Temporal Variation in the Estrogenicity of a Sewage Treatment Plant

Oct 12, 2007 - Boulevard, Duluth, Minnesota, 55804. Received April 04, 2007. Revised manuscript received. August 31, 2007. Accepted September 05, 2007...
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Environ. Sci. Technol. 2008, 42, 3421–3427

Temporal Variation in the Estrogenicity of a Sewage Treatment Plant Effluent and Its Biological Significance ´ , * ,† DALMA MARTINOVIC JEFFREY S. DENNY,‡ PATRICIA K. SCHMIEDER,‡ GERALD T. ANKLEY,‡ AND PETER W. SORENSEN† Department of Fisheries, Wildlife, and Conservation Biology, 1980 Folwell Avenue, University of Minnesota, St. Paul, Minnesota, 55108, and U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, Minnesota, 55804

Received April 04, 2007. Revised manuscript received August 31, 2007. Accepted September 05, 2007.

Daily variation in the estrogenic activity of effluent released by a modern sewage treatment plant (STP) was measured and its effects on the physiology, behavior, and reproductive success of male fish were evaluated. As measured by an estrogen receptor binding assay, the daily estrogenic activity of this effluent was both high and extremely variable (42 ( 25.4 [mean ( SD] ng 17β-estradiol (E2) equivalents/L; n ) 18). Liver VTG mRNA expression in male fathead minnows (FHM) covaried with the binding assay estimates, suggesting that these fluctuations are biologically relevant. Tests which exposed male FHMs to either fluctuating levels of E2, a constant concentration of E2 (time-weighted to reflect average concentrations), or control (no E2) demonstrated that while the estrogenic activity of this effluent was detrimental to male spawning success, the fact that its concentration varied in a daily manner was without additional influence. The variability of the effluent’s estrogenicity suggests that studies concerned with the effects of STP effluents should collect multiple daily samples and then test them on an appropriate time-weighted basis.

Introduction Sewage treatment plants (STPs) release environmental estrogens (EEs) to aquatic ecosystems, where they can have detrimental effects on fish sexual maturation and reproduction (e.g., 1–4). Although it appears that most STP effluents have at least some estrogenic activity (2, 5, 6), the overall level of such activity and its biological significance remain poorly understood because most studies utilize temporally sparse sampling schemes and lack in vivo based quantification of estrogenicity. There is some indication that temporal fluctuations in estrogenic activity may be significant, at least on a monthly scale (7, 8). The fluctuations are a concern for * Corresponding author tel: 218-529-5115; fax: 218-529-5003; e-mail: [email protected]. † University of Minnesota. ‡ U.S. Environmental Protection Agency. 10.1021/es0708013 CCC: $40.75

Published on Web 10/12/2007

 2008 American Chemical Society

two reasons. First, existing estimates of estrogenicity of STP effluents could be misleading due to overly simplistic sampling schemes. Second, the biological effects of estrogenic effluents on fish and wildlife could be underestimated if EEs, like many other toxicants (9, 10), have augmented effects when their exposure concentrations are varied. Indeed, the only study which we know to have evaluated the effects of pulsatile EE exposure on fish (but which did not mimic natural exposure regimes), found that highly intermittent exposure to 17β-estradiol (E2) caused greater vitellogenin (VTG) induction than did exposure to an equivalent, time-adjusted continuous dose (11). Notably, in vitro studies of the effects of estrogen on cancerous cells also suggest that variable exposure regimes can be unexpectedly potent (12, 13). Clearly, to understand the ecological risks posed by STP effluents, fine-scale variation in their estrogenicities and the biological effects of variable exposure regimes need to be understood. In this study we examined temporal variations in the estrogenic activity of a large, modern STP that processes municipal and industrial (pulp mill) wastewater, both of which are potential sources of EEs. In situations such as this, where unknown EEs may be present, biological analyses that quantify estrogenic activity of the whole effluent without knowledge of specific EEs can be superior to chemical analyses (14). Accordingly, we measured the estrogenic activity of the effluent using both an in vitro and two in vivo assays to provide an integrative measure of the estrogenic nature and potential of the effluent. Specifically, we used an in vitro competitive estrogen receptor (ER) binding assay (15) to provide a rapid and precise daily measure of effluent estrogenicity over a several week period. Recognizing that this assay could not distinguish between ER agonists and antagonists and might not accurately reflect the biological availability of estrogens, and thereby could overestimate estrogenic activity of the effluent, we concurrently measured in vivo production of VTG mRNA and/or protein in a limited number of male fish. These in vivo end points reflect the resultant agonistic activity of ER ligands, and do not ascribe estrogenic activity to compounds that bind to ER without activating the receptor. As such, in vivo transcriptional and translational assays usually represent more conservative measurements of estrogenicity than receptor binding studies, and thus yield lower estrogenicity estimates. Another advantage of in vivo measurements is that they account for processes of metabolic transformation (16, 17). In a final component of this study we used a model estrogen (E2) to determine the effects of different exposure regimes on male reproductive biology and success in a competitive spawning assay which we previously found to be highly sensitive and predictive of population-level responses (4). The overarching objective of the present study was to determine whether fluctuating exposure regimens (similar to those encountered in the wild) exert effects on fish behavior and reproduction comparable to constant regimens commonly used for laboratory assessment of EE effects.

