Human Health Risks from Endocrine Disruptors in Reclaimed Water

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Title:
Human Health Risks from Endocrine Disruptors in Reclaimed Water
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1 online resource (79 p.)
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english
Creator:
Sidhu, Harmanpreet S
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Soil and Water Science
Committee Chair:
O'connor, George A
Committee Co-Chair:
Wilson, Patrick Christopher
Committee Members:
Thomas, John E

Subjects

Subjects / Keywords:
assessment -- chemicals -- dislodgeable -- disrupting -- endocrine -- human -- reclaimed -- residues -- risk -- turfgrass -- water
Soil and Water Science -- Dissertations, Academic -- UF
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Soil and Water Science thesis, M.S.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Endocrine disrupting chemicals (EDCs) occur in reclaimed water (RW) and constitute unknown risks to humans. The presence of EDCs in reclaimed water used to irrigate turf and in nearby water retention ponds was determined, and used in the first step of an assessment of risk to a child playing on recently irrigated turf and subjected to dislodged EDC residues.Five EDCs (estrone, 17ß-estradiol, 17a- ethynylestradiol, bisphenol A and nonylphenol) were quantified in 28 samples of reclaimed waters (wastewater treatment plant effluents) and 88 samples from residential ponds. St. Augustine turf grass was irrigated with spiked RW to study dislodgement of the five EDCs using hand swipe and drag-sled methods. Grass clippings were analyzed to relate masses of EDC on grass with masses dislodged. EDCs were detected in all RW and pond water samples. Bisphenol A was detected most frequently and in greater amounts in both RW and pond samples. Maximum masses of EDCs were dislodged immediately after irrigation. Dislodged masses of estrone, 17ß-estradiol and 17a- ethynylestradiol decreased rapidly and were below detection limits 4 hours after application. Dislodged bisphenol-A and nonylphenol decreased more slowly and uniformly, but were not detected 6 hours after application. The human health risk associated with EDCs in RW and retention ponds, and dislodged residues of EDCs from turf irrigated with RW was minimal.
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Harmanpreet S Sidhu.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: O'connor, George A.
Local:
Co-adviser: Wilson, Patrick Christopher.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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Applicable rights reserved.
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lcc - LD1780 2013
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UFE0045812:00001


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1 HUMAN HEALTH RISKS FROM ENDOCRIN E DISRUPTORS IN RECLAIMED WATER By HARMANPREET SINGH SIDHU A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Harmanpreet S. Sidhu

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3 To my late p arents, may they rest in peace

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4 ACKNOWLEDGMENTS nor, for his continuous support and faith in me. He has the attitude and substance o f a genius and I have learnt a lot from him. Without his guidance and support this thesis would not have been possible. I am extremely thankful to him for the opportunity h e provided me to learn, work and progress under his esteemed supervision. I would also like to express my gratitude to my co advisor Dr. Chris Wilson, who provided me with resources to carry out my research. His help throughout my research made my success possible. I learned valuable techniques and research method s with his help. I would also like to thank my committee member, Dr. John Thomas for I have had the pleasure of learning from him. I would also like to thank Dr. Jian Lu and Dr. Jun wu for provi d ing me technical guidance about methods and i nstruments used in my research and Youjian Lin who accompanied me during sample collection. A final thank to my pare nts for it is their teaching that encourage d me to achieve success at every moment of my life I would not be who I am and where I am today without them.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Reclaimed Water ................................ ................................ ................................ .... 13 Benefits ................................ ................................ ................................ ............ 14 Concerns ................................ ................................ ................................ .......... 14 Sources of EDCs in Wastewater ................................ ................................ ...... 15 Potential Impacts of ED Cs ................................ ................................ ...................... 16 Ecological Impacts ................................ ................................ ........................... 16 Impacts on Human s ................................ ................................ ......................... 16 EDCs in RW ................................ ................................ ................................ ..... 18 EDCs in Surface Water Retention Ponds ................................ ......................... 20 Human Exposure Scenario/ Research Problem ................................ ..................... 20 Overview of Human Health Risk Assessment ................................ ......................... 21 Research Objectives ................................ ................................ ............................... 25 EDCs Selected for Study ................................ ................................ ........................ 25 Estrone ................................ ................................ ................................ ............. 26 Estradiol ................................ ................................ ................................ ........... 27 Ethynylestradiol ................................ ................................ ................................ 27 Bisphenol A ................................ ................................ ................................ ...... 28 Nonylphenol ................................ ................................ ................................ ..... 28 Toxicological Data on the Five EDCs ................................ ................................ ..... 32 Research Approach ................................ ................................ ................................ 33 2 MATERIALS AND METHODS ................................ ................................ ................ 35 Sites and Site Conditions ................................ ................................ ........................ 35 Water Samples ................................ ................................ ................................ ....... 37 Sample Collection ................................ ................................ ............................ 37 Sample Extraction and Preparation ................................ ................................ .. 38 Dislodged EDCs and EDCs on Grass Surface ................................ ....................... 39 Sample Collection ................................ ................................ ............................ 39 Sample Extraction and Preparation ................................ ................................ .. 43

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6 Sample Analysis ................................ ................................ ................................ ..... 45 Quality Assurance/Quality Control (QA/QC) ................................ ........................... 46 Method Detection Limit, Practical Quantitation Limit and Percent Recoveries ........ 47 3 RESULTS AND DISCUSSION ................................ ................................ ............... 49 EDCs in Water Samples ................................ ................................ ......................... 49 Comparison to Published Data ................................ ................................ ......... 53 Likely Sources ................................ ................................ ................................ .. 53 Possible Fate(s) of EDCs ................................ ................................ ................. 53 EDCs in Dislodged Residue Samples and Grass Clippings ................................ .... 55 Human Health Risk Assessment ................................ ................................ ............ 59 Water Samples ................................ ................................ ................................ 59 Dislodgeable Residues Exposure ................................ ................................ ..... 61 Assessment of Combined Risk (E2 Equivalent) from the five EDCs ................ 64 Water samples ................................ ................................ ........................... 64 Dislodged residues ................................ ................................ .................... 65 Risk Comparison to Published Data ................................ ................................ 66 Uncertainty ................................ ................................ ................................ .............. 66 Risk Communication/Management ................................ ................................ ......... 67 4 SUMMARY AND CONCLUSIONS ................................ ................................ .......... 68 LIST OF REFERENCES ................................ ................................ ............................... 70 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 79

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7 LIST OF TABLES Table page 1 1 Concentrations (ng/L)* of hormones in effluents of WWTPs (Ying et al., 2002a). ................................ ................................ ................................ ............... 19 1 2 Concentrations (g/L) of nonylphenol (NP) and bisphenol A (BPA) in effluents of WWTPs. ................................ ................................ ........................... 19 1 3 Concentrations ( g/L) of EDCs reported in septic effluents ............................... 20 1 4 Properties of the five EDCs studied. ................................ ................................ .. 30 1 5 Human NOAEL/LOAEL (oral dose) values of EDCs ................................ .......... 32 2 1 Masses of chemicals applied per sampling area. ................................ .............. 40 2 2 MDL, PQL and percent recoveries of the EDCs, E2 acetate, coprostanol and sucralose from different methods used. ................................ .............................. 48 3 1 Concentrations (SD) of EDCs in RW (n=28), ponds near RW use (64), and ponds near septic systems (n=24). ................................ ................................ ..... 5 2 3 2 Median concentrations of EDCs obtained from the study of RW and concentrations reported in literature. ................................ ................................ .. 52 3 3 EDC and sucralose masses (ng) dislodged, via drag sled, with time. ............... 56 3 4 DWELs and safety factor of EDCs when consumed in drinking water. .............. 61 3 5 RQs for the five EDCs: Acute and chronic toxicities. ................................ ......... 63 3 6 Relative potencies of the five EDCs (Clayton, 2011; Thorpe et al., 2003). ........ 65

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8 LIST OF FIGURES Figure page 2 1 Retention times and m/z ratios of EDCs analyzed using GC MS. ..................... 46 3 1 Concentrations of EDCs and coprostanol in RW (n=28), ponds near RW use (n=64) and ponds near septic systems (n=24). ................................ .................. 50 3 2 Frequency of detection of EDCs and coprostanol in RW (n=28). ...................... 50 3 3 Frequency of Detection of E Sample Collection DCs and coprostanol in all ponds (n=88). ................................ ................................ ................................ ..... 51 3 4 EDC masses (ng) af ter spraying with time (hours) ................................ ............ 57 3 5 Percent of EDCs and sucralose dislodged from gras s surface with time (hours). ................................ ................................ ................................ ............... 59

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9 LIST OF ABBREVIATIONS ADI Allowable Daily Intake BPA Bisphenol A BSTFA+TMCS Bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane CSF Cancer Slope Factor CO 2 Carbon di oxid e DWEL Drinking Water Equivalent Level E1 Estrone E2 17 estradiol EDC Endocrine Disrupting Chemical EE2 ethynylestradiol EFED Environmental Fate and Effects Division EC 50 Median Effective Concentration GC Gas Chromatograph GC MS Gas Chromatograph Mass Spectrometer HEI Highly Exposed Individuals HPLC MS High Performance Liquid Chromatograph Mass Spectrometer K ow Octanol Water Partitioning Coefficient LOAEL Lowest Observable Adverse Effect Level MDL Method Detection Limit NOAEL No Observable Adverse Effect Level NP Nonylphenol

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10 OPP Office of Pesticide Programs PCB Polychlorinated Biphenyl PPCP Pharmaceuticals and Personal Care Product PQL Practical Quantitation Limit RfD Reference Dose RQ Risk Quotient RSD Risk Specific Dose RW Reclaimed Water SD Standard Deviation SPE Solid Phase Extraction UF Uncertainty Factor USEPA United States Environmental Protection Agency WWTP Wastewater Treatment Plant

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillm ent of the Requirements for the Master of Science HUMAN HEALTH RISKS FROM ENDOCRIN E DISRUPTORS IN RECLAIMED WATER By Harmanpreet Singh Sidhu August 2013 Chair: Co chair: Patrick C. Wilson Major: Soil and Water Science Endocrine disru pting chemicals (EDCs) occur in reclaimed water (RW) and constitute unknown risks to humans. The presence of EDCs in reclaimed water used to irrigate turf and in nearby water retention ponds was determined, and used in the first step of an assessment of ri sk to a child playing on recently irrigated turf and subject ed to dislodged EDC residues. Five EDCs (estrone, 17 ethy ny lestradiol, bisphenol A and nonylphenol) were quantified in 28 samples of reclaimed waters (wastewater treatment plant effluents) and 88 samples from residential ponds. St. Augustine turf grass was irrigated with spiked RW to study dislodgement of the five EDCs using hand swipe and drag sled methods. Gras s clippings were analyzed to relate masses of EDC on grass with masses dislodged. EDCs were detected in all RW and pond water sample s. Bisphenol A was detected most frequently and in greater amounts in both RW and pond samples. Maximum masses of EDCs were dislodg ed immediately after irrigation. Dislodged masses of estrone, 17 ethynylestradiol decreased rapidly and were below detection limits 4 hours after application Dislodged bisphenol A and nonylphenol decreased more slowly and uniformly, bu t were not detected 6 hours after application The human health risk

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12 associated with EDCs in RW and retention ponds, and dislodged resid ues of EDCs from turf irrigated with RW was minimal.

