Estrogenic Activity Measurements in Water Using Diffusive Gradients

Wei Guo†‡§, Kersten Van Langenhove†§, Michael S. Denison∥, Willy ... Beijing city (China) showed comparable results to conventional spot (gr...
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Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Estrogenic Activity Measurements in Water Using Diffusive Gradients in Thin-Film Coupled with an Estrogen Bioassay Wei Guo,†,‡,§ Kersten Van Langenhove,†,§ Michael S. Denison,∥ Willy Baeyens,† Marc Elskens,† and Yue Gao*,† †

Analytical, Environmental and Geo-Chemistry (AMGC), Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussel, Belgium School of Environmental Science and Engineering, North China Electric Power University, Beijing, 102206, China ∥ Department of Environmental Toxicology, University of California, Davis (UC Davis), One Shields Avenue, Davis, California 95616, United States ‡

S Supporting Information *

ABSTRACT: A novel type of diffusive gradients in thin-film (DGT) was combined with a chemically activated luciferase gene expression bioassay (CALUX) to measure estrogens in aquatic systems. The performance of this novel method was assessed with 17β-estradiol (E2) as the model steroid hormone, XAD 18 resin gel as the binding phase in the DGT method and VM7Luc4E2 cells (formerly BG1Luc4E2) for the Estrogen Responsive Element (ERE)-CALUX bioassay. The measured effective diffusion coefficient of E2 in agarose diffusive gel was 4.65 ± 0.37 × 10−6 cm2 s−1 at 25 °C. The detection limit of this combined DGT/ERE-CALUX method for 1 day of sampling (0.026 ± 0.003 ng L−1 of E2) is significantly lower than that obtained by spot sampling combined with GC-MS/MS or LC-MS/MS analysis (0.1−7.0 ng L−1). The method is independent of pH (5−8), ionic strength (0.001−0.5 M), and dissolved organic matter (DOM; concentrations up to 30 mg L−1). Field applications of this novel DGT in effluents of three sewage treatment plants in Beijing city (China) showed comparable results to conventional spot (grab) sampling. This study demonstrates that the combined DGT/ERE-CALUX approach is an effective and sensitive tool for in situ monitoring of estrogenic activity in waters and wastewaters.

E

Generally, steroid hormones are present in the aquatic environment at very low concentrations ranging from pg L−1 to ng L−1,10,11 which requires very sensitive detection techniques and/or high-performance sample preconcentration methods. Spot samples (i.e., grab samples) are collected on site and transported back to the laboratory for analysis, which may lead to analyte contamination, induce postsampling alterations and result in loss of time and increased costs.12 However, spot sampling allows determination of spatial gradients and short time changes in the aquatic system. Passive sampling techniques are complementary to these spot sampling methods as they allow for preconcentration of the analyte in situ, thus, limiting contamination and postsampling alterations. Passive samplers such as ChemCatcher13 and polar organic chemical integrative sampler (POCIS)14 for polar organic compounds are, however, flow-rate dependent and require additional measurement of water flow to obtain a time-weighted average concentration. Flow-independent passive samplers consisting of a ceramic diffusive barrier have been successfully used for PAH and Dioxin analyses.15−17 A similar technique, called diffusive

ndocrine disrupting chemicals (EDCs) constitute a class of compounds of major concern due to various adverse health effects on humans and wildlife.1,2 EDCs include a wide range of organic species, among which are steroid hormones, both natural (estrone (E1), 17β-estradiol (E2), estriol (E3)) and synthetic (17α-ethinylestradiol (EE2)), present in human and animal excreta.3 These steroid hormones belong to the group of steroidal EDCs, which possess extremely high estrogenic potency, about 10000−100000 times higher than that of other EDCs such as organochlorine aromatic compounds, alkylphenols and bisphenols.4,5 Studies have shown that E2 concentrations in the range of 1−10 ng L−1 exhibit adverse health effects on aquatic species and the exposure to a combination of E1 and E2 is more detrimental than exposure to the individual hormone.6 Due to the (i) incomplete removal of these substances by conventional wastewater treatments, (ii) their continuous and recurrent discharges into the aquatic environment causing pseudo persistence, and (iii) their potential to disrupt endocrine homeostasis in wildlife,7,8 steroid hormones are considered emerging pollutants of concern or EPOCs.9 Hence, the concentration determination of these steroid hormones in aquatic ecosystems and subsequent risk assessment have become essential for future actions on their use. © XXXX American Chemical Society