Materials and Methods Experimental Design. The Western Lake Superior Sanitary District’s (WLSSD) wastewater treatment facility in Duluth (MN) was used for this study. This large modern plant processes both domestic sewage and paper mill wastewater (see study site description in the Supporting Information). Daily fluctuations in the estrogenic activity of the effluent were evaluated using an in vitro competitive binding assay for a three-week period. During the same period several VOL. 42, NO. 9, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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groups of male fathead minnows (FHMs) were simultaneously exposed to the effluent to assess estrogenic activity in vivo (liver VTG mRNA expression and circulating levels of VTG protein). Results of the in vitro and in vivo assays (results were expressed as estrogen equivalents relative to the E2 response (EEQs)) were then compared to confirm the biological relevance of the first measure. Once an assessment of the estrogenic activity of the effluent had been completed, we addressed the relevance of these fluctuations by exposing male FHMs to E2, matching the EEQs measured in the effluent. One group of fish was exposed to fluctuating levels of E2, which mimicked those measured in the effluent, while a second group was exposed to a constant concentration of E2 that represented a time-weighted average of the respective fluctuating dose. After 21 d of exposure, effects on gonadosomatic index (GSI), circulating VTG, reproductive behavior, and reproductive fitness of the fish were assessed using a competitive spawning assay (4). The FHM used for this study were bred and reared at the University of Minnesota, St. Paul, MN. At the time of testing, fish were 6–8 months old and sexually mature. Experiment 1A. In vitro Assessment of the Estrogenic Activity of the Effluent. Effluent collections and FHM exposures were conducted at the WLLSD facility. Effluent samples were collected daily for 18 d from August 9 to August 26, 2003. Daily composite samples (6-L) were generated by continuously pumping effluent into sampling tanks over a 6-h period each day between 1100 and 1700 h. To evaluate hourly variations in estrogenic activity an additional set of 6-L grab samples of effluent was collected every 2 h between 1000 h and 2000 h on July 25, 2004. During this period, a 36-L grab sample was also collected to estimate measurement error due to sample processing and interassay variability. This particular sample was divided into six 6-L subsamples, to three of which 50 ng of E2/L was added, while the other three samples were used as controls (i.e., no E2 added). All effluent samples were extracted using C18 solid-phase extraction columns (Waters Corp., MA) by passing 500 mL of each sample through 3-mL columns at a rate of 5 mL/min. Columns were eluted with 6 mL of 100% methanol, and the extracts were stored at -20 °C. An independent test using 3H-E2 found extraction efficiency to be 97% ( 8; n ) 3. The estrogenic activity of the effluent was measured using an established rainbow trout estrogen receptor (rtER) in vitro binding assay (rtERA) (15). The rtERA measurements for effluent samples were performed in duplicate on five different occasions. Briefly, extracts were concentrated by drying them under a stream of nitrogen at 37 °C to a volume of 100–200 µL on the morning of analysis. A range of dilutions was then prepared by diluting the extracts with methanol. Effluent samples were analyzed in duplicate at five different dilutions to generate competitive binding curves. Competitive binding curves and IC50 values (the concentration that caused a 50% inhibition in binding of 3H-E2) for E2 standards and effluent samples were generated using nonlinear regression and a one-site competition equation (Prism 3.0, GraphPad Software, San Diego, CA). For each effluent curve, the resulting IC50 represented the concentration at which the effluent sample had the same estrogenic activity as the E2 standard at its IC50. To calculate effluent EEQs (ng/L) we divided the IC50 for E2 by the IC50 of the effluent. To determine assayrelated measurement error, the standard deviation of the estrogenic activity of samples collected over the 18 d was calculated and compared to the standard deviation of unspiked and E2-spiked samples using an F-test. Differences between daily and hourly variation were compared using F-tests. An F-test was also used to determine if hourly variations were significantly different from variations associated with measurement error. Results were considered significant at p < 0.05. 3422

