Environ. Sci. Technol. 2005, 39, 2287-2293
Fate of Wastewater Effluent hER-Agonists and hER-Antagonists during Soil Aquifer Treatment OTAKUYE CONROY,† DAVID M. QUANRUD,‡ WENDELL P. ELA,† DANIEL WICKE,§ KEVIN E. LANSEY,| AND R O B E R T G . A R N O L D * ,† Chemical and Environmental Engineering, The University of Arizona, Tucson, Arizona 85721, Office of Arid Lands Studies, The University of Arizona, Tucson, Arizona 85719, Technical University, Berlin, Germany, and Civil Engineering and Engineering Mechanics, The University of Arizona, Tucson, Arizona
Estrogen activity was measured in wastewater effluent before and after polishing via soil-aquifer treatment (SAT) using both a (hER-β) competitive binding assay and a transcriptional activation (yeast estrogen screen, YES) assay. From the competitive binding assay, the equivalent 17Rethinylestradiol (EE2) concentration in secondary effluent was 4.7 nM but decreased to 0.22 nM following SAT. The YES assay indicated that the equivalent EE2 concentration in the same effluent sample was below the method-detection limit (90%) of estrogenic activity observed using an in vitro bioassay technique (20,31). The effects of multiple estrogens present simultaneously were assumed to be additive, and anti-estrogenic activity was neglected. Nevertheless, a variety of other estrogenic contaminants such as alkylphenols and phthalates are present in municipal wastewater effluent (21,33), and myriad unidentified organics in such chemically complex waters increase the likelihood that additional discoveries will be made. There have been only a few efforts to measure attenuation of known estrogenic compounds, anti-estrogens, or estrogenic activity in waters that are percolated for groundwater recharge (32). The relative merits and demerits of in vitro tests for detection of estrogenic activity in water have received considerable attention (34-36). Much of this is motivated by the need to screen large numbers of individual chemicals (37). There are dozens of recognizably different in vitro test methods for estrogenic activity. These are generally classified as receptor-binding, transcriptional-activation, or cellproliferation assays (36). Receptor-binding assays for estrogenic activity suffer from a potential lack of physiological relevance inasmuch as measured affinity for the hormone receptor protein is independent of transport impediments that may be present in whole-cell in vitro or in vivo bioassays. Furthermore, binding assays cannot distinguish between the activities of hormone agonists and antagonists so that physiologically relevant information is again inaccessible. Transcriptional activation assays, including the yeast bioassay employed here, respond directly to estrogen agonists and can detect receptor antagonists when the test chemicals or organics derived from environmental samples are added to positive controls (37). While more sensitive than most alternative in vitro methods, cell proliferation assays are also more time-consuming and demanding in terms of skill and effort. None of the cell proliferation assays was considered for development of a standard method in the recent ICCVAM review of in vitro screening methods (37). VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. SRF site plan. The Roger Road Wastewater Treatment Plant produces a conventional secondary effluent based on treatment in a biotower. A portion of the chlorinated secondary effluent is infiltrated for temporary storage and recovery in the local unconfined aquifer. Water samples utilized in this study represented chlorinated secondary effluent that was directly recharged to RB1 and RB8. Infiltrate samples were taken from wells at about 15 feet and 130 feet below land surface in RB1. (On the basis of a graphic provided by the City of Tucson, Tucson Water Department, Research and Technical Support Section.)
Here we combined the results of receptor binding and transcriptional activation assays to show that chemically complex waters such as secondary effluent can contain both estrogen agonists and antagonists. Estrogenic and antiestrogenic activities in secondary effluent were separated chromatographically and appear to be differentially removed during rapid infiltration of effluent for groundwater replenishment.
