the quantification processes. This latter case, although it is probably the most appropriate approach, requires an important investment due to the high cost of acquisition, and often are not commercially available, as occured with several of our analytes (e.g., alkylphenolic polyethoxylated compounds). A brief description of each method is given in the following sections. Figure 1 shows the pathways undertaken to analyse the e-EDCs in all of the fractions. 2.4.1. Analysis of e-EDCs in Liquid Samples. The accurate and simultaneous determination of the selected e-EDCs dissolved in the wastewater samples was undertaken first. The analytical method consisted of an on-line SPE step followed by the determination of the selected compounds via ultrahigh performance liquid chromatography coupled to a triple quadrupole mass spectrometry detector (UHPLC-MS/MS) (Waters, Milford, MA, USA). This method enabled us to considerably improve the limits of detection and quantification relative to the off-line SPE methods. Moreover, it significantly reduces both the global analysis time and background noise and noticeably improves the reproducibility of the results. The on-line SPE protocol was performed using two Oasis HLB extraction columns (20 𝜇m, 2.1 mm ×50 mm) working in parallel. Chromatographic separation was accomplished using an ACQUITY BEH C18 chromatographic column (1.7 𝜇m, 2.1 mm ×50 mm). Both the chromatographic and extraction columns were acquired from Waters (Milford, MA, USA). The mobile phase consisted of both water and methanol containing 0.1% NH3to promote both the proper ionisation of the compounds in the electrospray interface (ESI) and the formation of ammonium adducts. The gradient elution consisted of a 50 : 50 (v : v) mixture of water:methanol BioMed Research International 5 Sewage sludge samples MAE extract Liquid samples UPLC column Activated with After elution Ultrasonic-assisted extraction (UAE) Evaporate to dryness Reconstituted Injected into Injected into Sewage sludge samples Wastewater samples Stirred to homogenize and air-dried 1g of sample to each PTFE vessel Microwave-assisted extraction (MAE) Dissolved phase samples Particulate matter samples On-line SPE Oasis-HLB columns Oasis column re-equilibration Milli-Q water (0.5% acetic acid) Weak clean-up 1st elution 0.3 mL·min−1 Strong clean-up (Hex: act: MeOH) 1:1:1 UHPLC-MS/MS On-line SPE Oasis-HLB columns Filtered through Retained fraction in the filters (100 𝜇L MeOH) Dilution step: 1/20 (UAE extract/milli Q-water (v/v)) On-lineSPE clean-up On-line SPE clean-up Dilution step: 1/20 (MAE extract/milli Q-water (v/v)) ∙Room temperature ∙12 hours in the dar k ∙10mL of MeOH 0.45 𝜇m (H2O/MeOH 9/1 (v/v)) UHPLC-MS/MS 2mL·min−1 2mL·min−1 2mL·min−1 2mL·min−1 On-line SPE Oasis-HLB columns ∙10 min ∙300W ∙5mL of MeOH ∙10 min Injected into UHPLC-MS/MS Figure 1: Flow scheme of the analytical methodologies that have been undertaken for the chemical analysis of the selected e-EDCs in each fraction. that was linearly increased to 100% methanol (B) over 4 minutes [19]. 