Tabata Bagatim , Kean Steeves2, Steve Wiseman2, Natacha ... · Saskatoon 260,600 Pop Regina 232,890...
1
Saskatoon 260,600 Pop Regina 232,890 Pop Guelph 134,894Pop Kitchener 231,488 Pop Quebec City 321,221 Pop Montreal 1,900,000 Pop Map 1 – Locations of wastewater treatment plants (WWTPs) in Quebec, Ontario and Saskatchewan, and the population (Pop) they are serving. Tabata Bagatim 1 , Sara Hanson 2 , Kean Steeves 2 , Steve Wiseman 2 , Natacha Hogan 2,3 , Alice Hontela 4 , Paul Jones 2 , John Giesy 2,5 , Bragg Leslie 6 , Dhiyebi Hadi 6 , Mark R. Servos 6 , Gauthier Charles 7 and Markus Hecker 1,2 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 • The main objective of this study was to investigate 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. • 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 from wastewater treatment plants (WWTP’s) to environmental endocrine disruption in Canadian. surface waters is incomplete at best. Filtration: • 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). • The results illustrated are very preliminary, and additional bioassay analyses are ongoing. • In vitro 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. • The detection of specific biological in vitro potentials will inform targeted chemical analysis of suspected EDCs with the aim to pinpoint causative agents in wastewater. • 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. ACKNOWLODGEMENT: 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 Fig 1. Particulate Filtration Fig 2. SPE - Eluding Fig 3. SPE - Reconstitution 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). Effect Type Cell Line/Test Toxicity Endpoints Method Cytotoxicity (Mosman et al. 1983) MTT (T47D, MDA, H295R) 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. (Anti-)Estrogenicity (Wilson et al. 2004) T47D-Kbluc Estrogen receptor- mediated 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. (Anti-)Androgenicity (Wilson et al. 2002) MDA-KB2 Androgen receptor- mediated 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. Disruption of Steroidogenesis (Hecker et al. 2006 & 2011) H295R Modulation of steroidogenesis 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 enzyme- linked immunosorbent assay (ELISA). Table 1. In vitro test systems for the characterization of endocrine potentials of WWTP effluents and influents. Bioassays: 0 1 2 3 4 5 6 7 8 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 Fold Change Estradiol • Mosman et al. 1983 - J Immunol Methods 65:55-63 • Wilson et al. 2002 - Toxicol Sci 66:69-81 • Wilson et al. 2004 - Toxicol Sci 81:69-77 • Hecker et al. 2006 - Toxicol Appl Pharmacol 217:114-124 • Hecker et al. 2011 – Environ Sci Pol Res 18:503-515 • Characterize (anti-)estrogenic, (anti-)androgenic and steroidogenesis disruption potentials of influents and effluents from selected WWTPs in Ontario, Quebec and Saskatchewan. • Determine the efficiency of different WWTPs with different treatment steps to remove chemicals with endocrine disrupting properties. • Investigate factors such as season and temperature on the efficiency to remove EDCs from raw sewage.
Transcript of Tabata Bagatim , Kean Steeves2, Steve Wiseman2, Natacha ... · Saskatoon 260,600 Pop Regina 232,890...
Saskatoon 260,600 Pop
Regina 232,890 Pop
Guelph 134,894Pop
Kitchener 231,488 Pop
Quebec City 321,221 Pop
Montreal 1,900,000 Pop
Map 1 – Locations of wastewater treatment plants (WWTPs) in Quebec, Ontario and Saskatchewan, and the population (Pop) they are serving.
Tabata Bagatim1, Sara Hanson2, Kean Steeves2, Steve Wiseman2, Natacha Hogan2,3, Alice Hontela4, Paul Jones2, John Giesy2,5, Bragg Leslie6, Dhiyebi Hadi6, Mark R. Servos 6, Gauthier Charles7 and Markus Hecker1,2
1School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada; 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada; 3 Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada; 4Department of Biological Science, University of Lethbridge, Lethbridge, AB, Canada; 5Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada; 6 Biology Department, University of Waterloo, Watereloo, ON; 7INRS-ETE et UQTR, Quebec, QC
• The main objective of this study was to investigate 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.
• 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 from wastewater treatment plants (WWTP’s) to environmental endocrine disruption in Canadian. surface waters is incomplete at best.
Filtration: • 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).
• The results illustrated are very preliminary, and additional bioassay analyses are ongoing.
• In vitro 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.
• The detection of specific biological in vitro potentials will inform targeted chemical analysis of suspected EDCs with the aim to pinpoint causative agents in wastewater.
• 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.
ACKNOWLODGEMENT: 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
Fig 1. Particulate Filtration Fig 2. SPE - Eluding Fig 3. SPE - Reconstitution
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). Effect Type Cell Line/Test Toxicity Endpoints Method
Cytotoxicity (Mosman et al. 1983) MTT (T47D, MDA, H295R)
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.
(Anti-)Estrogenicity (Wilson et al. 2004)
T47D-Kbluc
Estrogen receptor-mediated 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.
(Anti-)Androgenicity (Wilson et al. 2002)
MDA-KB2
Androgen receptor-mediated 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.
Disruption of Steroidogenesis
(Hecker et al. 2006 & 2011)
H295R Modulation of steroidogenesis
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 enzyme-linked immunosorbent assay (ELISA).
Table 1. In vitro test systems for the characterization of endocrine potentials of WWTP effluents and influents.
Bioassays:
0
1
2
3
4
5
6
7
8
DMSO Forskolin1.0µL
Forskolin10µL
Prochloraz0.3µL
Prochloraz3.0µL
Isooctane Media BlankSample 5x
WaterSampler 5x
ReginaEffluent 5x
ReginaInfluent 5x
ReginaInfluent 1x
SaskatoonEffluent 5x
SaskatoonInfluent 5x
SaskatoonInfluent 1x
Fold
Cha
nge
Estradiol
• Mosman et al. 1983 - J Immunol Methods 65:55-63 • Wilson et al. 2002 - Toxicol Sci 66:69-81 • Wilson et al. 2004 - Toxicol Sci 81:69-77 • Hecker et al. 2006 - Toxicol Appl Pharmacol 217:114-124 • Hecker et al. 2011 – Environ Sci Pol Res 18:503-515
• Characterize (anti-)estrogenic, (anti-)androgenic and steroidogenesis disruption potentials of influents and effluents from selected WWTPs in Ontario, Quebec and Saskatchewan.
• Determine the efficiency of different WWTPs with different treatment steps to remove chemicals with endocrine disrupting properties.
• Investigate factors such as season and temperature on the efficiency to remove EDCs from raw sewage.