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13 CHAPTER 1 INTRODUCTION Increasing use of reclaimed water (RW) for crop irrigation, groundwater recharge, and landscape irrigation, increases the risk of human exposure to potentially harmful s, occur in surface and reclaimed water at concentrations ranging from below detection limits to several ng/ L (Dur an Alvarez et al., 2009, Liu et al., 2009). The human health risk associated with even small concentrations of EDCs is, however, uncertain. T he development of more advanced analytical techniques [e.g. gas chromatographs coupled with mass spectrometers (GC MS) and high performance liquid chromatographs coupled with mass spectrometers (HPLC MS) ], allows detection of even lower concentrations. Be tter detection technology heightens public concern about harmful chemicals as the mere presence of a chemical in the environment may pose some risk to humans. Reclaimed W ater Exponential population growth and rapid development places enormous stress on p otable water supplies, making more efficient water management crucial. Use of treated wastewate r (instead of potable freshwater) for irrigation of landscapes, lawns, and agricultural fields is an increasingly popular approach and is expanding rapidly. Trea ted wastewater effluent reused for various purposes is called reclaimed or recycled water (RW). Secondarily treated wastewater effluents are the source of reclaimed water in most cases (USEPA, 2012 a ). Irrigation of turf grass is one of the major water cons uming activities in the United States and use of RW to irrigate t urf is increasing

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14 Florida leads the U.S. in terms of reusable water production as well as use. Out of 2740 million liters reclaimed water produced daily in F %) are used for irrigating golf courses, residen tial turf and parks (F L DEP, 2012 ). Benefits Wastewater reuse not only partially counters freshwater scarcity, but also helps to maintain --Environmental sustainability : by r educing pollutant loads and discharge into receiving water bodies, Economic efficiency : through improved water conservation, promotion of water efficiency, waste reduction and harmonizing long term water demand and water supply, Food securit y: (for some c ountries) through application of certain essential plant nutrients via treated wastewater reuse (Indian Institute of Technology Team 2011). Concerns Despite the many benefits of using RW, there are some potential disadva ntages (Toze, 2006) including -Yu ck Factor : Many people are squeamish about using recycled water. Campaigns are sometimes necessary to educate people about the potential advantages and safety of RW. More and more people are, h owever, becoming more accepting of RW use. Presence of Pathog ens : Reclaimed water may contain greater number s and species of certain enteric pathogens than potable freshwater. Presence of contaminants : Chemicals like pharmaceuticals and p ersonal care products (PPCPs), polychlorinated biphenyls (PCBs), f lame retar dants, steroids etc. can occur in wastewater effluents (Desbrow et al., 1998; Kuch and Ballschmiter, 2001;

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15 Ternes et al., 1999a; Duran Alvarez et al., 2009). Wastewater treatment plants are not fully efficient in removal of such organic chemicals from wast ewater streams (Roberts and Thomas, 2006). As a result, trace organic chemicals are discharged into surface waters or are applied to agricultural fields, recreational areas, etc. with the reclaimed water. Many of the trace organic chemicals are endocrine d isrupting chemicals (EDCs), and have been detected in reclaimed water up to several nanograms per liter (Liu et al., 2009). EDCs are natural and/or synthetic substances that when absorbed into body, from an outside source, can overstimulate/block and/or in terfere with normal functioning of endocrine system of an organism producing adverse developmental, reproductive, neurological, an d/or immune effects (Jobling et al., 1998; 2004). Many EDCs have e strogenic activity and either mimic or aid in excess product ion of body estrogens (Gore, 2007). Estrogenic activity of a chemical is a measure of potency of a chemical in terms of ability to stimulate normal endocrine system to produce estrogen (particularly, estradiol). Sources of EDCs in Wastewater A wide variety of chemicals including pharmaceuticals and personal care products (PPCPs), polychlorinated biphenyls (PCBs), DDT and other pesticides, and plasticizers such as bisphenol A are considered endocrine disruptors, and are common in everyday products like plast ic bottles, metal food cans, detergents, flame retardants, food, toys, cosmetics, and pesticides (NIEHS, 2011; Naz, 2005). Many pharmaceuticals and dietary supplements containing EDCs are only partially digested by the body and excr eted intact through urin e and f eces (Redderson et al., 2002). Natural hormones produced by mammals (especially sex hormones like testosterone and estradiol) and their metabolites are also excreted in urine. Excreted hormones and pharmaceuticals

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16 then enter the environment through effluents discharged by wastewater treatment plants (WWTPs) and wet weather run off (Shore et al., 1988; Shore and Shemesh, 2003). Partial removal or formation of an active form during the process of sewage treatment results in release of endocrine disrupt ors in surface waters and/or reusable water (Gomez et al., 2006). Other sources of EDCs in the environment include but are not limited to industrial solvents or lubricants and their byproducts. Potential Impacts of EDCs EDCs not only contaminate environment but also affect organisms (especially aquat ic) and possibly humans ( Lewis, 1991; Joblin g et al., 1998; Clayton, 2011 ). Ecological Impacts Some EDCs like stero idal hormones (e.g. ethynylestradiol) can cause biological effects on aquatic organisms at parts per tr illion concentrations (Weber et al., 2005). Male fish held in cages exposed to sewage effluent undergo feminization effects, and rivers receiving sig nificant amounts of sewage effluent contain significan t numbers ( Jobling et al., 1998, 2004; Sumpter, 2005; Rodgers Gray et al., 2000). Floridian alligators suffer from under developed male gonads in j uvenile male alligators due to the presence of estrogenic like compounds in the Florida everglades (Guillette et al., 1994). Impacts on Human s Concentrations of EDCs in surface water bodies, reclaimed water s, and septic effluents are typically low (on the order of parts per billion or trillion). The risk of human exposure is, however, intensified as EDCs may have adverse effect s on humans even at such low concentrations (Routledge et al., 1998). EDCs are thought to be linked with

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17 the decline of sperm counts in adult males, increasing incidence of bre ast and testicular cancer, and the decrease in the age o f puberty onset (Xiao et al., 2001; Hotchkiss et al., 2008). The endocrine disruptions produced can cause cancerous tumors, birth defects, and other developmenta l disorders (Lewis, 1991). O ther suppos ed adverse effects of EDCs include decreased thyroid function and increased parathyroid hormone act ivity (NRC 2006; Stewart et al., 2003; Zoeller, 2007), reproductive abnormalities (Guillette et al., 1994; Diamanti Kandarakis et al., 2009), decreased sper m count in men (Carlsen et al., 1992), effects on brain of developing fetuses (McCally, 1997) and early puberty in females (Toppari and Juul, 2010). The mere presence of EDCs in the environment, however, does not necessarily indicate significant risk. The extent of adverse effects of any chemical depends upon hazard and exposure dose relationship. Further, some effects are also species dependent (Clayton, 2011). Thus, the risk from EDCs to humans is uncertain The UK Environment Agency (2000) commented tha laboratory conditions and do not necessarily reflect the responses which would occur in suggests that the concentrations of EDCs to w hich human beings are subjected in the environment pose negligible to low risk of adverse health effects (Clayton, 2011). For example, the data suggesting a decline in male sperm concentration in adu lt males reported by Carlsen et al., (1992) were not conf irmed by subsequent studies (Lukachk o, 1999; J rgensen et al., 2001; Clayton, 2011). However, with the increasing populations, increasing production and use of PPCPs, and new chemicals being developed on a daily basis, the risk from EDCs is likely to rise.

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18 Risk assessment of EDCs is complicated because of many factors including the complex nature of effects produced, potential delayed on set of effects, potential lack of a threshold for effect, suggested no relationship between dose and response, and effe cts at very low doses (NTP, 2001). Some EDCs can also be toxic and/or carcinogenic and some are suspected carcinogens, further increasing speculation about risk and thus increasing the importance of more research on EDCs. EDCs in RW Current wastewater tre atment plants (WWTPs) use primary and secondary treatments to produce effluents from which EDCs are in completely removed (Deblonde et al., 2011). Secondarily treated wastewater effluents are the source of reclaimed water in most cases (USEPA, 2012 a ). Terti ary treatments like ozonation, active carbon adsorption and catalytic oxidation show high EDC removal efficiencies, but ar e rarely used in WWTPs (Yoon et al., 2006; Broseus et al., 2009). As a result, EDCs have been detected in effluents of wastewater trea tment plants in many countries (Baronti et al., 2000; Belfroid et al., 1999; Desbrow et al.,1998; Kuch and Ballschmiter, 2001; Nasu et al., 2001; Snyder et al., 1999; Ternes et al., 1999a; Weber et al., 2005). Reuse of wastewater effluents containing EDCs can, thus, have potential adverse effects on human beings and/or the environment. Fortunately, there are restrictions on RW use depending upon the level of treatment (primary, secondary, and tertiary) undergone (e.g. secondary treated effluents are not pot able). However, there are no federal regulations regarding the maximum load of EDCs allowable in effluents (Weber et al., 2005; USEPA, 2012 a ). Concentrations of var ious EDCs detected in effluents wo rldwide are given in Tables 1 1 and 1 2

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19 Table 1 1 Con centrations (ng/L)* of hormones in effluents of WWTPs (Ying et al., 2002a). Location Number of Sample Estrone (E1) Estradiol (E2) Ethynylestradiol (EE2) Italy 30 2.5 82.1 (9.3) 0.44 3.3 (1.0) <0.9 1.7 (0.45) Netherlands 6 <0.4 47 (4.5) <0.1 5.0 (<0.1 ) <0.2 7.5 (<0.2 ) Germany 16 <1 70 (9) <1 3 (<1 ) <1 15 (1) Canada 10 <1 48 (3) <1 64 (6) <1 42 (9) UK 21 1.4 76 (9.9) 2.7 48 (6.9) <0.2 7 (<0.2) Japan 27 X 3 3.2 55 (14) b <1 43 (13) c 0.3 30 (14) d USA 5 0.477 3.66 (0.9) <0.05 0.759 (0.248) Germany 16 <0.1 18 (1.5) <0.15 5.2 (0.4) <0.10 8.9 (0.7) Concentration range; median in parentheses. b = summer sampling. c = autumn sampling. d = winter sampling. Table 1 2 Concentrations ( g/L) of nonylphenol (NP) and b isphenol A (BPA) in effluents of WWTPs. Location NP (Ying et al., 2002b) BPA Number of Samples Concentration range (median) N umber of Samples Concentration range (median) (reference) Canada 8 0.8 15.1 (1.9) 36 0.07 1.68 (0.41) (Mohapatra et al., 2011). UK 16 <0.2 5.4 (0.5) Switzerland 2 5 11 Spain 3 6 343 Japan 10 0.08 1.24 USA 13 <0.1 37 (<.5) > 50 0.0036 50 ( 0.01) (USEPA, 2010) Germany 16 <0.05 0.77 (0.111) 18 0.018 0.702 (0.03) (Spengl er et al., 2001). Italy 12 0.7 4 (1.8)

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20 EDCs i n Surface Water Retention P onds Residential water retention po nds (present in many residential areas) may be contaminated w ith EDCs from sources like run off and drainage of RW after irrigation and/or EDCs leach ing from septic systems. RW used for irrigating lawns can collect in nearby water retention ponds along with run off from other surfaces. Septic system effluen ts can al so contain EDCs (Table 1 3) (Carrara et al., 2008) that leach through soil (both vertically and horizontally) and pollute nearby water retention ponds. Sandy soils of south Florida may increase leaching of EDCs from septic systems while the underlying car bonates (less permeable) may facilitate lateral movement of septic effluents containing EDCs into the ponds. Unfortunately, no literature is available on concentrations of EDCs in residential water retention ponds. Table 1 3 Concentrations ( g/L) of EDCs reported in s eptic effluents Chemical Number of Samples Concentration range (reference) Estrone 42 > 2 (Sulleabhain et al., 2009) 17 estradiol 42 > 2 (Sulleabhain et al., 2009) 17 ethynylestradiol 26 0.1 0.36 (Stanford and Weinberg, 2010). Bisphenol A 5 0.11 1.7 (Rudel et al., 1998) Nonyl phenol > 25 10 16 (Swartz et al., 2006). Human Exposure S cenario/ Research Problem Use of RW to irrigate turf grass may increase risk of human exposure to EDCs. Dislodgment of EDCs from RW irrigated turf poses a potential pathway of exposure to

