Occurrence and estrogenic potency of eight bisphenol analogs in sewage sludge from the U.S. EPA targeted national sewage sludge survey

Accepted Manuscript Title: Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey Author: Xiaohua Yu Jingchuan Xue Hong Yao Qian Wu Arjun K. Venkatesan Rolf U. Halden Kurunthachalam Kannan PII: DOI: Reference:

S0304-3894(15)00541-5 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.07.012 HAZMAT 16939

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

6-5-2015 3-7-2015 6-7-2015

Please cite this article as: Xiaohua Yu, Jingchuan Xue, Hong Yao, Qian Wu, Arjun K.Venkatesan, Rolf U.Halden, Kurunthachalam Kannan, Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S.EPA Targeted National Sewage Sludge Survey, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.07.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Elsevier Editorial System(tm) for Journal of Hazardous Materials Manuscript Draft Manuscript Number: HAZMAT-D-15-01872R1 Title: Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey Article Type: Research Paper Keywords: BPA; bisphenols; WWTP; sewage sludge; estrogenic; biosolids Corresponding Author: Dr. Kurunthachalam Kannan, Ph.D. Corresponding Author's Institution: State University of New York at Albany, School of Public Health, First Author: Xiaohua Yu Order of Authors: Xiaohua Yu; Jingchuan Xue; Hong Yao; Qian Wu; Arjun K Venkatesan; Rolf U Halden; Kurunthachalam Kannan, Ph.D. Abstract: As health concerns over bisphenol A (BPA) in consumer products are mounting, this weak estrogen mimicking compound is gradually being replaced with structural analogs, whose environmental occurrence and estrogen risks are not well understood yet. We used high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of eight bisphenol analogs in 76 sewage sludge samples collected by the U.S. Environmental Protection Agency (EPA) in 2006/2007 from 74 wastewater treatment plants (WWTPs). Bisphenols were detected at the following concentration ranges (ng/g dry weight) and detection frequencies: BPA (6.5 4700; 100%); bisphenol S (BPS; <1.79 - 1480; 84%); bisphenol F (BPF; <1.79 - 242; 68%); bisphenol AF (BPAF; <1.79 - 72.2; 46%); bisphenol P (BPP; <1.79 - 6.42; <5%), bisphenol B (BPB; <1.79 - 5.60; <5%), and bisphenol Z (BPZ; <1.79 - 66.7; <5%). The calculated 17β-estradiol equivalents (E2EQ) of bisphenols present in sludge samples were 7.74 (0.26-90.5) pg/g dw, which were three orders of magnitude lower than the estrogenic activity contributed by natural estrogens present in the sludge. The calculated mass loading of bisphenols through the disposal of sludge and wastewater was <0.02% of the total U.S. production. This study establishes baseline levels and estrogenic activity of diverse bisphenol analogs in sewage sludge.

Cover Letter

July 2, 2015 Gerasimos Lyberatos, PhD Editor Journal of Hazardous Materials Dear Editor: Attached please find herewith the REVISED manuscript titled, “Occurrence and

Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey” for favor of publication in JHM. We thank the reviewers for their comments and itemized list of changes made in response to review is given below. Reviewer #1: The manuscript entitled "Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey" mainly deal with the concentration detection of eight bisphenol analogs in 76 sewage sludge samples collected by the U.S. Environmental Protection Agency (EPA) in 2006/2007 from 74 wastewater treatment plants (WWTPs). Moreover, the estrogenic activity of bisphenols were also assessed and the calculated 17-estradiol equivalents (E2EQ) of bisphenols present in sludge samples were 7.74 (0.26-90.5) pg/g dw, which were three orders of magnitude lower than the estrogenic activity contributed by natural estrogens present in the sludge. Thus, the manuscript may contain some interesting results about these weak estrogen mimicking compound bisphenol analogs. However, there still many points need to be answered before the manuscript can be published in this journal. 1. The authors should give more convincing reasons to emphasize the novelty of this work because there already have many similar work reported, for example reference 40. 2. The authors should give more detail discussion on your obtained data, especially for the estrogenic activity of these new compounds. In summary, the manuscript can be published after major revision.

1st comment: The authors should give more convincing reasons to emphasize the novelty of this work because there already have many similar work reported, for example reference 40. Response: Although a similar study was conducted in China, our work has its own originalities and novelties. To begin with, this is the first study about the occurrence of the eight bisphenol analogues in the USA. Limited information about the group of chemicals in sewage sludge from USA was available before this work. Secondly, archived sludge samples from U.S.

EPA TNSSS survey were used in this study. It allowed us to obtain a comprehensive and reliable and nationally representative data on the occurrence of these substances. Last but not the least, estimates of emission and estrogenic potentials of this group of chemicals in the sludge were reported in this study, providing further information about the risk of these substances. In the USA, land-application is the major use of sludge. Therefore, it is necessary to carefully evaluate the potential risks associated with the agriculture use of sludge. As bisphenol A (BPA) is replaced with BPS and BPF in many consumer products, the occurrence and estrogen risks of the latter are not well understood. There is a considerable interest in evaluating the occurrence of toxicity of bisphenol substitutes and in recent years environmental and government organizations request the need for such data. In this study, BPA, BPS and BPF were determined for the first time in sewage sludge from the USA. Although reference 40 is a similar survey conducted in China, our study shows that the levels and patterns of bisphenols are different from what was reported in China. Thus, data from China can no longer be used for generalizing the issues faced in the USA. In fact, our lab is the first to describe the occurrence of bisphenol substitutes in various human and environmental samples and our current study is an extension of our previous studies on BPA substitutes. We also estimated environmental emission and estrogenic activities of bisphenols compared with other naturally occurring estrogens in sludge.

2nd comment: The authors should give more detail discussion on your obtained data, especially for the estrogenic activity of these new compounds.

Response: More detail discussion about the estrogenic activity of these substances were given from line 308 in page 15 to line 316 in page 16. “However, BPA can stimulate the responses of the cellular even at a very low concentration through non-genomic mechanisms and those mechanisms involve membrane-associated forms of the estrogen receptors [56-57]. A variety of adverse health effects related with low dose BPA exposure have been observed in animals, such as female reproduction tract abnormalities, early puberty, reduced sperm count, and brain effects, especially when this exposure occurs before and shortly after birth [58]. Thus, although the estrogenic activity contributed by bisphenol analogs in sludge samples is much lower than that for 4-NP and natural estrogens present in the matrix, considerable concern is still need for these substances.” We have added some new refrences including the one published recently in environmental health perspectives titled, Bisphenol S and F: A Systematic Review and Comparison of the Hormonal Activity of Bisphenol A Substitutes (reference number 59) which again supports the novelty of our study describing the need for environmental analysis of substitutes of BPA.

