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Impact of Wastewater Treatment Configuration and Seasonal Conditions on Thyroid Hormone Disruption and Stress Effects in Rana catesbeiana Tailfin Pola Wojnarowicz, Olumuyiwa Ogunlaja, Chen Xia, W. J. Parker, and Caren Helbing Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 01 Nov 2013 Downloaded from http://pubs.acs.org on November 3, 2013

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Environmental Science & Technology

Impact of Wastewater Treatment Configuration and Seasonal Conditions on Thyroid Hormone Disruption and Stress Effects in Rana catesbeiana Tailfin Pola Wojnarowicz1, Olumuyiwa O. Ogunlaja2, Chen Xia2, Wayne J. Parker2, Caren C. Helbing1* 1

Department of Biochemistry & Microbiology, University of Victoria, P.O. Box 3055 Victoria, British Columbia, V8W 3P6, Canada

2

Department of Civil and Environmental Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada

KEYWORDS Wastewater treatment; Rana catesbeiana; Thyroid hormone; Endocrine disruption; Activated sludge; Biological nutrient removal; Amphibia; Stress

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ABSTRACT

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Improved endocrine disrupting compound (EDC) removal is desirable in municipal wastewater

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treatment plants (MWWTPs) although increased removal does not always translate into reduced

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biological activity. Suitable methods for determining reduction in biological activity of effluents

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are needed. In order to determine which MWWTPs are the most effective at removing EDC

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activities, we operated three configurations of pilot sized biological reactors (conventional

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activated sludge – CAS, nitrifying activated sludge – NAS, and biological nutrient removal –

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BNR) receiving the same influent under simulated winter and summer conditions. As frogs are

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model organisms for the study of thyroid hormone (TH) action, we used the North American

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species Rana catesbeiana in a cultured tadpole tailfin (C-fin) assay to compare the effluents. TH-

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responsive (thyroid hormone receptors alpha (thra) and beta (thrb)) and stress-responsive

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(superoxide dismutase, catalase, and heat shock protein 30) mRNA transcript levels were

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examined. Effluents infrequently perturbed stress-responsive transcript abundance but thra/thrb

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levels were significantly altered. In winter conditions, CAS caused frequent TH perturbations

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while BNR caused none. In summer conditions however, BNR caused substantial TH

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perturbations while CAS caused few. Our findings contrast other studies of seasonal variations of

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EDC removal and accentuate the importance of utilizing appropriate biological readouts for

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assessing EDC activities.

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Environmental Science & Technology

INTRODUCTION

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Downstream of municipal wastewater treatment plants (MWWTPs), the potential impacts of

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emerging contaminants on aquatic biota are a growing concern. MWWTPs are major point

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sources of contaminants such as pharmaceuticals and personal care products (PPCPs),

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surfactants, plasticizers, pesticides, and flame-retardants.1-3 Many of these contaminants are

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known to be endocrine disrupting compounds (EDCs).4-6 Estrogens and estrogen-active

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compounds in particular are the most heavily studied EDCs in MWWTP effluents.7 In contrast,

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very little is known regarding the efficacy of removal of thyroid hormones (THs) and TH-active

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EDCs in MWWTPs.8 In a recent study of TH-levels in Finnish waters, Svanfeldt et al.8 found

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levels of up to 22 ng/L T4 (one form of TH) in MWWTP effluent; a level that is highly

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bioactive.

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THs are important in all vertebrates to control diverse cellular processes such as cellular

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differentiation and metabolism.9 In humans, proper TH signaling is important throughout life and

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is particularly important during the perinatal period for normal growth and development of the

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central nervous system. In amphibians, THs drive the metamorphosis of a tadpole into a juvenile

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frog. The fundamental mechanisms of TH action are conserved among vertebrates. Thyroxine

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(T4) is released from the thyroid gland into circulation where it can be converted to the more

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bioactive form, T3 (3,5,3’-triiodothyronine) by deiodinase enzymes in target tissues.10,

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primarily exerts its effects by binding to nuclear thyroid hormone receptors (TRs) to mediate

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transcription of TH-responsive genes.12, 13 As TH is so crucial in all vertebrates, there is potential

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for permanent deleterious effects caused by exposure to TH-active compounds in municipal

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wastewater effluents entering receiving environments, even at parts per trillion levels.

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T3

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EDCs, in the context of wastewater effluents, pose complex toxicological challenges.