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21 humans present in the vicinity of the turf grass. C hildren playing on the contaminated turf could be at particularly high risk from the harmful effects of EDCs exposure. Knowledge of dislodgment rates of a particular chemical f rom this environmental niche is therefore important in assessing exposure of chemicals to humans. The presence of EDCs in resid ential surface water retention ponds is another cause of concern. Water retention ponds may act as a potential source for exposure of EDCs, either directly via contact with water or indirectly through contamination of drinking water sources recharged by th e ponds, or consumption of contaminated fish etc. grown in the ponds. Overview of Human Health Risk Assessment The US Environmental Protection Agency (USEPA, 2004) defines risk as the chance of harmful effects to human health or to ecological systems resul ting from exposure to an environmental stressor, a stressor being any physical, chemical or biological entity capable of inducing an adverse response. Risk is also defined as: Risk= Exposure X Hazard. The risk assessment process involves five principle ste ps: Hazard Identification: Identification of hazard/contaminant, its properties (e.g., form, partitioning in the environment, concentrations in environmental niche of concern, mode of action, behavior in an organism, toxicity etc.) and other potential ad verse environmental and/or human health impacts. Exposure Assessment: Calculation of maximum amounts of the hazard to which an individual or a group of individuals may be exposed. Exposure assessment involves determination of concentration s of a chemical to which an individual can be exposed identification of highly exposed individuals (HEIs) (the population which is

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22 most susceptible to exposure either due to environment in which they live/work or by virtue of their habits, developmental stages etc.), pat hways of exposure (e.g., air, water, soil, food etc.), routes of exposure (e.g., ingestion (food, water and hand to mouth ingestion), dermal absorption, and inhalation), and amount, duration and frequency of exposure. Dose response assessment: Reaction of the target organism, organ, tissue or cell after the hazard reaches inside the target. Dose response assessments involve toxicological and epidemiological studies, plus the use of models to assess adverse response by/effect on the target. Widely used appr oaches for assessment of non carcinogenic effects include: No observable adverse effect levels (NOAELs): Highest dose of a hazard administered without a statistically significant increase in adverse response. Lowest observable adverse effect levels (LOAELs ): Lowest dose of the hazard at which adverse response is significant/ unacceptable. For carcinogens, acceptable risk (level or chance of damage/ adverse effect deemed tolerable by an individual or population. For instance, risk of death in an airplane cra sh is less than 1 in 50,000) is u sed, as there is no threshold exposure. Risk Characterization: Quantification of risk and/or probability of risk in mathematical terms like additional risk to 1 individual per 10 0 ,000 individual s Risk characterization in volves determining risk factors, increased risk populations etc. and answers questions like whether there is considerable risk, how severe the risk is, and the consequences of risk.

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23 Many approaches to risk assessment of environmental chemicals have been p roposed, but the fundamental concept of a risk quotient (RQ) is common to all: RQ = Exposure (Concentration of contaminant that may reach inside a target organism)/Toxicity (NOAEL etc.) The Risk Quotient Method is used by the Office of Pesticide Program s (OPP) and Environmental Fate and Effects Division (EFED) of the US EPA for screening and higher level probabilistic risk assessment. Risk Quotients (RQs) are calculated by dividing exposure estimates by the acute and chronic ecotoxicity values (USEPA, 200 4). RQ values are compared to levels of concern (LOCs) or concentrations of the chemical in question. LOCs are criteria used by OPP to indicate potential risk to organisms and the need to consider regulatory action. The LOC for acute toxicity to mammal s is 0.5 and for chronic toxicity is 1 (USEPA, 2004). If RQ >LOC, then there is a risk of adverse effect from exposure to the chemical in question and a higher level probabilistic risk ass essment is warranted Calculation s of risk quotients are essential to evaluate risk from chemical exposure. Other important criteria used in risk assessment include: Risk Ratio or (Relative Risk)= Incidence of disease in population with risk factor/ Incidence of disease i n population without risk factor. Reference dose (RfD): The RfD is a benchmark dose operationally derived from the NOAEL and used to set regulations for the concentration of a hazard to which an organism can be exposed daily for a long time without signifi cant adverse effects.

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24 As NOAELs and LOAELs are obtained from studies on animals, uncertainty factors (UF, usually 10 1000 times) are used to extrapolate data for humans. Thus, RfD= NOAEL/ UF. Drinking Water Equivalen t Levels (DWELs): DWELs (mg/L) are the maximum safe concentration s of chemical s a person can drink in water, daily, without non carcinogenic adverse effects, over a life time of exposure. DWEL= (RfD* body weight)/ daily water intake. there is no dose response relationship for adverse effects. Conceptually, e ven a negligible dose can result in increased risk of cancer. Cancer slope factors (CSFs or SFs) are used to calculate acceptable/maximum increased risk of cancer via exposure to a carcinogen. A Cancer Slope Factor is an age averaged lifetime excess cancer incidence rate per unit intake of a carcinogen (or unit exposure for external exposure pathways) and is used to convert the intake to a cancer risk (USEPA, 1989). The u nit of CSF i s ( mg/kg day) 1 From CSF one can obtain a risk specific dose (RSD) that defines the acceptable concentration of carcinogen an individual ca n be exposed to ( per day ) with a predetermined acceptable risk of developing cancer. RSD is expressed as risk/CSF. For instance, if the CSF of a chemical is 20/mg/kg day and the acceptable risk of cancer is one in 1 000,000 then RSD= (0.000001/20)=0.05 ng of carcinogen/kg day. If the concentration of carcinogen an individual is exposed to is more than 0.05 ng/kg day fo r 70 years, the additional cancer risk exceeds the acceptable risk. Risk management and communication: Review of risk among peers and development and implementation of necessary steps for risk mitigation. Risk

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25 communication is the purposeful exchange of i nformation about the existence, nature, form, severity, or acceptability of risk to alert the public and/or decision makers of a significant risk or to calm concerns about a small risk perceived as more serious and risk management refers to the actions tak en to prevent or reduce risk thorough exposure co ntrol a nd/or monitoring etc Research Objectives 1) Determine the occurrence of EDCs in reclaimed water and residential surface water retention ponds. Hypothesis: EDCs will be detected in RW and residential su rface water retention ponds, but at very low concentrations (ng/L). 2) Assess the risk associated with exposure of humans to EDCs from direct consumption of RW and pond water, and indirectly from EDC dislodgement from turf irrigated with reclaimed water. Hypo thesis: Risk associated with exposure to EDCs in RW is negligible owing to very low EDC concentrations and minimal exposure. EDCs S elected for Study To address the issues related to EDCs in RW, five EDCs: estrone (E1), 17 ethynyl est radiol (EE2), bisphenol A (BPA) and nonylphenol (NP) were selected. E1 and E2 are estrogenic steroidal hormones naturally produced in bodies of humans and animals. Excreted in urine, t he chemicals are introduced directly into the environment through munic ipal wastewater treatment pl ant (WWTP) effluents (Daughton and Ternes, 1999; Norris, 2007). EE2 is a synthetic estrogenic compound

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26 used in oral contraceptives pills; some passes through the body and may contaminate the environment. BPA and NP are synthetic chemicals used to make plastics and surfactan ts, respectively. BPA belongs to the chemical class diphenylmethane derivatives and bisphenols whereas NP is an alkylphenol. The three steroidal hormones, BPA, and NP were selected because they are amongst the m ost studied EDCs and the relative availability of data on their occurrence and fate in the environment. Various properties of the five chemicals are given in Table 1 4 Estrone Estrone (E1) is a nat urally occurring estrogen produced in the ovaries and the adr enal glands. The International Union of Pure and Applied Chemistry nomenclature (IUPAC name) of E1 is 3 hydroxy 13 methyl 6,7,8,9,11,12,13,14,15,16 decahydrocyclopenta[a]phenanthren 17 one. Estrone is a lso a primary estrogenic component in variety of pharmaceuticals (Petrovic et al., 2004) u sed in the treatment of estrogen deficiency. Incomplete metabolism by the users results in significant concentrations of E1 in wastewater s and surface waters at concentrations greater than 1 ng/L and as high as 80 ng/L with median concentration of less than 10 ng/L (Thomas and Colburn, 1992; Ying et al., 2002a). E1 may be linked to birth defects, infertility, immune system suppression, deformities of reproductive organs, and various other health problems (Thomas and Colborn, 1992). Estrone is the least abundant of the three natural esterogenic hormones; estrone, estradiol and estriol. E1 is important to health because it can be converted to estrone

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27 sulfate, a long lived derivative, which can be converted to the more active estradiol. Estrone sulfate is commonly found in wastewater, primarily from urine. Estradiol Estradiol (E2) is a natural estrogen produced from estrone conversion metabolized by the liver and excreted from the body through urine (Gore, 2007). E2 is the predominant sex hormone found in females and the most abundant of the three natural estrogens. Estradiol is also present in males but at much lower levels. estra 1,3,5(10) triene 3,17 diol. estradiol is more prominent and was used in this study. Estradiol is the most biologically active naturally occurring estrogen. I t is also an active ingredient in many medications for birth control, hormone replacement therapy, infertility treatments and vaginal infections. Adverse effects of estradiol range from minor health problems such as nausea, migraines and dizziness to major health problems such as breast cancer, strokes and heart attacks. E2 has also been linked to birth defects and deformities of the reproductive organs. E1 and E2 are inter convertible and have been reported i n wastewater effluents (Ying et al., 2002a). Eth ynylestradiol Ethy nylestradiol (EE2) is a synthetic, steroidal estrogen commonly used as the active ingredient in birth control medications. The IUPAC name of EE2 is 19 n or pregna 1,3,5(10) trien 20 yne 3,17 diol, and the chemical is synthesized from e strone. With an estrogenic activity of 246 (with respect to 100 for estradiol), EE2 is one of the most potent EDCs found in wastewaters (Balaguer et al. 1999; Pillon et al., 2005). EE2 c oncentrations as high as 42 ng/L with median of less than 10 ng/L have been reported in effluents (Ying et al., 2002a).