Reviewer #2: General Comments The manuscript entitled "Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey" by Xiaohua Yu, Jingchuan Xue, Hong Yao, Qian Wu, Arjun K. Venkatesan, Rolf U. Halden and Kurunthachalam Kannan covers a very important aspect of the ecologic impact of Bisphenols, one class of the so called emerging pollutants. The approach is sound and there are sufficient support presented in the manuscript allowing to make a fair enough judgment of the quality. In my opinion the

authors have succeeded to achieve the goals they set up. I found the paper novel, interesting and informative. My recommendation is to accept the paper in its current form.

Response: Thank you for the kind comments!

Thanking you and looking forward to hearing from you. Sincerely,

K. Kannan, Ph.D. [email protected]

Highlights (for review)

HIGHLIGHTS 

BPA, BPS and BPF were determined in 76 sewage sludge from the USA



Bisphenols were found at concentrations ranging from 12.8 to 4730 ng/g dw in sludge



Only 0.02% of the total BPA production volume was emitted through sludge disposal



The estrogenic activity of BPA was lower than that of natural estrogens in sludge

*Novelty Statement

Novelty statement: As bisphenol A (BPA) is replaced with BPS and BPF in many consumer products, the occurrence and estrogen risks of the latter are not well understood. We determined eight bisphenol analogs in sewage sludge collected in 2006/2007 from 74 wastewater treatment plants. BPA, BPS and BPF were determined for the first time in sewage sludge from the USA. We also estimated environmental emission and estrogenic activities of bisphenols compared with other naturally occurring estrogens in sludge. This is a first study of this kind and we strongly believe that this study will have a great impact.

*Abstract

ABSTRACT: As health concerns over bisphenol A (BPA) in consumer products are mounting, this weak estrogen mimicking compound is gradually being replaced with structural analogs, whose environmental occurrence and estrogen risks are not well understood yet. We used high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of eight bisphenol analogs in 76 sewage sludge samples collected by the U.S. Environmental Protection Agency (EPA) in 2006/2007 from 74 wastewater treatment plants (WWTPs) in 35 states. Bisphenols were detected at the following concentration ranges (ng/g dry weight) and detection frequencies: BPA (6.5 - 4700; 100%); bisphenol S (BPS; <1.79 - 1480; 84%); bisphenol F (BPF; <1.79 – 242; 68%); bisphenol AF (BPAF; <1.79 – 72.2; 46%); bisphenol P (BPP; <1.79 – 6.42; <5%), bisphenol B (BPB; <1.79 – 5.60; <5%), and bisphenol Z (BPZ; <1.79 – 66.7; <5%). Bisphenol AP (BPAP) was not detected in any of the samples (<1.79 ng/g dw). Concentrations of BPA in sewage sludge were an order of magnitude higher than those reported in China but similar to those in Germany. The calculated 17β-estradiol equivalents (E2EQ) of bisphenols present in sludge samples were 7.74 (0.26-90.5) pg/g dw, which were three orders of magnitude lower than the estrogenic activity contributed by natural estrogens present in the sludge. The calculated mass loading of bisphenols through the disposal of sludge and wastewater was <0.02% of the total U.S. production. As the usage of BPA is expected to decline further, environmental emissions of BPS, BPF, and BPAF are likely to increase in the future. This study establishes baseline levels and estrogenic activity of diverse bisphenol analogs in sewage sludge.

*Response to Reviewers

Responses to the comments First, we thank the reviewers for their time to review the manuscript. The comments were considered carefully and manuscript has been changed in the light of reviews. An itemized list of changes is shown below. Reviewer #1: Reviewer #1: The manuscript entitled "Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey" mainly deal with the concentration detection of eight bisphenol analogs in 76 sewage sludge samples collected by the U.S. Environmental Protection Agency (EPA) in 2006/2007 from 74 wastewater treatment plants (WWTPs). Moreover, the estrogenic activity of bisphenols were also assessed and the calculated 17-estradiol equivalents (E2EQ) of bisphenols present in sludge samples were 7.74 (0.26-90.5) pg/g dw, which were three orders of magnitude lower than the estrogenic activity contributed by natural estrogens present in the sludge. Thus, the manuscript may contain some interesting results about these weak estrogen mimicking compound bisphenol analogs. However, there still many points need to be answered before the manuscript can be published in this journal. 1. The authors should give more convincing reasons to emphasize the novelty of this work because there already have many similar work reported, for example reference 40. 2. The authors should give more detail discussion on your obtained data, especially for the estrogenic activity of these new compounds. In summary, the manuscript can be published after major revision. Thank you for the kind comments!

1st comment: The authors should give more convincing reasons to emphasize the novelty of this work because there already have many similar work reported, for example reference 40.

Response: Although a similar study was conducted in China, our work has its own originalities and novelties. To begin with, this is the first study about the occurrence of the eight

bisphenol analogues in the USA. Limited information about the group of chemicals in sewage sludge from USA was available before this work. Secondly, archived sludge samples from U.S. EPA TNSSS survey were used in this study. It allowed us to obtain a comprehensive and reliable and nationally representative data on the occurrence of these substances. Last but not the least, estimates of emission and estrogenic potentials of this group of chemicals in the sludge were reported in this study, providing further information about the risk of these substances. In the USA, land-application is the major use of sludge. Therefore, it is necessary to carefully evaluate the potential risks associated with the agriculture use of sludge. As bisphenol A (BPA) is replaced with BPS and BPF in many consumer products, the occurrence and estrogen risks of the latter are not well understood. There is a considerable interest in evaluating the occurrence of toxicity of bisphenol substitutes and in recent years environmental and government organizations request the need for such data. In this study, BPA, BPS and BPF were determined for the first time in sewage sludge from the USA. Although reference 40 is a similar survey conducted in China, our study shows that the levels and patterns of bisphenols are different from what was reported in China. Thus, data from China can no longer be used for generalizing the issues faced in the USA. In fact, our lab is the first to describe the occurrence of bisphenol substitutes in various human and environmental samples and our current study is an extension of our previous studies on BPA substitutes. We also estimated environmental emission and estrogenic activities of bisphenols compared with other naturally occurring estrogens in sludge.

2nd comment: The authors should give more detail discussion on your obtained data, especially for the estrogenic activity of these new compounds.

