Environ. Sci. Technol. 2010, 44, 1137–1143
Profiles and Some Initial Identifications of (Anti)Androgenic Compounds in Fish Exposed to Wastewater Treatment Works Effluents E L I Z A B E T H M . H I L L , * ,† KERRY L. EVANS,† JULIA HORWOOD,† PAWEL ROSTKOWSKI,† FRANCIS OLUMIDE OLADAPO,† RICHARD GIBSON,† JANICE A. SHEARS,‡ AND CHARLES R. TYLER‡ Department of Biology and Environmental Science, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QJ, United Kingdom, and School of Biosciences, University of Exeter, Exeter, EX4 4PS, United Kingdom
Received June 22, 2009. Revised manuscript received December 17, 2009. Accepted December 21, 2009.
Exposure of fish to wastewater treatment works (WwTWs) effluents can result in reproductive anomalies consistent with exposure to estrogenic compounds. However, UK WwTWs effluentsalsocontaincompoundswithandrogenreceptoractivities which may contribute to reproductive dysfunction in fish. A toxicity identification and evaluation (TIE) approach was used to profile (anti)androgenic compounds in bile of fish exposed to two WwTWs effluents. Extracts of bile from exposed fish and effluent were fractionated by liquid chromatography and tested for (anti)androgenic activity using a yeast androgen receptor transcription screen (YAS). A number of bile fractions contained (anti)androgenic activity unique to the effluentexposed fish. Some of these fractions contained di(chloromethyl)anthracene or dichlorophene, and these contaminants showed antagonistic activity in the YAS when tested as pure compounds. No androgenic activity was detected in the effluents, but TIE analysis of bile revealed a number of androgenic fractions which contained testosterone metabolites that were unique to effluent-exposed fish. This is the first work reported on the nature of some of the (anti)androgenic compounds that bioaccumulate in fish from WwTWs effluents and indicates that other contaminants, besides estrogenic substances, need to be considered for their potential to contribute to the disruption of reproductive system of fish in UK waters.
Introduction Wildlife and humans are exposed to complex mixtures of environmental chemicals. Some of them can interact with the endocrine system and so disrupt the normal functioning of reproductive, neurological, immune, or developmental systems of animals (1). Some aquatic animals, such as fish, are highly susceptible to the effects of endocrine disrupting * Corresponding author phone: 44-01273-67-8382; fax: 44-01273877586; e-mail:
[email protected]. † University of Sussex. ‡ University of Exeter. 10.1021/es901837n
2010 American Chemical Society
Published on Web 01/07/2010
chemicals as they are continually exposed to these contaminants through discharges of wastewater treatment works (WwTWs) effluents, and these exposures can be lifelong. In the UK, discharges of treated effluents are often poorly diluted in the receiving waters and this has been shown to be associated with widespread sexual disruption and feminization of wild fish inhabiting downstream sites (2). Feminized males exhibit histological changes in the gonads characterized by the presence of developing eggs (known as intersex) and/ or an ovarian cavity and a reduction of milt volume and sperm density (3), and there is concern that a high incidence of intersexuality, together with other environmental stressors, may have population level consequences (4). Many of the pathologies of feminized fish have been associated with exposure to estrogenic chemicals present in WwTWs effluents (5) and some of the phenotypes have been induced in the laboratory through controlled exposures to some of the identified estrogens (e.g., (6)). However, experimental evidence with rodents suggests that the presence of male reproductive disorders in the human population, including declining sperm counts, could be caused by exposure to contaminants that block the action of androgens in the developing fetus (7, 8). Indeed, exposure of fish to model antiandrogens has been shown to result in reduced sperm counts, suppression of secondary sexually features and courtship behavior, and disruption of a number of molecular pathways associated with biosynthesis and action of sex steroids (9-11). A survey of UK WwTWs has revealed that the majority of the effluents sampled contain antiandrogenic as well as estrogenic activity (12). Modeling of the association between the antiandrogenic and estrogenic activities of 51 UK WwTW effluents and the feminization of wild fish (roach, Rutilus rutilus) in downstream waters has demonstrated that feminization of fish was best correlated with its predicted exposure to both antiandrogens and estrogens or to antiandrogens alone (13). Collectively, these studies highlight the importance of identifying the (currently unknown) structures of antiandrogenic chemicals in WwTW effluents to determine their contribution to the observed reproductive dysfunction in freshwater and coastal fish populations. Many biologically active contaminants, such as the synthetic estrogen 17R-ethinylestradiol (E2), can be present in WwTW effluents at subnanogram per liter concentrations. This poses difficulties in the extraction of enough sample to allow for purification and identification of steroid receptor active contaminants. However, a wide range of bioavailabile xenobiotics have been shown to bioconcentrate in fish bile, either as the parent compound or as glucuronide/sulfate conjugates, thus allowing the analysis of contaminants at concentrations tens of thousands greater than in the effluent itself (e.g., 14, 15). This approach has been utilized effectively to identify hormone replacement pharmaceuticals present in WwTW effluents that contribute to the estrogenic burden of contaminated fish (16). In this study we exposed juvenile trout to two WwTW effluents, or reference river or tap water conditions, and compared the concentrations of sex steroids receptor activities in the effluents and in the bile of effluent-exposed or reference fish. We used a toxicity identification and evaluation (TIE) method to compare the profiles of (anti)androgens in bile and effluents. Samples were fractionated using highperformance liquid chromatography (HPLC) and (anti)androgenic activity was detected using a yeast recombinant androgen receptor transcription screen (YAS). The identity VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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of androgen receptor active chemicals was investigated using gas chromatography-mass spectrometry (GC-MS).