Received: August 30, 2017 Accepted: November 19, 2017 Published: November 20, 2017 A

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Bio-Rad Laboratories (Spain). Dulbecco’s Modified Eagle Medium (DMEM without phenol red (Gibco)), sodium pyruvate (100 mM, sterile-filtered), α-Minimal Essential Medium (α-MEM (Gibco)), penicillin−streptomycin, fetal bovine serum (FBS), charcoal-stripped FBS, L-glutamine (200 mM), trypsine (0.5% (Gibco)), phosphate buffered saline (PBS, 1 X, pH 7.4), and trypsin without phenol red (10×, 0.5% (Gibco)) were purchased from Life Technologies (United Kingdom). Luciferine reagent and lysis reagent were obtained from Promega (The Netherlands). Stabilizing buffer A, B, and NucleoCounter cartridges were purchased from Chemometec (Denmark). DGT Preparation and Functioning. DGT holders were fabricated in-house using Teflon (PTFE) as the inert material of choice. A Teflon base (2.5 cm diameter) was stacked with resin gel, diffusive gel, and filter membrane (HVLP Durapore from Millipore consisting of hydrophilic PVDF: polyvinylidene fluoride, 0.45 μm) and then a Teflon cap tightly closed the sampler, leaving a 2.0 cm diameter window. The diffusive and resin gels of DGT were prepared as previously described.31 In summary, a 1.5% (w/v) agarose diffusive gel solution was prepared by dissolving 0.3 g agarose in 20 mL of Milli-Q water and then heated to 80 °C until the solution became transparent. The hot gel solution was immediately injected into a preheated, gel-casting assembly, consisting of two glass plates separated by a plastic spacer with a defined thickness (range 0.025−0.125 cm) and left to cool down to its gelling temperature (∼36 °C). For the resin gel (0.05 cm), 0.5 g (wet weight) of methanol conditioned XAD 18 was mixed with 1.5% (w/v) warm agarose solution (>80 °C) and pipetted into a preheated glass assembly and left to cool for 1 h. The XAD 18 resins were purified by ultrasonic extraction with n-hexane and acetone, followed by vortex conditioning using methanol (5 min) prior to use. The formed gels were peeled off from the assembly and hydrated in Milli-Q water for 24 h. The hydrated gels were cut into disks and stored in 0.03 M NaCl solution with a shelf life of approximately 6 months. Sample Extraction. All glassware used in the experiment was rinsed 3−5 times with Milli-Q water and capped with aluminum foil prior to baking at 450 °C for 8 h and stored until further use. Aqueous samples were extracted with Oasis HLB cartridges (5 cc, 200 mg, glass cartridge, Waters, U.S.A.), while XAD18 resin gels were extracted automatically using the Accelerated Solvent Extraction unit (ASE 200 from Dionex, Thermo Scientific). More details on the extraction procedures are available in the Supporting Information. In the last step, all extracts (∼60 mL) were evaporated to dryness using a vacuum centrifuge (Genevac MiVac Quattro) and resuspended into a defined volume of hexane. ERE-CALUX Bioassay. VM7Luc4E2 cells (formerly known as BG1Luc4E2 cells32) were used to carry out the ERE-CALUX bioassay, and the method was performed according to the XDS LUMI-CELL agonist protocol33 and OECD TG 455 guidelines,34 but with certain modifications.35,36 The response of those genetically modified cells toward estrogenic ligands is of biological/toxicological nature and ultimately yields photochemical signals directly proportional to the administered dose or compound concentration in the assay.25 Routine cell cultivation, seeding and dosing in 96-well plates, and analysis parameters are described in the Supporting Information. The calibration curve for these types of assays consists of a fourparameter sigmoidal equation and is depicted in Figure 1(for each parameter, the average values along with their

