U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2011–1132

Prepared in cooperation with the University of Arizona, Department of Chemical and Environmental Engineering, 

AECOM Technology Corporation, and the Water Environment Research Foundation

Pharmaceuticals, Hormones, Anthropogenic Waste 

Indicators, and Total Estrogenicity in Liquid and 

Solid Samples from Municipal Sludge Stabilization 

and Dewatering
Pharmaceuticals, Hormones, Anthropogenic 

Waste Indicators, and Total Estrogenicity 

in Liquid and Solid Samples from Municipal 

Sludge Stabilization and Dewatering

By Edward T. Furlong, James L. Gray, David M. Quanrud, Sondra S. Teske, 

Stephen L. Werner, Kathleen Esposito, Jeremy Marine, Wendell P. Ela, 

Steven D. Zaugg, Patrick J. Phillips, and Beverley Stinson

Prepared in cooperation with the University of Arizona,

Department of Chemical and Environmental Engineering, 

AECOM Technology Corporation, and the 

Water Environment Research Foundation

Open-File Report 2011–1132

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

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Suggested citation:

Furlong, E.T., Gray, J.L., Quanrud, D.M., Teske, S.S., Werner, S.L., Esposito, Kathleen, Marine, Jeremy, Ela, W.P., 

Zaugg, S.D., Phillips, P.J., and Stinson, Beverley, 2012, Pharmaceuticals, hormones, anthropogenic waste indicators, 

and total estrogenicity in liquid and solid samples from municipal sludge stabilization and dewatering: U.S. Geological 

Survey Open-File Report 2011–1132, 8 p.
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Purpose and Scope ..............................................................................................................................2

Study Sites and Sampling Design ...............................................................................................................2

Analyses of Pharmaceuticals, Hormones, and Anthropogenic Waste Indicators .............................2

Pharmaceutical Analysis.....................................................................................................................3

Hormone Analysis.................................................................................................................................3

Anthropogenic Waste Indicator Analysis.........................................................................................4

Determination of Total Estrogenicity ..........................................................................................................4

Centrifugation and Filtration ...............................................................................................................4

Microwave-Assisted Extraction.........................................................................................................4

Separation on C–18 Resin ...................................................................................................................5

Yeast Estrogen Screen (YES) Bioassay.............................................................................................5

KBluc Bioassay Summary ...................................................................................................................5

Standard Data Reduction Method for Bioassays............................................................................5

Data for Liquid and Solid Samples..............................................................................................................6

Acknowledgments.........................................................................................................................................6

References......................................................................................................................................................6

Tables

[Click on table title to view in Excel format]

1. Compound name, typical use, Chemical Abstracts Service registry number, lowest 

reporting level, and National Water Quality Laboratory schedule or laboratory code 

used for samples determined at the NWQL during this study

2. Concentrations of estrogens, androgens, progestins, and other related compounds 

in liquid and solid samples from Wastewater Treatment Plant A, collected between 

March 2006 and January 2007

3. Concentrations of estrogens, androgens, progestins, and other related compounds

in liquid and solid samples from Wastewater Treatment Plant B, collected between

December 2005 and May 2007

4. Concentrations of estrogens, androgens, progestins, and other related compounds 

in liquid and solid samples from Wastewater Treatment Plant C, collected between

December 2005 and July 2006

5. Concentrations of estrogens, androgens, progestins, and other related compounds

in liquid and solid samples from Wastewater Treatment Plant D, collected between

March 2006 and December 2006

6. Concentrations of anthropogenic waste indicators (AWIs) in liquid and solid samples 

from Wastewater Treatment Plant A, collected between March 2006 and January 2007

7. Concentrations of anthropogenic waste indicators (AWIs) in liquid and solid samples 

from Wastewater Treatment Plant B, collected between December 2005 and 

January 2007

8. Concentrations of anthropogenic waste indicators (AWIs) in liquid and solid samples 

from Wastewater Treatment Plant C, collected between December 2005 and July 2006

9. Concentrations of anthropogenic waste indicators (AWIs) in liquid and solid 

samples from Wastewater Treatment Plant D, collected between March 2006 and 

December 2006
iv

10. Estrogenicity of liquid and solid samples from Wastewater Treatment Plant A, 

collected between March 2006 and January 2007

11. Estrogenicity of liquid and solid samples from Wastewater Treatment Plant B, 

collected between December 2005 and January 2007

12. Estrogenicity of liquid and solid samples from Wastewater Treatment Plant C, 

collected between December 2005 and July 2006

13. Estrogenicity of liquid and solid samples from Wastewater Treatment Plant D, 

collected between March 2006 and December 2006

14. Estrogenicity of liquid and solid samples from Wastewater Treatment Plant D, 

collected June 2006

15. Concentrations of pharmaceuticals in liquid and solid samples from Wastewater 

Treatment Plant A, collected between March 2006 and January 2007

16. Concentrations of pharmaceuticals in liquid and solid samples from Wastewater 

Treatment Plant B, collected between December 2005 and January 2007

17. Concentrations of pharmaceuticals in liquid and solid samples from Wastewater 

Treatment Plant C, collected between December 2005 and July 2006

18. Concentrations of pharmaceuticals in liquid and solid samples from Wastewater 

Treatment Plant D, collected between March 2006 and December 2006

Conversion Factors

SI to Inch/Pound

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 3.281 foot (ft)

meter (m) 1.094 yard (yd)

