Correspondence Comment on “Estrogen Receptor Agonist Fate during Wastewater and Biosolids Treatment Processes: A Mass Balance Analysis” Recently, Holbrook et al. (1) published a mass balance analysis of estrogen receptor agonist during wastewater and biosolids treatment processes. Their study is a valuable attempt to understand the fate of estrogenic substances in the above system and to possibly enhance the elimination of these emerging contaminants. However, some important aspects are unfortunately ignored in the study. First of all, the description of “estrogen receptor agonist” is unscrupulous since Holbrook et al. (1) employed the widely used recombinant yeast estrogen screen (YES) assay (2, 3) for the estrogenic activity determination. In fact, the rationale of the YES assay renders itself responsive to both agonist and antagonist hormonal chemicals. In brief, gene sequence of the human estrogen receptor was stably integrated into the yeast (Saccharomyces cerevisiae) genome. This strain also contained expression plasmid carrying estrogen responsive elements regulating the expression of the reporter gene Lac Z (encoding the enzyme of β-galactosidase). Thus, when an active ligand (in this case, estrodiol or an estrogenic substance) bound to the receptor, β-galactosidase was synthesized and could be quantitatively detected by the later color reaction. On the other hand, however, when an inactive ligand (e.g., antagonist or antiestrogenic substances) bound to the receptor, no or less β-galactosidase was expressed correspondingly. The applications of the same recombinant yeast strain to study the antiestrogenic activity of chemicals were widely reported (4-8). Likewise, other yeast-based assays also demonstrated that antiestrogenic response could be observed, as the basic principal of all of them was nearly the same (estrogen receptor binding and reporter gene transcription activation) (9-14). However, it seems Holbrook et al. (1) completely ignored this issue and the complexity of the environmental samples. They performed the mass balance analysis of estrogen receptor agonist based on the results of YES assay. Actually, the estrogenic activity determined in their study was the “comprehensive estrogenic activity” of their samples, as the extracts of the samples were complex mixtures that may contain agonist, antagonist, and even inhibitory compounds (refer to the compounds that can interfere the response of YES assay). Thus, the results of YES assay just represented the final response of agonist, antagonist, and inhibitory compounds (if any). Moreover, according to previously published reports, antiestrogenic compounds or antagonists and inhibitory effects did occur when estrogenic activity of environmental samples was investigated using the yeastbased assay (16-19). It is also highly probable that the hexane extracts of samples, which Holbrook et al. (1) tested for estrogenic activity, contain the known antiestrogenic compounds or inhibitory factors, such as some polynuclear aromatic hydrocarbons (9, 13), several polychlorinated biphenyls (15), phytochemicals (10), some pyrethroid insecticides (12), and humic acid (17). The unknown antagonists and inhibitory factors should be concerned too. Unfortunately, Holbrook et al. (1) did not perform any effort to exclude or explain the above considerations before they conducted the mass balance analysis of estrogen 10.1021/es030436p CCC: $25.00 Published on Web 09/18/2003
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receptor agonist. This could be a serious conceptional mistake. It will mislead other researchers to overlook the occurrence and complicacy of antiestrogenic substances and inhibitory factors in the environmental samples. Additionally, as discussed above, based on YES assay alone, it is nearly impossible to determine the estrogen receptor agonist in water samples as well as in biosolids. On the other hand, even we accept that the YES assay gives us “comprehensive estrogenic activity” of the environmental samples, the mass balance analysis is still very hard to perform and may not present any practical significance. One reason is that, during the wastewater and biosolids treatment processes, the estrogenic or initial nonestrogenic substances keep changing, which in turn change the overall estrogenic potency. For instance, nonylphenol polyethoxylates can be degraded to estrogenic breakdown products such as nonyl and octylphenol (20). Another reason may be attributed to the matrix effects of the samples. The antiestrogenic compounds and inhibitory factors differ in the samples collected from different treatment stages. So the response of yeast assay will be masked in various extents correspondingly. In addition, toxicity effect is also a potential problem in the application of yeast assay to determine estrogenic activity in the environmental samples (17, 19). These phenomena suggest that the fate study of estrogenic activity in environmental samples may have to rely on the chemical analysis and