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Identification and Quantification of Estrogen Receptor Agonists in Wastewater Effluents
Identification and Quantification of Estrogen Receptor Agonists in Wastewater Effluents
Identification and Quantification of Estrogen Receptor Agonists in Wastewater Effluents
Total concentrations of several known xenobiotic estrogen in fish exposed to municipal wastewater treatment plant
effluents (11, 12). Nonylphenol (NP), nonylphenol poly-
receptor (ER) agonists and natural and synthetic estrogen
ethoxylates (NPEs), octylphenol (OP), and synthetic and
were measured in water by use of a combination of natural steroids were targeted in this investigation because
instrumental and bioanalytical approaches. Samples from
Downloaded via UNIV OF PADOVA on April 21, 2022 at 14:16:17 (UTC).
VOL. 35, NO. 18, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3621
125 µL per well (15 000-20 000 cells per well) using a predictions (29). To make an accurate comparison, it was
repeating pipet. To ensure homogeneity, the cell solution necessary to address the potential for antagonistic and
was continuously mixed during seeding. The 36 exterior wells synergistic interactions. This was done by the fine fraction-
of each microplate were filled with 125 µL of medium. Cells ation of samples, by which compounds known to be
were dosed after an overnight incubation to allow for cell antagonistic to the measurement of EEQ were separated from
attachment. Extracts or fractions were dissolved in stripped the active compounds.
medium to yield a final concentration of 1.0% extract. A 3-fold
dilution of each extract or fraction was also prepared, yielding Results and Discussion
a concentration of 0.33% extract. Test wells were dosed with ER Agonist Activity. None of the F1 fractions induced a
125 µL 1.0% or 0.33% extract in medium to yield final in-well significant response in the MVLN assay (Figure 1). Nonpolar
concentrations of 0.50% and 0.165% extract. Solvent control compounds such as PAHs, PCBs, and most organochlorine
wells were dosed with 125 µL of medium spiked with 1.0% (OC) pesticides, if present, would have been contained in F1
of the appropriate solvent to yield a final in-well concentration (Figure 1) (26). Certain OC pesticides and PAHs such as
of 0.50% solvent. Blank wells received 125 µL of the chrysene, benz[a]anthracene, and benzo[a]pyrene have been
appropriate media. Each plate tested included a minimum reported to cause weak ER-mediated responses in vitro (23,
of three solvent control wells, three blank wells, and three 31). Based on the method detection limit (MDL) for E2 in the
replicates of each fraction tested (at both 0.50% and 0.165% MVLN assay, concentrations of ER agonists were present in
levels). Dosed cells were exposed for 72 h at standard F1 at a concentration less than 0.55 ng EEQ/mL. These results
incubation conditions. support the conclusion that concentrations of nonpolar ER
Each test plate was inspected visually and differences in agonists in the surface waters and effluents examined were
cell numbers and condition relative to control wells and small.
conditions normally observed during routine culturing were Weak ER agonists such as NP and OP were present in F2
noted for each well. Culture medium was then removed, and (26). No F2 extracts elicited a significant response in the
each well was rinsed twice with phosphate buffered saline MVLN assay (Figure 1), despite the confirmed presence of
(PBS) supplemented with 1.0 mM Ca2+ and Mg2+ using an NP and OP (Table 1). These results suggest that the
eight channel vacuum manifold. Plates were inspected for compounds present in F2 contributed less than 0.55 ng EEQ/
cell loss during washing. Following inspection, 75 µL PBS mL. ER agonist potencies of NP and OP, relative to E2, for
supplemented with Ca2+ and Mg2+ was added to each well, luciferase induction in MVLN cells have been reported to be
followed by 75 µL Luc-lite reagent (Packard Instruments). 1.25 × 10-5 and 1.9 × 10-5 for NP and OP, respectively (25).