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Primarily, many EDCs are known to have non-monotonic dose response curves,14, 15 which can

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result in unexpected effects at low, environmentally relevant concentrations. Additionally,

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aquatic organisms are virtually never exposed to one single EDC. Receiving waters are a

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complex milieu of potential EDCs in dynamic flux dependent on daily influents. Because of

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these two factors, high dose lab exposures of individual contaminants of concern are of limited

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utility when extrapolating to expected effects of effluents on organisms in receiving

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environments.

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Chemical analyses as the sole endpoints determining the efficacy of emerging contaminant

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removal in MWWTPs should be treated with caution. Even in systems that have a seemingly

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high percentage of removal of EDCs, effluents are still capable of causing biological effects.16

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Additionally, comparing full scale MWWTPs has proven challenging, as effluents are dependent

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on influent composition, which fluctuates considerably between cities, seasons, and dates. In

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order to accurately compare the removal efficiencies of secondary treatment processes and to

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determine optimal operational parameters of those processes, Miège et al.7 suggested that pilot

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studies with carefully controlled influents as well as coordinated operational parameters, such as

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controlled alterations in sludge retention times (SRTs) and temperature, are necessary.

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Limited in vitro approaches to studying TH-disruption by wastewater effluent extracts have

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been reported17-20 but methods that study extract effects on protein fragments or synthetic

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reporter gene expression in non-native systems such as yeast are biologically limited with a high

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likelihood of false-negatives. A few studies have investigated the effects of wastewater effluents

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on amphibians21-23 linking effluent exposures to morphological abnormalities, alterations in

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mRNA transcript levels, and/or metamorphic rates, but such assays require large animal numbers

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Environmental Science & Technology

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and are costly and time-consuming. We recently developed the cultured tailfin (C-fin) assay24 to

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rapidly and effectively identify disruption of TH signaling and/or identify a stress response. The

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C-fin assay assesses gene transcript abundance within standardized tailfin biopsies from the

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globally-distributed North American bullfrog (Rana catesbeiana) tadpole. One tadpole yields

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multiple biopsies that are individually cultured and exposed to a variety of conditions designed to

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establish 1) TH-responsiveness on a per-animal basis, 2) the ability of effluent to affect key gene

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transcripts important in TH- and stress-related signaling, and 3) the ability of effluent to disrupt a

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TH-dependent tissue response. Therefore with multiple biological replicates, a powerful repeated

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measures approach for assessing exposure to MWWTP effluents can be taken. The C-fin

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approach has successfully been applied previously to a variety of chemical compounds,24-27 but

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application to complex mixtures has not yet been done.

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The present study is part of a larger initiative aimed at determining the efficacy of emerging

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contaminant removal within existing treatment trains relevant to Canadian operating conditions.

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Herein, we compare the effluents from three pilot secondary MWWTP process configurations

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representing three commonly-used, established technologies receiving the same municipal

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influent by investigating the biological effects of the resultant effluents on Rana catesbeiana

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tadpole tailfin tissues. Gene transcript abundance measurements using quantitative real time

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polymerase chain reaction (QPCR) were used to assess the biological impact of wastewater

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effluent exposure. Two pivotal genes, thra and thrb, that encode TH receptor alpha and TH

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receptor beta proteins, have been used as indicators of TH disruption.17, 21, 24-27 As wastewaters

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are complex mixtures of pathogens, excessive nutrients, pesticides, heavy metals,

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pharmaceuticals and personal care products, it is reasonable to expect that stress responses in

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biota of receiving environments might be significantly affected by effluent exposure. Changes in

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the abundance of heat shock protein 30 (hsp30) mRNAs are an indicator of general stress28-30

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whereas oxidative stress is frequently indicated by mRNAs encoding superoxide dismutase (sod)

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and catalase (cat).25-27, 31 The latter two stress-indicators have been used previously in effluent

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exposure studies32, 33 but rarely are these endpoints studied in non-fish species.

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MATERIALS AND METHODS

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Pilot Wastewater Treatment Plants. Three pilot wastewater treatment plants situated at the

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Environment Canada Wastewater Technology Centre (WTC) were employed for the research.

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The WTC receives authentic city of Burlington municipal wastewater via the Burlington Skyway

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Wastewater treatment plant. This wastewater is considered typical since it is primarily from

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residential sources with limited commercial, institutional, and industrial inputs. Schematics of

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the pilot plants used for the study are shown in Figure 1, the design parameters in Supplemental

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Table S1, and operating conditions in Table 1. The conventional activated sludge (CAS),

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nitrifying activated sludge (NAS), and biological nutrient removal (BNR) pilots were all fed

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from a common primary clarifier that received the raw wastewater.