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28 Bisphenol A Bisphenol A (BPA), 4,4' (propane 2,2 diyl)diphenol, is used in the production of plastics and related materials. Globally, more than six billion kilograms of BPA are produced annually (Welshons et al., 2006). BPA has been detected at concentrations as high as 4090 ng/L in wa stewater effluents (Deblonde et al., 2011). Median concentration, however, range between 350 400 ng/L (Mohapatra et al., 2011 ; Spengler et al., 2001 ). BPA is considered as an the ice caps of the arctic and a ntarctic circles (Fu and Kawamura, 2010) Concentrations of BPA up to 2550 ng/L have been report ed in US groundwater (Barnes et al., 2008). Chapin et al. (2008) reported no data on human developmental exposure to BPA, but studies with rats suggest that doses of 0.01 0.2 mg/kg/day can cause neural and behavioral changes related to normal sex differences in male and female rats. NOAEL values with respect to reproductive toxicity of BP A on sub chronic or chronic (long term) exposures have been calculated to be 47.5 mg/kg/day in female and 4.75 mg/kg/day in male rats ( Chapin et al., 2008 ). Nonylphenol Nonylphenol (NP), 4 (2,4 dimethylheptan 3 yl)phenol, is an anaerobic breakdown product of the nonionic surfactant nonylphenol ethoxylate (NPEO). The surfactant is widely used in domestic liquid laundry detergents, industrial liquid soaps and cleaners, cosmetics, paints, pesticides and herbicides (A lkylphenols and Ethoxylates Research Council 2001). Nonylphenol occurs in surface water and wastewater samples worldwide in concentrations up to 1 g/L (Blackburn and Waldock, 1995; Ahel et al., 1996; Rudel et al., 1998; Snyder et al., 1999; Potter et al., 1999; and Kuch an d Ballschmiter, 2001; Soa res et al., 2008). Air concentrations of NP range from 2.2 to 70

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29 ng/m 3 (Dachs et al., 1999; and Van Ry et al., 2000) suggesting that volatilization of NP should be considered when characterizing risk. However, the amou nt volatilized is very small (v apor pr essure of NP is ~10 4 atm). Nonylphenol has been implicated as an endocrine disruptor in higher animals (Soto et al., 1991; and Gi meno et al., 1997) and mimic s estradiol by competing for the binding site of the estrogen receptor (Lee and Lee, 1996; White et al., 1994; Ying et al., 2002b).

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30 Table 1 4 Properties of the five EDCs studied. Estrone 17 estradiol ethynyl estra diol Bisphenol A Nonylphenol Chemical Formula C 18 H 22 O 2 C 18 H 24 O 2 C 20 H 24 O 2 C 15 H 16 O 2 C 15 H 24 O CAS Number 53 16 7 50 28 2 77538 56 8 80 05 7 25154 52 3 Molecular Weight (g/mol) 270.37 272.38 296.41 228.29 220.35 Solubility (mg/L) in water (20 0 C) 30 13 4.8 120 270 5 l og Kow 3.6 4.03 3.94 3.67 4.15 3.32 5.76 p Ka 10.77 10.71 10.4 10.3 10.7 Half life in aerobic water (days) 0.1 1.8 0.5 2 17 <2 2.5 10 Photolytic Half life (clear water) (hours) 0.67 2 8 2 8 5 15

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31 Table 1 4. Continued. Chemical Properties Estrone 17 estradiol 17 ethynyl estradiol Bisphenol A Nonylphenol Estrogenic activity w.r.t E2 2.5 E2=100 246 0.66 0.32 Potential toxicity Probable human carcinogen; possible teratogen. Known human carcinogen (CSF 0 =39), human reprod uctive system disruptor. Human carcinogen, human reproductive system disruptor. May increase cancer risk, possible adverse effects on human reproductive system. Possible human reproductive disruptor, possible human neuro disruptor. Major Routes of Expos ure Ingestion, dermal absorption. Ingestion, dermal absorption Ingestion, dermal absorption. Ingestion, dermal absorption. Ingestion, Inhalation, D ermal absorption. Structure CSF 0 = Cancer Slo pe Factor for oral dose (mg/kg day) 1 (Source: The National Library of Science ; Hazardous Substance Database). (Est rogenic activities: Balaguer et al. 1999; Pillon et al., 2005).

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32 Toxicological Data on the Five EDCs Oral acute median effective concentrations (EC 50 rats ) for the adverse endocrine effects of the five EDCs range from around 500 g/kg (Estrogens) to around 2 mg/kg (BPA and NP) in rats (WHO, 2011; National Library of Science, Hazardous Substance Data Base; Spehar et al., 2010). Assuming a safety factor of 1000 the EC 50 values for human toxicity become 500 ng/kg for estrogens and 2 g/kg for BPA and NP. The CSF 0 for oral dose of E2 is 39/mg/kg day ( US EP A, 2009). Bisphenol A does not exhibit significant carcinogenicity ( US EPA, 2010). CSFs and other carcinogenicity data on the remaining three EDCs are not available. Estimated NOAEL/LOAEL values, for humans, of the five EDCs, and uncerta inty factors (UF) are given in Table 1 5. Table 1 5. Human NOAEL /LOAEL (oral dose) values of EDCs Chemical Reference NOAEL (Endocrine)* (g/kg/day) LOAEL (Endocrin e)* (g/kg/day) Estrone Mashchak et al., 1982 4 ---------17 estradiol JEFCA, 2000 5 ---------ethynylestradiol Mashchak et al., 1982 -------0.1 Bisphenol A Tyl et al., 2002 50 ** ---------Nonylphenol Tyl et al., 2006 50** ----------* NOAELs/LOAELs for other toxicities are higher than the values given ** Extrapolated from rats by using UF of 100 for BPA and 30 for NP (Synder et al., 2008).

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33 Research Approach Research was carried out in South Florida during the summer of 2012. Res earch consisted of: Firstly, d etermining the occurrence and concentrations of the f ive EDCs in representative WWTP effluents in south Florida. Secondly, d etermin ing the occurrence and concentrations of the five EDCs in several residential surface water re tention ponds in south Florida via water sampling surveys. Comparisons between occurrence of EDCs in water retention ponds in neighborhoods using reclaimed water for lawn irrigation and in water retention ponds in the vicinity of neighborhoods having septi c systems were also conducted Presence of Coprostanol ( used as an indicator of human contamination in RW and pond water samples) (Mudge and Lintern, 1999; Reeves and Patton, 2005) was also determined in the RW and pond samples to determine human contamination as the likely source of EDCs Third ly, i rrigating St. Augustine turf grass (Stenotaphrum secundatum ( I nternational C ode of N omenclature for algae, fungi and plants ) ) with RW spiked with EDCs and relating dislodgement of the five EDCs from the turf grass to total concentrations of EDCs prese nt on the leaf surface Fourthly, Assessing Risk: The risk quotient method and comparison of exposure content to NOAELs/LOAELs/EC50 of the EDCs were used to determine the risk of human exposure to EDCs present in RW. NOAELs, LOAELs and other data available on toxicity of EDCs along with concentration s dislodged from turfgrass, routes of exposure, pathways of exposure (from RW to turfgrass to skin and/or from RW to

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34 turfgrass to mouth via licking and/or from RW to turfgrass to air to nose) were used to calcul ate risk. NOAELs/LOAELs and RQchronic were used to calculate long term (chronic ) risk whereas EC50 (extrapolated from rats) and RQacute were used to calculate acute risk. Dermal EC50 data for the EDCs are not available, so EC50 values for oral dose were u sed assuming dermal EC50 values would be equal to or less than oral values. Risk to highly exposed individuals (HEI) was estimated Children, because of their small body mass and nature of playing, rolling and/or even li cking the irrigated grass, were cons idered HEIs for risk assessment. Total exposure was calculated using the USEPA Exposure Factors Handbook ( US EPA, 2011) as a guide to calculate amount, duration and frequency of exposure. Risk from direct drinking of secondarily treated RW as well as water from residential pond s was evaluated as a worst case scenario of EDC exposure to humans.

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35 CHAPTER 2 MATERIALS AND METHODS Estrone, 17 ethy ny lestradiol, bisphenol A, nonylphenol, 5 coprostan 3 ol, 17 estradiol acetate, Sucral ose, Bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane ( BSTFA+TMCS ) and Pyridine were purchased from Sigma Aldrich Inc. (St. Louis, MO) Preliminary experiments for c ompound extraction and analysis were conducted. Ten replicates were perfor med using a spiking mixture of estrone, 17 ethy ny lestradiol, bisphenol A, nonylphenol and 5 coprostan 3 ol, in acetonitrile, at 1 g/mL concentration each. For each replicate, a n aliquot (100 L) of the spiking mixture was added to 1 liter of double deionized water s o that the final concentration of each chemical in water was 100 ng/L. An aliquot (100 L) of a standard 1 g/mL 17 estradiol acetate solution was also added to serve as an analytical surrogate. Percent recovery, method detection l imit (MDL), and practica l q uantita tion limit (PQL) were then calculated from the ten replicates after chemical extraction from the double deionized water using methylene chloride. Sites and Site C onditions WWTPs : Effluents were collected from six WWTPs that produce and supply RW i n South Florida. All WWTPs use primary and secondary treatment processes before releasing effluents to be used as RW. In these WWTPs, RW is produced either daily (4 WWTPs) or twice a week (2 WWTPs) and production ranges from 400,000 liters to 11 million li ters per production day. Effluents are chlorinated with 5 15 mg of sodium or calcium hypochlorite per liter.

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36 Retention p onds : Eight retention ponds were chosen in South Florida. Ponds 1, 4 and 5 are in communities that use reclaimed water to irrigate lawn s. Irrigati on usually occurs 2 3 times a week depending upon observed grass requirements and rainfall received. Grass mowing occurs weekly. Pond cleaning did not occur during the survey period, except once for pond 5. For operational and sampling purposes, ponds 1, 4 and 5 were categorized as relatively low (<5 houses around the pond) medium (5 15 houses) and high (>15 houses), respectively, in housing density. Ponds 2 and 3 are surface water retention ponds in the vicinity of public schools using recla im ed water for grass irrigation. The ponds are larger and accumulate greater run off load s than ponds 1, 4 and 5 because of more irrigation and irrigation frequency. Ponds 6, 7 and 8 are in neighborhoods that do n ot use reclaimed water, but are servic ed by s eptic systems, which are expected to affect retention pond water quality via leaching of EDCs. Ponds 6, 7 and 8 are relatively low, medium and high in housing density, respectively. Run off from various surfaces like sidewalks etc. is also collected in the se ponds along with run off from nearby turf. Turf p lots : St. Augustine grass ( Stenotaphrum secundatum ( International Code of Nomenclature for algae, fungi and plants ) ) plots were located at the UF Indian River R esearch and Education Center at Fort Pierce, FL. The plots were mowed 2 days before treatments began (clippings remove d ) to an average height of 3 cm. The experimental area was a n aesthetic lawn irrigated with ground water An average of less than one weed per 2.5 cm 2 and 20 weeds per 1 m 2 (from a tot al of fifty 2.5 cm 2 sections and ten 1 m 2 sections) were present in the experiment al plot. Such low weed densities are indicative of typical St. Augustine grass lawns. Three sets of experiments w ere

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37 performed to study dislodgeable residues of the EDCs from turf (described later) Heavy rain occurred the night before the first set of experiments. It was sunny throughout the time the experiment was conducted and the plot was directly exposed to sun. No stagnant water was detected, but the grass was moist prio r to EDC application For the second and third sets of experiment, weather was clear and sunny throughout, but the grass was moist prior to applications due to morning dew Water Samples O ne hundred and sixteen water samples (28 from WWTPs an d 88 from ponds) were collected and analyzed for the five EDCs and coprostanol. Sample Collection A t otal of 28 effluent samples (four from each of the six WWTP s and four additional samples from one randomly selected WWTP) were collected in two weeks between 10 am and 3 pm during December 2012. The four a dditional RW samples were used as matric spike samples for quality assurance and quality control. One liter samples of RW were collected in one liter amber glass bottles from effluent discharge outlets with extreme caution to avoid contamination. Bottles were capped and put in ice immedia tely after sample collection. A t otal of 88 water samples fr om retention ponds (64 from the various pond s and 24 additional samples from one randomly selected pond) were collect ed in one liter amber glass bottles. Bottles were thoroughly cleaned with Nalgene L900 liquid d etergent (obtained from Thermo Scientific Inc., Waltham, M A) and double deionized water, rinsed with acetone and dried prior to sample collection. The 24 a dditional samples were used as sample duplicate and matric spike samples for quality assurance and quality control during each run Samples were col lected weekly