gradients in thin-films (DGT), has been widely used for in situ measurement of labile inorganic species in the aquatic environment.18,19 Recently, this DGT technique has also been applied to detect polar organic compounds (Log Kow < 5), such as pharmaceuticals, pesticides, and bisphenols in water,20−22 using a smaller diffusive barrier, thereby aiding in faster sampling rates of the aquatic environment. LC-MS/MS or GC-MS/MS are, in general, coupled with various sampling techniques to allow detection of very low levels of steroid hormones in water (0.1−7.0 ng L−1).23 However, this type of analysis has two drawbacks: (1) concentrations instead of estrogenic effects (biological and toxicological) of the selected compounds are measured and (2) there is no information about EDCs that may be present in the sample but were not specifically selected for analysis. Therefore, sensitive biological tests, addressing those drawbacks,24 could be of help. The ERE (estrogen responsive elements)-CALUX (chemically activated luciferase gene expression) bioassay is an in vitro bioassay based on a recombinant cell line containing an estrogen receptor responsive luciferase reporter gene,25,26 and this method has been widely used to assess the estrogenic potency of extracts of several environmental matrices.27−29 The aim of this study was to develop a novel time integrated monitoring method for steroid hormones in water. The combination of DGT and ERE-CALUX bioassay is ideally suited to get a general view of estrogenic activity in an aquatic system, but without details of individual compounds or small scale spatial and temporal variations. The DGT component allows for spatial and temporal integration while the bioassay encompasses the estrogenic activity of the individual compounds present in any given study area. In this study, E2 was used as the single and sole target compound since it is often used as the reference chemical for estrogen activity and potency5 and due to its recent status as a watch list chemical under the EU Water Framework Directive (WFD, 2013/39/ EU).30 The following laboratory experiments were carried out: (1) adsorption of E2 on DGT holders, diffusive gels and membrane filters; (2) uptake and capacity of E2 by the XAD 18 resin; (3) determination of diffusion coefficient of E2 in the diffusive gel of the DGT; and (4) analysis of effects of pH, ionic strength and dissolved organic matter (DOM) concentration on the adsorption characteristics of E2 on the XAD 18 resin. After this validation study, the DGT devices were deployed in the effluents of various sewage treatment plants from Beijing city (China) and the measured estrogenic activities were compared with those from conventional spot sampling. To date, and to the best of our knowledge, no study in literature has reported the determination of steroid hormones using a combination of flow-independent DGT passive samplers with CALUX determination.

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EXPERIMENTAL SECTION Chemicals. Hexane (minimum 96.0%) and acetone (minimum 99.9%) were purchased from Biosolve (The Netherlands). 17β-Estradiol (E2, minimum 98.0%), methyl tert-butyl ether (MTBE, HPLC grade), methanol (MeOH, HLPC grade), hydrochloric acid (HCl), humic acid (H1675−2, Lot#131078), glass fiber thimbles (Whatman, 19 × 90 mm), and D-glucose (minimum 99.5%) were purchased from Sigma− Aldrich (Germany). Dimethyl sulfoxide (DMSO, minimum 99.7%) and sodium hydroxide (NaOH) were purchased from Merck (Germany). Amberlite XAD18 was obtained from Rohm and Haas Company (U.S.A.) and agarose was obtained from B