2.4.2. Analysis of e-EDCs in Solid Samples Sludge Samples. The concentrations of these substances in the sewage sludge samples were obtained using a microwaveassisted extraction (MAE) technique [28], followed by the On-Line-SPE-UHPLC-ESI-MS/MS protocol described in the previous section [19] to purify the extracts and determine the concentration of each analyte. To summarise the MAE procedure, 1 g of the sludge was transferred to a polytetrafluoroethylene (PTFE) vessel. Next, 5 mL of an extractant (methanol) was added to the sample, andthevesselswereclosedandplacedsymmetricallyona rotor. Once the rotor was placed in the microwave oven, a power of 300 W was used for 10 min. Particulate Matter. The extraction of the e-EDC from the particulate phase was carried out according to the following extraction methodology [29]. The 0.45 𝜇m membrane filters with the retained particulate matter (between 0.1 and 0.2 g for all samples) were immersed for 10 min in an ultrasonic bath containing 10 mL of methanol. The methanol extract was collected in a flask, evaporated to dryness under a gentle nitrogen stream, and reconstituted in 100 𝜇L of methanol. The final extracts were analysed separately, and the concentrations of the dissolved and particulate phases are reported separately for each sample. The determination of the analytes was accomplished via On-Line-SPE-UHPLC-ESI-MS/MS [19]. 2.5. Estrogenic Potential Calculations. The estrogenicity of the sampleswasevaluatedintermsoftheestradiolequivalent concentrations (EEQs) [15,16]. EEQs can be defined as follows: EEQ𝑖=𝐶 𝑖×EEF𝑖,(1) where 𝐶𝑖is the concentration of compound 𝑖in the sample, and EEF𝑖is the estradiol equivalency factor of compound i. 2.5.1. Estradiol Equivalency Factors. The estradiol equivalency factor (EEF) is defined by the following expression: EEF𝑖=EC50E2 EC50𝑖,(2) where EC50E2is the concentration that yields half of the maximum response for the estradiol, and EC50E2is the concentrationthatyieldshalfofthemaximumresponsefor compound i. A large number of in vitro tests have been developed for the rapid and sensitive screening of endocrine disrupting chemicals [34]. However, the most widely employed in vitro tests for estimating the estrogenic potential and thus 6BioMed Research International Table 2: Analytical parameters obtained for the developed methodology. On-Line-SPE-UHPLC-MS/MSe Compound LODa(ng L−1)LOQb(ng L−1)RSDc(%, peak area) Recoveryd(%) 5ngL −1(𝑛=6)50ngL −1(𝑛=6)10ngL −1(𝑛=6)500ngL −1(𝑛=6) NP 1.3 4.3 1.7 5.2 93.1 95.3 OP 1.8 6.0 2.4 9.8 89.9 95.0 NPEOs 0.3–1.8 1.0–6.0 1.1–3.9 2.0–8.1 83.1–100.2 84.1–104.0 OPEOs 0.7–2.1 2.3–7.0 0.9–4.8 1.5–7.3 86.2–104.2 85.3–95.7 BPA 1.9 6.3 5.0 8.5 90.7 88.4 E2 1.0 4.0 6.4 5.7 89.7 88.4 E1 1.3 4.3 2.1 4.9 84.5 98.3 E3 1.3 4.3 7.7 10.1 86.9 85.4 EE 0.9 3.0 6.0 9.1 88.3 90.9 DES 0.6 2.0 7.5 6.2 102.5 90.3 MAE-On-Line-SPE-UHPLC-MS/MS Compound LODa(ng g−1)LOQb(ng g−1)RSDc(%, peak area) Recoveryd(%) 5ngg −1(𝑛=6)250ngg −1(𝑛=6)5ngg −1(𝑛=6)250ngg −1(𝑛=6) NP 0.1 0.3 8.9 9.2 79.5 78.2 OP 0.1 0.3 7.0 8.1 82.6 88.1 NPEOs 0.1–0.4 0.3–1.3 1.3–7.1 2.1–8.7 74.3–99.7 76.7–102.0 OPEOs 0.1–0.6 0.3–2 0.9–5.7 1.9–6.3 82.5–100.7 80.3–95.1 BPA 0.2 0.7 3.3 3.3 79.3 82.2 E2 0.3 1.0 5.0 8.1 95.3 90.4 E1 0.5 1.7 4.8 5.7 75.1 80.9 E3 0.1 0.2 2.3 4.0 88.8 83.9 EE 0.3 1.0 8.3 5.9 98.1 97.2 DES 0.1 0.3 5.8 5.4 92.9 97.0 UAE-On-Line-SPE-UHPLC-MS/MS Compound LODa(ng g−1)LOQb(ng g−1)RSDc(%, peak