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38 between 10 am and 3 pm for eight weeks during summer 2012. The sampler was a 3 meter long aluminum shaft, and was used to collect samples from up to 2.5 meter from the side of the ponds, 0.5 meter below the water surface. Each bottle was rins ed with the respective pond water prior to sampling, and the samples were capped and put in ice immediately after collection. Samples were extracted within 4 5 hours after collection. Sample Extraction and Preparation Two extractions per water sample wer e done on automated shakers operated at 120 rpm. Sixty mL methylene chloride was used for each sample extraction Each sample was shaken for 30 min and 20 min for 1st and 2nd extraction s respectively. Methylene chloride extracts were collected in glass fl asks and the volume was reduced in a water bath adjusted at 68.8 0 C and then transferred to graduated glass tubes and completely dried in a Labconco rapidvap (Labconco Inc., Kansas City, Mo) vacuum evaporation system. Compounds in the dried samples were der ivatized by addition of 50 L BSTFA+TMCS (99:1) plus 50 L pyridine as a stabilizer ( Knapp, 1979). Following addition, extracts were covered with parafilm and derivatized for 40 min in the water bath at 68.8 0 C, after which samples were again dried using t he nitrogen gas evaporation system. Hexane (200 L) was added to each sample, thoroughly mixed by shaking and then the extract was transferred to GC analysis vials. The same sample extraction and preparation method was applied for preliminary water experim ents and the RW samples and retention pond water samples. However, the RW and retention pond water samples required an addition step of de emulsification after extraction. De emulsification involved passing the emulsified methylene chloride extract through screening filters (0.2 microns) using syringes, followed by addition of an additional 10

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39 mL of methylene chloride to minimize or reduce chemical loss during filtering. Samples were analyzed for th Coprostanol Dislodged EDCs and EDCs on Grass S urface Preliminary experiments to study the amounts of the five EDCs dislodged from the turf grass were conducted by swiping turf grass from the residential lawns of water retention pond survey sites 1, 2, 4 and 5. Cotton facial pu ffs wetted with double deionized water were used to rigorously swipe the grass and the EDCs from puffs were extracted using 30 mL (two times) methylene chloride in an ultrasonic clean er. Up to 20 ng of E1, 40 ng of EE2 and 100 ng of BPA were dislodged from turf grass. Sample Collection Three sets of experiments were per formed to study EDC dislodgement from turf. The experimental design for first set of experiments was a randomized block, consisting of 5 replicate plots of 2 meter by 1.2 meter each. Another 2 meter by 1.2 me ter plot in the sa me area was used as a control. Two liter s of double deionized water spiked with the five EDCs was used to simulate irrigation of the block. For ease of detection and to simulate a worst case scenario, the upper (higher) reported concentra tions of estrone (80 ng/L), 17 ethy ny lestradiol (42 ng/L) and environmentally relevant values (reported median concentrations) for bisphenol A (400 ng/L) and nonylphenol (400 ng/L) in reclaimed water were chosen as spiking co ncentrati ons for the spraying. Both hand sw ipe method and drag sled methods ( USEPA 1998 ) were used to collect samples for the first set of experiment. The hand swipe method involved use of circular polyvinyl pipes 12.7 cm in radius and 5 cm in height to m ark the sampling area of 506 cm 2 Kendall Excilon (Drain and I.V.) sponges (Covidien Inc., Mansfield, MA) were used as the swiping material. Prior to swiping, each

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40 sponge was wetted with simulated sweat (70:30 v/v Phosphate Buffer: acetonitrile, phosphate b uffer = 0.1M Sodium acid Phosphate). The hand swipe method provided a 2 Sampling was done in zig zag pattern by exerting nearly equal pressure for 3 0 seconds per sampling area. In the drag sled method sleds consisted of baking pa ns (19.05 cm*19.05 cm e ach). Un dyed cotton denim cloth was attached at the bottom of the pans, and was used for sampling. Sand bags (13.6 kilograms) w ere used as weight over each sled (approximating the weight of a 3 4 year child). Sleds were dragged in t he irrigated replicates to cover an area of ~ 553 cm 2 The sled was moved forward 10 cm, then back 10 cm and then forward 10 cm again. R eclaimed water w as the sole source of irrigation and an irrigation depth of 2 cm was applied. T he volume of water requir ed to irrigate the plot area of 2 meter 5 (replicates) 1.2 meter per application was calculated to be 240 L. Since 1 L reclaimed water (RW) contain ed 80 ng of estrone, 240 L RW contain ed 19 2 00 n g. S imilarly, 240 L RW contain ed 15400 ng of 17 estradiol 10100 ng of e thynylestradio l, and 96000 ng each of BPA and NP (Table 2 1). Initial masses of EDCs (EDCs applied) present per sampling area of 506cm 2 for hand swipe and 553 cm 2 for drag s led method s are given in Table 2 1. Table 2 1 Masses of chemicals applied per sampling area. Chemical Mass per sampling area of 506 cm 2 (hand swipe) Mass per sampling area of 553 cm 2 (drag sled) E1 81 ng 89 ng E2 65 ng 71 ng EE2 43 ng 47 ng BPA 405 ng 442 ng NP 405 ng 442 ng Pl ot area=2*100*1.2*100*5=120000 cm 2

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41 Two liters of double deionized water was spiked with 19200 ng estrone, 15400 ng 17 ethynyl estradiol, 96000 ng BPA and 96000 ng NP and sprayed uniformly over the entire plot with a compressed CO 2 backpack sprayer, adjusted to 207 kilopascal pressure and yielding an average sprayer flow rate of 25 mL/second (n=10). Application was done at a uniform height of 0.5 m above the ground. The control plot was sprayed with double deionized water. Spraying was done in the morning around 8 a.m. Samplings were conducted before spraying, immediately after spraying, 4 hours after spraying, 8 hours after spraying and after rewetting the plot with double deionized water 8 hours after spraying. One sample per replicate per sampling was collected for each method in 250 mL glass bottles. Bottles were ca pped air tight immediately after sample collection and placed in ice before extraction. Results using the hand swipe method were inconsistent and highly variable, possibly due to the inconsistent pressure with which the grass was swiped. Due to wide differences in results and inability t o set a standard pressure, the hand swipe method was determined to be unsuitable for measuring amount of EDCs dislodged from turf grass, and results will not be discussed. The remaining two sets of experiment were conducted using only the drag sled metho d with slight modifications to obtain more reliable results. The same plot size (2 X 1.2 meters) and number of repli cates (5 + Control) were used. The s ame drag s led method was followed. H owever, the area sampled per sampling was increased to 844 cm 2 to co llect more d islodged mass and to improve detection reliability Experiments were conducted on two consecutive days. Actual RW (instead of double deionized

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42 water) was spiked with EDCs and applied to simulate real irrigation conditions A portion of RW (1 L) was analyzed for EDC concentrations and the concentrations were found to be less than 20 ng/L for steroidal hormones and less than 60 ng for BPA and NP. The spiked EDC concentrations were adjusted accordingly. The backpack sprayer was adjusted to yield m aximum possible droplet size to better simulate lawn sprinkler droplet size. Water droplets from sprinklers, by virtue of their large size and high release pressure may displace prior water droplets present on the turf grass. This may result in decre ased p otential for EDC dislodgement as the EDC in the displaced droplets may be sorbed to soil or grass thatch The average sprayer flow rate was calculated to be 48 mL/sec (n=10). Initial masses of the EDCs for sampled area of 844 cm 2 were calculated to be 135 ng for estrone, 108 ng for 17 ethy ny lestradiol, 675 ng for BPA and 6 75 ng for NP. Sucralose (142200 n g) was also added to the spiking mixture to obtain a mass of 1000 ng for sucralose per sampled area. Sucralose was added as a co nservative tracer to check the method and its reliability. Sucralose has a half life on the order of months and is relatively resistant to degradation (Soh et. al., 2011) and thus can be used to check the method as sucralose detection by GC MS if the EDCs are not detected suggest discrepancies in the method. Samples were collected before spray ing immediately after spray ing and 2 and 4 hours after spray ing. Additional samplings occurred following rewetting of grass with double deionized water S amples were collected 2 hours after rewetting (6 hours after initial spray ing ) and 4 hours after rewetting (8 hours after initial spray). Subsequently, the p lot was again rew etted with double deionized water 8 hours after the initial spray ing, and a last set of sampl es w as collected.

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43 Along with dislodged residue samples, grass clippings were collected from each replicate before the initial spray ing immediately after initial spray ing and before the first and second rewetting s. For each grass sampling grass clippings (0.5 0.7 cm above the soil) were collected from 506 cm 2 area which corresponded to thirty grams (301 g) of grass (area to weight calculations determined from more than 200 samples). Sample bottles were capped and placed in ice immediately after sample c ollection. Initial masses of EDCs applied to 506 cm 2 of sampling area were calculated to be 81 ng for E1, 65 ng for E2, 43 ng for EE2, 405 ng for BPA, 405 ng for NP and 600 ng for sucralose. Samples were stored in a refrigerator for 8 10 hours before extr action. Sa mple Extraction and Preparation For dislodged residue samples, two extractions per sample were performed using methylene chloride. 150 mL and 30 mL methylene chloride we re used per extraction for drag sled and hand swipe methods, respectively. E xtractions were done in an ultrasonic clean er ( 15 3 35 222, Fisher Scientific Waltham, MA) 20 min for first extraction and 15 min for second extraction per sample. After extraction, the methylene chloride extracts were collected in glass flasks and the vo lume was reduced in a water bath adjusted at 68.8 0 C. The r est of the extract preparation procedure was similar to that used for the water sample preparation. A d ifferent method was used to extract EDCs on the surface of grass clipping s since methylene chl oride or any other organic solvent to extract EDCs would have resulted in extraction of interfering compounds from the leaves (e.g. pigments, lipids, etc.). Modification of a method developed by Iwata et al. (1977) was used to determine total available E DC residues on foliage. D ioctyl sulfosuccinate sodium salt (96%) w as

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44 purchased from Sigma Aldrich Inc. (St. Louis, MO) and 1 gram was dissolved in 49 mL of double deionized water to get a 1:50 dilution of salt (Iwata et. al., 1977). One L of double deioni zed water was added to 30 grams of grass clippings coll ected in glass jars. Thirty drops (~ 6 mL) of diluted dioctyl sulfosuccinate sodium salt were added to the bottle. Bottle ingredients were rigorously mixed for an hour using a vortex mixer. Solid phase extraction (SPE) was used (instead of liquid liquid extraction) to extract sucralose from water along w ith the EDCs. Because of the greater solubility of sucralose in water than in organic solvents, liquid liquid extraction was not suitable for sucralose extraction. Samples were then subjected to solid phase extraction using 500 mg/ 6 mL Enviro C lean ECDVB 156 extraction cartridges (UCT Inc., Bristol, PA) The ECDVB 156 c artridge is capable of extracting both hydrophilic and hydrophobic chemicals. Each ext raction c artridge was placed over each port of a 12 port vacuum manifold. The vacuum pressure was set at 25 bars, and the system was made air tight. C artridges were cleaned twice with 6 mL (each time) of acetone. Water from each glass bottle containing EDC extracts was then passed through the respective extraction cartridges The system was allowed to run for 5 hours to remove all water from the extraction cartridges Extraction c artridges retai ned the EDCs, whereas water passed through the vacuum and colle cted in a waste bottle attached to the vacuum. When the system was stopped, graduated concentration tubes were placed inside the manifold such that each extraction c artridge corresponded to a single concentration tube. Vacuum was run again. The extraction c artridges were washed three times with 6 mL ( each time ) of methylene chloride. The methylene chloride (containing the EDCs) was collected in concentration tubes until the SPE cartridges appeared to be completely dry.

