Environ. Sci. Technol. 2010, 44, 4348–4354
Effects of Advanced Treatments of Wastewater Effluents on Estrogenic and Reproductive Health Impacts in Fish A M Y L . F I L B Y , * ,† J A N I C E A . S H E A R S , † BRIANE E. DRAGE,‡ JOHN H. CHURCHLEY,§ AND CHARLES R. TYLER† School of Biosciences, University of Exeter, Exeter EX4 4PS, United Kingdom, AquaRite Ltd., Worcester WR5 3QG, United Kingdom, and Severn Trent Water Ltd., Coventry CV3 6SD, United Kingdom
Received February 23, 2010. Revised manuscript received April 19, 2010. Accepted April 26, 2010.
Whether the implementation of additional treatments for the removal of estrogens from wastewater treatment works (WwTWs) effluents will eliminate their feminizing effects in exposed wildlife has yet to be established, and this information is crucial for future decisions on investment into WwTWs. Here, granular activated carbon (GAC), ozone (O3), and chlorine dioxide (ClO2) were investigated for their effectiveness in reducing steroidal estrogen levels in a WwTW effluent and assessments made on the associated estrogenic and reproductive responses in fathead minnows (Pimephales promelas) exposed for 21 days. All treatments reduced the estrogenicity of the standard-treated (STD) effluent, but with different efficacies; ranging between 70-100% for total estrogenicity and 53-100% for individual steroid estrogens. In fish exposed to the GAC- and ClO2- (but not O3-) treated effluents, there was no induction of plasma vitellogenin (VTG) or reduction in the weight of the fatpad, a secondary sex character in males, as occurred for fish exposed to STD effluent. This finding suggests likely benefits of employing these treatment processes for the reproductive health in wild fish populations living in rivers receiving WwTW discharges. Exposure of pair-breeding minnows to the GAC-treated effluent, however, resulted in a similar inhibition of egg production to that occurring for exposure to the STD effluent (34-40%). These data, together with a lack of effect on egg production of the estrogen, ethinylestradiol (10 ng/L), alone, suggest that chemical/physical properties of the effluents rather than their estrogenicity were responsible for the reproductive effect and that these factor(s) were not remediated for through GAC treatment. Collectively, our findings illustrate the importance of assessing integrative biological responses, rather than biomarkers alone, in the assessment and improvement of WwTW technologies for the protection of wild fish populations. * Corresponding author phone: +44 (0)1392 263752; fax: +44 (0)1392 263700; e-mail:
[email protected]. † University of Exeter. ‡ AquaRite Ltd. § Severn Trent Water Ltd. 4348
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 11, 2010
Introduction Estrogenic chemicals in effluents discharged from wastewater treatment works (WwTWs) are a principal cause of feminized responses observed in wild fish populations worldwide, including intersexuality and induction of the female-specific protein vitellogenin (VTG) (1, 2). Importantly, reduced fertility has been shown in some of the more severely affected fish (3), highlighting the potential for population-level effects. Moreover, laboratory exposures of fish to the individual estrogens responsible for the majority of the estrogenicity of WwTW effluents (particularly the synthetic steroid 17Rethinylestradiol (EE2)) has highlighted other effects which could potentially impact on the genetic diversity of populations (4-6). Conventional WwTW processes have variable performance in removing estrogens, and other endocrine disrupting chemicals (EDCs), and the possibility of installing additional treatments to ensure a more complete and consistent removal, minimizing their entry into water courses, is currently receiving considerable attention in both the scientific and the regulatory communities (7, 8). An example of this is the £40 M U.K. Endocrine Disruption Demonstration Programme (EDDP), running from 2005-2010, which is assessing the efficacy of various technologies for the removal of environmental estrogens (9). Advanced treatment technologies offering particular promise for estrogen removal include adsorption onto granular activated carbon (GAC) and chemical transformation using ozonation (O3) and chlorination (10-13). The significant investment and operational costs of these technologies, together with other concerns such as the formation of toxic byproducts (7, 14), have likely been factors preventing their widespread application to date. However, a lack of knowledge as to whether these advanced technologies will actually reduce the effects of effluents in exposed fish is also apparent. To date, most studies in this area have used analytical chemistry only to measure estrogen removal and/or based their conclusions on in vitro responses (e.g., 15). Few studies have integrated chemical and in vivo biological testing and, where this has been done, responses in fish have been restricted to biomarkers of exposure. In vitro methods do not account for differences in bioconcentration/pharmacokinetics, and are thus not necessarily predictive of in vivo potencies (16), and biomarker measures (17, 18) do not necessarily inform directly on fish health. Integrated biological responses in intact fish are essential for determining whether the investment required to incorporate advanced treatments into WwTW protocols would be justified. Here, studying a U.K. WwTW effluent, we directly compared the effects of three different advanced treatments, GAC, O3, and chlorine dioxide (ClO2), on estrogen reduction (via analytical chemistry, in vitro analyses, and in vivo biological responses in exposed fish). Our in vivo analyses included biomarker responses (VTG induction, male secondary sex characters (SSCs), gonad development) and, for the first time, reproduction, as a population relevant end point. For this work, two fish test systems were employed, namely, the fathead minnow (Pimephales promelas) screening and pair-breeding assays to maximize the sensitivity for detecting estrogenic stimulation and effects on reproductive output, respectively (19, 20).
Methods Test Organisms. Fathead minnows were obtained as juveniles from breeding stocks at Brunel University, Uxbridge, U.K., 10.1021/es100602e
2010 American Chemical Society
Published on Web 05/05/2010
TABLE 1. Estrogenicity of the Effluent and Removal Rates of This Activity with the Different Advanced Treatmentsa E2EQ
removal
E1
removal
E2
removal
EE2
removal
effluent
(ng/L)
(%)
(ng/L)
(%)
(ng/L)
(%)
(ng/L)
(%)
STD GAC O3 ClO2
1.48 ( 0.87 0.21 ( 0.11 0.18 ( 0.08