Response: More detail discussion about the estrogenic activity of these substances were given from line 308 in page 15 to line 316 in page 16. “However, BPA can stimulate the responses of the cellular even at a very low concentration through non-genomic mechanisms and those mechanisms involve membrane-associated forms of

the estrogen receptors [56-57]. A variety of adverse health effects related with low dose BPA exposure have been observed in animals, such as female reproduction tract abnormalities, early puberty, reduced sperm count, and brain effects, especially when this exposure occurs before and shortly after birth [58]. Thus, although the estrogenic activity contributed by bisphenol analogs in sludge samples is much lower than that for 4-NP and natural estrogens present in the matrix, considerable concern is still need for these substances.” We have added some new refrences including the one published recently in environmental health perspectives titled, Bisphenol S and F: A Systematic Review and Comparison of the Hormonal Activity of Bisphenol A Substitutes (reference number 59) which again supports the novelty of our study describing the need for environmental analysis of substitutes of BPA.

Reviewer #2: General Comments The manuscript entitled "Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey" by Xiaohua Yu, Jingchuan Xue, Hong Yao, Qian Wu, Arjun K. Venkatesan, Rolf U. Halden and Kurunthachalam Kannan covers a very important aspect of the ecologic impact of Bisphenols, one class of the so called emerging pollutants. The approach is sound and there are sufficient support presented in the manuscript allowing to make a fair enough judgment of the quality. In my opinion the authors have succeeded to achieve the goals they set up. I found the paper novel, interesting and informative. My recommendation is to accept the paper in its current form.

Response: Thank you for the kind comments!

*Revised Manuscript Click here to view linked References

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Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey

Xiaohua Yu1, 2, Jingchuan Xue1, Hong Yao2, Qian Wu1, Arjun K. Venkatesan3, Rolf U. Halden3 and Kurunthachalam Kannan1,4*

1

Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA 2

Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing 100044, China 3

Center for Environmental Security, The Biodesign Institute, Global Security Initiative, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287, USA 4

Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

*Corresponding author: K. Kannan Wadsworth Center Empire State Plaza, P.O. Box 509 Albany, NY 12201-0509 Tel: +1-518-474-0015 Fax: +1-518-473-2895 E-mail: [email protected]

For submission to:

Journal of Hazardous Materials

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ABSTRACT: As health concerns over bisphenol A (BPA) in consumer products are mounting, this weak estrogen mimicking compound is gradually being replaced with structural analogs, whose environmental occurrence and estrogen risks are not well understood yet. We used high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of eight bisphenol analogs in 76 sewage sludge samples collected by the U.S. Environmental Protection Agency (EPA) in 2006/2007 from 74 wastewater treatment plants (WWTPs) in 35 states. Bisphenols were detected at the following concentration ranges (ng/g dry weight) and detection frequencies: BPA (6.5 - 4700; 100%); bisphenol S (BPS; <1.79 - 1480; 84%); bisphenol F (BPF; <1.79 – 242; 68%); bisphenol AF (BPAF; <1.79 – 72.2; 46%); bisphenol P (BPP; <1.79 – 6.42; <5%), bisphenol B (BPB; <1.79 – 5.60; <5%), and bisphenol Z (BPZ; <1.79 – 66.7; <5%). Bisphenol AP (BPAP) was not detected in any of the samples (<1.79 ng/g dw). Concentrations of BPA in sewage sludge were an order of magnitude higher than those reported in China but similar to those in Germany. The calculated 17β-estradiol equivalents (E2EQ) of bisphenols present in sludge samples were 7.74 (0.26-90.5) pg/g dw, which were three orders of magnitude lower than the estrogenic activity contributed by natural estrogens present in the sludge. The calculated mass loading of bisphenols through the disposal of sludge and wastewater was <0.02% of the total U.S. production. As the usage of BPA is expected to decline further, environmental emissions of BPS, BPF, and BPAF are likely to increase in the future. This study establishes baseline levels and estrogenic activity of diverse bisphenol analogs in sewage sludge.

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KEYWORDS: BPA, bisphenols, WWTP, sewage sludge, mass loading, estrogenic, biosolids

INTRODUCTION Bisphenol analogs (hereafter ‘bisphenols’) are a class of chemicals with two para-hydroxyphenyl functional groups, with bisphenol A (BPA) as the prototype compound. Bisphenols are widely used in industrial and commercial applications including inner coating of food cans, dental sealants, electronic equipment, thermal papers, and polycarbonate plastics [1, 2]. As the concern over the safety of BPA continues to grow, this compound is gradually replaced with other analogs, such as bisphenol F (BPF) and bisphenol S (BPS), which are structurally similar to BPA [3], in many applications. BPA, BPS, and BPF have been shown to be toxic in many laboratory animal studies, and most notably, these compounds elicit weak estrogenic activities [4-9]. Human exposure to BPA has been linked to endocrine disorders and obesity [10, 11]. Bisphenols have been frequently found in consumer products such as thermal receipt papers [3], currency bills [3, 12], personal care products [13], foodstuffs, and polycarbonate plastic bottles [5, 14-16]. Sources and pathways of human exposure to BPA have been widely studied. The occurrence of BPA in blood, urine, breast milk, placenta, and hair has been reported in many countries [5, 17-21]. Studies have also reported the occurrence of BPA in environmental matrices such as air, water, soil, sediment, sewage sludge (SS) and indoor dust [18, 22, 23]. However, very few studies have reported the occurrence of bisphenols other than BPA in

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environmental matrices [23, 24]. Several studies have reported the occurrence and fate of BPA in wastewater treatment processes. The removal efficiency of BPA in wastewater treatment varies widely, from 1% to 77%, and a considerable fraction of BPA is sorbed to sludge and the sorption is influenced by the degree of nitrification and hydraulic retention time [25]. Sewage sludge can be a good matrix for monitoring environmental contaminants, as this can reflect the usage pattern of many chemicals derived from domestic and industrial activities [26]. Sewage sludge, as defined by the U.S. Environmental Protection Agency (EPA), is nutrient-rich organic residuals that when treated and processed, may be recycled and applied as fertilizer [27]. Land-application of SS is an inexpensive option for disposal and can provide valuable nutrients to the soil, which may enhance soil properties and plant yield [28]. In 2004, approximately 50-60% of 6.5 million tons (dry weight) of municipal SS produced in the U.S. was land-applied [29]. While land-applied SS offers a source of rich nutrients, they can also release pollutants that become sequestered into the terrestrial food chain or leached into groundwater [30, 31]. A few studies have reported the occurrence of artificial sweeteners, pharmaceuticals, and illicit drugs in agricultural soils following the land application of SS [32-34]. The EPA initiated the Targeted National Sewage Sludge Survey (TNSSS) in the mid-2000s to characterize pollutants that may be present in SS generated by the nation’s publicly owned treatment works (POTWs) to develop regulations on the use or disposal of SS [35]. In that study, SS samples were collected from 74 POTWs that employ secondary treatment or better in 2006/2007 [35]. Several pollutants have been analyzed