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Experiment 1B. In vivo Assessment of the Estrogenic Activity of the Effluent. To determine whether the compounds that bound to the rtER were bioavailable and functioning as ER agonists, parallel in vivo exposures were conducted in which we measured expression of liver VTG mRNA or plasma concentrations of VTG protein. Mature male FHM were exposed for either 24 h or 7 d, to either the WLSSD effluent or a range of E2 concentrations. Effluent exposures were conducted at the WLSSD facility at the same time as the daily effluent collection was being conducted in 2003. For VTG mRNA-based estimates of estrogenic activity, six tanks (one male per tank) were filled with either 10 L of well (control) water or 10 L of the composite effluent sample. The effluent used for the VTG mRNA in vivo tests was collected in the same manner as effluent used in the rtER binding analyses. Fish were exposed to control water or effluent (25 °C) for 24 h on three randomly selected days. For VTG proteinbased estimates of estrogenic activity, we exposed two groups of six males to the effluent for 7 d (the exposure regimen was longer because a reliable measurement of VTG protein is difficult to achieve in 24 h). Each individual was held in a separate exposure tank and exposure tanks were supplied with constant flow of aerated 25 °C effluent (200 mL/min; dissolved oxygen >6.0 mg/L; mean un-ionized ammonia concentrations were 0.09 ( 0.06 (SE) mg/L, well below toxic levels (18)). Fish were fed brine shrimp daily. No overt effects on health or behavior of fish were observed. A final experiment was conducted to develop concentration–response curves for calculating VTG mRNA- and VTG protein-based EEQs. To accomplish this, we exposed groups of six males (in separate 10-L tanks) to either control (0.0) or 12.5, 25.0, or 50.0 ng E2/L for either 24 h (for VTG mRNA) or for 7 d (for VTG protein). Exposures were conducted at the University of Minnesota where a peristaltic pump delivered a continuous flow (1.0 mL/min) of E2 stock solution (containing 0.005% ethanol) into E2-exposure aquaria and 0.005% ethanol solution into control aquaria. The E2exposure and control aquaria received a constant flow of well water (200 mL/min) and supplemental aeration. Temperatures were maintained at 25 °C under a16:8 L:D photoperiod and the animals were fed brine shrimp daily. Upon completing exposures, fish were euthanized with 2-phenoxy-ethanol following protocols approved by the University of Minnesota Institutional Animal Care and Use Committee. Blood was collected from the caudal sinus using heparinized microcapillary tubes, and plasma was isolated by centrifugation and stored at -20 °C for VTG protein analyses using an enzyme-linked immunoassay (ELISA) with a homologous FHM antibody (19). For mRNA analyses, livers were collected, homogenized with TRI reagent (Sigma, St. Louis, MO), and total mRNA was isolated using the protocol supplied by the manufacturer (Sigma). Isolated mRNA was stored in ethanol at -80 °C until analyzed. Liver VTG mRNA was quantified using a VTG mRNA hybridization protection assay (Molecular Light Technology, Cardiff, UK) (20) (see Supporting Information for details). Effluent EEQs were calculated by comparing liver VTG mRNA abundance and plasma VTG protein induction values measured in effluent-exposed fish to those of a matching set of fish exposed to E2. Standard curves for liver VTG mRNA and circulating protein VTG induced by E2 exposure were generated using simple linear regression (Prism 3.0, GraphPad Software, San Diego, CA); EEQs were calculated from the resultant equation. To determine the proportion of rtERbased estrogenic activity explained by agonistic activity, FHM liver VTG mRNA EEQs were divided by rtER binding assay EEQs for the three corresponding sampling days. To evaluate circulating VTG protein EEQ estimates, we first calculated mean estrogenic activity for each 7-d exposure period by averaging daily binding assay estimates. VTG protein-based