Experimental Section Site Description. Samples were collected from the City of Tucson’s SRF (Figure 1) in southern Arizona. This site consists of the Sweetwater Wetlands and eight recharge basins (RB1-8) that receive chlorinated secondary effluent from the Roger Road Wastewater Treatment Plant, operated by the Pima County Department of Wastewater Management. Chlorination is based on sodium hypochlorite addition to produce a combined chlorine residual. The SRF began operation in 1990 and provides short-term (6-12 months) aquifer storage of reclaimed water for nonpotable reuse. RB1 contains two monitoring wells located at 4.5 m (MW-5) and 37 m (WR-199A) below land surface. Sample Preparation and Preliminary Analysis. Samples were collected in April 2003 in 1-L amber borosilicate glass bottles. Bottles were previously washed with 0.1 N HCl and baked at 550 °C for 5 h. Samples from RB8, MW-5, and WR199A were filtered using 0.45-µm cellulose nitrate filters (Millipore) on the day of collection. Infiltration basins, including RB8, are operated by alternating wet and dry 2288
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periods. During wet periods, the basins are flooded with chlorinated secondary effluent. During subsequent dry periods, desiccation of surface sediments restores infiltration capacity. Wet and dry periods of operation are normally several days long. The RB-8 sample was obtained from ponded water during a wet period. Hydrophobic organics were extracted from the filtered samples using 47-mm C-18 disks (3M Empore) that were preconditioned with two 10mL volumes of 100% ethyl alcohol (Aaper) and 10 mL of Nanopure (Nanopure Infinity) water. Retained organics were eluted with two 10-mL portions of 100% ethyl alcohol. Eluates were dried under nitrogen, and the dry residual materials were redissolved in sufficient buffer (competitive binding assay) or Nanopure water (yeast estrogen screen) to yield concentration factors of 200×, assuming that separations and recoveries were completely efficient. DOC was analyzed using a combustion technique with a Shimadzu TOC-5000 total organic carbon analyzer. Samples were acidified to pH 2 using 2 N HCl, sparged for 4 min using ultrapure hydrocarbon-free air, then analyzed 4-6 times to produce a coefficient of variation e 0.02. Standard solutions (made using potassium hydrogen phthalate) were included in each instrument run, and reported values were derived from a standard curve that was obtained via linear regression analysis. The lower level of detection and practical quantitation limit for DOC measurement with this analyzer were 0.2 and 0.5 mg/L, respectively. hER-β Competition Binding Assay. A competitionbinding fluorescence polarization assay (Invitrogen) was used to measure total estrogenic activity in water samples. This test establishes the affinity of a specific organic compound, or collective affinity of a mixture of organics in a complex sample, for a recombinant human estrogen receptor, hER-β, in the presence of a competing fluorescent ligand, ES2. The amount of fluorescent ligand displaced by the environmental sample is quantified via fluorescence polarization (38). The 200× concentrates were re-suspended and serially diluted in the assay buffer to produce final volume fractions ranging from 0.50 to 1 × 10-4. In addition to sample dilutions, assay mixtures contained 10 nM hER-β and 1 nM ES2. Mixtures were allowed to react for 2 h in the dark. A positive control was developed in the same manner by serially diluting EE2 from 1 µM to 1 × 10-5 µM. A process blank was developed by concentrating organics from Nanopure water on a C-18 disk, eluting in alcohol, etc. A blank without ES2 and hER-β was also prepared for each sample dilution to correct for fluorescence derived from the organic matrix. Fluorescence polarization was measured using a Beacon 2000 Fluorescence Polarization Machine (Invitrogen). An IC50 value was defined for environmental samples as the volume fraction of sample in the assay mixture that resulted in displacement of 50% of the bound ES2 molecules from hER-β. Results corresponding to chemically complex samples are usually reported as an equivalent EE2 concentration. Yeast Estrogen Screen (YES) Bioassay. The YES bioassay of Routledge and Sumpter (39) was also used to measure estrogenic and anti-estrogenic activity in RB-8, MW-5, and WR-199A water samples. The published procedure was modified as described by de Boever et al. (40). The modified procedure for measurement of total estrogenic activity was carried out by serially diluting each (200×) sample concentrate across 10 wells in a 96-well plate (Costar). Each dilution series was initiated by placing 100 µL of the sample concentrate in the first well of a single row. Fifty microliters was transferred to the second column and mixed with 50 µL of Nanopure water (2× dilution per step). The process was repeated across the row to produce an overall dilution of 29. Fifty microliters of 200× concentrate derived from Nanopure water was added to wells 11 and 12 of each row to serve as (negative) process controls. All eight rows of the plate were