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in these samples and the results have been published [29, 36-39]. However, no earlier study has reported the occurrence of bisphenols in SS from nationally representative samples. The aims of the present study were to investigate the occurrence and profiles of bisphenols in SS samples, and to estimate the mass loading of bisphenols through the application of SS to land. The estrogenic activity contributed by several bisphenols in SS was calculated using the reported estrogenic potency values. We analyzed eight bisphenols, including BPA, BPF, BPS, 4,4-(hexafluoroisopropylidene)-diphenol (BPAF), 2,2-bis(4-hydroxyphenyl)butane (BPB), 4,4’-cyclo-hexylidenebisphenol (BPZ), 4,4’-(1,4-phenylenediisopropylidene)bisphenol (BPP), and 4,4-(1-phenylethylidene)bisphenol (BPAP) in 76 archived SS samples collected by the EPA in 2006/2007 using high performance liquid chromatography−tandem mass spectrometry (HPLC-MS/MS).

MATERIALS AND METHODS Standards and Reagents Analytical standards of 2,2-bis(4-hydroxyphenyl)propane (BPA; purity ≥97%), BPAF (~97%), BPAP (~99%), 4,4-sulfonyldiphenol (BPS; ~98%), BPP (~99%), BPZ (~98%), and 4,4’-dihydroxydiphenylmethane (BPF; ~98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Analytical standard of BPB (~98%) was obtained from TCI America (Portland,

OR, USA). 13C12-BPA was purchased from Cambridge Isotope Laboratories (≥99%; Andover, MA, USA). Formic acid (98.2%) was from Sigma-Aldrich, and HPLC-grade methanol was from

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Mallinckrodt Baker (Phillipsburg, NJ, USA). Milli-Q water (18.2 MΩ) was prepared from an ultrapure water purification system (Barnstead International, Dubuque, IA, USA). Stock solutions of bisphenols and 13C12-BPA were prepared at 1 mg/mL in methanol and stored at −20°C.

Sample Collection A total of 76 sewage sludge samples were collected by the EPA from 74 POTWs in 35 states that are representative of the nation’s 3337 POTWs during August 2006 and March 2007. The selection criteria for POTWs and sampling procedure were provided in detail in the Sampling and Analysis Technical Report [35]. Briefly, each POTW in full operation in 2002 and/or 2004, and treated more than 1 million gallons per day (MGD), with secondary or better treatment was selected. Two samples were collected at ten POTWs, either because the facility had more than one treatment system and produced two types of final SS, or for quality assurance purposes, as well as one single grab sample at the remaining 64 facilities (Table S1). After the completion of TNSSS, the samples were acquired by the Halden Laboratory and archived at -20oC as part of the Human Health Observatory (H2O) and National Sewage Sludge Repository (NSSR) at Arizona State University [36]. Eight samples were either missing or broken during shipment and handling. A total of 76 samples were shipped in glass jars to Wadsworth Center, New York State Department of Health, Albany, New York, and were stored at -20ºC until chemical analysis.

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Sample Preparation Sewage sludge samples were freeze-dried using a freeze drier (Labconco, Kansas City, MO, USA). Sludge extraction and cleanup procedures were similar to those previously reported, with minor modifications [23]. Briefly, freeze-dried sample (0.1-0.2 g) was spiked with 20 ng of 13

C12-BPA and extracted with 5 mL of methanol/water mixture (5:3, v/v) by shaking in an orbital

shaker (Eberbach, Ann Arbor, MI, USA) at 250 oscillations/min for 30 min. After centrifugation at 4800 g for 5 min (Eppendorf Centrifuge 5804, Hamburg, Germany), the supernatant was transferred into a 15 mL glass tube. The extraction was repeated twice, and the extracts were combined and concentrated to ~4 mL under a gentle stream of nitrogen. After dilution to 10 mL with 0.2% formic acid (pH 2.5), the extract was purified with an Oasis MCX cartridge (60 mg/3 cm3; Waters, Milford, MA, USA). The cartridge was preconditioned with 5 mL of methanol and 5 mL of water. After loading the sample, the cartridge was washed with 15 mL of 25% methanol in water and 5 mL of water, and then eluted with 5 mL of methanol. The eluate was transferred into a vial and vortex mixed before HPLC-MS/MS analysis.

Instrumental Analysis The chromatographic separation was carried out using a Shimadzu Prominence Modular HPLC system (Shimadzu Corporation, Kyoto, Japan), consisting of a system controller, a binary pump and an automatic sampler. Identification and quantification of target analytes were performed with an Applied Biosystems API 3200 electrospray triple quadruple mass spectrometer

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(ESI-MS/MS; AB SCIEX, Framingham, MA, USA). A Betasil C18 column (2.1 mm × 100 mm, 5 µm; Thermo Fisher Scientific, Waltham, MA, USA) serially connected to a Javelin guard column (Betasil C18, 2.1 mm × 20 mm, 5 µm; Thermo Fisher Scientific) was used. The injection volume was 10 µL, and the mobile phase comprised methanol (A) and Milli-Q water that contained 1% (v/v) ammonium hydroxide (B). The target compounds were separated by gradient elution at a flow rate of 300 µL/min starting at 15% (v/v) A, held for 2 min; increased to 75% A within 3 min (5th min), held for 2 min; then further increased to 99% A within 3 min (10th min), held for 4 min (14th min); and reverted to 15% A at the 14.5th min that was held for 5.5 min (20th min), with a total run time of 20 min. The MS/MS was operated in the multiple reaction monitoring (MRM) negative ionization mode and ion pairs are shown in Table S2. The compound specific MS/MS parameters are shown in the Supplementary Material (Table S3). Nitrogen was used as both a curtain and a collision gas. The electrospray ionization voltage was set at -4.5kV. The curtain and collision gas flow rates were set at 25 and 2 psi, respectively, and the source heater was maintained at 650°C. The nebulizer gas (ion source gas 1) was set at 20 psi, and the heater gas (ion source gas 2) was set at 70 psi. The scan speed was 80 ms and full width at half maximum (FWHM) value for resolving power was 0.70.