EEQs were then divided by the mean binding assay EEQs for the corresponding 7-d sampling period. ANOVA was used to assess if effluent exposure induced significant induction of liver VTG mRNA or VTG protein in males, and whether it varied among three sampling dates. Experiment 2. Determining the Reproductive Consequences of Fluctuating versus Constant Exposure. Male fathead minnows were exposed to two fluctuating concentrations of E2: one which mimicked the EEQs measured in the effluent using the binding assay (Experiment 2A; a “high” estimate of estrogenicity), and the other which mimicked the values of VTG mRNA analysis, (Experiment 2B; a “low”, conservative estimate of estrogenicity). At the same time, independent groups of male fish were exposed to one of two time-weighted constant levels of E2 and a carrier control. After exposures, VTG and GSI were quantified and behavioral/ reproductive performance was evaluated using competitive spawning assay (4). This assay pairs male fish and makes them compete for access to a spawning habitat, a realistic scenario in the wild. Here, we compared the competitive abilities of fish that were exposed to fluctuating E2 or no E2, fluctuating or constant levels of E2, and constant levels of E2 or no E2. For these exposures, groups of 15 male FHMs were randomly distributed among 12 80-L exposure aquaria (for a total of four replicates per treatment). After a 7-d acclimation period, fish were exposed to well water (control), to constant, or to fluctuating concentrations of E2 for 21 d. Basic test conditions were the same as those described above in experiment 1B. Two experiments were conducted: one based on EEQ values from the rtER binding data (Experiment 2A), and the other based on the VTG mRNA EEQs (Experiment 2B). In Experiment 2A the nominal constant concentration was 40 ng/L, while the fluctuating exposure regime had a nominal mean of 40 ng/L (SD ) 25 ng/L) and concentrations changed daily (see Figure S1 in the Supporting Information). In Experiment 2B the nominal constant test concentration was 13 ng/L while the fluctuating exposure regime had a nominal mean of 13 ng/L (SD ) 8.5 ng/L) and changed daily (Figure S1). Concentrations of E2 selected for Experiment 2B were approximately 67% lower than those in Experiment 2A, based on findings from Experiment 1B (Figure S1). The control fish in both experiments were exposed to well water that contained the same final concentration of ethanol (carrier solvent) as the E2 treatments. Concentrations of E2 in the control and exposure tanks were measured on seven randomly selected dates using a commercially available E2 ELISA (Cayman Chemicals, Ann Arbor, MI). The relationship between nominal and measured concentrations was evaluated using simple linear regression. After 21 d of exposure, we sampled blood and gonads of 10 males from each treatment for VTG and GSI measurements while the other fish were used for behavioral assays (see below). Blood collection procedures and VTG protein analyses were identical to those in Experiment 1 while GSI was calculated by expressing gonad wet weight as a percentage of the total body wet weight. We used a competitive behavioral assay to assess the effects of EEs on male reproductive fitness (4). For this assay, two males with different exposure histories were marked with latex paint, then placed into the same tank with two females and a single nest where they had to compete for spawning opportunities. We tested the following scenarios for both high and low EEQs (Experiments 2A, 2B): (1) a control male vs. a male exposed to constant levels of E2; (2) a control male vs. a male exposed to fluctuating levels of E2; and (3) a male exposed to constant vs. a male exposed to fluctuating levels of E2. In each case, competitive assays continued for 5 d, during which we recorded the identities of the nest-holder(s). After 5 d we collected, counted, and hatched the eggs to evaluate reproductive success. Because