The activated sludge

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reactors were segmented into six cells (60 L each) to simulate pseudo-plug flow and coarse

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bubble aerators were used for aeration and mixing (in a cyclic pattern; high air flow at 40 L/min

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and low air flow at 10 L/min) in the CAS, NAS, and the aerated zones of the BNR. An insulated

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water jacket with an automatic temperature controller was installed around the bioreactors to

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control the temperatures of the bioreactors. Di-potassium phosphate (K2HPO4) (11 g/L at the rate

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of 14.4 L/d) and sodium bicarbonate (NaHCO3) (22 g/L at the rate of 14.4L/d) were added to the

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clarified influent stream to ensure the system was not phosphorus limited and to provide a pH

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buffer for the system.

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The CAS pilot was a single sludge aerobic system operated at SRTs of 6 and 3 days (winter

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and summer respectively) to facilitate biochemical oxygen demand removal without nitrification.

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The NAS pilot was a single sludge aerobic system operated at an SRT of 20 and 10 days during

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the winter and summer conditions, respectively. These SRTs enable the proliferation of

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autotrophic nitrifying bacteria for consistent nitrification. The BNR process consisted of

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anaerobic, anoxic, and aerobic zones and was operated at an SRT of 40 and 20 days during

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winter and summer conditions, respectively. This mode of operation was designed to achieve

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nitrification, denitrification, and enhanced biological phosphorous removal. To achieve an

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appropriate range of chemical oxygen demand to phosphorus (COD/P) ratio that will support a

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healthy BNR activated sludge, the clarified influent wastewater stream to the BNR bioreactor

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was augmented with an exogenous source of readily biodegradable organic substrate (34.36 g/L

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sodium acetate at the rate of 14.4 L/d).

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The pilot plants were operated for over 365 days with consistent monitoring for over 180 days

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to ascertain stable operation in terms of biomass and effluent characteristics so as to enable

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efficient comparison among the three treatment trains. Stable plant performance was ascertained

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by monitoring the following parameters: SRT, Temperature, COD, NH3-N, total phosphorus

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(TP), soluble PO4-P, NO3-N, total suspended solids (TSS), volatile suspended solids (VSS), pH,

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mixed liquor suspended solids (MLSS), and mixed liquor volatile suspended solids (MLVSS).

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Water Sampling and Handling. Composite samples were collected from all three systems

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over four dates (November 8, November 22, December 1, and December 9, 2010 – sampling

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points 1-4) for winter conditions and three dates (June 14, June 23, and July 19, 2011 – sampling

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points 5-7) for summer conditions. The samples were immediately filtered with a 1.5 µm glass

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microfiber filter (Whatman, Toronto, ON, Canada), concentrations of ammonia, nitrite, and

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nitrate were measured as per Standard Methods34, and sent on wet ice by courier to Victoria, BC

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for C-fin assays preformed the following day. Samples were sterilized with a 0.2 µm nylon filter

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(Nalgene – Thermo Fisher, Rochester, NY, USA) before dilution and application to the organ

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cultures.

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Experimental Animals. Premetamorphic Taylor and Kollros (TK)35 stage VI–VIII Rana

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catesbeiana tadpoles were caught locally (Victoria, BC, Canada). The care and treatment of

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animals was in accordance with the guidelines of the Canadian Council on Animal Care under

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the guidance of the Animal Care Committee, University of Victoria. Animals were housed in the

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University of Victoria aquatics facility, and maintained in a 100-gallon tank with recirculated

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water at 14oC with exposure to natural daylight. Tadpoles were fed daily with spirulina (Aquatic

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ELO-Systems Inc., FL, USA).

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Organ Culture of Tailfin Biopsies. Tailfin biopsy cultures were prepared according to the

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methods described previously for the C-fin assay.24 One complete C-fin assay was performed per

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secondary wastewater treatment type per collection day. Each C-fin assay used eight tadpoles.

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Six circular 4 mm tailfin biopsies (Miltex, Plainsboro, NJ, USA) were taken from each animal in

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order to test six different treatment conditions outlined below. Biopsies were cultured in

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individual wells at 25oC in air for 48 h in 96-well multi-well culture plates (BD Falcon,

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Mississauga, ON, Canada) containing 200 µL of 1X culture medium. This medium was

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comprised of 70% strength Leibovitz’s L15 medium (Gibco – Life Technologies Inc.,

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Burlington, ON, Canada) supplemented with 10 mM HEPES pH 7.5, 50 units/ml penicillin G

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sodium, 50 ug/ml streptomycin sulfate (Gibco – Life Technologies Inc.), 50 µg/ml neomycin

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(Sigma-Aldrich, Oakville, ON, Canada), and 2 mM L-glutamine (Sigma-Aldrich).