Quality Assurance and Quality Control For the evaluation of extraction efficiency, 8 samples were randomly selected and a third extraction (after the first two extractions as mentioned above) was performed with 4 mL of

8

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methanol/water mixture (5:3, v/v). No quantifiable amounts of bisphenols were detected in the third extraction, which suggested that the first two extractions were sufficient for the extraction. Quantification of bisphenols was performed by the isotope dilution method, with the response relative to 13C12-BPA. Instrumental calibration was verified daily by the injection of 10 calibration standards at concentrations ranging from 0.05 to 500 ng/mL, and the linearity of the calibration curve (r) was >0.99. The limits of quantification (LOQs), calculated from the lowest acceptable calibration standard and a nominal sample weight of 0.14 g, were 1.79 ng/g dw for all bisphenols (Table S4). Retention times of native and labeled compounds had to be within ±12 s (0.2 min) of the respective retention time established in the calibration curve. For each batch of 12 samples, a procedural blank, and a procedural blank duplicate were analyzed. A mid-point calibration standard was injected after every 10 samples as a check for the drift in instrumental sensitivity. Pure solvent (methanol) was injected periodically as a check for carryover of bisphenols from sample to sample. A replicate sample, a matrix spike, and a matrix spike duplicate were analyzed for every 25 samples to evaluate precision and accuracy of the analysis. Matrix spike recoveries were 101±8 % for BPA, 103±10 % for BPAF, 92±6 % for BPAP, 94±10 % for BPB, 98±5 % for BPF, 78±16 % for BPP, 84±20 % for BPS, 98±8 % for BPZ (Table S2). Relative percentage difference (RPD), calculated using the following formula, was used to express the precision of analysis.
% 

|      |  100      2

Where   and   are the concentrations detected in the original sample and in 9

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the duplicate, respectively. The concentrations of bisphenols in SS samples are reported on a dry weight (dw) basis.

Data Analysis Among the 76 sludge samples collected from 68 POTWs, 4 were duplicate samples (we report average of the two values) and 8 were selected from 4 POTWs with two different treatment systems (the results were not averaged for these samples). Concentrations below the LOQ were substituted with a value equal to LOQ divided by 2 (LOQ/2) for the calculation of geometric mean (GM) and median. All data were log-transformed for statistical analyses. To compare median concentrations of bisphenols among different regions, the data were dichotomized at the pooled median to obtain a 2×2 table, and then a Pearson’s Chi-squared test was used. Spearman correlation analysis was also employed to determine the associations among bisphenols. The percent 17β-estradiol equivalent (E2EQ%) contributed by each analog was estimated by the following equation.  %     /"  × 100 Where Csi is the measured concentration of individual bisphenol analog in sludge samples (ng/g dw) and EEFi is the estradiol equivalency factor, calculated as the ratio between the half maximal effective concentration (µM) of E2 and the individual bisphenol analog [40].

Statistical analysis

was performed with statistical package R (version 3.1.0) and Microsoft Excel 2007, and the statistical significance was set at p <0.05 level.

10

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RESULTS AND DISCUSSION Occurrence of bisphenols in sewage sludge Concentrations of individual bisphenols and the sum concentration of eight bisphenols (ΣBisphenols) are summarized in Table 1. BPA, BPS, BPF, and BPAF were the most abundant bisphenols found in SS samples, and the respective geometric mean (GM) and median concentrations (ng/g dw) were; 177 and 222, 5.59 and 5.80, 6.30 and 8.16, and 2.13 and
11

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The relative contribution of individual bisphenol analog to GM concentrations of ΣBisphenols is shown in Figure 1. BPA was the predominant analog in SS and the percentage contribution of BPA varied from 89% to 92% depending on the geographic region. BPF was the second most abundant bisphenol analog (1.47% to 4.90%), followed by BPS (2.19% to 4.64%), and BPAF (0.76% to 1.52%). BPAP, BPB, BPP, and BPZ accounted for <0.5 % of the total ΣBisphenols concentrations. Correlations between the concentrations of bisphenol analogs in SS were examined by Spearman correlation analysis (Table S6). Significant positive correlations (r=0.27, p<0.05) between BPA and BPF were found, which may indicate a common source of release for the two compounds. No significant correlations were found for other bisphenols in SS, which suggested the existence of different sources of origin for these analogs. Detailed information (such as wastewater type, population served, hydraulic retention time) on the WWTPs were not available, except for treatment capacity, to further characterize the factors that affect BP concentrations in sludge (Table S1). In general, high (median) concentrations of BPA and ΣBisphenols were found in WWTPs that treated >100 MGD, followed by WWTPs with a treatment capacity of between 10 and 100 MGD (Figure 2, Table S7). It is likely that industrial discharges contribute to high concentrations of BPA in large treatment plants. Studies have shown that wastewater collected from paper mills, especially those that produced thermal receipt papers, and landfill leachate from solid-waste disposal facilities contained elevated concentrations of BPA [41-44].

12

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Comparison of BP concentrations in sewage sludge from several countries Several studies have reported the occurrence of bisphenols, especially BPA, in SS from Germany [45, 46], Canada [47, 48], Greece [49], Australia [50], China [40], and South Korea [26]. A concentration range of 70 to 770 ng/g dw was reported for BPA in three SS samples collected in Southwestern Germany in 2001 [45]. Another study in 2002 reported a BPA concentration range of 4 to 1363 ng/g dw in 38 SS samples collected in Germany [46]; these concentration ranges are comparable to those found in the U.S. (this study). BPF was also reported in that study at a concentration range of 4.2 to 181 ng/g dw [46], which is similar to that found in our study. A BPA concentration range of <20 to 36700 ng/g dw and 3.78 to 74.4 ng/g dw, was reported in two studies conducted in Canada with a sample size of 50 and 4, respectively [47, 48]. Median concentrations of BPA reported in SS samples from Greece [49] and Australia [50] were 280 and 365 ng/g dw, respectively, which were higher than those found in samples analyzed in our study (222 ng/g dw) (Figure 3). Studies conducted in China [40] and South Korea [26] also reported the occurrence of other bisphenols in SS samples. Median concentrations of BPA, BPS, and BPF in SS analyzed in our study were 24, 2, and 4 times, respectively, higher than those found in the samples from China (Figure 3). However, median concentrations of BPA and BPF in SS from the U.S. were approximately 2 and 30 times lower than those reported from South Korea, respectively, although the median concentration of BPS in SS from South Korea was 2 times lower than that found in the U.S. These results reflect the difference in the usage patterns of bisphenols in various countries. The U.S. accounts for 22.9%

13

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of the global BPA production, whereas China accounts for 3.6% of the global BPA production [18]. High concentration of BPA in South Korean SS samples was associated with the release from paper and textile industries in that country [26]. High concentrations of BPF in SS samples in South Korea suggest the use of this analog in certain industrial activities.

