Characterization of Estrogenic Characterization of Estrogenic Endocrine Disrupting Endocrine Disrupting Chemicals in Source Waters Chemicals in Source Waters of Central Arizona of Central Arizona Nikole Fales, Absar Alum, and Morteza Abbaszadegan Arizona State University
What is an Endocrine What is an Endocrine Disruptor? Disruptor? � As defined by the EDSTAC As defined by the EDSTAC � � “An exogenous substance that “An exogenous substance that � changes endocrine function and changes endocrine function and causes adverse effects at the level of causes adverse effects at the level of the organism, its progeny, and/or the organism, its progeny, and/or (sub) populations of organisms” (sub) populations of organisms”
Partial List of Synthetic and Natural Partial List of Synthetic and Natural Compounds With Suspected/Known Compounds With Suspected/Known Estrogenic Activity Estrogenic Activity Synthetic Compounds Natural Compounds Synthetic Compounds Natural Compounds Synthetic hormones EE2 EE2 Sex steroids E2 E2 Synthetic hormones Sex steroids Organochlorine Compounds Phytoestrogens Organochlorine Compounds Phytoestrogens Chlordecone, Endosulfan, Chlordecone, Endosulfan, Phenolics Phenolics Nonyl- Nonyl - and octylphenols and octylphenols Alkylphenol Alkylphenol polyethoxycarboxylates polyethoxycarboxylates Bisphenol A Bisphenol A
Effects of EDCs Effects of EDCs � Mimic natural hormones Mimic natural hormones � � Alters natural hormonal balance Alters natural hormonal balance � � Block hormone receptors Block hormone receptors � � Prevents action of normal hormones Prevents action of normal hormones � � Alter the synthesis and metabolism of Alter the synthesis and metabolism of � natural hormones natural hormones � Affects the concentration of natural hormones Affects the concentration of natural hormones � in the body in the body
Mechanism Animation Mechanism Animation Normal Hormonal Function Normal Hormonal Function HORMONE RECEPTOR REACTION CELL
Mechanism Animation Mechanism Animation Hormone mimicking Hormone mimicking EDC Endocrine disruptor gives a stronger than normal signal RECEPTOR REACTION CELL
Mechanism Animation Mechanism Animation Hormone mimicking Hormone mimicking EDC Endocrine disruptor gives a weaker than normal signal REACTION CELL
Mechanism Animation Mechanism Animation Hormone blocking Hormone blocking HORMONE Endocrine disruptor prevents natural hormone from sending EDC signal NO REACTION CELL
Potential Sources Potential Sources � Activities in Watershed Activities in Watershed � � Agriculture Agriculture � � Recreation Recreation � � Indigenous Flora and Fauna Indigenous Flora and Fauna �
Endocrine Disruption is a Endocrine Disruption is a complicated science . . . complicated science . . .
Metabolites formed upon incubation Metabolites formed upon incubation with human flora under aerobic and with human flora under aerobic and anaerobic conditions (72h) anaerobic conditions (72h) Substrate Metabolite Substrate Metabolite Estrone Estradiol Estrone Estradiol Estradiol Estrone Estradiol Estrone 16a- -Hydroxyestrone Hydroxyestrone Estriol 16a Estriol 16- -Oxoestradiol Oxoestradiol 16- -Epiestriol Epiestriol 16 16 15a- - - -Hydroxyestrone Hydroxyestrone 15a- -Hydroxyestrone Hydroxyestrone 15a 15a Estriol No metabolites Estriol No metabolites Scubert 1964
Chemical structures of Bisphenol Bisphenol Chemical structures of A and the chlorinated A and the chlorinated derivatives derivatives Fukazawa, et al., 2001
BPA and its chlorinated and its chlorinated derivatives derivatives BPA Compound EC x10 Relative Compound EC Relative x10 Activity Activity (nM) (nM) Bisphenol A 4200 1 Bisphenol A 4200 1 3- -CIBPA CIBPA 550 8 3 550 8 3,5- -diCIBPA diCIBPA 520 8 3,5 520 8 3,3’- -diCIBPA diCIBPA 110 38 3,3’ 110 38 3,3’,5- -triCIBPA triCIBPA 210 20 3,3’,5 210 20 3,3’,5,5’- -tetraCIBPA tetraCIBPA 1500 3 3,3’,5,5’ 1500 3 EC x10 = Concentration at which ratio of signal of sample to that of blank was 10 Fukazawa, et al., 2002
Objectives Objectives � Investigate occurrence of estrogenic Investigate occurrence of estrogenic � endocrine disrupting chemicals in source endocrine disrupting chemicals in source water in central Arizona water in central Arizona � Develop an estrogenicity profile of source Develop an estrogenicity profile of source � waters waters � Evaluate effect of chlorination on raw Evaluate effect of chlorination on raw � water water