FIGURE 1. (A) Daily fluctuation in the estrogenic potential of a sewage treatment plant effluent estimated by a rainbow trout receptor binding assay (rtERA) (hatched) (Days 1–18) and by a VTG mRNA (solid) (Days 1, 8, 11). The relationship between rtERA and VTG mRNA estrogen equivalent (EEQ) estimates is presented in the upper right corner. (B) Hourly fluctuation in the estrogenicity of the effluent on one sampling date. Columns represent estrogen equivalents (ng/L) derived from EC50 values for effluent samples vs. a positive control (17β-estradiol). our previous studies have demonstrated that among fish tested under these conditions only nest-holders attract females and fertilize their eggs, paternity was assigned based on the identities of the nest holders (which we knew not to change readily (4)). For both experiments, differences in plasma VTG concentrations were assessed using Kruskal–Wallis ANOVAs, followed by post hoc Dunn’s test. Differences in GSI values between control and constant versus fluctuating-exposed males were tested using ANOVA. To determine if there was a difference in daily nest-holding scores we counted the number of control, constant-, and fluctuating-exposed males that held nests on each day. Differences in the daily nestholding values for each of three male combinations were compared using Fisher’s Exact Test. Finally, differences in the cumulative number of hatched larvae sired by males from the control, constant, and fluctuating treatments were assessed with t tests. All tests were two-tailed, and differences were considered significant at p < 0.05.

Results Experiment 1A. In vitro Assessment of the Estrogenic Activity of the Effluent. The rtERA demonstrated that WLSSD effluent contained compounds that bind to ERs in a competitive manner. Complete binding displacement curves were obtained for all effluent samples (i.e., >80% displacement of 3H-E2). Assuming that all chemicals measured by the binding assay were ER agonists, the daily estrogenic activity of WLSSD effluent (n ) 18) was both high (mean ) 42 ng EEQs/L) and variable (SD ) 25.4; range 10.7–93.8 ng EEQs/L) (Figure 1). The daily change in estrogenic potential ranged from a minimum of 2% dayto-day change to a maximum of 622%. Daily variation in estrogenic activity was significantly higher than variation due to assay measurement error, which was low for both unmodified effluent samples (SD ) 4.8 ng EEQs/L, n ) 3) and E2-enriched effluent samples (SD ) 3.4 ng EEQs/L, n ) 3). Although the estrogenicities of the 2-h composite samples varied over the course of a day (SD ) 11.3 ng EEQs/L, n ) VOL. 42, NO. 9, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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5), the variation was not significantly different from the variation associated with the measurement error (Figure 1). Experiment 1B. In vivo Assessment of Estrogenic Activity of Effluent Using VTG Induction. Exposure of male FHMs to E2 resulted in a concentration-dependent expression of VTG mRNA in the liver after 24 h (Figure S2). The VTG mRNA concentrations measured in males exposed to effluent on 3 different days were not significantly different (Figure S2). The in vivo VTG mRNA-derived daily estrogenic activity estimates (Day 1 ) 8.59 ( 3.24; Day 8 ) 13.9 ( 8.62; Day 11 ) 9.6 ( 4.43 ng EEQs/L) were on average 67.33% lower (SD ) 4.25%) than estimates derived from the rtER binding assay. Exposure to E2 in the laboratory also resulted in concentration-dependent VTG protein production (Figure S2). The VTG levels in fish exposed to effluent for 7 d were significantly higher than those of control fish (Figure S2). When the VTG-based EEQs were compared to the mean rtERbased EEQs for corresponding 7-d exposure periods, they were consistently lower (42.8% on average) (Days 8–14 ) 15.3 ( 1.49; Days 11–17 ) 20.88 ( 3.11 ng EEQs/L). Experiment 2. Reproductive Effects of Fluctuating vs. Constant Exposure. There was a significant positive correlation between the nominal and measured E2 concentrations in the 21-d test (R2 ) 0.99); however, delivered concentrations were consistently lower than the nominal concentration, ranging between 65 and 70% of target concentrations (Figure S3). Exposure to both constant and fluctuating concentrations of E2 induced a significant (p < 0.01) elevation in plasma VTG concentrations compared to controls in both experiments (Figure S4). However, there were no significant differences in VTG concentrations between males subjected to a constant versus fluctuating exposure in either experiment. Males from control, constant, and fluctuating treatments did not exhibit significant differences in the GSI in either experiment (data not shown). In Experiment 2A, exposure to 40 ng E2/L reduced the ability of males from both constant and fluctuating treatments to acquire nests when competing with control males (Figure 2, Table 1). As a result, control males had much greater reproductive success (more eggs) (Table 1). When placed into direct competition with each other, males from the constant versus fluctuating E2 treatments acquired nests at similar rates and had comparable reproductive success (Figure 2, Table 1). In Experiment 2B, exposure to the lower level of E2 did not appear to strongly influence the ability of males exposed to either constant or fluctuating concentrations of E2 to obtain nests in the presence of control males (Table 1, Figure S5). Also, these males reproduced as successfully as control males (Table 1). When tested in direct competition against each other, constant- and fluctuatingE2 exposed males acquired the nests equally well (Table 1, Figure S5), and had similar reproductive success (Table 1).

