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Available online at www.sciencedirect.com

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Changes in hormone and stress-inducing activities of municipal wastewater in a conventional activated sludge wastewater treatment plant Pola Wojnarowicz a, Wenbo Yang b, Hongde Zhou b, Wayne J. Parker c, Caren C. Helbing a,* a

Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada School of Engineering, University of Guelph, Guelph, Ontario N1G 2W1, Canada c Department of Civil and Environmental Engineering, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada b

article info

abstract

Article history:

Conventional municipal wastewater treatment plants do not efficiently remove contami-

Received 8 March 2014

nants of emerging concern, and so are primary sources for contaminant release into the

Received in revised form

aquatic environment. Although these contaminants are present in effluents at ng-mg/L

11 August 2014

concentrations (i.e. microcontaminants), many compounds can act as endocrine disrupt-

Accepted 12 August 2014

ing compounds or stress-inducing agents at these levels. Chemical fate analyses indicate

Available online 3 September 2014

that additional levels of wastewater treatment reduce but do not always completely remove all microcontaminants. The removal of microcontaminants from wastewater does

Keywords:

not necessarily correspond to a reduction in biological activity, as contaminant metabolites

Thyroid hormone

or byproducts may still be biologically active. To evaluate the efficacy of conventional

Endocrine disruption

municipal wastewater treatment plants to remove biological activity, we examined the

Amphibian

performance of a full scale conventional activated sludge municipal wastewater treatment

C-fin

plant located in Guelph, Ontario, Canada. We assessed reductions in levels of conventional

Microcontaminant

wastewater parameters and thyroid hormone disrupting and stress-inducing activities in wastewater at three phases along the treatment train using a C-fin assay. Wastewater treatment was effective at reducing total suspended solids, chemical and biochemical oxygen demand, and stress-inducing bioactivity. However, only minimal reduction was observed in thyroid hormone disrupting activities. The present study underscores the importance of examining multiple chemical and biological endpoints in evaluating and monitoring the effectiveness of wastewater treatment for removal of microcontaminants. © 2014 Elsevier Ltd. All rights reserved.

Abbreviations: BOD5, biological oxygen demand; C-fin, cultured; cat, catalase; COD, chemical oxygen demand; EC, emerging contaminant; EDC, endocrine disrupting compound; eef1a, eukaryotic translation elongation factor 1 alpha; hsp30, heat shock protein 30; MWWTP, municipal wastewater treatment plant; ON, Ontario; PPCP, pharmaceutical and personal care product; PPT, parts per trillion; qPCR, quantitative real time polymerase chain reaction; rpl8, ribosomal protein L8; rps10, ribosomal protein S10; sod, superoxide dismutase; T3, triiodothyronine; TH, thyroid hormone; thra, thyroid hormone receptor alpha; thrb, thyroid hormone receptor beta; TP, total phosphorous; TSS, total suspended solids. * Corresponding author. Tel.: þ1 250 721 6146; fax: þ1 250 721 8855. E-mail address: [email protected] (C.C. Helbing). http://dx.doi.org/10.1016/j.watres.2014.08.035 0043-1354/© 2014 Elsevier Ltd. All rights reserved.

266

1.