other combination methods. As a matter of fact, to date, most of the applications of yeast assay in environmental samples focused on the studies of reduction or occurrence of the estrogenic activity (6, 16-19, 21, 22). Secondarily, Holbrook et al. (1) might not be aware of the importance of the samples dose-response study (16, 18, 19, 21, 22). The entire sample extracts tested in their work seemed to be taken from the single concentration factor, as no further data was shown or later mentioned in the paper. This design could not be able to observe any dose-response information of the samples. The attempt to establish the dose-response relationship is to meet the basic requirement of toxicology. Briefly, the potency of estrogenic activity cannot be judged by one arbitrary concentration. For example, when a water sample was concentrated or extracted in different concentration factors, the extract would show a different potency of estrogenic activity in yeast assay depending on the factors. Thus, the line between “positive” and “negative” results became blurred because the positive result will be questioned at the too high concentration factor and the negative one may due to the low concentration factor. To solve this problem, the toxicological concept of EC50 (median effective concentration) might have to be introduced to this issue (18, 19). Or at least, the potency of the estrogenic activity in environmental samples should be checked under several different concentration factors (16, 21, 22). However, Holbrook et al. (1) did little attempt to study the doseresponse relationship of the samples. The results of yeast assay they obtained may not be representative for the potency of the estrogenic activity in samples, if extracts came from the single concentration factor. Finally, it was noted that only natural and synthetic estrogens were included when Holbrook et al. (1) performed a comparison of their results with those of other studies. This judgment would be acceptable because the previous opinions suggested that natural and synthetic estrogens contribute the highest proportion to the overall estrogenic activity of effluent from wastewater (18, 23, 24). But, it has VOL. 37, NO. 20, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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to be pointed out that recent reports suggested the alternative considerations, especially when Holbrook et al. (1) did not perform related chemical analysis to identify the estrogenic substances in their work. The weak xenoestrogens may also play a key role in contributing the overall estrogenic activity of the environmental samples. For instance, a high correlation between the concentration of alkylphenolic compounds in water and sediments samples with the presence of plasma vitellogenin in male carp was observed by Petrovic et al. (25). Similarly, Todorov et al. (26) found that phenolic xenoestrogens may contribute significantly to estrogenic activity observed in the effluents from three sewage treatment plants serving New York City. Sheahan and other co-workers (27) demonstrated that the xenoestrogens are the major contributors to the estrogenic activity of the River Aire (Yorkshire, U.K.). Moreover, the health concerns of weak xenoestrogenes on human and wildlife became more controversial when joint action and additive combination effects of them were reported recently (28, 29). These findings suggest that it cannot completely rule out the weak xenoestrogens from the assessment of the estrogenic activity in environmental samples in the future study. To date, there is no ideal standard protocol available to assess the estrogenic activity of the environmental samples. Nevertheless, one must comprehend the rationale and the limitation of the selected assay before it is applied to the specified research objectives. The existing arguments will further disclose the complexity of this emerging issue.
Acknowledgments The author acknowledges the financial support of the National University of Singapore and the Singapore Millenium Foundation.
Literature Cited (1) Holbrook, R. D.; Novak, J. T.; Grizzard, T. J.; Love N. G. Environ. Sci. Technol. 2002, 36, 4533-4539. (2) Routledge, E. J.; Sumpter, J. P. Environ. Toxicol. Chem. 1996, 15, 241-248. (3) Beresford, N.; Routledge, E. J.; Harris, C. A.; Sumpter, J. P. Toxicol. Appl. Pharmacol. 2000, 162, 22-33. (4) Sohoni, P.; Sumpter, J. P. J. Endocrinol. 1998, 158, 327-339. (5) Routledge, E. J.; Sumpter, J. P. J. Biol. Chem. 1997, 272, 32803288. (6) Taneda, S.; Hayashi, H.; Sakushima, A.; Seki, K.; Suzuki, A. K.; Kamata, K.; Sakata, M.; Yoshino, S.; Sagai, M.; Mori, Y. Toxicology 2002, 170, 153-161. (7) Zierau, O.; Bodinet, C.; Kolba, S.; Wulf, M.; Vollmer, G. J. Steroid Biochem. Mol. Biol. 2002, 80, 125-130. (8) Tyler, C. R.; Beresford, N.; van der Woning, M.; Sumpter, J. P.; Thorpe, K. Environ. Toxicol. Chem. 2000, 19, 801-809. (9) Tran, D. Q.; Ide, C. F.; Mclachan, J. A.; Arnold, S. F. Biochem. Biophys. Res. Commun. 1996, 229, 102-108.