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45 Methylene chloride was then completely dried in a Labconco rapidvap vacuum evaporation system (Labconco Inc., Kansas City, Mo) derivatized and prepared for GC MS analysis following the same method used for dislodged residues. Percent recovery, method detection limit (MDL), and practical quant it ation limit (PQL) were calculated prior to the analysis of actual samples. Sample Analysis All samples were analyzed using GC MS consisting of an Varian 3800 gas chromatograph conn ected to Varian 4000 ion trap mass spectrometer with an electron ionizat ion (EI) source (V arian, USA). Ionization energy was 70 eV electron multiplier voltage was 1.19 kV, and ionization temperature was 200 C. The target compounds were separated on a capillary column Rxi thickness) (Restek C o., Bellefonte, PA, USA). Helium was the carrier gas and was maintained at a constant flow rate of 1 mL min 1 The temperatures of the trap and injected in splitless mode at a n inlet temperature of 280 C. The column oven temperature program was as follows: the initial temperature of 130 C was set and then in creased at a rate of 10 C min 1 up to 280 C. The final temperature was held for 5 min. The temperature of the transfer line was 270 C. The solvent delay was set to 5 min. The characteristic ion fragments of the analytes were selected for q uantification and confirmed in selected ion monitoring mode. The GC MS was checked for air water in the system c alibration gas level s, trap frequencies, and was used for analysis after it passed the auto tune check. Retention times and monitored ion fragments ( m/z ) ratios of the five EDCs analyzed us ing GC MS are shown in Figure 2 1.

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4 6 Fig ure 2 1. Retention times and m/z ratios of EDCs analyzed using GC MS. Quality Assurance/Quality Control (QA/QC) Water s amples : For QA/QC purpos e, 4 samples per sampling event were collected from one of the WWTPs and 4 from the retention pond site 2 (one sample, one sample duplicate, one matric spike s ample and one matric spike sample duplicate). An aliquot (100 L) of the spiking mixture (1 g/ mL) was added to the matric spike sample and the matric spike sample duplicate. An aliquot (100 L) of stock solution of 17 estradiol acetate (1 g/mL) was add ed to each sample as an extraction surrogate to maintain extraction process quality as severe reduction or increase in surrogate recovery indicates something wrong with the extraction procedure. Method blanks were run along with the samples each time. Two addition al 1 L double deionized water samples spiked with the spiking mixture (1 g/ mL) at concentrations of 45 ng/L and 100

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47 ng/L were also run each time to check for contamination/problem s in extraction or GC MS analysis processes Grass s amples : Sample s were collected before application A control plot was sampled along with each sampling of spiked plot s Method blanks and additional spiked samples (at masses of 45 ng and 100 ng ) were run with each set of samples. An aliquot (100 L) of stock solution o f 17 estradiol acetate (1 g/mL) was added to each sample as a surrogate to check the extraction process Sucralose was added as a conservative tracer to check the method and its reliability. Method Detec tion Limit Practical Quantit ation Limit and Percen t R ecoveries Method detection limit (MDL ), practical quantification limit (PQL ) and percent recoveries of the five EDCs, E2 acetate, coprostanol and sucralose for water samples, dislodged residue samples and grass clippings are given in Table 2 2. MDLs and PQLs were calculated from the 10 replicates using EPA method (40 CFR 136, 2013 ). Reduced recoveries of EDCs from spiked water samples, as compared to those from double deionized water, may have been due to matrix hindrances against extraction such as sorp tion of spikes onto colloidal organic matter, chemical reactions between spiking mixture and RW and the extra filtration of the samples to break emulsions when in RW R 2 values for the calibration curves, for all samples, were greater than 0.99.

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48 Table 2 2 MDL, PQL and percent recoveries of the EDCs, E2 acetate, coprostanol and sucralose from different methods used. Water Samples Dislodged Residues Grass Clippings MDL (ng) PQL (ng) % recovery MDL (ng) PQL (ng) % recovery Denim (Methylene Chloride) MDL (ng) PQL (ng) % recovery Surfactant Double deionized water Spiked sample E1 5 20 85 63 5.6 21.8 73 6.7 28.7 81 E2 7.3 29 96 70 7.5 30.2 71 5.8 30.1 79 EE2 7.3 29 98 72 7.7 29.8 75 5.4 29.3 83.5 BPA 7.7 31 98 74 7.2 26.7 79 7.3 31.2 92 NP 4.9 19 95 69 6.3 22.5 80 7.9 31 88 E2 acetate 7.4 30 97 67 7.4 30.5 67 7.8 25.7 83 Copr ostanol 5.7 23 87 66 ---------------------------------------------------Sucralose -----------------------5.8 23.8 82 5 21.9 94

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49 CHAPTER 3 RESULTS AND DISCUSSION EDCs in Water Samples Detection and concentrations in RW and p onds : A ll five EDCs were detected in RW and ponds at various times during the survey. BPA was detected in greater amo unts than the other EDCs (Table 3 1, Figure 3 1 ) and with the greatest frequencies (> 96% for RW and 76% for all ponds Figures 3 2 and 3 3. ). Co ncentrations of all EDCs and coprostanol (not corrected for recovery percentages) were significantly lower (t test, in ponds near RW use than in reclaimed water samples indicating attenuation (degradation, dilution or adsorption ) during drainage and/or runoff of RW (Figure 3 1). Results suggest that the irrigation of turf grass with RW can play an imp ortant role in the transport and accumulation of EDCs in surface water ponds The concentrations of EDCs were statistically similar in the water retention ponds 1, 4 and 5 suggesting housing density does not play a major role in the occurr ence of EDCs in the retention ponds. Concentr ations of EE2, BPA and c oprostanol were significantly higher (t in retention ponds near homes with septic systems than near homes using RW for turf irri gation (Figure 3 1 Table 3 1 ) and were comp arable to those in RW consistent with less removal of the chemicals in septic systems than in WWTPs, or contributions from other ( unidentified ) sources. BPA concentrations were more variable than other EDCs in all water types (i.e, RW, ponds near RW use a nd ponds near septic systems). Higher variability in BPA concentrations is in agreement with literature on the occurrence and fate of BPA (Belfroid et al., 2002), and may be attributed to variable initial concentrations in wastewater du e to variable use in households

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50 Figure 3 1. Concentrations of EDCs and coprostanol in RW (n=28) ponds near RW use (n=64) and ponds near septic systems (n=24) Figure 3 2. Frequency of d etection of EDCs and copr ostanol in RW (n=28)

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51 Figure 3 3. Frequency of Detection of EDCs and coprostanol in all p onds (n=88)

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52 Table 3 1. Concentrations (SD) of EDCs in RW (n=28), ponds near RW use (64) and ponds near septic systems (n=24). Avg. conc. in RW (ng/L)SD Highest conc. in RW (ng/L) Avg. conc. in Ponds near RW use (ng/L)SD Avg. conc. in Ponds near septic systems (ng/L)SD Highest conc. in Ponds (all) E1 2315 47 14*10 15*10 35 E2 23*18 51 8*7 22*14 34 EE 2 28*14 49 16*13 3315 58 BPA 6731 138 3193 7398 303 NP 2915 52 7*14 14*18 46 *= concentration more than MDL but less than PQL. SD= Standard Deviation. Table 3 2. Median concentrations of EDCs obtained from the study of RW and concentrations rep orted in literature. Chemical Median concentration found (ng/L) Number of Samples Median concentration reported (ng/L) E1 26 104 <10 (Ying et al., 2002a) E2 26 185 <14 (Ying et al., 2002a) EE2 31 104 <10 (Ying et al., 2002a) BPA 57 104 350 400 ( Mohap atra et al., 2011 ; Spengler et al., 2001) NP 31 50 4 00 500 ( Ying et al., 2002b ) *= Median more than MDL but less than PQL

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53 Comparison to Published Data All EDC concentrations were in ng/L. The m edian concentration of E1 was higher than reported in literat ure. Median concentrations of E2 and EE2 were similar to literature values, wherea s those of BPA and NP were less than the published data (Table 3 2). Also, t he range s (Tables 1 1, 1 2 and 3 1) were similar to peer reported concentration range s The concen trations of EDCs obtained in this study are thus, in general agreement with and are representative of p reviously published data Likely Sources 5 Coprostanol was detected in more than 90% of the RW and 75% of all residential pond samples, suggesting human contamination in both RW and water retention ponds (Mudge and Lintern, 1999; Reeves and Patton, 2005). Human wastes in wastewater collected from houses and treated in WWTPs are the most likely source of EDCs in RW as WWTPs cannot fully remove such or ganic chemicals (Roberts and Thomas, 2006). Run off and drainage of contaminated water from RW irrigated turf to nearby retention ponds likely contributes to detection of EDCs in retention ponds. Similarly human wastes directed to septic systems are the likely sources of EDCs in retention ponds in the vicinity of neighborhoods using septic systems. Wastes from septic systems contain E DCs (Carrara et al., 2008), which could leach from the septic drainage fields consisting of high permeability sandy soils. I mpermeable spodic and argilic horizons as well as bed rock materials below sandy surface layers could enhance lateral movement of the EDCs to retention ponds. Possible Fate(s) of EDCs Adsorption: EDCs from RW strongly adsorb on organic components of soil s (when sprayed on land), on grasses (when irrigated) and on sediments (when

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54 discharged to surface water). EDCs in ponds may be bio accumulated by fishes and adsorbed on sediments. Degradation: Half lives of the five EDCs, especially photolytic half lives are on the order of a few h ours (Table 1 4) suggesting the possibility of rapid degradation. Frequent use of RW for turf irrigation and its run off / drainage however, results in pseudo persistence (maintenance of concentrations of otherwise quickly and /or easily degradable chemicals due to continuous release) of EDCs in surface water retention ponds. Similarly, the c ontinuous leaching of EDCs from septic tanks could maintain chemical presence in nearby retention ponds. Contamination of groundwater : W ater containing EDCs, especially from retention ponds, may leach to the groundwater and/or contaminate the drinking water supply. The expected large dilution of leachate by uncontaminated groundwater however, likely reduces EDC concentrations below detect ion limits. Human exposure: Humans can be exposed to EDCs present in RW either directly (e.g. voluntary/involuntary consumption, swimming/ bathing etc.) or indirectly (e.g. eating contaminated fish grown in ponds etc ). Results confirm that EDCs are pre sent in RW as well as residential surface water retention ponds and that attention to their fates and human risk assessment is appropriate. By association, turf grass irrigated using RW is expected to come in contact with EDCs present in the water. Evaluat ion of the potential exposure to humans in contact with the grass following irrigation is warranted.