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used a concentration of 16 ng L−1, which is almost 4 times higher than in the adsorption experiments, as well as a sufficiently large volume (9 L). Aliquots (10 mL) of the aqueous phase were collected before and after DGT deployment in the solution and were extracted with HLB cartridges as described above. The XAD 18 resin gel samples were peeled off from the DGT devices, dried gently, and extracted using the ASE 200 unit and extracts analyzed by ERE-CALUX. Diffusion Boundary Layer (DBL) Measurements. The same experiments used for the determination of the diffusion coefficient (see paragraph here above) were also used to determine the DBL thickness under laboratory conditions. DBL thickness measurements in the field were carried out at station GBD-2 at the Gaobeidian sewage treatment plant in Beijing. In that effluent, DGTs with 3 different diffusive gel thickness (0.05, 0.075, and 0.10 cm) were deployed for 6 h. All detailed information is included in the Supporting Information. Effects of pH, Ionic Strength, and DOM. DGT samplers including XAD 18 resin gel (0.05 cm), agarose diffusive gel (0.075 cm), and the filter membrane (0.017 cm) were deployed in 1 L of 16 ng L−1 E2 solution for 4 h at different pH, ionic strength, and DOM concentration, respectively. The pH of natural fresh water is usually in the range of 5−8, ionic strength is generally tested in the range of 0.001 to 0.5 M NaCl, and DOM concentration ranges from 0 to 30 mg L−1.21,31,38 Thus, the pH of the E2 solution was adjusted to cover a range of 5−8, with 1 M HCl or 1 M NaOH. Ionic strength of the E2 solution was adjusted to cover a range of 0.001−0.5 M with 1 M NaCl (pH 7.0). The DOM concentration covered a range of 0−30 mg L−1 (pH 7.0) by dilution of a humic acid stock solution (464 mg L−1, determined using a TOC analyzer). The humic acid stock solution was prepared according to literature.39 Field Trial. Discharge from urban sewage treatment plant is the main source of estrogenic hormones entering the aquatic environment.7 A field trial was conducted in March 2017 in the effluents of three typical sewage treatment plants from Beijing city (China). The three sewage treatment plants are respectively Gaobeidian sewage treatment plant (GBD; 1000000 m3 d−1; activated sludge + biological filtration system), Qinghe recycling water plant (QH; 550000 m3 d−1; activated sludge + ultrafiltration system combined with ozone oxidation), and Liangxiang sewage treatment plant (LX; 90000 m3 d−1; oxidation ditch + sand filtration system). Considering the difference in secondary and tertiary treatment in these sewage treatment plants, the DGT devices were deployed in the effluent water of the secondary sink basin and in the downstream filtration system of each sewage treatment plant. Triplicate DGT devices were deployed at each site, 30 cm below the water surface over a period of 6 h (from 10 am to 4 pm). The average temperature of the water during deployment was 16 ± 2 °C. At the end of the deployment, the DGT devices were retrieved, rinsed with Milli-Q water, and transported to the laboratory for further experimental work. Water samples in the amount of 1L were collected at the start and at the end of the DGT deployment. Both water samples were mixed in the laboratory and then about 500 mL (1/4 of the original sample volume) was extracted with Oasis HLB cartridges. Detailed extraction and analytical methods of XAD gels in DGT samplers and water samples were as described in the Supporting Information. Statistical Analysis. All DGT experiments were carried out at triplicate and the results were expressed as the mean ± standard deviation. Statistical analysis was performed with

Figure 1. Concentration−response curve of E2 standard solutions measured with the ERE-CALUX bioassay (n = 15). The four parameters of the sigmoid Hill plot are indicated as the background (y0), the maximum induction (m), the effective concentration at halfmaximal induction (EC50) in pM, and corresponding to an induction percentage equal to [y0 + (1/2 × (m − y0))]. The hill parameter (h) equals the slope of the sigmoid curve at the inflection point (which equals the EC50).