FIGURE 2. Agonist and antagonist responses in a positive control (EE2) curve from the yeast-based reporter gene assay. Presence of estrogen agonists will cause an increased response in the lower limb of the curve. Presence of antagonists will cause a decreased response in the upper limb of the curve, as shown. filled in an identical manner (same sample and dilution program) to generate estimates of experimental error. The recombinant strain of Saccharomyces cerevisiae used here was provided by John Sumpter of Brunel University, Oxbridge, U.K. Yeast cells were grown in the Routledge/ Sumpter medium to OD630 ) 1.0 cm-1. The culture was then diluted in the same medium to OD630 ) 0.133 cm-1, and 150 µL of the diluted suspension was added to each well of the 96-well plate (total volume ) 200 µL at this point). The resultant OD630 value in each well was about 0.10 cm-1. Plates were then incubated for 24 h at 32 °C for growth of S. cerevisiae and estrogen-dependent expression of lacZ. At that point, 50 µL of a cycloheximide/CPRG (chlorophenol red β-D-galactopyranoside) solution consisting of 3 mL of minimal medium (40), 2 mL of 10 mg/mL cycloheximide, and 200 µL of 10 mg/mL CPRG was added to each test well. After an additional 24 h for β-galactosidase-dependent color development, absorbance was measured at 570 nm (β-galactosidase activity) and 630 nm (turbidity). The A570 contribution due to celldependent light scattering was determined by measuring the ratio of A570/A630 (here defined as R) in negative control wells. Corrected β-galactosidase activity was determined as A570 - RA630. The positive control series was developed in a similiar manner to yield test concentrations of EE2 ranging from 1.0 × 10-7 to 5.0 × 10-12 M. IC50 was defined as the concentration of EE2 that produced a half-maximal test response (Figure 2). Results derived from environmental samples were converted to an equivalent concentration of EE2 based on
EE2(equivalent) )
IC50,EE2 (FS)(CF)
where FS is the volume fraction of sample in the dilution that produced a half-maximal test response and CF is the sample concentration factor (here 200×). Agonist/Antagonist Bioassays. Estrogen agonist/antagonist activities were evaluated using the YES bioassay by determining the effect of sample organics on the positive (EE2) control series. The dilution procedure for the positive control was modified slightly to produce a 1:2 dilution series (EE2 stock in Nanopure water) and final EE2 concentrations ranging from 5 pM to 100 nM. The remainder of the procedure was as described. Agonist effects were evident if the environmental sample raised the lower limb of the S-shaped EE2 curve. Anti-estrogens depressed the upper limb of the positive control curve (Figure 2). Sample additions (constant over a specific EE2 dilution series) produced final sample concentrations factors of 5, 10, or 20× in the test mixtures. To show that the modified YES bioassay can measure anti-estrogenic activity, trans-tamoxifen (Aldrich) or trans4-hydroxytamoxifen (4-OHT, Calbiochem) was added at final
FIGURE 3. Antagonist experiment with 4-OHT. Anti-estrogenic activity was observed at 3.0 × 10-7 M 4-OHT and higher concentrations. Results were dose-dependent. Results with transtamoxifen (not shown) were similar, although anti-estrogenic effects were not observed at dosages below 6.0 × 10-7 M. concentrations from 1.0 × 10-7 to 1.0 × 10-6 M to the EE2 positive control series. Tamoxifen is widely regarded as a partial antagonist of estrogen activity. That is, the estrogendependent response is blocked by tamoxifen in certain human tissues (e.g., breast) although tamoxifen is an estrogen agonist in uterine tissues (41-43). It was once widely held that estrogen antagonism could not be reproduced in yeastbased transcription activation assays (44) leading to questions regarding the suitability of yeast-based platforms for measurement of anti-estrogenic assays. However, Routledge and Sumpter (45) observed antagonism involving 17β-estradiol and 4-OHT in the bioassay system used here. To assure ourselves regarding the ability of our cells to respond to antiestrogens such as 4-OHT and trans-tamoxifen, we reproduced those studies prior to carrying out work with environmental samples that were hypothesized to contain anti-estrogenic activity. 4-OHT proved to be anti-estrogenic at concentrations ranging from 1.0 × 10-7 to 1.0 × 10-6 M (Figure 3). Lower dosages had little effect and may have enhanced cell response to EE2. Results were dose dependent at 4-OHT concentrations g 1.0 × 10-7 M. Results of experiments involving transtamoxifen (not shown) were similar, although agonist effects were not observed below a dose of 6.0 × 10-7 M.
Results and Discussion Competitive binding assay results for the contemporary (200×) pond/perched water/aquifer samples indicated that estrogenic activity in the complex water samples was attenuated by more than an order of magnitude during transport from the infiltration pond to the unconfined aquifer immediately beneath pond RB1 (Figure 4). The estradiol equivalent for the concentrate derived from water in infiltration basin RB8 (unchlorinated secondary effluent) was 4.7 nM. The well WR-199A concentrate had an EEQ of 0.22 nM (95% reduction). None of the binding assay controls (ethanol process control, 200 Nanopure concentrate, and resuspension buffer; not shown) exhibited estrogenic activity. The perched water sample taken at MW-5 was also improved relative to the pond sample in terms of DOC and estrogenic activity (EEQ ) 1.7 nM, Table 1). Previous boron isotope measurements (46) have shown that samples derived from WR-199A are of wastewater origin. The raw samples were independently reconcentrated for a second round of analyses, again using the competitive binding assay with essentially the same result (data not shown). The results of similar depth-dependent measurements in RB1, reported previously for the SRF, were essentially identical (32). When the three sample concentrates were analyzed using the yeast-based reporter-gene assay, only the WR-199A VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Displacement of fluorescent ligand (ES2) from hER-β as a function of fractional (percent) content of the sample concentrate (200×) in assay mixtures. Results show that 10 times more of the well WR-199A concentrate was required for equivalent displacement of the fluorescent ligand, indicating that the WR-199A sample was less estrogenic than the MW-5 and infiltration pond (RB8) samples.
TABLE 1. Summary of Estrogenic Activities Determined Using the Competitive Binding and Reporter-Gene Assays IC50 (nM) sample or compd tested
DOCc (mg/L)
EE2 RB-8 MW-5 WR-199A
20.7 13.9 1.93
binding assay
reportergene assay
10
0.3
equivalent EE2 concentration (nM)a,b binding assay
reportergene assay
4.7 1.7 0.22