Discussion This study demonstrates that the estrogenic activity of a typical STP effluent can vary substantially on a daily basis and should be carefully considered in STP sampling strategies. However, despite the high amplitude of these fluctuations, they do not appear to complicate integrated measures of fitness such as behavior and reproductive success when tested in the laboratory. Based on our EEQ estimates, the daily estrogenic activity of the WLSSD effluent ranged from concentrations with no expected effects (∼5 ng/L) to concentrations which can impair reproductive behavior, fertility, and development of fish (4, 21–23). Hourly fluctuation was not significantly higher than assay measurement error (p ) 0.069); statistical analyses suggest that we could have dramatically increased the power to detect differences (from 43% to over 90%) if we had used sample size comparable to that used for evaluations of daily fluctuations. Although this 3424

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FIGURE 2. Daily nest-holding success for males exposed to control, constant, and fluctuating dose of 17β-estradiol at an average value of 40 ng/L (Experiment 2A). Males were paired in the following competitive scenarios: (A) Control (white) vs. Constant (hatched); (B) Control (white) vs. Fluctuating (hatched); (C) Constant (white) vs. Fluctuating (hatched). Significant differences are marked with an asterisk (*). Data indicate the percent of males from each treatment that became nestholders when they had to compete for limited number of nests. Each scenario was replicated 10 times. suggests that hourly variation may also have been significant, further studies are needed to confirm this possibility. Our estimates of estrogenicity based on in vivo VTG mRNA assays tracked the patterns of variation measured by the rtER binding assay, suggesting that ER agonists in the effluent are bioavailable and likely responsible for the observed fluctuations. Finally, our results suggest that studies of the effects of STP effluents on fish should be careful to use appropriate (integrative) sampling schemes to evaluate EE release and their biological effects. Although estrogenic activity indicated by the rtER assay was higher than that observed in vivo, together they suggest that the actual estrogenicity of the effluent ranged from 4 to 33 ng EEQ/L. Differences in the magnitude of estrogenic activity estimates between in vitro and in vivo assays are not unusual (24–26). One cause of discrepancies between in vitro and in vivo measurements could have been a difference in the binding properties of FHM ERs versus rainbow trout ERs. Previous studies have demonstrated that relative binding affinities for many estrogens are similar for both species, but in some cases (EE2, nonylphenol) they can differ up to 10-fold (27). Other reasons for lower in vivo activity of the effluent could include a lesser comparative bioavailability of com-

TABLE 1. Reproductive Success and Nest-Holding Ability of Males Exposed To Control, Fluctuating, or Constant Concentrations of 17β-Estradiol When Tested in Combination with Control Fish or versus One Anothera Scenario 1. Control vs. Constant

Scenario 2. Control vs. Fluctuating

Scenario 3. Constant vs. Fluctuating

(n ) 10)

(n ) 10)

(n ) 10)

control experiment 2A nest-holding reproduction experiment 2B nest-holding reproduction

constant

control

fluctuating

constant

fluctuating

70* 50.5*(0, 83)

10 0 (0, 0)

80* 31.5*(0, 65)

20 0 (0, 0)

50 0 (0, 41)

50 6.5 (0, 97)

40 17.5 (0, 45)

50 18.5 (0, 102)

30 0 (0, 41)

60 16 (0, 65)

40 5 (0, 41)