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Introduction

Municipal wastewater treatment plants (MWWTPs) are a source of emerging contaminants (ECs) discharging into receiving waters. Conventional treatment at MWWTPs typically consists of three levels: a preliminary screen of wastewater influent, a primary to remove solids, and secondary biological treatment to biodegrade unwanted compounds (Onesios et al., 2008; Ternes et al., 2004). Although significant improvements to MWWTP technologies have enhanced the quality of effluents, anthropogenic contaminants persist in wastewater and these “microcontaminants” have been detected at nanogram or microgram per liter (ng-mg/L) concentrations in surface waters impacted by MWWTP discharges (Blair et al., 2013; Fent et al., 2006; Holeton et al., 2011; Kolpin et al., 2002). Many of the microcontaminants originate as pharmaceuticals and personal care products (PPCPs), plastics, and flame-retardants (Kolpin et al., 2002; Ratola et al., 2012). These microcontaminants are chemicals of emerging concern, not only because they are often recalcitrant to conventional MWWTP systems, but also because many are known endocrine disrupting compounds (EDCs). Much EDC research has concentrated on estrogenic and androgenic compounds and only in recent years has disruption of other crucial hormone systems such as thyroid hormone (TH) received attention in wastewater effluent and receiving waters (Castillo et al., 2013; Ishihara et al., 2009; Jugan et al., 2009; Kusk et al., 2011; Metcalfe et al., 2013; Murata and Yamauchi, 2008; Searcy et al., 2012; Sowers et al., 2009; Svanfelt et al., 2010; Wojnarowicz et al., 2013). EDCs are of particular concern because they are bioactive at low, environmentally relevant concentrations and possess non-monotonic doseeresponse curves (Kendig et al., 2010; Vandenberg et al., 2012). Even at parts per trillion (i.e. ng/L) levels, EDCs have the potential to cause potent long-term effects on aquatic organisms (Palace et al., 2006, Kidd et al. 2007). EDCs in municipal wastewater effluents in particular pose additional complexity to environmental risk management as organisms in receiving water bodies are exposed to more than one compound at a time. Wastewaters are complex mixtures that contain a variety of components that can act as EDCs individually or together in ways that are not necessarily predicted by additive or synergistic effects models (Crofton et al., 2005; Kortenkamp, 2007; Rajapakse et al., 2004). Chemical analyses to determine the efficacy of removal and fates of select EDCs in activated sludge systems indicate that a small portion of highly lipophilic compounds are removed via sorption during primary treatment, while secondary treatment is the most effective process for removing microcontaminants by biodegrading the compounds (Carballa et al., 2004; Onesios et al., 2008; Oulton et al., 2010; Ternes et al., 2004). Biological degradation however has the potential to create more bioactive metabolites in final effluents (Joss et al., 2004) while preliminary treatments have been known to increase estrogenic activity via deconjugation events (D'Ascenzo et al., 2003). In addition, although high efficacies for microcontaminant removal are found in some MWWTPs,

efficient removal does not guarantee reduced biological effects (Osachoff et al., 2014). The present study is part of a larger initiative aimed at determining the efficacy of removal of contaminants of emerging concern within existing treatment trains relevant to Canadian operating conditions. The objective of the present study was to examine the performance of a full scale activated sludge municipal wastewater treatment plant operating in Guelph, Ontario, Canada for reducing the levels of conventional wastewater parameters, while also evaluating, at three stages along the treatment train, changes in biological activity related to endocrine and stress responses. Biological activity was assessed using a cultured tail fin biopsy assay (i.e. C-fin assay) designed to evaluate molecular markers for thyroid hormone and stress signaling pathways.

2.

Materials & methods

2.1.

MWWTP sampling

Wastewater samples were collected as 24 h composites from the MWWTP serving a population of ~130,000 in the City of Guelph, ON, Canada on three dates of October 2011, and August and November, 2012. The plant treats a mixture of domestic, commercial, and industrial wastewater. A schematic of the full-scale MWWTP is shown in Fig. 1. The operating conditions were obtained from the MWWTP and are summarized in Table 1. In short, this plant has four parallel treatment trains of which one was selected for intensive study. This train included primary sedimentation and secondary nitrifying conventional activated sludge treatment units. Water samples collected from the points along the treatment train indicated in Fig. 1 were immediately filtered through a 1.5 mm glass microfiber filter (Whatman, Toronto, ON, Canada). Filtered samples were then sent overnight on ice for subsequent analyses.

2.2.