Materials and Methods WwTW Sites. Two WwTWs, A and B (see Table S1 in Supporting Information for details), were chosen for this study, each of which received primarily domestic waste and had previously been shown to produce effluents containing estrogenic activity. The estrogenic activity of WwTW A effluent has been reported at levels between 2 and 18 ng estradiol equivalents (E2eq)/L and the antiandrogenic activity between 348 and 382 µg flutamide equivalents (FEq)/L (12, 16). The population equivalent of WwTW B was 3 fold higher than WwTW A, and the estrogenic activity of the effluent has been reported previously at between 26 and 99 ng E2eq/L (16), however there was no information on levels of antiandrogenic activity for this effluent. Grab samples (1-2.5 L) of final effluent or river water were collected in solvent-rinsed glass containers. Methanol (final concentration 3%) and acetic acid (final concentration 1%) were added to the samples which were stored overnight at 4 °C prior to solid-phase extraction (SPE) using Oasis HLB cartridges (200 mg, Waters, Massachusetts). Effluent extracts were sequentially eluted from SPE with 5 mL each of methanol, ethyl acetate, and hexane. The eluates were combined, the solvents were evaporated under vacuum, and the samples were redissolved in ethanol (1 mL) for receptor activity assays, or in methanol-water (1:1) for HPLC fractionation. Fish Exposure. Female immature rainbow trout (Oncorhynchus mykiss) were obtained from local fish farms. Mean ( SEM length and weight of fish pre-exposure were 25.0 ( 0.3 cm and 200.0 ( 8.4 g, respectively, for the WwTW A experiment, and 22.8 ( 0.2 cm and 132.9 ( 3.6 g, respectively, for WwTW B experiment. Prior to the exposures, fish were anesthetized, tagged, and a blood sample taken to measure baseline concentrations of plasma vitellogenin (VTG), a biomarker of estrogen exposure. This baseline (time 0) concentration was compared to that measured after exposure in the control and effluent groups simply to assess the relative biological estrogenic potency of the effluents for these studies that were primarily focused on assessments on (anti)androgenic activities. In WwTW A experiment, trout were either caged in one tank within the effluent discharge (19 fish) or kept in a tank with flow-through tap water (18 fish) at 15 °C for 10 days. In the WwTW B exposure study, trout were exposed in a single tank (1 m3, flow rate of 10 L/min) to either undiluted effluent (29 fish) or to charcoal-filtered river water abstracted upstream of the effluent discharge point (30 fish). After 10 days, 16 fish from both the effluent and control tank were sacrificed. The remaining 13 fish in the effluent exposure were transferred to another tank containing charcoal-filtered river water to investigate the effect of depuration. Of these fish, 6 were harvested after 4 days of depuration (day 14 of the study), and 7 were harvested after 11 days depuration (day 21 of the study). A similar number of control fish held in river water were also harvested at the same time points (14 and 21 days of the study period). Fish were fed daily on a commercial trout food, but food was withheld 3 days prior to harvesting to maximize bile production. At the end of the exposure periods, fish were anesthetized, sampled for blood, sacrificed, and the bile sacs were removed. Fulton’s condition factor (K ) body weight (g) × 100/total length cm3), a measure of body form, and the hepatosomatic index (HSI) were calculated for individual fish. Heparintreated blood samples were centrifuged at 3000g and the plasma VTG concentrations were quantified using a previously validated rainbow trout ELISA (17). 1138