60 12 (0, 54)

a Nest-holding (percent of males from each treatment that were holding nests on the last day of competitive behavioral test) and reproductive success (reproduction; median number of fertilized eggs with upper and lower quartiles) are shown. Differences (p < 0.05) are marked with an asterisk (*). Comparisons were made within each scenario only.

pounds that bind ERs, in vivo metabolism (deactivation) of possible ER agonists, and/or the presence of compounds that bind to ERs but do not elicit estrogenic activity downstream from receptor (i.e., antagonists (25)). Furthermore, lower in vivo activity could have been a result of the nonendocrine active components of the effluent reducing the ability of the liver to express VTG mRNA and protein. Whatever the reason for the higher in vitro indication of estrogenic potential, the correlation between in vitro and in vivo data suggests it was reasonable to adjust the former downwards by a consistent percent to yield daily estimates. These adjusted estimates indicate that the estrogenic activity of the WLSSD effluent ranges from approximately 4 to 33 ng EEQs/L, comparable to that reported for other North American and European STP effluents (5, 25, 28, 29). In our experiments, the rtER binding assay proved suitable for initial screening of the fine temporal variation in estrogenic activity of effluents. Although the VTG mRNA hybridization protection assay was apparently not sensitive enough to detect daily variation in estrogenic activity, it did provide a reliable estimate of in vivo estrogenic activity. It should be noted that the inability of the VTG mRNA assay to detect statistically significant differences in estrogenic activity of the effluent on the three selected dates could have been due to the fact that estrogenic activities of the effluent on those dates were very similar (22, 23, and 31 ng EEQs/L based on rtER binding). While we cannot fully explain the causes of the observed fluctuations in estrogenicity, the WLSSD operators have noted that volumes of influents from municipal sources and the paper mill (both potential sources of estrogenic chemicals) varied substantially on a daily basis (ca. 12–26%, respectively) over the course of our study (Tim Tuominen, WLSSD, personal communication). Furthermore, the input of organic matter (as measured by biological oxygen demand (BOD)) and total suspended solids (TSS) was highly variable in both influents as well as in final STP effluent (Figure S6, data provided by Tim Tuominen, WLSSD). Because estrogenic chemicals may bind to particulate matter, fluctuations in the TSS could affect estrogenic activity from a bioavailability perspective. Furthermore, efficiency of the BOD (ca. 86–95%) and TSS removal (ca. 96–99%) was also variable during the study period, suggesting that the processing efficiency of the STP could contribute to fluctuations. However, the fluctuations observed during the study period were well within levels normally observed in this and other STP effluents. There are other potential sources of the fluctuations in estrogenicity/ estrogenic chemicals, but without identification of chemicals responsible for estrogenicity and extensive monitoring it is not possible to ascertain root causes of the phenomenon. The variation we observed in estrogenic potential of the WLSSD effluent served as a basis for investigating the effects