Experimental animals

All care and treatment of animals used in the tadpole tailfin biopsy culture (i.e. C-fin) assays was in accordance with the guidelines of the Canadian Council on Animal Care under the guidance of the Animal Care Committee, University of Victoria. Rana catesbeiana (TK stage VI-VIII; premetamorphic) (Taylor and Kollros, 1946) tadpoles were caught locally (Victoria BC, Canada) and housed in the University of Victoria aquatics facility. Tadpoles were kept in a recirculated-water, 100-gallon tank at 14  C with exposure to natural daylight. Animals were fed daily with spirulina (Aquatic ELO-Systems Inc., FL, USA).

2.3.

Tadpole tailfin biopsy culture (C-fin) assay

C-fin assays were performed according to methods previously described by Wojnarowicz et al. (2013). Water samples were sterilized with a 0.2 mm nylon filter (Nalgene e Thermo Fisher, Rochester, NY, USA) before application to the organ cultures. Each sterilized wastewater sample was tested in a C-fin assay in the presence or absence of thyroid hormone at 20% and 50%

w a t e r r e s e a r c h 6 6 ( 2 0 1 4 ) 2 6 5 e2 7 2

267

Fig. 1 e Process flow schematic of the full-scale MWWTP treatment train monitored in the present study. Stars indicate where composite water samples were collected along the treatment train.

serial dilutions. These dilutions were used as it was determined through separate work to be non-lethal to intact animals (Ings et al., 2011). Specifically, six 4 mm tailfin biopsies (Miltex, Plainsboro, NJ, USA) were taken from each animal (n ¼ 8 animals/C-fin assay) and individually cultured in medium for 48 h to test a total of six treatment conditions divided into two groups: with 10 nM 3,5,30 -triiodothyronine (T3 SigmaeAldrich) in 400 nM NaOH and three treatments without T3 (400 nM NaOH only; T3-vehicle control). In this way the C-fin assesses: 1) each individual animal's response to the endogenous ligand T3, 2) the ability of wastewater to alter the natural state of the premetamorphic tadpole tissue, and 3) the ability of wastewater to alter the response of the tadpole tissue to a T3 challenge.

2.4. Isolation of RNA and quantitation of gene transcript abundance C-fin biopsy tissues were processed for the isolation of RNA and cDNA synthesis according to methods described by Wojnarowicz et al. (2013). In brief, RNA was isolated using TRIzol reagent (Invitrogen e Life Technologies Inc., Burlington, ON, Canada) as described by the manufacturer after mechanical disruption of tissues using a Retsch MM301 Mixer Mill (Fisher Scientific, Ottawa, ON, Canada) with a 1 mm diameter tungsten-carbide bead in a 0.5 ml microcentrifuge tube. RNA yield was maximized by addition of 20 mg glycogen (Roche Diagnostics, Laval, QC, Canada) prior to isopropanol precipitation. RNA was resuspended in 10 ml diethyl pyrocarbonate (DEPC)-treated (SigmaeAldrich) RNase-free water and stored at 80  C. Total RNA concentration and quality were determined using a Nanodrop ND-1000 (Thermo Fisher Scientific Inc., Nepean, ON, Canada). The High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems e Life Technologies Inc., Burlington, ON, Canada) was

Table 1 e Mean operating conditions for the full-scale nitrifying conventional activated sludge MWWTP serving the city of Guelph, Ontario, Canada during the months when samples of wastewater were collected. HRT ¼ Hydraulic retention time; SRT ¼ solids retention time. Sampling period

HRT (h)

SRT (d)

Average flow (106 L/d)

Temperature  ( C)

October 2011 August 2012 November 2012

N/A 5.6 4.9

12 12 12

8.10 7.39 8.43

18.7 22 17

N/A, not available.