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Preparation of Bile Samples. Metabolites in bile samples were deconjugated and extracted by Oasis HLB 60-mg cartridges according to Gibson et al. (18). Cartridges were conditioned with 5 mL of methanol followed by 5 mL of acetic acid (1% solution). Bile samples (10-100 µL of bile) were applied to the cartridges, which were washed with water (2 mL) and organics were eluted with methanol (5 mL), followed by 3 mL of ethyl acetate and 3 mL of hexane. The eluents were combined and the solvents were removed under vacuum and reconstituted in either 1 mL of ethanol for analysis in steroid-receptor bioassays, or methanol/water (2:1) (100 µL) for HPLC. HPLC Fractionation. Samples were fractionated using a C18 Nova Pak column (4 µm particle size, 4.6 × 250 mm) with water (0.2% acetic acid) and acetonitrile at a starting ratio of 69:31. Samples were separated at room temperature (flow rate 1.0 mL/min) with a linear gradient program of 0 min (69:31), 35 min (65:35), 50 min (0:100) holding for up to 25 min. Retention times of standards were 17β-estradiol (E2) 27-29 min, 17R-ethinylestradiol (EE2) 37-39 min, estrone (E1) 41-43 min, 4-technical nonylphenol (4-NP) 55-56 min, 11-ketotestosterone (11-KT) 10-11 min, and testosterone (T2) 32-34 min. HPLC fractions were collected every minute for analysis in receptor assays. The identity of chemicals in fractions with (anti)androgenic activity was investigated after further purification by HPLC, and analysis by time-of-flight MS and GC-MS (see Supporting Information pp S2-S3 for details). Steroid Receptor Transcription Screens. (Anti)estrogenic or (anti)androgenic activity of bile or effluent samples were quantified using recombinant yeast screens that contain either the human estrogen (ERR) (YES) or androgen receptor (YAS) and that have been validated previously for a range of estrogenic or androgenic contaminants (19, 20). Samples (20 µL) were serially diluted in ethanol, and the solvent was evaporated to dryness before addition of culture media. Samples with estrogenic activity were quantified as E2 equivalents (E2Eq), and for androgenic activity as dihydrotestosterone, (DEq) equivalents. To test for receptor antagonist activity, the relevant agonist (either E2 or D) was added to the yeast medium at a concentration giving a 65-80% submaximal response of the assay. Samples were quantified as hydroxytamoxifen equivalents (HEq) for antiestrogenic activity, or flutamide equivalents (FEq) for antiandrogenic activity. Samples showing toxicity which resulted in poor yeast growth (monitored at 620 nm) were not quantified. Samples showing receptor antagonist activity were checked for inhibition of the reporter gene product β-galactosidase by incubation with a solution of commercial enzyme from Escherichia coli (Grade VI lyophilized powder, 0.0352 units in 100 µL of yeast cell media). The limits of detection (LOD) of receptor activity measurements in the bile, water, or effluent samples were calculated from the EC50 values of the standards used to calibrate the screen, the volume of sample extracted, and the concentration factor of the sample extract.
Results Fish Condition, Hepatosomatic Index, and Vitellogenin Synthesis during Exposure Experiments. There was no significant change in the condition factor of the fish exposed to either WwTW A or B at the end of the exposure period (Tables S2 and S3 in the Supporting Information). Fish exposed to WwTW A, but not WwTW B, exhibited an apparent small increase in the HSI from a mean ( SEM of 0.91 ( 0.02 to 1.11 ( 0.05 (Table S2). This may reflect increased VTG synthesis in the liver as a result of exposure of the juvenile trout to estrogens present in the effluent (21), or it may be a result of inflammation from uptake of toxic components in the effluent. In the WwTW A exposure study, pairwise
TABLE 1. Comparison of (Anti)Estrogenic and (Anti)Androgenic Activities in Bile of Juvenile Trout Exposed to Either Tap Water or WwTWs Effluents for 10 Daysa sample WwTW A study extracts of WwTW A effluent bile from reference fish exposed to tap water bile from fish exposed to WwTW A effluent WwTW B study extracts of reference river water extracts of WwTW B effluent bile from reference fish exposed to river water bile from fish exposed to WwTW B effluent
estrogenic activity E2Eq 11.3 ( 1.4 ng/L
antiestrogenic activity HEq
androgenic activity DEq
-
-
19.5 ( 2.2 ng/mL
150 ( 70 µg/mL
235 ( 20 ng/mL
320 ( 150µg/mL
0.57 ( 0.04 ng/L 15.2 ( 1.2 ng/L
antiandrogenic activity FEq 1392 ( 39 µg/L