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The six treatment conditions were divided into two sets: three treatments with 10 nM 3,5,3’-

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triiodothyronine (T3; #T2752, Sigma-Aldrich) in 400 nM NaOH, and three treatments without T3

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(400 nM NaOH only; T3-vehicle control). T3 was prepared as a 10-5 M stock in 400 µM NaOH

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and was applied at 1 µL/mL of media. Within each set, treatments were further subdivided such

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that each sterilized wastewater sample was tested in the presence or absence of T3 at 20% and

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50% serial dilutions. The final concentration of medium components was maintained at 1X. Each

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individual animal’s biopsies were exposed to the six treatment conditions in order to establish 1)

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that each animal was competent to respond to T3 alone, 2) how wastewater effluent alone could

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affect the tissues, and 3) how wastewater effluent could alter the normal T3 induced response of

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each animal. At the end of the 48 h incubation period, each biopsy was stored in 100 µL of

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RNAlater (Ambion – Life Technologies Inc., Burlington, ON, Canada) for 24-48 h at 4°C and

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then transferred to -20°C until processed for total RNA.

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Isolation of RNA and Quantitation of Gene Transcript Abundance. RNA was isolated

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using TRIzol reagent as described by the manufacturer (Invitrogen – Life Technologies Inc.,

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Burlington, ON, Canada). Mechanical disruption utilized 300 µL TRIzol reagent, a 1 mm

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diameter tungsten-carbide bead, and a safe-lock Eppendorf 0.5 ml microcentrifuge tube in a

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Retsch MM301 Mixer Mill (Fisher Scientific, Ottawa, ON, Canada) at 20 Hz two times for 3

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min, with the chambers rotated in between cycles. Twenty µg glycogen (Roche Diagnostics,

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Laval, QC, Canada) were added prior to isopropanol precipitation to maximize RNA yield.

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Isolated RNA was subsequently resuspended in 10 µL diethyl pyrocarbonate-treated (Sigma-

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Aldrich) RNase-free water and stored at -80°C.

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cDNA was synthesized from 5 µL prepared RNA (representing ~1 µg total RNA) as per

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manufacturer’s protocol using the High-Capacity cDNA Reverse Transcription Kit with RNase

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Inhibitor (Applied Biosystems – Life Technologies Inc., Burlington, ON, Canada).

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products were diluted 20-fold prior to QPCR amplification and stored at -20°C.

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cDNA

Transcript abundance of thyroid hormone receptors alpha and beta (thra and thrb), Cu/Zn

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superoxide dismutase (sod), catalase (cat), and heat shock protein 30 (hsp30) was analyzed using

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a MX3005P real-time QPCR system (Agilent Technologies Canada, Inc., Mississauga, ON)

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using primers and conditions as described in Hammond et al.36 and in Supplemental Table S2

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with the exception that eukaryotic translation elongation factor 1-alpha (eef1a) and ribosomal

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protein S10 (rps10) reactions consisted of a thermocycle program of 95°C (9 min) followed by

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40 cycles of 95°C denaturation (15 s), 60°C annealing (30 s), and 72°C elongation (30 s). The

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mRNA levels were normalized to the geometric mean of the transcript levels of ribosomal

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proteins L8 (rpl8) and rps10, and eef1a using the ∆∆Ct method.37 These three normalizer

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transcripts were deemed to be suitable normalizers using RefFinder

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(http://www.leonxie.com/referencegene.php?type=reference).

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Statistical Analysis. Statistical analyses were performed using R-studio software (R-studio

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Inc., Boston, MA, USA). The data were log-transformed and evaluated for normality using a

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Shapiro-Wilk test. Since the data were nonparametric, a Wilcoxon signed-rank test for repeated

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measures data was used. Repeated measure comparisons were as follows: first, the ability of the

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tissues to respond to T3 treatment was assessed for each animal; second, wastewater treatment

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effects were examined relative to the vehicle control (NaOH); third, alteration of the normal

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response to T3 by wastewater was assessed. All statistical analyses were performed on log2

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transformed data and were deemed significant at p