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1 School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada; 2 Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada; 3 Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada; 4 Department of
Biological Science, University of Lethbridge, Lethbridge, AB, Canada; 5 Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada; 6 Biology Department, University of Waterloo, Watereloo, ON; 7 INRS-ETE et UQTR, Quebec, QC
• There has been increasing concern regarding municipal wastewater effluents (MWWEs) as a major source for endocrine disrupting chemicals (EDCs) in surface waters.
• EDCs have been shown to have the potential to adversely affect the endocrine system of man and wildlife, resulting in impacts on reproduction, growth, and/or development.
• Conventional wastewater treatment technologies are frequently incomplete or inefficient at removing EDCs such as pharmaceuticals, naturally occurring hormones and other toxicants from wastewater.
• Although Canada has initiated first steps with the aim to establish standardized testing and monitoring criteria for EDC’s in the environment, our understanding of the contribution of effluents
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• from wastewater treatment plants (WWTP’s) to environmental endocrine disruption in Canadian. surface waters is incomplete at best.
The
effectiveness and efficiency of WWTPs across Canada to remove
EDCs, and to characterize the contribution of MWWEs to emerging contaminant loads in receiving surface waters.
Characterize properties. of this study was to investigate the
(anti-)estrogenic, (anti-)androgenic from selected WWTPs in Ontario, Quebec and Saskatchewan. and steroidogenesis disruption potentials of influents and effluents
• Determine the efficiency of different WWTPs with different treatment steps to remove chemicals with endocrine disrupting
Investigate factors such as season and temperature on the efficiency to remove EDCs from raw sewage.
• Effluent and influent samples were/will be collected from selected WWTPs during winter, spring, summer and fall 2014/2015 (Map 1).
• Samples were/will be filtered using glass microfiber filters 0.6µm to remove particulates (Fig 1).
• Samples undergo Solid Phase Extraction (SPE) to retain hormones, pharmaceuticals and other EDCs using Oasis mixed cation exchange (MCX) and Oasis hydrophobic lipophilic balanced (HLB) cartridges
(Fig 2), resulting in a 5,000x concentrate (Fig 3).
Fig 1.
Particulate Filtration
Fig 2. SPE - Eluding
Table 1.
In vitro test systems for the characterization of endocrine potentials of WWTP effluents and influents.
Effect Type
Cytotoxicity
( Mosman et al. 1983 )
Cell Line/Test
MTT (T47D, MDA, H295R)
(Anti-)Estrogenicity
( Wilson et al. 2004) T47D-Kbluc
(Anti-)Androgenicity
( Wilson et al. 2002) MDA-KB2
Map 1 – Locations of wastewater treatment plants (WWTPs) in Quebec, Ontario and Saskatchewan, and the population (Pop) they are serving.
Disruption of
Steroidogenesis
( Hecker et al. 2006 &
2011 )
H295R
Fig 3. SPE - Reconstitution
Toxicity Endpoints Method
Cellular metabolic activity via NAD (P) H-dependant cellular oxidoreductase
Cells were/will be exposed to 5x and
1x concetratied samples for 48h in triplicate in 96-well plates. WST-1 assay Dye Solution was/will be added to each well and incubated for 2h and absorbance test will determine cell viablity.
Estrogen receptormediated luciferase reporter gene assay
Cells will be exposed to increasing sample concentrations for 48h in quadruplicate in a 96-well luminometer plates and luciferase activity will be determined.
Androgen receptormediated luciferase reporter gene assay
Modulation of steroidogenesis
Cells will be exposed to increasing sample concentrations for 48h in quadruplicate in a 96-well luminometer plates and luciferase activity will be determined.
Cells were/will be exposed increasing sample concentrations for 48h in triplicate in a 24-well plate, medium was removed and production of hormones determined by enzymelinked immunosorbent assay (ELISA).
Canadian Water Network, University of Saskatchewan, City of Saskatoon Wastewater, Saskatoon, SK, City of Regina Wastewater Treatment Plant, Regina, SK, University of Waterloo, Waterloo, ON, INRS-ETE et UQTR, Quebec, QC, Hongda Yuan, Bryan Sarauer, Shawn Beitel
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DMSO Forskolin
1.0µL
Forskolin
10µL
Prochloraz
0.3µL
Prochloraz
3.0µL
Isooctane Media Blank
Sample 5x
Water
Sampler 5x
Regina
Effluent 5x
Regina
Influent 5x
Regina
Influent 1x
Saskatoon
Effluent 5x
Saskatoon
Influent 5x
Saskatoon
Influent 1x
Graph 1 : Fold-change changes in 17beta-estradiol production by H295R Cells after exposure to extracts of influent and effluent samples collected from the Regina and Saskatoon WWTPs. Results are represented as mean SD (n=2).
Asterisks indicate that treatment groups are significantly different from solvent control (DMSO; p<0.05) using one-way ANOVA followed by LSD post-hoc test (SPSS Statistics, V20).
• Exposure to Forskolin and Prochloraz resulted in increased and decreased production of Estradiol, respectively, which is in accordance with previously reported responses of H295R cells to the exposure with these compounds (Hecker et al. 2006).
• Exposure to Regina Influent, Saskatoon Influent and Saskatoon Effluent showed a significant increase in estradiol production suggesting that these samples contain compounds with the ability to disrupt steroidogenesis.
• Exposure to the Water Sampler showed a significant 1.7-fold increase in
E2 production, suggesting the samplers may be contaminated by compounds with the ability to disrupt steroidogenesis, and decontamination should be conducted before using them.
(Note: this sampler is new and WWTP samples were taken by a different apparatus that had been rinsed thoroughly over the past years).
• The detection of specific biological chemical analysis of suspected EDCs with the aim to pinpoint causative agents in wastewater. analyses are ongoing.
potentials will inform targeted
• Through the proposed bioassay-directed analysis (Table 1), this project will characterize the efficiency of current WWTP technologies across
Canada to eliminate emerging contaminants of concern, and thus, will inform future needs for developing more efficient, economical and effective approaches for removing EDC’s from wastewater.
• The results illustrated are very preliminary, and additional bioassay
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analysis include the T47D-KBluc ((anti-)estrogenicity), MDAkb2
((anti-)androgenicity), and H295R Steroidogenesis (steroidogenesis disruption) assays.
• Sample collection will continue throughout 2014 and 2015 during different seasons and locations in Ontario and Quebec.