Emission estimates through sewage sludge disposal and WWTP discharges and estrogenic activities of bisphenols Environmental emission of bisphenols through land-application of SS was assessed in this study. Annual generation volume of SS (dry weight) was reported to be 6 513 586 metric tons in 2004 in the U.S. [51]. On the basis of mean concentrations of eight bisphenols determined in this study, the nationwide annual emission inventory through SS was estimated to be 3390 kg/y (Table 1). The proportion of SS applied on land was approximately 50-60% per year [29]. Therefore, approximately 1695 to 2034 kg bisphenols were released every year, and 88% (i.e., 1490– 1790 kg) of which was BPA. Based on the adsorption rate of BP analogs onto the sludge, and the concentrations determined in sludge, the proportion of bisphenols released through wastewater discharges from WWTPs was estimated. The adsorption rates of BPA, BPAF, BPAP, BPB, BPF, BPP, BPS, and BPZ on the sludge were predicted using the U.S.EPA’s EPISuite and the values were 9.39, 53.9, 72.1, 35.9, 4.88, 92.2, 1.93, and 77.0%, respectively (Table S8). The estimated amount of bisphenols released through wastewater discharges was 45,900 kg per year (Table 1).

14

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Aggregated national production volume of BPA in the U.S. was reported to be 453, 592, 370 kg [52]. Assuming that a 50% of the annual production volume was used every year in the U.S., the proportion of bisphenols emitted through WWTPs was <0.02% of the production. Thus, SS and wastewater discharges constitute a small fraction of the environmental emission of bisphenols. The estrogenic activity contributed by bisphenols in SS was assessed, based on the relative estradiol equivalency factors (E2EF) reported for BPA, BPS, BPF, and BPAF in an in vitro estrogenic assay using MVLN cell lines [40]. The mean 17β-estradiol equivalents of each bisphenol analog in sludge samples ranged from 0.15 to 6.29 pg/g E2EQ dw (Table 1). Among the four bisphenols, BPA was the major contributor to the estrogenic activity in sludge accounting for 81% of the total estrogen equivalents, followed by BPAF (13%), BPS (3.5%), and BPF (1.9%). The total 17β-estradiol equivalents (E2EQ) of bisphenols in sludge samples were 7.74 (range: 0.26-90.5) pg/g dw, which were 1000 times lower than the estrogenic activity contributed by natural estrogens (E1+E2+E3; 27.9-63.8 ng/g dw) present in the sewage sludge samples [53]. Moreover, based on the concentrations of 4-nonylphenol (4-NP) measured in the SS [54], the 17β-estradiol equivalent of 4-NP (E2EF was 0.0001 [55]) was 53.4 (range: 40.5-86.1) ng/g, which was three orders of magnitude higher than that calculated for bisphenols. However, BPA can stimulate cellular responses even at low concentrations through non-genomic mechanisms and membrane-associated estrogen receptors [56-57].

A variety of adverse health

effects related with low dose BPA exposure have been observed in animals, especially when the exposures occurred in utero and early life stages [58]. BPS and BPF have been shown to possess

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

estrogenic activities similar to that of BPA [59]. Although the estrogenic activity contributed by bisphenol analogs in sludge samples is much lower than that for 4-NP and natural estrogens present in the matrix, further studies are needed for these substances. This study establishes baseline values for eight bisphenol analogs in SS, which can inform risk assessments and will enable evaluation of future trends in environmental releases of these compounds. Further studies are needed to assess the sources and fate of bisphenols in the environment.

* Supporting Information Available; Additional information as noted in the text (Tables and Figures). Notes The authors declare no competing financial interest.

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20

1 2

177

222

6.48-4700

100

2990

31800

13.7

6.29

GM

Median

Range

DF

Emission through SS

Emission through WWTPs

E2EF

E2EQ

Chemical structure

12.9

774

SD

1.03

172

72.6

39.2

46

<1.79-72.2

<1.79

2.13

6.01

459

BPAF

Mean

BPA

-

-

-

-

0

<1.79

<1.79

0.90

0


BPAP

21

-

-

17.6

6.32

1.4

<1.79-5.6

<1.79

0.92

0.55

0.97

BPB

0.15

8.60

2300

112

-

-

7.49

6.90

4.2

<1.79-6.4

<1.79-24 2 68

<1.79

0.96

0.85

1.06

BPP

8.16

6.30

32.9

17.2

BPF

0.27

7.82

11600

225

84

<1.79-148 0

5.8

5.59

174

34.5

BPS

-

-

15.3

11.8

1.4

<1.79-66.7

<1.79

0.96

7.70

1.81

BPZ

-

-

-

45900

3390

-

12.8-473 0

265

238

798

521

∑BPs

Table 1. Concentrations (ng/g dw), detection frequency (DF %) and estrogenic potency of bisphenol analogs in sewage sludge collected from the United States, and the estimated emission (kg per year) through sewage sludge disposal and WWTP discharges.

pg/g dw -

10-6

kg

kg

%

ng/g dw

ng/g dw

ng/g dw

ng/g dw

ng/g dw

Units

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

10

9

8

3 4 5 6 7

2

1

80

85

BPAF

90

BPB

BPF

Composition (%)

BPAP

BPP

95

BPS

BPZ

100

22

Figure 1. Composition profiles of bisphenol analogs in sewage sludge from the United States. The value within parentheses on the Y-axis representss the number of samples analyzed in this study.

Northeast (20)

Midwest (20)

South (25)

West (11)

All (76)

BPA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

16

15

14

13

10 11 12

9

8

7

0

200

2500

Northeast

A

1

Midwest

South

3

2

West

4

0

500

1000

3000

4000

5000

MGD > 100

B

10 < MGD < 100

1 < MGD < 10

6

5

23

Figure 2. Concentrations of bisphenols (reported in units of ng/g dw; as total bisphenol concentrations) in U.S. sewage sludge stratified by (A) geographic regions and (B) treatment capacity of the wastewater treatment plants (WWTPs). MGD stands for million gallons per day. Whiskers represent maxima.