area) Recoveryd(%) 25 ng g−1(𝑛=6)250ngg −1(𝑛=6)25ngg −1(𝑛=6)250ngg −1(𝑛=6) NP 1.3 4.3 3.7 3.1 71.5 73.2 OP 0.9 3.0 6.1 8.4 77.7 80.9 NPEOs 0.3–1.8 1.0–6.0 0.9–5.0 0.7–4.2 81.2–102.6 77.3–96.5 OPEOs 0.6–1.9 2.0–6.3 1.2–6.1 0.5–4.2 79.9–103.5 80.9–98.4 BPA 0.5 1.3 3.3 6.1 85.2 90.0 E2 1.3 4.3 5.0 4.6 94.3 96.4 E1 1.0 4.0 4.8 8.0 85.1 92.4 E3 0.9 3.0 2.3 7.3 100.2 98.6 EE 1.9 6.3 8.3 4.9 91.4 87.3 DES 0.5 1.3 5.8 3.5 86.1 90.2 aLimit of detection; blimitofquantification;crepeatability (intraday assays) expressed as relative standard deviation (RSD); drecovery percentages obtained for different matrices spiked at two different concentration levels; edata already published in Vega-Morales et al., 2012 [19]. the estradiol equivalency factors can be summarised as follows. (i) Receptor Binding Assays.Measurethebindingaffinityof asubstancetoahormonereceptor(e.g.,oestrogenreceptor (ER)). (ii) Cell Proliferation Assays. Measure the ability of a substance to stimulate the growth of a hormone responsive cell (e.g., MCF-7, and E-screen). (iii) Reporter Gene Assays. Measure the ability of a substance to activate the transcription of a reporter gene construct in cells (e.g., yeast oestrogen screen (YES) or mammalian cell assays). All of these in vitro tests are able to satisfactorily estimate the estrogenicity of several e-EDCs in a large variety of environmental matrices such as the wastewater samples. However, the EEFs reported in the literature for the same e-EDC vary significantly depending on the in vitro test BioMed Research International 7 Table 3: Estradiol equivalency factors values of 17𝛽-estradiol, estrone, estriol, 17𝛼-ethinylestradiol, diethylstilbestrol, bisphenol-A, nonylphenol, octylphenol, and their short-chained ethoxylates. E2 E1 E3 EE DES BPA NP NP1EO NP2EO OP OP1EO OP2EO EEFa 1b0.38b2.4 × 10−3b1.19b2.6l1.1×10−4b2.5 × 10−5b2.0 × 10−7c6.0 × 10−6j7.8 × 10−6b4.0 ×10−6g0d 1d0.01c0.01c1.62b3.6× 10−5b1.0 × 10−5d1.3 × 10−5c5.7 × 10−6k4.9 × 10−4c40 ×10−6g 1e0.05d0.08c0.8c2.5×10−3b5.1 × 10−4e3.8 × 10−6d1.1 ×10−6k1.0 × 10−5c 1f0.02e0.34e1.9c5.0×10−5c4.0 ×10−4f4×10−6d5.7 × 10−4d 1g0.14f0.8d1.0× 10−5d5.0 × 10−4g1×10−6d2.1 × 10−4e 1h0.096g1.20d7.8 × 10−6d2.1 × 10−4h1.6 ×10−4f 1i0.1k—2.4×10−5e2.3 × 10−4i3.0 × 10−5g Average 1 0.11 0.11 1.25 2.6 3.9 ×10−42.7 ×10−44.4 ×10−64.3 ×10−62.1 ×10−44.0 ×10−62.0 ×10−6 aEstradiol equivalency factor; bRutishauser et al., 2004 [17]; cCampbell et al., 2006 [14]; dMurk et al., 2002 [20]; eSun et al., 2008 [21]; fPurdom et al., 1994 [22]; gLegler et al., 2002 [23]; hSong et al., 2006 [24]; iVigan` oetal.,2008[25]; jDuft et al., 2003 [26]; kBrix et al., 2010 [18]; lPojana et al., 2004 [27]. employed [20,23,35]. In addition, the reported EEFs sometimes vary significantly for the same in vitro test applied by different researchers [14]. Table 3 showstheEEFspreviously reported for the selected e-EDCs in the literature. Given the observed variability, the EEFs reported in the literature were averaged, and then, applied in (1)tocalculate the individual estradiol equivalent concentrations (EEQ𝑖). 