Each plate was incubated for 10 min at 30 °C and then scanned When concentrations of NP and OP present in the samples
with an ML 3000 microplate reading luminometer (Dynatech (Table 1) were multiplied by their corresponding relative
Laboratories, Chantilly, VA). Following the luminometer scan, potencies and summed, it was concluded that these two
125 µL of 1.08 mM fluorescamine (Sigma) in acetonitrile compounds contributed less than 0.075 ng EEQ/mL for 15
(ACN) was added to each well, and plates were assayed for of the 16 samples tested. Thus, the general lack of significant
protein after a 15 min incubation at room temperature (28). induction of the MVLN cells was consistent with the known
Plates were scanned using a Cytofluor 2300 (excitation 400 concentrations of ER agonists (alkylphenols) present in F2.
nm, emission 460 nm), and responses were compared to a The BV-effluent sample extract contained approximately 1.90
standard curve consisting of six concentrations of bovine ng of EEQ/mL, which would correspond to approximately
serum albumin (BSA) (Sigma) ranging from 1.5 to 50 µg per 8.5 fmol EEQ/well in the MVLN bioassay. Based on regression
well. against an E2 standard curve, this dose could have elicited
All data were collected electronically and imported into a response as great as 53% E2-max. However, F2 extract of
a spreadsheet (Excel 7.0, Microsoft Inc., Seattle, WA) for data BV-effluent failed to induce a significant response in the
analysis. Protein content per well was calculated by regression MVLN bioassay (Figure 1a). This suggests that F2 of the BV-
against the BSA standard curve. Protein data were used as effluent sample might have contained interfering compounds
an index of cell number to detect outliers that were not that suppressed the ER agonist potency of NP and OP. The
apparent by visual inspection. Relative luminescence units F2 extract was fractionated further, and fine fractions were
(RLU) were not adjusted for protein. Sample responses in analyzed in the MVLN bioassay (Supporting Information,
RLU were expressed as a percentage of the mean maximum Figure 2). No significant ER-mediated responses were
response observed for standard curves developed on the same observed in the fine fractions. Thus, the hypothesized
day (% E2-max) (29). The greatest response of the two extract interfering compounds, if present, must have properties
dilutions was reported. However, for each significant re- similar to NP. The same fine fractionation was applied to the
sponse, the greatest response came from the greater extract F2 extract from Black Lagoon (Supporting Information, Figure
concentration (0.5% in the well). 3). Once again, no significant estrogen-like activity was
Potency balance analyses were conducted by comparing observed. In general, responses of MVLN cells to F2 samples
observed bioassay response magnitudes to those predicted were in agreement with the potency expected based on the
based on the concentrations of known ER agonists present known concentrations and relative potencies of these
in an extract (30). Instrumentally determined concentrations compounds.
of individual compounds were multiplied by their assay- F3 samples caused the greatest magnitude of ER agonist
specific relative potencies. The sum of the products for all response in the MVLN bioassay. Six of the 16 F3 samples
target compounds present in an extract provided an estimate elicited significant ER-mediated responses in the MVLN
of the 17β-estradiol equivalents (EEQ) in the extracts. Linear bioassay (Figure 1). The greatest magnitudes of response for
regression against a 17β-estradiol (E2) standard curve was F3 extracts (≈80% E2-max) were observed for samples
used to predict the bioassay response magnitude for the collected from the LV Wash and LV Bay in April 1997 (Figure
sample. Variability in the predicted bioassay response 1c). However, samples from LV Wash and LV Bay collected