ΣBisphenols (ng/g dw)

5000

ΣBisphenols (ng/g dw)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

10 11 12

9

8

7

6

5

4

3

2

1

0

10

500

1000

USA

China South Korea Greece Australia Canada

BPA BPS BPF

24

Figure 3. Comparison of median concentrations (ng/g dw) of major bisphenol analogs in sewage sludge samples from the United States (this study) and other countries (reported in the literature; Kate et al., 2011; Stasinakis et al., 2008; Lee et al., 2015; Song et al., 2014; Lee and Peart, 2000).

Concentration (ng/g dw)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Supplementary Material

SUPPLEMENTARY MATERIAL Occurrence and Estrogenic Potency of Eight Bisphenol Analogs in Sewage Sludge from the U.S. EPA Targeted National Sewage Sludge Survey Xiaohua Yu1, 2, Jingchuan Xue1, Hong Yao2, Qian Wu1, Arjun K. Venkatesan3, Rolf U. Halden3 and Kurunthachalam Kannan1,4*

1

Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA

2

Department of Municipal and Environmental Engineering, Beijing Jiaotong University, Beijing 100044, China 3

Center for Environmental Security, The Biodesign Institute, Global Security Initiative, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287, USA 4

Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

*Corresponding author: K. Kannan Wadsworth Center Empire State Plaza, P.O. Box 509 Albany, NY 12201-0509 Tel: +1-518-474-0015 Fax: +1-518-473-2895 E-mail: [email protected] Pages: 11 1

Table S1. List of wastewater treatment plants (WWTPs) sampled for the targeted national sewage sludge survey by U.S. EPA.

Sugar Creek WWTP

Flow (F) in MGD 1
Aldridge Creek WWTP

1
Huntsville

AL

Phoenix WWTP

10
Phoenix

AZ

Valley Sanitary District STP

1
Indio

CA

San Francisco

F>100

San Francisco

CA

El Estero WWTP

1
Santa Barbara

CA

Santa Rosa

1
Santa Rosa

CA

Stockton Water Quality Plant

F>100

Stockton

CA

Los Angeles County Sanitation District

10
Whittier

CA

Boulder WWTP

1
Boulder

CO

South Windsor

1
South Windsor

CT

Three Oaks WWTF

1
Estero

FL

Orange County Northeast WRF

1
Orlando

FL

Tampa

1
Tampa

FL

Albany

10
Albany

GA

Americus-Mill Creek

1
Americus

GA

Boone STP

1
Boone

IA

Calumet Water Reclamation Plant

F>100

Chicago

IL

Plainfield WWTP

1
Plainfield

IL

Lake County DPW, New Century STP

1
Vernon Hills

IL

Dupage County-Knollwood STP

1
Wheaton

IL

Blucher Poole WWTP

1
Bloomington

IN

William Ross Edwin WWTP

10
Richmond

IN

Parsons

1
Parsons

KS

Topeka

10
Topeka

KS

Mayfield WWTP

1
Nayfield

KY

Eunice

1
Eunice

LA

Jefferson Parish East Bank WWTP

1
Marrero

LA

Nantucket

1
Nantucket

MA

Salisbury

1
Salisbury

MD

Mechanic Falls Treatment Plant

1
Mechanic Falls

ME

Benton Harbor-St. Joseph WWTP

1
St. Joseph

MI

Wixom WTP

1
Wixom

MI

Festus Crystal City STP

1
Crystal City

MO

Elizabeth City WWTP

1
Elizabeth City

NC

Hillsborough WWTP

1
Hillsborough

NC

Beatrice

1
Beatrice

NE

WWTP

2

City

State

Alexander City

AL

Wildwood Lower WTF

10
Cape May Court House

NJ

Middlesex County Utility Authority WRC

F>100

Sayreville

NJ

Verona TWP DPW

1
Verona

NJ

Buffalo

F>100

Buffalo

NY

Canajoharie WWTP

1
Canajoharie

NY

Geneva A-C Marsh Creek STP

1
Geneva

NY

NYC DEP-Jamaica WPCP

10
New York City

NY

North Tonawanda STP

1
North Tonawanda

NY

Clermont County Commissioners

1
Batavia

OH

Bedford

1
Bedford

OH

Metropolitan Sewer District Little Miami

10
Cincinnati

OH

Northeast Ohio Regional Sewerage District Southerly

F>100

Cleveland

OH

Delaware County Alum Creek WWTP

1
Delaware

OH

Mingo Junction STP

1
Mingo Junction

OH

Duncan Public Utilities Authority

1
Duncan

OK

City of Klamath Falls WWTF

1
Klamath Falls

OR

Western Westmoreland Municipal Authority

1
Irwin

PA

Allegheny County Sanitary Authority

1
Pittsburgh

PA

Greater Pottsville Area Sewer Authority

1
Pottsville

PA

Punxsutawney

1
Punxsutawney

PA

South Kingstown WWTF

1
Narragansett

RI

Plum Island WWTP

10
Charleston

SC

Lawson Fork WTP

1
Spartanburg

SC

Elizabethton

1
Elizabethton

TN

Amarillo

10
Amarillo

TX

Dallas Southside WWTP

F>100

Dallas

TX

Trinity River Authority of Texas

1
Ellis County

TX

Fredericksburg

1
Fredericksburg

TX

Odo J. Riedel Regional WWTP

1
Schertz

TX

Wagner Creek WWTP

1
Texarkana

TX

Tyler Southside WTP

1
Tyler

TX

Spanish Fork City Corporation

1
Spanish Fork

UT

Buena Vista

1
Buena Vista

VA

Everett City SVC Center MVD

10
Everett

WA

Beaver Dam

1
Beaver Dam

WI

Elkins WWTP

1
Elkins

WV

Huntington

10
Huntington

WV

3

Table S2. Formula and Ions Monitored for Eight Bisphenol Analogs in U.S. Biosolids Samples. Common name

Acronym

CAS

Formula

MW

MS/MS ions (m/z)

Bisphenol A

BPA

80-05-7

C15H16O2

228.29

227>212

Bisphenol AF

BPAF

1478-61-1

C15H10F6O2

336.23

335>265

Bisphenol AP

BPAP

1571-75-1

C20H18O2

290.36

289>274

Bisphenol B

BPB

77-40-7

C16H18O2

242.32

241>212

Bisphenol F Bisphenol S Bisphenol P Bisphenol Z

BPF BPS BPP BPZ

620-92-8 80-09-1 2167-51-3 843-55-0

C13H12O2 C12H10O4S C24H26O2 C18H20O2

200.23 250.27 346.46 268.35

199>93 249>108 345>330 267>173

4

Table S3. MS/MS Parameters Optimized for Analysis of Eight Bisphenol Compounds in U.S. Biosolids Samples.