2.5.2. Estrogenic Potential. As the additive estrogenic activity of e-EDCs has been proven [17], the EEQ can be defined as the sum of the concentrations for each individual e-EDC after normalising with their estradiol equivalency factors (EEFs).Thus,theconcentrationsobtainedfromtheanalytical methods employed (𝐶𝑖) for each target e-EDC and fraction studied (dissolved phase, particulate matter and sludge) were multiplied by their relative potency. Consider EEQ𝑡=∑EEQ𝑖, EEQ𝑡=∑[𝐶1+EEF1]+[𝐶 2+EEF2]+[𝐶 3+EEF3]⋅⋅⋅. (3) The estradiol equivalent concentration calculated from the sum of the individual compounds represents the overall endocrine-disrupting activity of the sample. 3. Results and Discussions 3.1.ConcentrationsofSelectedEDCs. The concentrations of the recorded e-EDCs for all of the samplings conducted (July 2010–July 2011) are highlighted in Tables 4and 5. Each table shows the concentration of the selected analytes in the dissolved phase fraction (samples are collected from the final effluents of the two WWTPs under study) and solid phases (particulate matter and sludge samples), respectively. 3.1.1. Natural and Synthetic Oestrogens. The concentration of both natural and synthetic oestrogens dissolved in the wastewater samples has consistently been in the low ng L−1 levels throughout the entire sampling time. Of the selected natural oestrogens, 17𝛽-estradiol (E2) and estriol (E3) were present in the highest concentrations in the two WWTPs studied with an average of 21.5 and 16.8 ng L−1, respectively. Moreover, both compounds were consistently detected in the liquid samples (93% of all cases). Estrone (E1) was also detected at low ng L−1 levels in these fractions; however, neither its average concentration (5.4 ng L−1)norits detection frequency (detected in only 50% of the samples) approached those reported for E2 and E3. The studied synthetic steroids, 17𝛼-ethinylestradiol (EE) and diethylstilbestrol (DES) were only detected in a few samples of this fraction (14% and 7% of the total samples, resp.). However, they were regularly found in both the particulate matter and sludge samples with observed concentrations higher than those of natural oestrogens. This phenomenon led us to hypothesise that, given the relatively high octanol/water partition coefficient and low water solubility of EE and DES, both substances were strongly associated with the solid fractions of the samples; however, they were not eliminated by the WWTP degradation processes. The natural and synthetic oestrogen concentrations observed in both the particulate matter and sludge samples were in the low ng g−1 levels, reaching concentrations close to 100 ng g−1 in a few cases. Attending to the obtained results, we can describe a common behavioural pattern for these substances; compounds with a relatively high log 𝐾ow were systematically detected in the solid fraction of the samples (e.g., EE, DES or E2), whereas compounds with a relatively low log 𝐾ow were not commonly detected in either the particulate matter or sludge samples (e.g., E3). The analytical results obtained in the present study for oestrogens in water are in agreement with those of previous studies [17,18,36]. 