used to synthesize cDNA from 5 mL prepared RNA (representing ~1 mg total RNA). cDNA was diluted 20-fold and stored at 20  C prior to quantitative real-time polymerase chain reaction (qPCR) amplification. Transcript abundance of thyroid hormone receptors alpha and beta (thra and thrb), superoxide dismutase (sod), catalase (cat), heat shock protein 30 (hsp30), eukaryotic translation elongation factor 1-alpha (eef1a), ribosomal proteins s10 (rps10) and ribosomal protein L8 (rpl8) was analyzed using a MX3005P real-time qPCR system (Agilent Technologies Canada, Inc., Mississauga, ON) using primers and conditions as described in Wojnarowicz et al. (2013) with the exception that eef1a and rps10 primers were run with SyBr green. The 15 mL eef1a and rps10 reactions contained 0.01% Tween 20, 0.8% glycerol, 10 mM TriseHCl (pH 8.3 at 20  C), 50 mM KCl, 3 mM MgCl2, 40,000-fold dilution of SYBR Green I (Molecular Probes, Invitrogen), 200 mM dNTPs, 69.4 nM ROX reference dye (Invitrogen), one unit of Immolase DNA polymerase (Bioline), 2 mL of diluted cDNA, and 5 pmol of each primer. The eef1a primer sequences are as described in Wojnarowicz et al. (2013) and rps10 primer sequences were: 50 -TTTGCYTGGCGKCACTTTT-30 (TAX7up) and 50 -ARCRGCACTGCGYCTGTA-30 (TAX7dn) to produce an amplicon of 213. The thermocycle program for eef1a and rps10 consisted of an enzyme activation step at 95  C (9 min), followed by 40 cycles of 95  C denaturation (15 s), 60  C annealing (30 s), and 72  C elongation (30 s). The mRNA levels were normalized to the geometric mean of the transcript levels of rps10, rpl8, and eef1a using the DDCt method (Livak and Schmittgen, 2001). All amplification reactions satisfied the DDCt criteria for efficiency as the slope of the line of log2 dilutions versus the DCt values (between the gene of interest and the normalizer gene rpl8) were between 0.1 and 0.1. The three normalizer transcripts were deemed to be suitable normalizers using RefFinder (http://www.leonxie. com/referencegene.php?type¼reference), a web-based program that uses a combination of software including geNorm (Vandesompele et al., 2002), Normfinder (Andersen et al., 2004), Bestkeeper (Pfaffl et al., 2004), and comparative DCt (Silver et al., 2006) to assess normalizer suitability.

2.5.

Statistical analysis

R-studio software (R-studio Inc., Boston, MA, USA) was used to perform a Wilcoxon signed-rank test for non-parametric, repeated measures for the C-fin data. We first determined that the tissues from each animal were responding as expected to T3 treatment by examining the responses of thra and thrb transcripts. Then, the effect of wastewater exposure was assessed relative to the vehicle control (NaOH), followed by a

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determination of whether or not wastewater exposure caused an alteration of the normal response to T3. All statistical analyses were performed on log2 transformed data and were deemed significant at p < 0.05.

3.

Results and discussion

3.1.

Conventional wastewater quality parameters

The performance of the MWWTP was initially examined with respect to conventional wastewater contaminants to establish its efficiency with respect to BOD/COD removal and nitrification. Table 2 presents the average wastewater quality parameters obtained from the MWWTP that were observed in the full-scale plant for approximately one month periods prior to sampling campaigns. The wastewater characteristics and performance of the MWWTP were stable across sampling events and consistent good BOD/COD removal was attained. In addition, the treatment train was nitrifying to achieve low final NH4eN concentrations and elevated NO3eN concentrations in the effluents.

3.2. Thyroid hormone responsive gene transcripts in Cfin assays Because of the repeated measures design of the C-fin assay, along with the internal positive controls in each exposure, it is possible to distinguish which animals respond to TH and to what extent. As previously observed, gene transcript levels for both thyroid hormone receptor alpha (thra) and thyroid hormone receptor beta (thrb) (Fig. 2A and B, respectively) were significantly increased (3- and ~30-fold, respectively) by addition of 10 nM T3 alone to establish the ability of each individual animal to respond to the hormone and, therefore, to establish competence of the animals to respond to compounds that may alter the inherent THresponse (Hinther et al., 2010a,b, 2011; 2012, Wojnarowicz et al., 2013). In the context of the present study, thra transcript levels were affected by exposure to municipal wastewater at all stages of the treatment train whereas thrb transcript levels were not. Exposure to wastewaters attenuated the thra response to the internal 10 nM T3 control by ~20e50% in each of the treatment train units. Secondary treatment however,