BPA

BPAF

BPAP

BPB

BPF

BPP

BPS

BPZ

Declustering potential (DP)

-40

-45

50

-52

-52

-58

-55

-55

Entrance potential (EP)

-10

-3

-6

-4

-4

-10

-3

-9

Collision energy (CE)

-30

-35

-35

-32

-30

-40

-38

-38

Collision cell exit potential (CXP)

-1

-1

-3

-1

-1

-1

-1

-1

5

Table S4. Analytical Method Performance of Eight Bisphenol Compounds in U.S. Biosolids Samples. 1

LOQ 2

3

101±8

3.19±2.35

31.5±24.3

103±10

18.3±28.2

97.7±85.4

1.79

92±6

0±0

0±0

1.79

94±10

0±0

0±0

0.05

1.79

98±5

6.30±10.9

15.5±18.2

BPP

0.05

1.79

78±16

0±0

0±0

BPS

0.05

1.79

84±20

31.0±32.6

52.6±57.3

BPZ

0.05

1.79

98±8

0±0

0±0

Analyte

1

ng/mL

ng/g d.w.

BPA

0.05

1.79

BPAF

0.05

1.79

BPAP

0.05

BPB

0.05

BPF

2

Recovery (%)

RPD (%)

3

RPD (%)

LOQ: Limit of Quantitation; RPD: Relative Percentage Difference; in procedural replicates; RPD field duplicates;

6

Table S5. Concentrations of bisphenol analogs (ng/g dw) in biosolids from various U.S. geographical regions.

BPA

BPAF

Northeast

Mean

465

4.64

(n=20)

Median

222


2

BPAP

BPB

BPF

BPP

BPS

BPZ

∑BPs

1



35.9


19.7


529



13.6


9.17


274

STD

691

9

0

Range

22.4-2760








DF (%)

100

44.4

0

0

77.8

0

88.9

0

Midwest

Mean

493

3.69



9.92

1.19

9.39


520

(n=20)

Median

122




6.18


3.52


200

3

754.68

13.03

0.00

Range

6.48-3360



DF (%)

100

42.1

South

Mean

423

(n=25)

Median

140

STD

0

0.00

59

0

37

0

15.10

707 38.7-2870

16.91

1.62

16.40

757.97






0

0

63.2

5.3

63.1

0

7.6


1.1

11.7

1.14

68.1

3.64

517

1.96



9.48


2.33


196

12.8-3390

946.32

14.75

0.00

0.96

12.80

0.19

301.07

0.00

996.23

Range

9.16-4700








27.9-4730

DF (%)

100

54

0

3

71

8

58

3

West

Mean

467

8.78



11.4


28.9


519

(n=11)

Median

292




8.09


13.1


360

STD

7

366.49

14.69

0.27

Range

18.6-1020








DF (%)

100

36.4

0

0

54.6

0

100

0

STD

0.11

1

10.03

0.05

68.18


8

2.04

353.05 48.8-1050

Table S6. Spearman correlation coefficients among concentrations of individual BPs measured in the U.S. biosolid samples. BPA

BPAF

BPAP

BPB

BPF

BPP

BPS

BPAF

0.187

*

*

*

*

*

*

BPAP

NA1

*

8

*

*

*

*

BPB

0.083

-0.103

NA

*

*

*

*

BPF

0.2702

-0.151

NA

0.154

*

*

*

BPP

0.012

0.203

NA

-0.025

0.136

*

*

BPS

0.204

-0.094

NA

0.021

0.144

-0.057

*

BPZ

0.151

0.072

-0.014

-0.142

-0.025

0.124

NA 1

2

NA: Not available; p<0.05;

9

Table S7. Concentrations of BPs in biosolids from treatment plants with different capacity in the U.S.

Flow Volume (Million Gallons per Day)

MGD > 100 (N=10)

10 < MGD < 100 (N=10)

1 < MGD < 10 (N=56)

Descriptive statistic

BPA

BPAF

BPAP

BPB

BPF

BPP

BPS

BPZ

∑BPs

GM

597

2.16

0.9

0.9

4.63

0.9

7.62

1.38

645

Mean

795

5.08

0.9

0.9

9.81

0.9

11.8

7.48

832

Median

639

1.43

0.9

0.9

6.84

0.9

12.5

0.9

655

Range

1252130








1652180

DF (%)

100

50

0

0

60

0

80

10

-

GM

230

3.66

0.9

0.9

7.28

1.08

6.74

0.9

286

Mean

321

12.8

0.9

0.9

16.4

1.37

9.99

0.9

364

Median

311

2.05

0.9

0.9

10.5

0.9

7.22

0.9

355

Range

46.7-785








74.6-873

DF (%)

100

70

0

0

70

10

90

0

-

GM

133.09

1.91

0.9

0.93

6.5

0.95

5.08

0.9

189.33

Mean

420.35

4.88

0.9

0.99

18.83

1.02

43.59

0.9

491.47

Median

117.65

0.9

0.9

0.9

8.55

0.9

4.17

0.9

178.43

Range

6.484700








12.84730

DF (%)

100

43

0

2

68

4

70

0

-

10

Table S8. Physiochemical properties of bisphenol analogs (predicted data from www.chemspider.com, based on USEPA’s EPISuiteTM).

a

Analyte

Log Kowa

Log Kocb

Solubility (mg/L) at 25°Cc

Removal in WWTP (%)d

Adsorption on sludge (%)d

Biodegradation (%)d

Half-life in sediment (h)e

BPA

3.64

4.88

172.7

9.55

9.39

0.16

8.10 x 103

BPAF

4.47

6.21

4.302

54.44

53.93

0.51

3.89 x 104

BPAP

4.86

6.26

3.758

72.77

72.13

0.64

8.10 x 103

BPB

4.13

5.17

29.23

36.33

35.96

0.37

8.10 x 103

BPF

3.06

4.47

542.8

5

4.88

0.12

3.24 x 103

BPP

6.25

7.11

0.113

92.97

92.2

0.77

1.30 x 104

BPS

1.65

3.88

3518

2.02

1.93

0.09

3.24 x 103

BPZ

5

5.78

3.782

77.7

77.02

0.68

8.10 x 103

b

c

d

e

TM

Based on KOWWIN v1.67 estimate; Based on PCKOCWIN v1.66; Based on WSKOW V1.41; Based on STPWINTM; Based on level-III fugacity model via WOVWIN .

11