Environ. Sci. Technol. 2003, 37, 4821-4822
Response to Comment on “Estrogen Receptor Agonist Fate during Wastewater and Biosolids Treatment Processes: A Mass Balance Analysis” Yuan (1) raised concern over several aspects of our study (2), which used results from the yeast-estrogen screen (YES) bioassay to perform estrogenic mass balances across biological wastewater and solids digestion processes. We interpret these concerns to be (i) failure to consider the impact of antagonist and inhibitory compounds, which can be coextracted from the environmental samples; (ii) inappropriate use of mass balance due to changes in estrogenic activity during biological wastewater treatment; (iii) use of single concentration factors in determining estrogenic response from extracted samples; and (iv) neglecting to discuss the role of weak xenoestrogens in wastewater, sludge, and biosolid samples. We discuss each of these concerns individually. (i) Influence of Co-extracted Compounds. We acknowledge that recombinant yeast cells are susceptible to both antagonistic and inhibitory compounds that may be present in the sample matrix (3), producing a lower response than would be expected from the calculated response based on compound potency and concentration (e.g., ref 4). Additionally, the YES assay may overestimate the presence of nonestrogenic compounds such as linear alkylbenzene sulfonates (LAS) (5). However, the original recombinant YES assay described by Routledge and Sumpter (6) has been used in many investigations concerning the estrogenic activity contained in both natural and wastewater systems; at the time this response was written, their paper had been referenced over 200 times. While use of a second analytical method like GC/MS or LC/MS would have been beneficial to our study, the YES assay is a widely accepted tool for characterizing both known and unknown estrogenic activity in environmental samples (e.g., refs 7 and 8). We concur that future studies should use multiple methods (e.g., ref 9) but believe that general screening methods such as the YES assay will continue to be a valid analytical tool for these types of studies because all the compounds that cause estrogenic activity (either in vitro or in vivo) are not known. Furthermore, screening techniques such as YES are likely to be used to generate widely available data sets for characterizing estrogenic activity because of their simplicity. What may be lost in terms of identifying individual compounds will likely be (at least partially) offset by the robustness of datasets that are likely to be much larger. (ii) Inappropriate Use of Mass Balance. Yuan (1) correctly asserts that estrogenic compounds can undergo transformations in the treatment bioreactor that were not accounted for in our simple mass balance, which considered influent and effluent activities and extractable fractions from biological solids. Other types of transformations, such as biodegradation, photodegradation, and nonextractable sorption, were not specifically considered. We stated in our paper that these transformations are known or are likely to occur. We believe that future studies should be directed toward developing our knowledge base so that a more rigorous mass balance approach can be completed within engineered biological treatment systems. Such a rigorous approach would consider all potential transformations of the target chemicals in the bioreactor; however, the current state of knowledge 10.1021/es030079i CCC: $25.00 Published on Web 09/18/2003
2003 American Chemical Society
regarding the extant partitioning factors and biodegradation rates for all key estrogen or estrogen-like chemicals is not sufficient to complete such a task. In our study, we were able to use the simple mass balance approach to point out that significant loss of estrogenic activity occurred in the bioreactors. Additionally, we were able to use it to compare the ability of different technologies treating the same wastewater to remove estrogenic activity. We concur with Yuan (1) that the next question is: What happened to the estrogenic activity? As analytical techniques evolve, it should be possible for the discipline to answer this question more thoroughly. (iii) Use of Single Concentration Factors. During the early phase of our study, a number of field samples were collected, extracted using several different sample volumes, and subjected to the YES assay for method validation purposes and in order to determine an appropriate concentration factor. This proved to be most important for the sludge and biosolids samples, as the estrogenic activity was somewhat higher than expected. We determined that multiple concentration factors of the same sample yielded statistically equivalent bioassay responses, indicating that the extraction method was working correctly. (iv) Discussing the Role of Weak Xenoestrogens. In framing the results of our study, we did not ignore the impact of estrogenic compounds relative to 17β-estradiol (E2) and 17R-ethinylestradiol (EE2). As previously discussed, our extraction method was not selective and, therefore, may have contained a number of estrogenic compounds, both known and unknown. Yuan (1) specifically mentions alkylphenolic compounds as a potential activator of the YES response. Indeed, Johnson and Sumpter (10) suggest that EE2 and the alkylphenol polyethoxylate metabolites octylphenol and nonylphenol may account for up to 90% of the in vivo estrogenicity of a typical wastewater effluent. Subsequently, there exists a significant amount of literature pertaining to the estrogenic activity of alkylphenolic chemicals, including their behavior in wastewater treatment facilities (11, 12), structural features which contribute to their estrogenic behavior (13), and detection (14-16) and persistence (17, 18) in the natural environment. In addition to alkylphenolic compounds, many anthropogenic compounds are estrogenic including, to name but a few, phthalates (19), bisphenol A (20), and flame retardants (21). Summary. We believe that bioassays are and will continue to be an important tool in assessing the role of biological wastewater treatment processes in reducing estrogenic activity. Thorpe et al. (22) recently suggested that we consider the total estrogenic load in receiving waters instead of individual estrogenic compounds, a task that possess a formidable challenge for conventional analysis methods (e.g., GC/MS) but that is routine for bioassay investigations. We hope that our research effort will help stimulate other investigators to conduct similar mass balances in an effort to understand the role of wastewater treatment processes in reducing the public health and environmental risks associated with estrogenic activity.