corresponding standard deviation (SD) were calculated). Based on 15 independent full concentration−response E2 standard curves, the mean EC50 value from these analyses amounted to 8.2 ± 1.2 pM (Figure 1). Adsorption of E2 on DGT Holder, Diffusive Gel, and Filter Membrane. The DGT technique is based on a simple device which accumulates solutes on a binding resin layer after passage through a diffusive gel which acts as a well-defined diffusion layer.37 Thus, for optimal measurement, DGT materials, including DGT holders, diffusive gels, and filter membranes, should not adsorb E2. DGT holders were immersed in a UV protecting bottle containing 1 L of 4.3 ng L−1 E2 solution (in Milli-Q water, pH 7.0, 23 °C) and were then shaken for 48 h. Diffusive gels and filter membranes were separately immersed in 40 mL of 4.3 ng L−1 E2 solution (in Milli-Q water, pH 7.0, 23 °C) and were also shaken for 48 h. Aliquots (10 mL) of each E2 solution were collected at 0 and 48 h and were extracted with an HLB cartridge, followed by ERE-CALUX bioanalysis to examine possible changes in E2 response of the spiked solution over time. Resin Capacity. Resin gels used in the DGT samplers should, ideally, present high adsorption capacities for target compounds. XAD 18 resin gels were immersed in a beaker containing 1 L of E2 solution (in Milli-Q water, pH 7.0, 23 °C) at various concentration levels (2, 4, 8, 16, 32, and 64 ng L−1) and were then shaken at room temperature for 48 h. Aliquots (10 mL) of the E2 solution were collected before and after the XAD 18 resin gel deployment and extracted with an HLB cartridge as described above. The XAD 18 resin gel samples were retrieved from the E2 solutions, gently dried and extracted using the ASE 200 unit and the extracts were analyzed by ERECALUX. Diffusion Coefficient Measurement. In the laboratory, DGT samplers with diffusive gels of different thickness (0.025, 0.05, 0.075, 0.10, and 0.125 cm) were deployed in 9 L of solution (in Milli-Q water, pH 7.0, 23 °C) containing 16 ng L−1 E2 in 0.03 M NaCl at a stir rate of 300 rpm for 4 h. To avoid a decrease of the E2 concentration during the experiment, we C

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diffusive gel) is used to calculate the time-integrated concentrations of organics in the bulk solution using Fick’s first law, see eq 1:31

GraphPad Prism 7.0 software. Analysis of variance (ANOVA) was performed to detect results that were significantly different at a level of α = 0.05.

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RESULTS AND DISCUSSION Adsorption by DGT Holder, Diffusive Gel, and Filter Membrane. Adsorption of our target compound on the DGT components was assessed by testing the variation of E2 concentration in solution when these DGT materials were immersed in a spiked solution. A small decrease of the E2 concentration in solution was observed after 48 h when DGT holders and agarose diffusive gels were immersed in the solution (lower than 5%), while a small increase (2.4%) was observed when the filter membranes were immersed. This corresponds to the relative adsorption (%) of E2 onto DGT holders, diffusive gels, or filter membranes shown in Figure 2. The adsorption/desorption effect onto the DGT has been always verified in the subsequent experiments but never influenced the obtained results.

Cw =

M(Δg + δ) DeAt

(1)

where Cw is the concentration of the compound (organic or metallic in nature) in the water, M is the mass of the compound accumulated on the resin gel, Δg is the thickness of the diffusion layer which includes the diffusive gel (0.025 to 0.125 cm) and filter membrane (0.017 cm), δ is the DBL thickness depending on the flow velocity of the ambient water40 which has been measured in laboratory and in the field, De is the effective diffusion coefficient of organics in the diffusive gel, A is the exposure area (3.14 cm2), and t is the exposure time. The following equation, transformed from eq 1, was used to calculate the effective diffusive coefficient (De) at the tested temperature: De =

M(Δg + δ) CwAt

(2)

The diffusion coefficient is dependent on the solution temperature; therefore, the following temperature-dependent equation37 was used to calculate the effective diffusion coefficient at 25 °C (De25): log De =

1.37023(t − 25) + (8.36 × 10−4)(t − 25)2 109 + t De25(273 + t ) + log 298

(3)

where De and De25 are the effective diffusion coefficient of the compound at the temperature t (°C) and at 25 °C, respectively. The diffusion coefficient of chemical compounds in water can be obtained via experimental and theoretical methods. Plotting the accumulated mass versus the inverse of the diffusive layer thickness (Figure 3) provides a slope, k (0.0025 ng·cm), and a diffusion coefficient, De, at 23 °C of 4.41 ± 0.35 × 10−6 cm2 s−1 via eq 3. The diffusion coefficient (De25) at 25 °C (eq 3) is then 4.65 ± 0.37 × 10−6 cm2 s−1, which is slightly

Figure 2. Relative adsorption (%) of E2 (4.3 ng L−1) onto DGT holders, diffusive gels, or filter membranes. Data are mean values ± standard deviation (n = 3).