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55 EDCs in Dislodged Residue Samples and Grass Clippings Detection and masses dislodged via drag sled method : Residues of all five EDCs were dislod ged from t he turf grass using the drag sled method. Average masses (not corrected for percent recoveries) of EDCs dislodged from the three sets of experiments (set 1 extrapolated to 844 cm 2 ) with time are shown in T able 3 3 and Figure 3 4 B Dislodged masses of E1, E2 and EE2 were very small (fe w ng) immediately after application a nd were below detection limits 4 hours after irrigation. Dislodged masses of BPA and NP were significantly greater and disappeared more slowly and uniformly (Table 3 3. ). Masses of all EDCs were, however, belo w MDLs 6 hours after irrigation All EDC masses before spraying and from control plots were below MDLs. The dislodged EDC masses decreased rapidly after spraying for E1, E2 and EE2 but changed more slowly for BPA, NP and sucralose with t ime (Figure 3 4 B ). BPA masses (St. Dev. 43) showed greater variability among replicates than masses of the other EDCs. More uniform dislodg ement of BPA, NP and sucralose with time may be attributed to their high initial masses, high water solubility o f BPA (270 mg/L) and sucralose (283 g/L ) and very low l og Kow of 1.00 ( USEPA, E stimation P rogram I nterface S uite) of sucralose. Greater half lives of sucralose (up to months) and NP (up to 10 days) likely also contributed to the more uniform dissipation o f these EDCs as compared to E1, E2 and EE2. The masses of EDCs dislodged via the drag sled method from set 1 (extrapolated to 844 cm 2 ), set 2 and s et 3 were statistically similar (ANOVA, suggesting reliability and consistency of the sampling method. No outliers were detected among the

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56 Rewetting of grass with double deionized water had no significant effect on the dis lodgment of the EDCs (Table 3 3 ). Dislodged masses of sucralose increased after the first rewetting but not after second rewetting The i ncrease in dislodged masses of sucralose after rew etting can be explained by its high water solubility (283 g/L) and su ggests that sucralose dislodgement depends upon availability of water on the leaf surface. Sucralose was detected in relatively high er amounts than other EDCs in samples collected at various times conf irming method reliability. Table 3 3. EDC and sucralos e masses (ng) dislodged, via drag sled, with time. E1 E2 EE2 BPA NP Sucralose Before application <5.6 <7.5 <7.7 <7.2 <6.3 <5.8 Immediately after application 21*10 399 8*12 23043 30313 63821 2 hours after application <5.6 12*8 < 7.7 7 97 19217 39121 4 hours after application < 5.6 < 7.5 < 7.7 535.5 9112 13027 After I st rewetting < 5.6 < 7.5 < 7.7 3310 9*15 14920 6 hours after application < 5.6 < 7.5 < 7.7 < 7.2 < 6.3 455 8 hours after application < 5.6 < 7.5 < 7.7 < 7.2 < 6.3 < 5.8 Af ter 2 nd rewetting < 5.6 < 7.5 < 7.7 < 7.2 < 6.3 < 5.8 *= above MDL but below PQL.

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57 Figure 3 4 EDC mass es (ng) after spraying with time (hours). A) On grass. B ) D islodged A B

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58 Masses of EDCs present on th e grass surface als o decreased with time (Figure 3 4 A ) and followed similar trend s as the masses dislodged (Figures 3 4 B ). E1, E2 and EE2 were detected on grass (not disl odged) four hours after application but at masses near MDLs. None of the five EDCs were detected on grass eight hours after application Figure 3 5 shows percent ages of EDC masses present on grass that dislodged at different times after application The p ercent ages of EDCs present on grass that actually disl odged immediately after applic ation were 50% for E2, 69% for BPA, 51% for NP and 80% for sucralose. Percentages are consistent with the respective m asses applied, l og Kow values of the EDCs and leaf area considerations. More initial mass/concentrati on of the chemical and smaller l og Kow values resulted in greater percentage s of the chemical s present on grass that was dislodged. Percentages of E1 (26%) and EE2 (19%) dislodged immediately after irrigation were much lower than other EDCs which suggesting less dislodgement than other EDC s The percent of sucralose dislodged decreased substantially to 26% four hours after application This sharp decrease suggests that dislodgement of sucralose dep ends on availability of water. The amount dislodged decreases significantly once the grass dri es as sucralose adheres more strongly to the leaf surface.

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59 Figure 3 5 Percent of EDCs and sucralose dislodged from grass surface with time (hours). Human Health Ri sk Assessment Risk was assessed using maximum EDC concentrations and masses ( corrected for percent recoveries ) obtained in the study Water Samples Worst case c ontaminated water used for drinking : To predict an extreme case of possible exposure, assume th at the water containing highest concentrations of EDCs found (from RW and ponds combined) is used as drinking water Drinking water equivalent levels (DWELs) for estro ne, 17 ethynyl estradiol, bisphenol A and nonylphenol are given in Table 3 4 (USEPA, 2012 b ). Maximum chemical concentrations and the safety factor s for non carcinogenic adverse effects are also shown. Safety factors represent the ratio of mass of chemical consumed when chemical concentration is at DWEL to mass consumed when chemical concentration is

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60 the maximum value found, and were calculated by dividing DWEL by maximum EDC concentration in water DWELs for carcinogens, assuming a 70 kg pe rson consumes two liters of water daily for 30 years over a 70 year lifetime can be estimated using the formula ( Synder et al., 2008 ): DWEL (g/L)= (acceptable risk X 70 kg X 70 yr X 1000 g/ mg)/ (CSF (/mg/kg day) X 2 L/day X 30 yr). (3 1) CSF for E2 is 39/mg/kg day. Assuming an acceptable risk of one in 100,000 (10 5 ), DWEL for carcinogenic effects of E2 becomes= (10 5 X 70 X 70 X 1000)/ (39 X 2 X 30) = 0.021 g/L. The h ighest concentration of E2 found in water samples was 0.073 g/L, approxi mately 3.5 times more than DWEL The data in Table 3 4 suggest that using water with such high EDCs concentration s for drinking purposes is not l ikely to cause non carcinogenic adverse effects e xcept for 17 ethynyl estradiol The safety factors and carcinogenic dose (for E2) were generated using the highest obtained concentrations of EDCs. Further, DWELs are calculated for continuous consumption o f a chemical in water, on daily basis, for 70 years (or 30 years for carcinogenic DWELs) S econdarily treated RW and/or retention pond water is not used for drinking and the incidence of even accidental drinking of the water is rare. Moreover, drinking wat er is more highly treated and concentrations of the EDCs are very low (e.g., <0.00 1 g/L f or EE2) (Synder et al., 2008), suggesting minimal non carcinogenic as well as carcinogenic (for E2) effects.

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61 Table 3 4 DWELs and safety factor of EDCs when consumed in drinking water. Dislodgeable Residues Exposure The greatest masses of EDCs were dislodged immed iately after application (Table 3 3 ). Maximum mass dislodged via drag sled method was 33 ng for E1, 51 ng for E2, 25 ng for EE2, 296 ng for BPA and 323 ng for NP; from an ar ea of 844 cm 2 Masses corrected for percent recoveries become 45 ng for E1, 72 ng for E2, 33 ng for EE2, 370 ng for BPA and 403 ng for NP. Values extrapolated to a household turf grass area of 100 m 2 become 53 g for E1, 85 g for E2, 39 g for EE2, 438 g for BPA and 478 g for NP. Assume a 3 year old child weighing 13.6 kg plays on contaminated turf (immediately after RW application) for an hour (USEPA, 2011) and d ra gs on the contaminated grass an average of 20 times covering an area of 2000 cm 2 each t ime. Further, assuming that the entire body of the child is exposed to the EDCs and all the EDCs are retained on the skin, t he total dislodge able masses of EDCs the child contacts become 2133 ng of E1, 3412 ng of E2, 1564 ng of EE2, 17536 ng of BPA and 19 100 ng of NP. Further, assume that only 5% of the adhering EDC masses are Chemical DWEL (g/L) (non carci nogenic) Max. conc. found (g/L) (corrected for recovery) Safety Factor E1 0. 46 0.075 6 E2 1.8 0.073 25 0.021 (carcinogenic) 0.3 EE2 0. 0 035 0.081 0. 04 BPA 1800 0.409 4400 NP 1800 0.075 24 000

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62 absorbed into the f exposure (Monteiro Riviere et al., 2000; Demierre et al ., 2012; Kao and Hall, 1987). Doses of th e EDCs become 107 ng for E1, 171 ng for E2, 78 ng for EE2, 877 ng for BPA and 955 ng for NP. NP has an additional route of possible exposure Inhalation. On an average, a 3 year old child inhales 10.1 m 3 of air/day ( USEPA 2011). Playing for an hour in the contaminated lawn, the child will inhale about 0.5 m 3 of air. Air concentrations of NP range from 2.2 to 70 ng/m 3 (Dachs et al., 1999; and Van Ry et al., 2000). Assuming a concentration of 70 ng/m 3 in the air surrounding the lawn, the child can inhale about 35 ng of NP through air. Thus, t he to tal dose for NP becomes (955+35=) 990 ng. T he child may also ingest 10 0 mg /day (around 4 mg/ 1hour) of the soil and dust ( USEPA, 2011). Child may ingest EDCs via licking contaminated grass and/or hands/ fingers. If 0.001% of the ingested material is EDC ( as mass of EDCs is g/ several hundred kg of turf negligible EDC volatilization and there is no direct ingesti on of grass or the soil below grass), 40 ng of each EDC will be ingested. The tot al dose of EDCs, thus, become 147 ng of E1, 211 ng of E2, 1 18 ng of EE2, 917 ng of BPA and 1030 ng of NP. Assuming an EC 50 of 500 ng/kg (6800 ng/ 13.6 kg) for E1, E2 and EE2; and 2 g/kg (27 g/ 13.6 kg) for BPA and NP, RQs (Dose/ EC 50 ) for acute endocrine related risks can be calculated ( Ta ble 3 5 ) RQs from chro nic exposure (Table 3 5 ) were calc ulated using NOAEL/LOAEL (X 13.6 kg) values. Dose was multiplied by an additional factor of 10, where LOAEL data was used (Dose/NOAEL or Dose X10 /LOAEL).

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63 Table 3 5 RQs for the five EDCs: Acute and c hronic toxicities. Ch E1 E2 EE2 BPA NP RQ acute 0.0 22 0. 0 31 0. 01 7 0. 0 34 0.0 38 RQ chronic 0.0 0 3 0.0 0 3 0. 87 0.0 0 13 0. 00 15 RQ values for acute exposure are less than LOC of 0.5 and RQ values of chronic exposure are less than LOC of 1 for all five EDCs, suggesting minima l acute or chronic risk associated with the EDCs masses that dislodged in this study. NOAEL/LOAEL and EC 50 values (endocrine related toxicity) are based on oral dose; thus the actual RQ values (from residues absorbed by skin) may be significantly less Mo reover the turf is irrigate d two times (most cases) a week For carcinogenic effect of E2, CSF 0 for E2 is 39/mg/kg day and risk specific dose for a cancer ris k of 1 in 100,000 is 0.26 ng/kg day or 3.54 ng/ 13 .6 kg day. Considering that the lawn is irrigate d twice every week, the per day E2 mass decreases to 36 ng and the dose becomes 2.7 ng/ 13.6 kg day. Thus, the dose of E2 is less than risk specific dose for cancer development if the acceptable risk is 1 in 100,000. The risk associated with dislodgement o f these EDCs is further reduced by the fact that it is not likely that the child plays in ju st irrigated (wet) turf. A s the child ages, his/her body weight increases, soil/dust ingestion decreases and habits suc h as rolling /dragging on wet grass likely de crease; decreasing the masses of EDCs to which he/she is exposed. The dislodged masses extrapolated to an area of 100 m 2 were used to calculate risk from an extreme case scenario (whole body of a 5 year old child is exposed to all the EDC mass available i n 100 m 2 of contaminated turf just after irrigation). Based on very low concentrations of EDCs (few g/100 m 2 ), the child is likely to come in contact