3.1.2. Alkylphenolic Compounds and Their Ethoxylates. Alkylphenolic ethoxylated surfactants (APnEOs) and raw alkylphenols (APs) (nonylphenol (NP) and octylphenol (OP)) were commonly found in all of the studied matrices in concentrations ranging from low parts per trillion (ppt) to low parts per billion (ppb). The distribution of the alkylphenolic ethoxylates varied significantly between the different matrices analysed. On the one hand, we detected the presence of a wide range of APnEOs in the dissolved samples, ranging from raw 8BioMed Research International Table 4: Dissolved phase concentrations (ng⋅L−1) for each target compound in the three WWTPS under study. Jul ’10 Sep ’10 Nov ’10 Jan ’11 March ’11 May ’11 Jul ’11 Frequency (%) Average WWTP 1 NPa88.5 ±0.7 18.1 ±1.1 71.0 ±5.0 209.1 ±15.3 77.0 ±0.9 89.4 ±9.7 110.0 ±9.0 100 94.7 OPa12.6 ±0.7 BQLc9.7 ±0.7 95.2 ±9.2 17.8 ±1.1 11.7 ±0.9 23.0 ±0.9 85.7 28.3 NPEOsa729.4 ±0.7 395.3 ±22.7 958.9 ±17.3 464.3 ±18.7 313.0 ±12.9 303.7 ±9.3 572.0 ±22.3 100 533.8 OPEOsa101.6 ±0.7 94.1 ±4.5 569.0 ±45.9 350.4 ±22.9 66.9 ±5.9 84.2 ±5.3 93.9 ±7.3 100 194.3 BPAa12.6 ±0.7 BQLcBQLc9.6 ±0.9 BDLbBQLc25.1 ±3.2 42.9 15.8 E2a21.3 ±0.7 18.2 ±0.3 39.2 ±2.9 65.4 ±3.1 BQLc8.3 ±0.5 14.7 ±1.3 85.7 27.9 E1a4.5 ±0.7 3.9 ±0.1 9.7 ±0.9 BDLb8.2 ±0.5 BQLcBDLb57.1 6.6 E3a16.4 ±0.7 31.9 ±2.1 42.1 ±2.0 18.7 ±0.9 19.2 ±1.9 7.7 ±0.3 12.8 ±0.9 100 21.3 EEaBQLc2.9 ±0.1 BQLcBQLcBQLcBDLbBQLc14.3 2.9 DESaBQLcBQLcBQLcBQLcBQLcBQLcBQLc0.0 0.0 WWTP 2 NPa31.4 ±3.1 49.3 ±3.3 12.7 ±0.3 95.8 ±7. 5 17.5 ±1.1 15.7 ±3.9 56.6 ±2.3 100 39.9 OPaBQLc12.9 ±0.7 3.1 ±0.1 14.9 ±0.8 11.4 ±0.4 18.5 ±1.1 BDLb71.4 14.4 NPEOsa698.0 ±53.5 230.1 ±13.3 534.4 ±11.9 302.5 ±19.6 94.0 ±4.1 95.8 ±6.9 178.6 ±12.9 100 304.8 OPEOsa187.1 ±11.7 94.4 ±7.9 19 5 .8 ±12.7 230.9 ±21.5 56.6 ±3.6 95.4 ±8.1 53.2 ±4.6 100 130.5 BPAa18.1 ±1.7 4.9 ±0.2 7.4 ±0.8 BQLcBQLc39.5 ±3.1 8.0 ±0.8 71.4 15.6 E2a9.1 ±0.1 14.8 ±0.7 19.5 ±1.1 26.9 ±1.5 14.5 ±1.5 13.5 ±1.3 7.1 ±0.6 100 15.1 E1a1.9 ±0.2 BQLc4.9 ±0.1 BQLc5.4 ±0.4 BQLcBQLc42.9 4.1 E3a16.1 ±1.3 8.3 ±0.1 7.2 ±0.3 18.5 ±2.1 8.9 ±0.2 18.0 ±1.5 BDLb85.7 12.2 EEaBQLcBQLc14.3 ±0.6 BQLcBDLbBQLcBQLc14.3 14.3 DESaBQLcBQLcBQLcBQLcBQLcBQLcBQLc0.0 0.0 aMean and standard deviation of three determinations; bconcentration below the limit of detection; cconcentration below the limit of quantification. nonylphenol and octylphenol to ethoxymers containing 1 to 12 ethoxylated units. On the other hand, we consistently determined the presence of nonylphenol, octylphenol, and short-chained APnEOs (𝑛<5) in the solid matrices; however, concentrations of the more water-soluble ethoxymers (𝑛>5) were only found in a small percentage of these samples. While it would be more logical to find higher concentrations of the more water-soluble compounds, such as long-chained APnEOs (𝑛>5),inthedissolvedphase, that was not the case for any of the WWTPs. Considering that we have been analysing samples of the final effluent, this phenomenon can be explained as a direct consequence of APnEO breakdown in the WWTPs, most likely during biological treatments [37]. These compounds progressively lose ethoxylated units, which form APs (a raw material for microorganisms), short-chain APnEOs, and other biotransformation products such as carboxylated and halogenated derivatives [38]. Therefore, as the degradation treatments progressed, the relative composition of the homologous mixture was further enriched with short-chained AP1-2EOs and APs, which are more toxic, more lipophilic, more estrogenic, and more persistent than the parent substances [39]. As explained above, these substances tend to be quickly adsorbed by the particulate matter present in the samples. We estimated that over80%ofthetotalNPand60%oftotalOPwerefound in the particulate phase, which indicates the importance of analysing these compounds in the solid fraction. 