Uptake and Capacity of XAD18 Resin Gel. The E2 binding characteristics of the XAD18 resin gel were tested at increasing E2 concentrations of 2, 4, 8, 16, 32, and 64 ng L−1 of E2 (with 64 ng L−1 as a maximum based on literature7). Both the water samples and resin gel were extracted (respectively using Oasis HLB SPE and Dionex ASE 200) and analyzed for E2 content. The residual mass of E2 in solution was approximately 2−5% of the initial mass in solution. The accumulated mass in the resin gel was approximately equal to the mass added to the original solution (see Figure S1). The uptake of E2 by XAD 18 resin gel increased linearly from 2 to 64 ng L−1 of E2 (R2 = 0.999) and was very close to the value of 100% recovery (Figure S1), the latter amount being the highest concentration of E2 reported in natural waters or in effluent waters of water treatment plants.7 Since saturation of the XAD 18 resin gel by E2 was never observed, even at the highest E2 concentration ever reported, it is reasonable to assume that a DGT with XAD 18 resin gel will be suitable for monitoring steroid hormones in natural waters without concerns of resin saturation by E2. Diffusion Coefficient. The total mass of analyte(s) accumulated on the binding phase after passing through all diffusive layers (diffusive boundary layer (DBL), filter, and

Figure 3. Mass (M) of E2 on XAD 18 resin gel as a function of the inverse of the diffusive gel layer thickness (Δg). Data are mean values ± standard deviation (n = 3). D

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Table 1. Physicochemical Properties7 of E1, E2, E3, and EE2 and Their Theoretical Diffusion Coefficients in Diffusive Gel at 25 °C

a

chemical

mol wta (Mw) g mol−1

Log Kowb

mol volc (V; Ǻ 3)

estrone (E1) 17β-estradiol (E2) estriol (E3) 17α-ethinylestradiol (EE2)

270.4 272.4 288.4 296.4

3.43 3.94 2.81 4.15

387.1 386.4 380.1 406.8

calcd diffusion coefficient (Dt; cm2 s−1) 5.17 5.17 5.20 5.08

× × × ×

10−6 10−6 10−6 10−6

Molecular weight. bOctanol−water partition coefficient. cMolecular volume.

lower than the corresponding literature value at the same temperature in water (4.88 × 10−6 cm2 s−1).22 Moreover, the diffusion coefficient of E2 in porous aqueous media can be theoretically calculated according to eq 4:41 Dt = pm Dw

The MDL is then the IDL of CALUX (0.024 pg E2) multiplied by the dilution factors during dosing (doseCALUX equal to 3.72) and the fraction of resuspended sample used during routine analyses (dosesample equal to 4). This MDLDGT corresponds to a mass M of 0.36 pg E2 on the DGT resin. This result can be converted to a concentration by considering eq 1 using De = 4.65 ± 0.37 × 10−6 cm2 s−1, Δg = 0.092 cm, A = 3.14 cm2, and t = 1 day as a reference deployment time, to ultimately obtain a DGT-MDL of 0.026 ± 0.003 ng L−1 of E2. Based on the proposed Environmental Quality Standards (EQS) for E2 (0.4 ng L−1 of E2; WFD, 2013/39/EU)30 we can provide a sufficiently low MDL to determine whether a sample would exceed this threshold and would be able to do so within a single day. This DGT-MDL is significantly lower than the limits reported for a spot sample analyzed with GC-MS/MS or LC-MS/MS (0.1−7.0 ng L−1 of E2).23 Effects of pH and Ionic Strength on DGT Trapping of E2. pH changes can influence the surface charge on adsorbent particles as well as the ionization potential of chemicals,45 leading to a change in the performance of DGT resins. As shown in Figure 4a, the ratio of DGT E2 concentration (CDGT) to the E2 concentration in the bulk solution (Cs) did not change notably within the tested pH range of 5−8 except for the value between pH 6.01 and pH 7.01 with a little difference (ANOVA, 0.01 < p < 0.05). Similar results were observed for antibiotic measurements using XAD 18 DGT31 and for E2 with other resins such as chitin, chitosan, ion-exchange resin, or a carbonaceous material.46 The pKa of E2, which is 10.5,23 is higher than the range of pH applied in this study, which indicates that E2 is mainly in neutral form and is more easily adsorbed than ionized species. The high log Kow value of E2 (3.94) also suggests that its hydrophobic form is more prominent and remains constant throughout the adsorption experiment.7 Ionic strength can also affect the adsorption of estrogens by DGT samplers. Higher ionic strength can enhance the estrogen adsorption47 because of (1) the “salting-out” effect reducing the solubility of organic compounds45 or (2) the screening effect of the surface charge and π−π interactions.48 As shown in Figure 4b, the ratio of CDGT/Cs shows no appreciable decrease, even at a very high ionic strength of 0.5 M, which suggests that in our experiments the “salting-out” effect did not influence the adsorption of E2 on XAD 18 gels. Similar observations are reported in literature;48,49 however, a study on antibiotics mentioned that the DGT determination was significantly influenced at high ionic strength.31 Effects of DOM on DGT Trapping of E2. The presence of DOM in aquatic environment may influence DGT trapping of EDCs on carbon materials due to (1) competition between EDCs and DOM for adsorption sites on XAD 18; (2) pore blockage of the diffusive gel reducing the number of open pores and thus the surface area available for adsorption; and (3) reaction between DOM and EDCs.8,48,50 Therefore, the