was only able to remove ~20% of the TH-disruptive activity detected in the influent by the thra transcript. Relatively few studies have investigated TH-activity of municipal wastewaters (Castillo et al., 2013; Inoue et al., 2011; Ishihara et al., 2009; Jugan et al., 2009; Metcalfe et al., 2013; Shi et al., 2012) and even fewer have investigated the effects on thra transcript levels specifically (Wojnarowicz et al., 2013). When Ings et al. (2011) investigated the same full-scale MWWTP as the present study using caged rainbow trout downstream of an additional tertiary treatment step, liver thrb transcript levels were found to be increased by up to 15-fold compared to an upstream reference site. The function of the proteins encoded by thrb and thra is overlapping yet distinctive and it has been suggested that thra may play a greater role in establishing tadpole competence to respond to TH (Buchholz et al., 2006; Hollar et al., 2011). Although the implications of the gene-specific TH-perturbations in bullfrog tailfin are unclear, the present data along with report on the effects of effluents from the same MWWTP on rainbow trout (Ings et al., 2011), indicate that there is potential for thyroid hormone disruption in municipal wastewater effluents treated using this typical nitrifying activated sludge system.

3.3.

Stress responsive gene transcripts in C-fin assays

Previous studies have measured oxidative stress endpoints as indicators of effluent exposure in fish and mussels (Carney  et al., 2006, 2011; Ings et al., 2011; Almroth et al., 2008; Gagne Sturve et al., 2008; Veldhoen et al., 2011). In rainbow trout caged downstream of an MWWTP in Sweden, oxidative damage and stress were identified by altered mRNA and protein levels of genes involved in antioxidant response (Carney Almroth et al., 2008). Although previous studies have implicated the importance of reactive oxygen species in THmediated amphibian metamorphosis (Johnson et al., 2013; Menon and Rozman, 2007), little is known about oxidative stress in amphibian species exposed to MWWTP effluents. In the present study, wastewater samples collected from every treatment train unit caused no major effects on either of the two “classical” oxidative stress-responsive gene transcripts (sod and cat e Fig. 3A and B, respectively). The results indicate that both untreated and treated wastewaters do not cause an oxidative stress response in R. catesbeiana tailfin tissues in the same way that these responses have been observed in other  et al., 2006). aquatic organisms, such as rainbow trout (Gagne

Table 2 e Average wastewater quality 30-days prior to sampling in the MWWTP for the city of Guelph, ON, Canada. Influent ¼ untreated wastewater; Primary ¼ treated wastewater after primary treatment; Secondary ¼ treated wastewater after secondary treatment. Date

Aug-12

Nov-12

Treatment unit

pH

Influent Primary Secondary Influent Primary Secondary

7.8 7.7 8.0 7.8 7.0 7.8

N/A, not available.

TSS

COD

BOD5

NH4eN

NO2eN

NO3eN

TP

(mg/L)

(mg/L)

(mg/L)

(mg/L)

(mg/L)

(mg/L)

(mg/L)