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(10) Collins, B. M.; McLachlan, J. A.; Arnold, S. F. Steroid 1997, 62, 365-372. (11) Coldham, N. G.; Dave, M.; Sivapathasundaram, S; McDonnell, D. P.; Connor, C.; Sauer, M. J. Environ. Health Perspect. 1997, 105, 734-742. (12) Saito, K.; Tomigahara, Y.; Ohe, N.; Isobe, N.; Nakatsuka, I.; Kaneka, H. Toxicol Sci. 2000, 57, 54-60. (13) Hirose, T.; Morito, K.; Kizu, R.; Toriba, A.; Hayakawa, K.; Ogawa, S.; Inoue, S.; Muramatsu, M.; Masamune, Y. J. Health Sci. 2001, 47, 552-558. (14) Takeyoshi, M.; Yamasaki, K.; Sawaki, M.; Nakai, M.; Noda, S.; Takatsuki, M. Toxicol. Lett. 2002, 126, 91-98. (15) Tran, D. Q.; Jin, L.; Chen, J.; McLachlan, J. A.; Arnold, S. F. Biochem. Biophys. Res. Commun. 1997, 235, 669-674. (16) Garcia-Reyero, N.; Grau, E.; Castillo, M.; de Alda, M. J. L.; Barcelo, D.; Pina, B. Environ. Toxicol. Chem. 2001, 20, 11521158. (17) Tanghe, T.; Devriese, G.; Verstraete, W.; J. Environ. Qual. 1999, 28, 702-709. (18) Matsui, S.; Hakigami, H.; Matsuda, T.; Taniguchi, N.; Adachi, J.; Kawami, H.; Shimizu, Y. Water Sci. Technol. 2000, 42, 173179. (19) Tanaka, H.; Yakou, Y.; Takahashi, A.; Higashitani, T.; Komori, K. Water Sci. Technol. 2001, 43, 125-132. (20) Johnson, A. C.; Sumpter, J. P. Environ. Sci. Technol. 2001, 35, 4697-4703. (21) Kirk, L. A.; Tyler, C. R.; Lye, C. M.; Sumpter, J. P. Environ. Toxicol. Chem. 2002, 21, 972-979. (22) Witters, H. E.; Vangenechten, C.; Berckmans, P. Water Sci. Technol. 2001, 43, 117-123. (23) Ko¨rner, W.; Bolz, U.; Su ¨ ssmuth, W.; Hiller, G.; Schuller, W.; Hanf, V.; Hagenmaier, H. Chemosphere 2000, 40, 1131-1142. (24) Snyder, S. A.; Villeneuve, D. L.; Snyder, E. M.; Giesy, J. P. Environ. Sci. Technol. 2001, 35, 3620-3625. (25) Petrovic, M.; Sole, M.; de Alda, M. J. L.; Barcelo, D. Environ. Toxicol. Chem. 2002, 21, 2146-2156. (26) Todorov, J. R.; Elskus, A. A.; Schlenk, D.; Ferguson, P. L.; Brownawell, B. J.; McElroy, A. E. Mar. Environ. Res. 2002, 54, 691-695. (27) Sheahan, D. A.; Brighty, G. C.; Daniel, M.; Jobling, S.; Harries, J. E.; Hurst, M. R.; Kennedy, J.; Kirby, S. J.; Morris, S.; Routledge E. J.; Sumpter, J. P.; Waldock, M. J. Environ. Toxicol. Chem. 2002, 21, 515-519. (28) Rajapakse, N.; Silva, E.; Kortenkamp, A. Environ. Health Perspect. 2002, 110, 917-921. (29) Silva, E.; Rajapakse, N.; Kortenkamp, A. Environ. Sci. Technol. 2002, 36, 1751-1756.
Tao Yuan* Center for Water Research Department of Civil Engineering National University of Singapore 10 Kent Ridge Crescent Singapore 119260 ES030436P