Sampling Sites Sampling Sites � Salt River Salt River � � Verde River Verde River � � CAP canal CAP canal � � Water Treatment Plant 1 Water Treatment Plant 1 � � Water Treatment Plant 2 Water Treatment Plant 2 �
Sampling Sites Sampling Sites Verde River at its intersection with SR-87 in the Fort McDowell indian reservation N Verde River Salt River Salt River just before the confluence of the Salt and Verde Rivers
Sample Collection and Assay Sample Collection and Assay Strategy Strategy Sample from Phoenix area source water Concentration by Site Selection Liquid-Liquid Extraction Cell Culture E-Screen Assay MCF-7 Cell Line CellTiter96 Aqueous One (Promega)
E- -Screen Assay Screen Assay E Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Extra Cells Day 7
Dose Response Curve of Dose Response Curve of 17 β β - -Estradiol Used to Determine Estradiol Used to Determine 17 Detection Limit (DL) Detection Limit (DL) 4500 4000 ber of Cells 3500 3000 2500 2000 Num 1500 1000 500 0 1 1 4 3 3 3 3 2 2 2 2 2 1 1 0 l s 0 0 o 1 1 1 1 1 1 1 1 1 1 1 1 1 r 0 0 r h - - - - - - - - - - - - - 0 0 t E E E E E E E E E E E E E 4 n 0 0 5 1 4 6 8 1 2 4 6 8 1 5 1 2 o 0 0 C 0 0 0 0 0 . 0 . 0 Concentration
Data Analysis Data Analysis � Graphs use Relative Proliferation Effect, or Graphs use Relative Proliferation Effect, or � the percent difference from control the percent difference from control � 0 0 – – 25% : Not highly estrogenic 25% : Not highly estrogenic � � 25 25 – – 70% : Moderately estrogenic 70% : Moderately estrogenic � � >70%: Highly estrogenic >70%: Highly estrogenic �
Seasonal variations in the Seasonal variations in the estrogenicity of Verde River water estrogenicity of Verde River water and effect of chlorination and effect of chlorination 100 80 60 RPE 40 20 0 Feb March April May June July Verde River Non-Chlorinated Verde River Chlorinated 2ppm for 30 minute
Seasonal variations in the Seasonal variations in the estrogenicity of Salt River water estrogenicity of Salt River water and effect of chlorination and effect of chlorination 100 80 60 40 20 0 Feb March April May June July Salt River Non-Chlorinated Salt River Chlorinated 2ppm for 30 minute
Seasonal variations in the Seasonal variations in the estrogenicity of CAP Canal water estrogenicity of CAP Canal water and effect of chlorination and effect of chlorination 100 80 60 RPE 40 20 0 Feb March April May June July CAP Canal Non-Chlorinated CAP Canal Chlorinated 2ppm for 30 minute
Summary of Results Summary of Results Percent Decrease in Estrogenicity Estrogenicity After Chlorination After Chlorination Percent Decrease in Verde River 51.2 % Verde River 51.2 % Salt River 44.5 % Salt River 44.5 % CAP canal 43.5 % CAP canal 43.5 %
Summary of Results Summary of Results Concentration of estrogenic chemicals as EEQ Concentration of estrogenic chemicals as EEQ Verde River 0.12 – – 12.0 12.0 ng ng/L /L Verde River 0.12 Salt River 0.09 – – 11.0 11.0 ng ng/L /L Salt River 0.09 CAP canal 0.17 – – 12.0 ng/L 12.0 ng/L CAP canal 0.17
Conclusions Conclusions � In general, estrogenic potency in Verde River, In general, estrogenic potency in Verde River, � Salt River, and CAP canal was higher in summer Salt River, and CAP canal was higher in summer months months � Chlorination of raw water from Verde River, Salt Chlorination of raw water from Verde River, Salt � River, and CAP canal resulted in significant River, and CAP canal resulted in significant decrease in estrogenicity (most cases <DL) decrease in estrogenicity (most cases <DL) � Chlorination gave 51.2, 44.5, and 43.5 % Chlorination gave 51.2, 44.5, and 43.5 % � removal, respectively removal, respectively � Chlorination byproducts were not highly Chlorination byproducts were not highly � estrogenic in nature estrogenic in nature
Acknowledgements Acknowledgements � Funding for this research was provided by Funding for this research was provided by � NSF Water Quality Center at ASU NSF Water Quality Center at ASU � We appreciate the help extended by the We appreciate the help extended by the � Salt River Project (SRP), Central Arizona Salt River Project (SRP), Central Arizona Project (CAP), and the two water Project (CAP), and the two water treatment plants. treatment plants.
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