345 79 8 308 58 6

456 268 44 544 281 30

202 110 N/A 240 82 N/A

29 N/A 0.3 26 N/A 0.1

N/A N/A 0.2 N/A N/A 0.4

N/A N/A 29 N/A N/A 30

7.5 N/A 0.4 6.4 N/A 0.2

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Fig. 2 e Transcript levels in Rana catesbeiana tadpole tailfin biopsies in treatments with municipal wastewater influent (i.e. untreated), primary treated wastewater, and secondary treated wastewater as determined by qPCR for: A) thyroid hormone receptor alpha (thra) and B) thyroid hormone receptor beta (thrb). Tissues from eight biological replicates were cultured for 48 h in the indicated wastewaters under two test conditions: with T3-vehicle control (400 nM NaOH e white boxplots), and with 10 nM T3 in 400 nM NaOH (gray boxplots). The bevel indicates 20% and 50% concentrations of wastewater added in each of the two test conditions. Relative transcript abundance is presented in comparison to the geometric mean of rpl8, eef1a, and rps10 transcripts. The median of the data is represented as a solid black line within each box, while the rest of the box indicates the 25th and 75th percentiles, and the whiskers denote the remaining 50% of the data. An asterisk indicates outlier values and an open circle indicates extreme values. “a”: significance of 10 nM T3 (T3only control) to 400 nM NaOH (T3-vehicle control) (p < 0.05; Wilcoxon signed-rank test). “b”: significance relative to nowastewater-added control (“0”) within a test condition (p < 0.05; Wilcoxon signed-rank test).

The indicator of general cellular stress (hsp30) however, was perturbed by exposure of fin biopsies to wastewaters (Fig. 4). In both test conditions with and without T3, hsp30 transcripts showed a notable increase due to exposure to

269

Fig. 3 e Transcript levels in Rana catesbeiana tadpole tailfin biopsies in treatments with municipal wastewater influent (i.e. untreated), primary treated wastewater, and secondary treated wastewater as determined by qPCR for: A) superoxide dismutase (sod) and B) catalase (cat). Refer to the Fig. 2 legend for details.

untreated and primary treated wastewater. However, this increase was not evident upon exposure to secondary treated effluents. There was a ~85% reduction of perturbations to hsp30 after secondary treatment indicating effective removal of this biological activity. The heat shock protein (HSP) family functions under cellular stress conditions to bind proteins and protect them from misfolding (Heikkila, 2010). These cellular stress conditions occur during the metamorphic process in amphibian tissues and hsp30 specifically is known to be TH-responsive (Helbing et al., 1996). Some members of the HSP family have been implicated in altered energy metabolism in fish exposed to MWWTP effluents (Ings et al., 2012, 2011). Chemical fate studies of microcontaminants in MWWTPs indicate that primary treatment is expected to have minimal impact on their removal while secondary treatment removes the majority of these contaminants via biodegradation (Oulton et al., 2010; Ternes et al., 2004). Secondary treatment of the wastewaters significantly abated the hsp30 effects induced exposure to

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Hammond, A. Carew, T. Brown, and S. Maher for their helpful discussions and technical expertise. This work was funded by the Canadian Municipal Water Management Research Consortium through the Canadian Water Network.

references

Fig. 4 e Transcript levels in Rana catesbeiana tadpole tailfin biopsies in treatments with municipal wastewater influent (i.e. untreated), primary treated wastewater, and secondary treated wastewater as determined by qPCR for heat shock protein 30 (hsp30). Refer to the Fig. 2 legend for details.

untreated or partially treated (i.e. primary) wastewaters, indicating that secondary treatment is important for reducing the biological effects relating to general stress.

4.

Conclusions

There was good reduction of all conventional wastewater parameters and stress-related biological responses from wastewater in the full-scale nitrifying conventional activated sludge plant monitored in the present study. Nevertheless, the ability to remove specific endocrine disrupting biological activities was not necessarily linked to removal of conventional parameters. The present data stress the need to include tests for determining biological effects to evaluate the potential impacts of contaminants of emerging concern in municipal wastewaters.

Acknowledgments We thank Drs. T. Sullivan, C. Metcalfe, O. Ogunlaja, T. Sultana, and M.E. Hoque for their insights and valuable discussions. We would also like to thank Dr. N. Veldhoen, T.-A. Ichu, S.A.

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Changes in hormone and stress-inducing activities of municipal wastewater in a conventional activated sludge wastewater treatment plant.

Conventional municipal wastewater treatment plants do not efficiently remove contaminants of emerging concern, and so are primary sources for contamin...
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