(4)

where Dt is the diffusion coefficient of E2 in porous aqueous media; Dw is the diffusion coefficient of E2 in water; p is the porosity of the porous media (0.98);20 and m is Archie’s law exponent, which in porous media usually ranges between 1.5 and 2.5.15 Dw can be calculated according to Stokes−Einstein equation (eq 5):42 Dw =

k bT 6πηr

(5)

where kb is the Boltzmann constant (1.3806 × 10−23 J K−1), T is the absolute temperature (K), η is the water viscosity (Pa·s), and r is the solute hard sphere molecular ratio. Assuming that the molecular volume of solute (V) corresponds to a spherical shape (V = 4/3πr3) and considering the molecular density as δ = Mw/V, eq 6 can be rewritten into eq 6: Dw =

k bTδ1/3 3/41/36ηπ 2/3M w1/3

(6)

Molecular volumes (V) were calculated with the quantitative structure−activity relationship (QSAR) software (Hyperchem 8.0 package software from Hypercube Inc.).43 Using eq 4, the diffusion coefficients of different steroid hormones, including E1, E2, E3, and EE2 (at 25 °C) can be calculated (Table 1). The measured effective diffusion coefficient of E2 (4.65 ± 0.37 × 10−6 cm2 s−1) was slightly lower than the theoretical calculated diffusion (Dt) coefficient (5.17 × 10−6 cm2 s−1), but was within 10%. The 16 ng E2 L−1 solutions were used for the determination of the E2 diffusion coefficient with a satisfactory recovery of 87.4 ± 2.3% for spiked water samples. Due to similarities in molecular structure and chemical characteristics, the Dt values of many steroid hormones (E1, E2, E3, and EE2) are very similar (Table 1), yielding an average value of 5.16 ± 0.05 × 10−6 cm2 s−1 based on four steroid hormones. Blank Values and Detection Limits. The MDLDGT (method detection limit of the DGT technique) is described as the value (mass or concentration) arising from the measurement of a blank sample going through the whole procedure, except for the deployment,44 while the IDLCALUX (instrument detection limit of the CALUX bioassay technique) is calculated based on the mean response from the solvent control sample (DMSO) and increased by 3*SD on this mean value. The method detection limit for our DGT sample was therefore calculated using the following equation: MDL DGT = IDLCALUXdoseCALUX dosesample

(7) E

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shows that DOM in the range of 0−31 mg L−1 has no significant effect (ANOVA, p > 0.05) on the E2 trapping by a DGT sampler. Even at a high concentration of 31 mg L−1, the ratio of CDGT/Cs (0.93 ± 0.15) was not significantly different from the CDGT/Cs ratio at the lowest DOM concentration (0.91 ± 0.04). At high DOM concentration (>15 mg L−1), the solution and the DGT filter show a much darker color (Figure 5b). However, this effect was not visible on the diffusive gels, indicating that the filter membranes prevent the diffusion of most DOM molecules, while E2 and the small molecular weight of DOM can effectively pass the diffusive gel and reach the adsorption sites of the XAD 18 resin gel. The adsorption reduction of E2 on XAD 18 is very small, which is consistent with the small effect of dissolved organic matter, DOM ( 0.05), which also suggests that the operating conditions and