magnitude was estimated based on the 95% confidence band in September of 1997 did not elicit significant responses in
for a first-order polynomial fit to the E2 standard curve (PlotIT, the MVLN assay (Figure 1d). The samples collected in
Scientific Programming Enterprises, Haslett, MI). Compari- September 1997 were obtained after a large storm event,
sons were predicated on the assumption that EEQs would which diluted the wastewater entering LV Wash and LV Bay
behave as if they were 17β-estradiol in the bioassay. Violation (26). The difference in bioassay responses for April and
of this assumption may have resulted in some error in the September samples was paralleled by decreases in EEQs in
3622 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 18, 2001
TABLE 1. Extract Concentrations and 17β-Estradiol Equivalents (EEQs) (ng/mL)c
location date NP OP NPE E2 EE2 NP/OP-EEQa E2/EE2-EEQb
Lake Mead
LV Wash 4/30/97 4560 172 36000 10.70 1.92 0.061 10.9
LV Bay 4/30/97 3000 108 19400 8.84 2.08 0.040 9.05
9/5/97 640 ND 12710 0.752 1.01 0.008 0.86
LV Marina 9/5/97 ND ND ND 1.08 ND NA 1.08
Saddle Island 4/30/97 ND ND ND ND ND NA NA
Callville Bay 9/5/97 ND ND ND ND ND NA NA
Trenton Channel
WWTP 8/30/97 1916 20 21600 4.26 ND 0.024 4.26
Chem. 8/30/97 3450 60 29200 3.64 ND 0.044 3.64
B. Lagoon 8/30/97 3740 264 34700 5.18 1.44 0.052 5.30
M. Creek 8/30/97 4740 324 71260 4.24 ND 0.066 4.25
WWTPs
BV-upstream 10/8/97 ND ND ND 2.50 ND NA 2.50
BV-effluent 10/8/97 148000 2350 1160000 14.6 3.04 1.90 14.9
MA-upstream 10/8/97 ND ND ND ND ND NA NA
MA-effluent 10/8/97 2065 64 19400 3.62 1.43 0.027 3.77
ER-upstream 10/8/97 ND ND ND ND ND NA NA
ER-effluent 10/8/97 680 ND ND 1.90 ND 0.009 1.90
a Nonylphenol and octylphenol-derived 17β-estradiol equivalents. NP/OP-EEQ ) (NP
relative potency(REP) × NPconcentration) + (OPREP × OPconcentration).
NPREP ) 1.25 × 10-5. OPREP ) 1.9 × 10-5. A REP estimate was not available for NPE; therefore, it was not considered when deriving EEQ estimates.
b Estradiol and ethynylestradiol-derived 17β-estradiol equivalents. E2/EE2-EEQ ) (E2
REP × E2concentratration) + (EE2REP × EE2concentration). E2REP ) 1.0.
EE2REP ) 0.10. c ND ) not detectable; NA ) not applicable
3624 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 18, 2001
Supporting Information Available (14) Belfroid, A. C.; Van der Horst, A.; Vethaak, A. D.; Schafer, A. J.;
Rijs, G. B. J.; Wegener, J.; Cofino, W. P. Sci. Total Environ. 1999,
Figures of RP-HPLC fine fractionation and fine fractionations 225, 101-108.
of BV WWTP and Black Lagoon, Trenton Channel F2 extract (15) Bennie, D. T. Water Qual. Res. J. Canada 1999, 34, 79-122.
and BV WWTP F3 and Black Lagoon, Trenton Channel F3 (16) Naylor, C. G.; Mieure, J. P.; Adams, W. J.; Weeks, J. A.; Castaldi,
extract. This material is available free of charge via the Internet F. J.; Ogle, L. D.; Romano, R. R. JAOCS 1992, 69, 695-703.
at http://pubs.acs.org. (17) Poland, A.; Knutson, J. C. Annu. Rev. Phamacol. Toxicol. 1982,
22, 517-554.
Literature Cited (18) Neff, J. M. Polycyclic Aromatic Hydrocarbons in the Aquatic
Environment: Sources, Fates, and Biological Effects; Applied
(1) Kendall, R. J.; Dickerson, R. L.; Giesy, J. P.; Suk, W. A. Principles Science: London, 1979.
and Processes for Evaluating Endocrine Disruptors in Wildlife; (19) Schecter, A. Dioxins and Health; Plenum Press: New York, 1994;
SETAC Press: Pensacola, FL, 1998. p 710.
(2) Ankley, G.; Mihaich, E.; Stahl, R.; Tillitt, D.; Colborn, T.;
(20) Krishnan, V.; Safe, S. Toxicol. Appl. Pharmacol. 1993, 120, 55-
McMaster, S.; Miller, R.; Bantle, J.; Campbell, P.; Denslow, N.;
61.
Dickerson, R.; Folmar, L.; Fry, M.; Giesy, J.; Gray, E.; Guiney, P.;
Hutchinson, T.; Kennedy, S.; Kramer, V.; LeBlanc, G.; Mayes, (21) Anderson, M. J.; Miller, M. R.; Hinton, D. E. Aquat. Toxicol.
M.; Nimrod, A.; Patino, R.; Peterson, R.; Purdy, R.; Ringer, R.; 1996, 34, 327-350.