of fluctuating vs. continuous exposure of fish exposed to a model estrogen, E2. Significantly, no differences between the two exposure scenarios were observed in VTG, GSI, behavior, and reproductive success of male FHMs. The only possible trend we observed was in Experiment 2A where there was a tendency toward increased VTG production in fish exposed to fluctuating E2 versus the constant exposure (P ) 0.12). Notably, Panter et al. (11) observed a similar phenomenon in VTG induction in some of their treatments, but it is difficult to compare their results to ours because the exposure regimens were very different. Specifically, we employed a fluctuating exposure with at least some E2 always present, while Panter et al. (11) used a regime of exposure to high E2 concentrations, interspersed with no exposure to the estrogen. Increased VTG concentrations under an intermittent scenario could be a result of differential kinetics of VTG mRNA production. Studies in birds and amphibians have documented that an initial exposure to estrogen can result in a comparatively slow rate of VTG mRNA production, which increases in magnitude with subsequent exposures (30, 31). In this study we saw no differences between exposure to constant and fluctuating levels of E2 on GSI. While our inability to detect differences between fluctuating and constant exposure could have been a result of the fact that the tested concentrations elicited maximal responses, this is unlikely. Korte et al. (19) exposed fish to a high dose of E2 by intraperitoneal injection (5 mg/kg), and reported plasma concentrations of VTG up to 20 times higher than those measured in the present study. Furthermore, longer-term exposures (21 d) to the same effluent (4) and a comparable concentration of E2 (32) also resulted in higher VTG levels in males than observed in the present study. Finally, in contrast to a finding of no effect on GSI in the present study (data not shown), pronounced reductions of GSI in FHM were observed upon exposure to a higher concentration of E2 (11), again suggesting that the concentrations used in the current experiments did not elicit maximal responses. Our data on male behavior and competitive reproductive success are especially relevant to understanding effects of the effluent on wild fishes which must normally compete for mates (4). In Experiment 2A, where male FHMs were exposed to 40 ( 25 ng/L E2 (mean ( SD) for 21 d, we observed an impaired ability of E2-exposed males to compete with control males for the nests and reproduce whether exposure regime was constant or variable. However, when constant- and fluctuating-exposed males were placed into direct competition with each other, they exhibited the same level of behavioral and reproductive impairment confirming that the dosing regimens exerted comparable adverse effects. The results of Experiment 2B, in which FHMs were exposed to lower E2 concentrations (13 ( 8.5 ng/L E2 [mean ( SD]) confirmed the finding that effects of fluctuating versus VOL. 42, NO. 9, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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constant E2 exposure were similar, in that neither treatment exerted adverse effects on behavior and reproduction under any scenario. The results of the present study directly support our earlier work (4) in which constant exposure to either intermediate levels of E2 (∼30 ng E2/L) or to an estrogenic STP effluent led to similar impairment of reproductive behavior and fitness of exposed vs. control males after 21 d. Furthermore, past in situ exposures to the WLSSD effluent have also resulted in a comparable induction of VTG and reduced competitive behavioral fitness in male FHMs (32), suggesting that this effluent has the potential to adversely affect fish living in its vicinity. Notably, all of these assays have also shown behavioral deficits associated with these exposures typically immeasurable by standard noncompetitive reproductive assays. However, in order not to overestimate or underestimate the magnitude of effects on fish populations it is important to first characterize typical ambient exposure scenarios, taking into account developmental stages of exposure, duration of exposure, and effluent dilution. Although our findings suggest that, at least within the range of variation observed in the WLSSD effluent, a constant E2 exposure regimen will result in the same biological effects as a fluctuating regimen, exposure to a wider range of concentrations could have yielded different results for several reasons. Exposure to very high concentrations of EEs even for a very brief time could exert toxic effects and reduce reproductive success. Additionally, exposure to EEs which accumulate more rapidly that E2 may have different effects (9). Further, if we had exposed fish at different developmental stages (e.g., undergoing sex determination and differentiation) we might also have observed more pronounced and dissimilar effects. In conclusion, our studies demonstrate that, over a 21-d period, the estrogenic potential of a relatively representative STP effluent can vary significantly on a daily basis. These variations were large enough that very different conclusions regarding possible adverse effects of this plant might be made depending on when a sample(s) happened to be collected. This highlights the need for rigorous temporal sampling to fully characterize the potential risk of EEs in effluents. Within the range of concentrations we tested, fish seemed to react to estrogen exposure in an integrated manner; effects could be predicted by averaging the concentrations to which the fish were exposed. Although we did not observe any differences between constant and fluctuating exposure to E2, it is important to examine this phenomenon at a wider range of concentrations, employing additional exposure scenarios (e.g., duration of exposure, exposures to diluted effluent) reflective of what might be found in the environment, and examining a variety of end points in organisms at different stages of development.

Acknowledgments We thank Dr. Tala Henry, Joe Korte, Mike Simone, and Molecular Light Technologies. We also thank Joe Stepun, Tim Tuominen, and the WLSSD staff and management. This work is the result of research sponsored by the Minnesota Sea Grant College Program supported by the NOAA office of Sea Grant, United States Department of Commerce, under grant NA03OAR4170048. This paper is journal reprint No. 535 of the Minnesota Sea Grant College Program.

Supporting Information Available Detailed descriptions of the STP, bioassays, exposure regimens, and additional VTG, VTG mRNA, and behavioral data. This material is available free of charge via the Internet at http://pubs.acs.org. 3426

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