Figure 5. (a) Effect of dissolved organic matter (DOM) on the ratio of the E2 concentration assessed with DGT (CDGT) to the bulk E2 concentration (Cs); (b) effect on the color of water solution, diffusive gel and DGT device. Low level DOM: 0−6 mg L−1; High level DOM: 15 and 31 mg L−1. Data are mean values ± standard deviation (n = 3). F

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functioning of the wastewater treatment plants were fairly stable during the DGT deployment period. The estrogenic activities in effluents from sewage treatment plants are significantly affected by the treatment process; for example, the estrogenic activity in the filtration system outlet of Qinghe recycling water plant is 95% lower than in the secondary sink outlet (see Figure 6). Therefore, one should be very careful when comparing our results with those of the literature. The estrogenic activities measured in this study are higher than those (0.8−29.7 ng E2-equivalents L−1) in effluents form sewage treatment plants in Finland,53 but comparable to those (2.1−48.2 ng E2-equivalents L−1) in effluents form sewage treatment plants in Slovenia.3

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: + 32 2 629 32 61. Fax: + 32 2 629 32 74. ORCID

Yue Gao: 0000-0002-0582-395X Author Contributions §

These authors contributed equally to this work. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financed by Belspo-FOD18, Belspo-NewSTHEPS and Innoviris Prospective Research for Brussels (PRFB). We would also like to thank David Verstraeten for the measurement of humic acid. The CALUX bioassay VM7Luc4E2, formerly known as BG1Luc4E2, cell line was developed with funding from the National Institute of Environmental Health Sciences Superfund Research Grant (ES04699) to M.S.D.

CONCLUSIONS A novel, combined time integrated monitoring method using DGT and the ERE-CALUX bioassay was developed for estrogen activity measurement in the aquatic environment. The DGT technique allows in situ preconcentration of the estrogens while the ERE-CALUX bioassay allows the determination of estrogen activity at very low concentrations (pg E2-equivalence L−1). The integrated DGT sampler and ERE-CALUX bioassay method has a Method Detection Limit of 0.026 ± 0.003 ng E2-equivalents L−1 for a 1 day in situ sampling. Theoretical diffusion coefficients of steroid hormones E1, E2, E3, and EE2 at 25 °C are very similar, with an average value of 5.16 ± 0.05 × 10−6 cm2 s−1, but slightly higher than the experimentally determined one (4.65 ± 0.37 × 10−6 cm2 s−1). This work showed that DGT components, diffusive gel and filter do not significantly interfere with the ERE-CALUX assessment of estrogenic potency. Moreover, the method was not affected by aqueous environmental parameters including pH (5−8), ionic strength (0.001−0.5 M) and DOM (0−30 mg L−1). This study demonstrates that DGT combined with ERECALUX is an effective tool for preconcentrating steroid estrogens and thus suited for the monitoring of estrogenic activity in wastewaters. The method is complementary to spot sampling combined with GC-MS/MS or LC-MS/MS for the detection of estrogenic activity in water, but is faster, easier, and more sensitive. While the latter methods allow the determination of spatial gradients and short-term fluctuations of a specific estrogenic compound, our method provides a time integrated response of total estrogenic activity in an aquatic system.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03537. The extraction procedure of aqueous samples; The extraction procedure of XAD 18 resin; VM7Luc4E2 cells cultivation, seeding, and dosing in 96-well plates; Diffusion boundary layer (DBL) measurements; Figure S1: The uptake characteristics of E2 on XAD 18 resin gel at different E2 concentrations; Figure S2: DGT deployments in the laboratory and in the field (sampling station GBD-2 in Gaobeidian sewage treatment plant in Beijing). 1/mass of E2 on XAD 18 resin gel is plotted versus the diffusive layer thickness (PDF). G

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Analytical Chemistry

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