Literature Cited (1) Yuan, T. Environ. Sci. Technol. 2003, 37, 4819-4820. (2) Holbrook, R. D.; Novak, J. T.; Grizzard, T. J.; Love, N. G. Environ. Sci. Technol. 2002, 36, 4533-4539. (3) Garcia-Reyer, N.; Gray, E.; Castillo, M.; de Alda, M. J. L. Environ. Toxicol. Chem. 2001, 20, 1152-1158. (4) Murk, A. J.; Legler, J.; van Lipzig, M. M. H.; Meerman, J. H. N.; Belfroid, A. C.; Spenkelink, A.; van der Burg, B.; Rijs, G. B. J.; Vethaak, D. Environ. Toxicol. Chem. 2002, 21, 16-23. VOL. 37, NO. 20, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(5) Miyamoto, N.; Tanaka, H.; Tamamoto, H.; Komori, K. Fractionation Method for Estimating the Cause of Estrogen-like Activity in Sewage. Proc. Water Environ. Fed. 75th Ann. Tech. Exposition Conf., Chicago, IL, 2002 [CD-ROM]. (6) Routledge, E. J.; Sumpter, J. P. Environ. Toxicol. Chem. 1996, 15, 241-248. (7) Kirk, L. A.; Tyler, C. R.; Lye, C. M.; Sumpter, J. P. Environ. Toxicol. Chem. 2002, 21, 972-979. (8) Witters, H. E.; Vangenechten, C.; Berckmans, P. Water Sci. Technol. 2001, 43, 117-123. (9) Beresford, N.; Routledge, E. J.; Harris, C. A.; Sumpter, J. P. Toxicol. Appl. Pharmacol. 2000, 162, 22-33. (10) Johnson, A. C.; Sumpter, J. P. Environ. Sci. Technol. 2001, 35, 4697-4703. (11) La Guardia, M. J.; Hale, R. C.; Harvey, E.; Matteson-Mainor, T. Environ. Sci. Technol. 2001, 35, 4798-4804. (12) Ahel, M.; Giger, W.; Koch, M. Water Res. 1994, 28, 1131-1145. (13) Routledge, E. J.; Sumpter, J. P. J. Biol. Chem. 1997, 272, 32803288. (14) Ahel, M.; Giger, W.; Schaffner, C. Water Res. 1994, 28, 11431152. (15) Rudel, R. A.; Melly, S. J.; Geno, P. W.; Sun, G.; Brody, J. G. Environ. Sci. Technol. 1998, 32, 861-869. (16) Ferguson, P. L.; Iden, C. R.; Brownawell, B. J. Environ. Sci. Technol. 2001, 35, 2428-2435.
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(17) Heinis, L. F.; Knuth, M. L.; Liber, K.; Sheedy, B. R.; Tunell, R. L.; Ankley, G. T. Environ. Toxicol. Chem. 1999, 18, 363-375. (18) Shang, D. Y.; Macdonald, R. W.; Ikonomou, M. G. Environ. Sci. Technol. 1999, 33, 1366-1372. (19) Harris, C. A.; Henttu, P.; Parker, M. G.; Sumpter, J. P. Environ. Health Perspect. 1997, 105, 802-811. (20) Kawagoshi, Y.; Tsukagoshi, Y.; Fukunaga, I. J. Environ. Monit. 2002, 4, 1040-1046. (21) Meerts, I. A. T. M.; Letcher, R. J.; Hoving, S.; Marsh, G.; Bergman, A.; Lemmen, J. G.; van der Burg, B.; Brouwer, A. Environ. Health Perspect. 2001, 109, 399. (22) Thorpe, K. L.; Cummings, R. I.; Hutchinson, T. H.; Scholze, M.; Brightly, G.; Sumpter, J. P.; Tyler, C. R. Environ. Sci. Technol. 2003, 37, 1142-1149.
R. David Holbrook, John T. Novak, Thomas J. Grizzard, and Nancy G. Love* Department of Civil and Environmental Engineering Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061-0246 ES030079I