3.1.3. Bisphenol-A. Bisphenol-A (BPA) was consistently found in the dissolved phase (65% of the samples conducted) in concentrations ranging from 4.9 ng L−1 to 39.5 ng L−1. However, its presence in the solid fractions seems to be much more erratic. We only observed BPA concentrations above the quantification limits in 57% of the sludge samples and 35% of the particulate matter samples analysed. The average BPA concentration in the particulate matter was 11.2 ng g−1, whereas its average concentration in the sludge samples was 5.8 ng g−1. This behaviour can be attributed to its relatively low octanol/water partition coefficient (see Table 1). 3.1.4. Evaluation of the Temporal Variability. Regarding the temporal evolution recorded over the 12 months of sampling, we can state that in most cases, it was not possible to link the fluctuations in the concentrations of the studied compounds with the changes of the physical parameters associated with seasonal variations, or with the volumes and characteristics of the inlet waters, or even with the different biological treatmentsemployedinbothWWTPs,havingbeenobserveda greatrandomnessintheresultsobtained.Thishighvariability can also be attributed to the different origins and uses of the selected compounds (both natural (e.g., E2 and metabolites) and anthropogenic (e.g., BPA, DES or EE)). 3.2. Estimation of Estrogenic Activity. The field experiment results showed us that a complex mixture of endocrinedisrupting compounds occurs in the effluent from the studied BioMed Research International 9 Table 5: Sludge and particulate matter (solid fractions) concentrations (ng⋅g−1) for each target compound in both WWTPS under study. Sludge samples Jul ’10 Sep ’10 Nov ’10 Jan ’11 March ’11 May ’11 Jul ’11 Frequency (%) Average WWTP 1 NPa216.1 ±9.1 479.1 ±22.7 52.7 ±2.1 211.3 ±5.2 324.5 ±6.7 175.9 ±12.3 22.1 ±0.2 100 211.7 OPa49.3 ±1.4 22.2 ±1.2 39.1 ±3.0 20.7 ±2.0 8.7 ±0.6 14.0 ±2.3 22.3 ±1.0 100 25.2 NPEOsa461.1 ±15.7 890.1 ±35.1 125.4 ±11.1 471.5 ±8.1 92.1 ±6.7 92.4 ±3.3 451.4 ±10.1 100 369.1 OPEOsa74.5 ±6.0 55.9 ±1.7 21.9 ±1.2 190.1 ±9.5 25.1 ±1.5 129.3 ±9.1 44.0 ±1.3 100 77.3 BPAa0.9 ±0.1 BQLc7.7 ±0.7 BQLcBQLc2.5 ±0.2 7.9 ±0.5 57.1 4.8 E2a10.1 ±1.0 5.1 ±0.3 14.1 ±0.9 BQLc17.9 ±1.4 20.6 ±1.6 39.1 ±1.1 85.7 17.8 E1aBQLcBQLc7.5 ±0.3 BQLc1.7 ±0.1 BQLc3.2 ±0.3 42.9 4.1 E3aBQLcBQLcBQLcBQLcBQLcBQLc0.3 ±0.0 14.3 0.3 EEa27.1 ±2.1 21.1 ±2.0 22.0 ±1.7 5.9 ±0.4 38.9 ±2.1 9.3 ±0.5 42.0 ±2.7 100 23.8 DESaBQLcBQLc3.0 ±0.4 BQLc0.9 ±0.1 11.5 ±0.7 BQLc42.9 5.1 WWTP 2 NPa91.0 ±5.0 101.2 ±7.9 370.2 ±26.3 250.1 ±9.3 27.7 ±2.1 63.0 ±1.1 32.0 ±1.8 100 133.6 OPa60.1 ±2.5 38.1 ±3.1 35.1 ±2.2 50.9 ±1.7 12.5 ±0.2 16.7 ±1.3 71.2 ±0.9 100 40.7 NPEOsa240.9 ±19.6 500.4 ±32.3 612.1 ±19.1 96.1 ±1.1 218.3 ±15.5 683.4 ±4.5 403.9 ±27.1 100 393.6 OPEOsa311.8 ±6.3 43.2 ±2.1 104.0 ±7.1 44.8 ±2.3 47.5 ±2.5 191.0 ±7.0 1 6 1 .6 ±3.3 100 129.1 BPAaBQLcBQLc15.9 ±0.9 2.7 ±0.2 1.9 ±0.1 BQLc7.6 ±0.7 71.4 6.8 E2a1.7 ±0.2 7.2 ±0.3 33.9 ±1.0 21.1 ±1.1 7.7 ±0.1 73.7 ±3.2 1.5 ±0.1 100 21.0 E1a4.3 ±0.2 BQLc5.0 ±0.3 BQLc4.5 ±0.1 1.5 ±0.1 BQLc57.1 3.8 E3aBQLcBQLcBQLcBQLcBQLcBQLcBQLc0 0.0 EEa111.1 ±9.3 56.1 ±2.1 121.7 ±1.3 13.1 ±0.4 25.8 ±2.1 83.1 ±3.9 55.9 ±3.3 100 66.7 DESaBQLcBQLc17.5 ±0.4 BQLcBQLcBQLc3.8 ±0.3 28.6 10.7 Particulate matter Jul ’10 Sep ’10 Nov ’10 Jan ’11 March ’11 May ’11 Jul ’11 Frequency (%) Average WWTP 1 NPa198.6 ±5.3 754.3 ±39.1 105.7 ±4.7 390.4 ±33.3 401.6 ±30.7 200.0 ±15.9 90.9 ±3.4 100 305.9 OPa22.7 ±0.9 50.0 ±2.9 19.9 ±1.1 120.4 ±8.0 68.1 ±4.3 30.5 ±0.9 11.6 ±1.0 100 50.1 NPEOsa618.3 ±40.2 391.8 ±19.5 93.6 ±6.1 290.5 ±17.4 214.2 ±16.1 183.5 ±12.0 207.9 ±10.6 100 285.7 OPEOsa99.2 ±3.9 69.6 ±3.1 BQLc78.9 ±7. 0 33 . 9 ±1.9 40.7 ±2.1 60.8 ±5.9 85.7 63.9 BPAa3.7 ±0.2 12.3 ±0.8 BQLcBQLcBQLcBQLc12.7 ±0.6 42.9 9.6 E2a21.5 ±2.7 9.0 ±0.7 51.7 ±0.6 29.5 ±3.3 7.1 ±2.0 31.8 ±1.3 50.3 ±3.0 100 28.7 E1a22.5 ±0.9 14.0 ±0.9 BQLc5.1 ±0.4 BQLc11.0 ±0.8 BQLc57.1 13.2 E3aBQLcBQLcBQLc3.1 ±0.1 BQLcBQLcBQLc14.3 3.1 EEa14.0 ±0.7 BQLc7.7 ±0.4 BQLc105.1 ±7.8 1 2. 9 ±1.0 19.1 ±0.5 71.4 31.8 DESa14.3 ±1.0 BQLc6.3 ±0.3 BQLc10.4 ±0.4 BQLcBQLc42.9 10.3 WWTP 2 NPa129.2 ±7.9 2 1 2. 4 ±3.9 209 ±9.6 301.8 ±19.1 63.6 ±2.9 331.9 ±20.1 99.5 ±1.8 100 192.5 OPa34.8 ±0.7 41.6 ±2.9 49.0 ±3.3 69.7 ±3.8 5.0 ±0.2 22.9 ±1.4 17.7 ±0.9 100 34.4 NPEOsa89.5 ±5.3 350.1 ±3.1 189.6 ±10.6 78.0 ±2.0 189.2 ±4.9 1003 ±61.9 106.8 ±14.7 100 286.6 OPEOsa129.6 ±11.8 51.0 ±2.1 128.4 ±5.1 77.1 ±0.9 31.7 ±0.9 59.7 ±5.1 184.6 ±3.9 100 100.4 BPAa21.5 ±1.5 BQLcBQLcBQLc3.9 ±0.6 BQLcBQLc28.6 12.7 E2a33.0 ±2.0 BQLc33.7 ±2.9 22.7 ±0.7 14.6 ±0.4 44.8 ±0.7 25.1 ±3.3 85.7 29.0 E1a13.5 ±0.3 5.9 ±0.3 21.3 ±2.0 BQLcBQLc32.7 ±0.8 5.5 ±0.2 57.1 15.8 E3aBQLcBQLcBQLcBQLcBQLcBQLcBQLc0 0.0 EEa33.9 ±0.9 12.0 ±0.1 75.9 ±2.7 29.0 ±3.1 91.8 ±2.9 29.4 ±0.5 BQLc85.7 45.3 DESa4.1 ±0.3 BQLc23.7 ±2.0 BQLcBQLc6.9 ±0.1 BQLc42.9 11.6 aMean and standard deviation of three determinations; bconcentration below the limit of detection; cconcentration below the limit of quantification. Submit your manuscripts at http://www.hindawi.com Pain Research and Treatment Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Toxins Journal of Vaccines Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Antibiotics International Journal of Toxicology Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Stroke Research and Treatment Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Drug Delivery Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Pharmacological Sciences Tropical Medicine Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Medicinal Chemistry International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Addiction Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 BioMed Research International Emergency Medicine International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Autoimmune Diseases Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Anesthesiology Research and Practice Scientifica Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Pharmaceutics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 MEDIATORS INFLAMMATION of