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64 with very small amounts of EDCs (assuming mass of 100m 2 grass+ upper 0.5 cm of soil (with bulk density of 1.33 g/cm 3 ) to be 670 k g (mass of 0.5 cm of soil=~666 k g), percent of 100 g of EDC, that a child can contact, would be less than 10 8 % on mass basis). If the child comes in contact with the entire 100 m 2 area and 1% of dislodged EDCs present in that are the total EDCs to which a 3 year old child could be dermally exposed become 530 ng of E1, 850 ng of E2, 390 ng of EE2, 4380 ng of BPA and 4780 ng of NP. If actually absorbed by the s kin, EDC doses become 27 ng for E1, 43 ng for E2, 20 ng for EE2, 219 ng for BPA and 239 ng for NP. If 40 ng of each EDC is ingested t hrough licking grass or contaminated hands/fingers (assuming 4mg/hour of ingestion in contaminated area and 0.001% of the i ngested material is EDC) and 35 ng of NP is inhaled, the total dose of EDCs would be 67 ng for E1, 83 ng for E2, 60 ng for EE2, 259 ng for BPA and 314 ng NP. These values are even lower that those calculated for exposure to dislodged residues and represent vanishingly small risk. Assessment of Combined R isk (E2 Equivalent) from the five EDCs For calculations of human risk from combined exposure to the five EDCs the concentrations and masses of EDCs were converted to E2 equival ents (Table 3 6 ) and summed. DWEL, NOAEL and EC 50 values for E2 were then used to calculate risk. Water s amples Maximum c o ncentrations of EDCs in water (Table 3 4) when converted to E2 equivalents and summed become 1724 ng. Th e DWEL for E2 is 1800 ng (Table 3 4 ). The concentratio n of E2 equivalents in water is less than DWEL, suggesting minimal

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65 risk of combined exposure from the five EDCs from consumption of contaminated water. RW may contain numerous other EDCs but their concentrations are likely to be similar to the ones studie d. Moreover, EE2 is one of the most potent EDCs present in RW (Clayton 2011; Thorpe et al., 2003). T he risk associated with the five EDCs was so small that it is unlikely that the risk from other EDCs would be considerable. Table 3 6 Relative potencies o f the five EDCs ( Clayton 2011; Thorpe et al., 2003). EDCs Potency E1 0.4 E2 1 EE2 20 BPA 0.0002 NP 0.0000045 Dislodged r esidues Ma ximum masses of EDCs dislodged (Table 3 3) correc ted for percent recoveries become 45 ng for E1, 72 ng for E2, 33 ng for EE2, 370 ng for BPA and 403 ng for NP. C onverted to E2 equivalents and summed the combined E2 equivalent mass become 750 ng. The total E2 equivalent exposure for a 3 year old child playing in recently sprayed grass (assuming the child drags in the con taminated turf 20 times, covering 2000 cm 2 each time ) becomes 35.5 g. Assuming that entire body of the child is dir ectly exposed to the EDCs, the E2 equivalent exposure becomes is, thus, 35.5 g. If 5% of the EDC, the child is exposed to, is absorbed by the E2 equivalent dose becomes ~ 1800 ng. Adding 857 ng for EDCs ingested with soil and dust (40 ng for each EDC converted to E2 equivalent and summed ), to tal dose would be ~26 60 ng. The EC 50 for E2 is 500 ng/kg ( 6800 ng/13.6 kg ) and NOAEL is 5000 g/kg/day ( 68000 g/13.6 kg/day ) The RQ acute becomes (2660/6800=) 0.39 and RQ chronic becomes (2660/68000=) 0.039

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66 The RQ for acute toxicity is less than the LOC of 0.5 and RQ for chronic toxicity is less than LOC of 1, suggesting minimal risk ( on E2 equivalent basis) from combine d masses of the five EDCs present in RW used for irrigating turf. Carcinogenic effects from long term exposure to these EDCs (when a person is exposed to several EDCs), however, may become a concern. Risk Comparison to P ublis hed Data Concentrations used for risk assessment were towards the upper end of the concentration range for the EDCs reported in the literature Results for risk assessment from human exposure to RW as well as dislodged residues of the EDCs, thus, represent a case where EDC concentrations are greater than reported me dian (Tables 1 1, 1 2 and 3 2) concentration s of the five EDCs. T he risk associated with human exposure to EDCs at environmen tally relevant concentrations should be minimal. Uncertainty A person can be exposed to numerous EDCs from numerous sources, both indoors and outdoors, which may increase the risk of adverse effects A lso, the cumulative effect of various EDCs on humans is largely unknown. The c ombined effect of numerous EDCs may be more dra stic (synergistic) than the effect from a single EDC. Ingestion (via food and/or drinking water), indoor and outdoor inhalation, and dermal absorption of hundreds of c hemicals (some of which are more toxic and/or carcinogenic than studied) used in everyday life could significantly increase the risk. Further, risk assessment is a dynamic field and involves many assumptions and uncertainties. For instance, this study lacked solid data on carcinogenicity of the EDCs. Further, many data points (for EDCs in wate r samples as well as dislodged residue samples) were below PQLs, so the true concentrations of the EDCs for these data points were not

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67 known. Uncertainty also includes the many assumptions made throughout. Thus, further research is needed about risks assoc iated with the combined intake as well as carcinogenicity of EDCs from various sources and about the combined effect s of various EDCs on human bodies. Risk Communication/Management To account for various uncertainties, t he risk assessment was conducted to represent a conservative approach using worst case scenarios and maximum concentrations/masses of EDCs measured. The results suggest minimal risk associated with exposure to the five EDCs at current environmental concentrations. Also, the metabolites (degr adation products) of the five EDCs are less estrogenic and less toxic than the parent compounds (Larcher et al. 2012; Kang et al. 2006; Hao et al., 2008). It is, however, prudent to wait for turf to dry before allowing children to play in the area irriga ted with RW.

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68 CHAPTER 4 SUMMARY AND CONCLUSION S Risk of human exposure to EDCs from rapidly increasing use of RW for turf irrigation is a possible concern. To address the issue, the occurrence of five EDCs (estron e, 17 estradiol ethynyl estradiol, bisphenol A and nonylphenol) was determined in RW and residential surface water retention ponds in South Florida. The information was then used to conduct a human health risk assessment for dislodged residues of the EDC s from St. A ugustine turf grass irrigated with RW. All five EDCs were detected in RW and water retention ponds at concentrations as high as 47 ng/L for E1, 51 ng/L for E2, 58 ng/L for EE2, 303 ng/L for BPA and 52 ng/L for NP. Concentrations of all compounds were simil ar to, or less than, concen trations reported by others. C oncentrations of E2, EE2, BPA and NP in retention ponds in neighborhood s likely impacted from RW use were significantly less than concentrations in ponds from areas serviced by se p tic systems EDCs w ere detected i n both RW and retention ponds, but at low ng/L levels (similar to published values), supporting the first hypothesis. Maximum masses of EDCs were dislodged from 844 cm 2 of RW irrigated turf immediately after application and were up to 33 ng for estrone, 51 ng for 1 estradiol, estradiol, 296 ng for bisphenol A, and 323 ng for nonylphenol. Dislodgement decreased rapidly with time after spraying, and was undetectable 6 hours after spraying. Re wetting the tur f failed to release significant amounts of additional chemicals. Human health risk assessments for the EDCs suggested minimal r isk associated with exposure to the EDCs support ing the second hypothesis.

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69 E nvironmentally relevant concentrations of E1, E2, EE2, BPA and NP in RW or retention ponds r epresent minimal risk of adverse health effects on humans. Even a 10 fold increase in EDC masses dislodged after turf irrigation with RW results in minimal non carcinogenic risk. Such a 10 fold increase in EDC concentration in RW ( long term) is highly unli kely. D ue to a lack of data on human toxicity, carcinogenicity and other adverse effects from the EDCs, the risk assessment conducted involved many assumptions. But the assumptions were very stringent and conservative uncertainty factors were applied to t he toxicological data. T oxicological da ta used to characterize risk were based on oral dose of EDCs. The entire r isk assessment approach was highly conservative and represent ed worst case scenarios. Nonetheless a person can be exposed to numerous EDCs fr om numerous other sources and a wide range of uncertainty exists regarding cumulative effects of various EDCs. M ore research regarding potential adverse effects of EDCs especially on toxicity and carcinogenicity in humans is needed to better assess the l ink between EDC concentration s and associated risk. But the risk associated with the five EDCs was so small that the risk from other EDCs should be minimal. Nonetheless cpncerned parents may wish to recently RW irrigated turf until the grass is dry.

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70 LIST OF REFERENCES Ahel, M., Schaffner, C., Giger, W.,1996. Behaviour of alkylphenol polyethoxylate surfactants in the aquatic environment -III. Occurrence and elimination of their persistent metabolites during infiltration o f river water to groundwater. Water Res. 30, 37 46. A lkylphenols and Ethoxylates Research Council (2001). Safety of APEs, Retrieved on July 31, 2001 from http://www.aperc.org/docs/safetyofapes.html Balaguer, P., Francois, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.C., Casellas, C., 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47 56 Barnes, K.K., Kolpin, D.W., Furlong, E.T., Zaugg, S.D., Meyer, M.T., Barber, L.B., 2008. A national reconnaissance of pharmaceuticals and other organic wastewater contaminants in the United States I. Groundwater. Sci. Total Environ. 402, 192 200. cia, A., Gentili, A., Samperi, R., 2000. Monitoring natural and synthetic estrogens at activated sludge sewage treatment plants and in a receiving river water. Environ. Sci. Tech nol 34, 5059 5066. Belfroid, A.C., Van der Horst, B., Vethaak, D., Schfer, A .J., Rijs, G.B.J., Wegener, J., Cofino, W.P., 1999. Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in the Netherlands. Sci. Total Environ. 225, 101 108. Belfroid, A.C., Van Velzen, M., Van der Horst, B., Vethaak, D., 2002. Occurrence of bisphenol A in surface water and uptake in fish: evaluation of field measurements. Chemosphere. 49, 97 103. Blackburn, M.A., Waldock, M.J., 1995. Concentrations of alkylphenols in rivers and estuaries in England and Wal es. Water Res. 29, 1623 1629. Broseus, R., Vincent, S., Aboulfadl, K., Daneshvar, A., Sauve, S., Barbeau, B., Prevost, M., 2009. Ozone oxidation of pharmaceuticals, endocrine disruptors and pesticides during drinking water treatment. Water Res. 43, 4707 47 17. Carlsen, E.A., Giwercman, A., Keiding, N., Skakkebaek, N.E., 1992. Evidence for decreasing quality of semen during last 50 years. Bri. Med. J. 305, 609 613. Carrara, C., Ptacel, C.J., Robertson, W. D., Blowes, D. W., Moncur, M. C., Sverko, E., Backus, S., 2008. Fate of pharmaceutical and trace organic compounds in three septic system plumes, Ontario, Canada. Environ. Sci. Technol.42, 2805 2811. Chapin, R.E., Adams, J., Boekelheide, K., Gray, L.E., Hayward, S.W., Lees, P.S., McIntyre, B.S., Portier, K.M. Schnorr, T.M., Selevan, S.G., Vandenbergh, J.G.,

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79 BIOGRAPHICAL SKETCH Harmanpreet Sidhu was born in 1987, in Northwest India. Coming from a magnificent region situated i n the foot hills of Himalayas, Harman learned to admire, s great spectacle Earth. After his s ophomore natural interest towards scient ific mysteries of the planet, his zeal and aim propelled him to take biology, physics and chemistry as the main subjects for his senior secondary exa mination After high school, Harman was admitted to Punjab Agricultural University, Ludhiana, India, degree in agriculture (h ons.). It was in that university, that his interest in environmental sciences encouraged him to pursue a ca reer in related fiel d. He then decided to major in s oils. Harman graduated from Punjab Agricultural University in 2011. Harman began his graduate career at the University of Flori da in August of 2011, pursuing m Department under the advisement of Dr. research focused on human risk assessment of endocrine disrupting chemicals in reclaimed water. Harman enjoys the work he does and hopes to continue a similar scientific research in his Ph.D. at University of Florida.