Thomas, P.; Touart, L.; Van Der Kraak, G.; Zacharewski, T. (22) Tran, D. Q.; Ide, C. F.; McLachlan, J. A.; Arnold, S. F. Biophys.
Environ. Toxicol. Chem. 1998, 17, 68-87. Res. Commun. 1996, 229, 102-108.
(3) U.S. EPA. Special report on environmental endocrine disrup- (23) Clemons, J. H.; Allan, L. M.; Marvin, C. H.; Wu, Z.; McCarry, B.
tion: an effects assessment and analysis; EPA/630/R-96/012; E.; Zacharewski, T. R. Environ. Sci. Technol. 1998, 32, 1853-
Office of Research and Development: Washington, DC, February 1860.
1997. (24) Sanderson, J. T.; Giesy, J. P. In Encyclopedia of Environmental
(4) Safe, S. H. Pharmacol. Ther. 1995, 67, 247-281. Analysis and Remediation; Meyers, R. A., Ed.; John Wiley and
(5) Ahlborg, U. G.; Lipworth, L.; Titus-Ernustoff, L.; Hsieh, C.-C.; Sons: New York, 1998; pp 5272-5297.
Hanberg, A.; Baron, J.; Trichopoulos, D.; Adami, H. O. Crit. Rev. (25) Villeneuve, D. L.; Blankenship, A. L.; Giesy, J. P. In Toxicant-
Toxicol. 1995, 25, 463-531. Receptor Interactions; Denison, M. S., Helferich, W. G., Eds.;
(6) Sharpe, R. M.; Skakkebaek, N. E. Lancet 1993, 341, 1392-1395. Taylor & Francis: 1998; pp 69-97.
(7) Carlsen, E.; Giwercman, A.; Keiding, N.; Skakkebaek, N. E. (26) Snyder, S. A.; Keith, T. L.; Verbrugge, D. A.; Snyder, E. M.; Gross,
Environ. Health Perspect. 1995, 130, 137-139. T. S.; Kannan, K.; Giesy, J. P. Environ. Sci. Technol. 1999, 33,
(8) Horwitz, K. B.; Jackson, T. A.; Rain, D. L.; Richer, J. K.; Takimoto, 2814-2820.
G. S.; Tung, L. Mol. Endocrinol. 1996, 10, 1167-1177. (27) Pons, M.; Gagne, D.; Nicolas, J. C.; Mehtali, M. Biotechniques
(9) Katzenellenbogen, J. A. Environ. Health Perspect. 1995, 103, 99- 1990, 9, 450-459.
101. (28) Kennedy, S. W.; Jones, S. P. Anal. Biochem. 1994, 222, 217-223.
(10) Gillesby, B. E.; Zacharewski, T. R. Environ. Toxicol. Chem. 1998, (29) Villeneuve, D. L.; Blankenship, A. L.; Giesy, J. P. Environ. Toxicol.
17, 3-14. Chem. 2000, 19, 2835-2843.
(11) White, R.; Jobling, S.; Hoare, S. A.; Sumpter, J. P.; Parker, M. G.
(30) Hilscherova, K. M.; Machala, M.; Kannan, K.; Blankenship, A.
Endo. 1994, 135, 175-182.
L.; Giesy, J. P. Environ. Sci. Pollut. Res. 2001, 7, 159-171.
(12) Bevans, H. E.; Goodbred, S. L.; Miesner, J. F.; Watkins, S. A.;
Gross, T. S.; Denslow, N. D.; Schoeb, T. Water-Resources (31) Soto, A. M.; Chung, K. L.; Sonnenschein, C. Environ. Health
Investigations Report 96-4266; 1996. Perspect. 1994, 102, 380-383.
(13) Snyder, S. A.; Snyder, E.; Villeneuve, D.; Kurunthachalam, K.;
Villalobos, A.; Blankenship, A.; Giesy, J. In Analysis of Environ- Received for review May 10, 2000. Revised manuscript re-
mental Endocrine Disruptors Keith, L. H., Jones-Lepp, T. L., ceived May 23, 2001. Accepted July 2, 2001.
Needham, L. L., Eds.; American Chemical Society: Washington,
DC, 2000; pp 73-95. ES001254N
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