Volume

liter (L) 33.82 ounce, fluid (fl. oz)

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

liter (L) 61.02 cubic inch (in3

) 

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

kilogram (kg) 2.205 pound, avoirdupois (lb)

Pressure

kilopascal (kPa) 0.009869 atmosphere, standard (atm)

kilopascal (kPa) 0.01 bar

kilopascal (kPa) 20.88 pound per square foot (lb/ft2

)

kilopascal (kPa) 0.1450 pound per square inch (lb/in2

)

Density

kilogram per cubic meter (kg/m3

) 0.06242 pound per cubic foot (lb/ft3)

gram per cubic centimeter (g/cm3

) 62.4220 pound per cubic foot (lb/ft3)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

°C=(°F–32)/1.8
v

Acronyms and Abbreviations

°C degrees Celsius

< less than

μL microliter

μm micrometer

AECOM AECOM Technology Corporation

APE alkylphenol ethoxylate

AWI anthropogenic waste indicator

CAS Chemical Abstracts Service

CF concentration factor

cm centimeter

CPRG chlorophenol red β-D-galactopyranoside

DCM dichloromethane

DCM/DEE dichloromethane/diethyl ether

DEE diethyl ether

EC50 concentration at which 50 percent of exposed organisms exhibit an effect

EDC endocrine-disrupting compound

EE2 17-alpha-ethnylestradiol

EEQ 17-alpha-ethnylestradiol equivalents

FBS fetal bovine serum

fM femptomole

g gram

GC/MS/MS gas chromatography/tandem mass spectrometry

GFF glass-fiber filter

HLB hydrophilic-lipophilic-balanced reversed-phase sorbent for acids, bases,

and neutrals

HPLC high-performance liquid chromatography

HPLC–ESI/MS high-performance liquid chromatography–electrospray ionization/ 

mass spectrometry

HRT hydraulic residence time

kPa kilopascals

L liter

MAE microwave-assisted extraction

MeOH methanol

mg milligram

mL milliliter

mm millimeter

MS mass spectrometer
vi

MSTFA N-methyl-N-(trimethylsilyl)trifluoroacetamide

ND not detected

nM nanomole

nm nanometer

NP nonylphenol

NPEO nonylphenol ethoxylate

RCF relative centrifugal force

RLU relative light units

SPE solid-phase extraction

SRT solids-retention time

TMS trimethylsilyl

TOC trace organic compound

USGS U.S. Geological Survey

v/v volume to volume

WERF Water Environment Research Foundation

WWTP wastewater treatment plant

YES yeast estrogen screen

Acronyms and Abbreviations—Continued
Pharmaceuticals, Hormones, Anthropogenic Waste Indicators, 

and Total Estrogenicity in Liquid and Solid Samples from 

Municipal Sludge Stabilization and Dewatering

By Edward T. Furlong, James L. Gray, David M. Quanrud, Sondra S. Teske, Stephen L. Werner, Kathleen Esposito, 

Jeremy Marine, Wendell P. Ela, Steven D. Zaugg, Patrick J. Phillips, and Beverley Stinson

Introduction

The common occurrence of pharmaceuticals and other 

emerging contaminants, also referred to as trace organic compounds (TOCs), in surface water (Kolpin and others, 2002; 

Glassmeyer and others, 2007) has resulted in research and 

monitoring efforts to identify TOC sources to surface waters 

and to understand the loadings of TOCs from these sources. 

Wastewater treatment plant (WWTP) discharges have been 

identified as an important point source of TOCs to surface 

water (Carballa and others, 2007; Dickenson and others, 

2009; Joss and others, 2004, 2005; Wick and others, 2009), 

and understanding the transport and transformation of TOCs 

through wastewater treatment processes is essential to controlling the introduction of TOCs to surface-water environments.

Whereas the effects of many TOCs are unknown, a number are classified as endocrine-disrupting compounds (EDCs); 

specific examples include natural and synthetic estrogens, 

bisphenol A, alkylphenol ethoxylates (APEs), particularly 

nonylphenol ethoxylates (NPEOs), and nonylphenol (NP), 

a persistent transformation product of NPEOs (Jobling and 

Sumpter, 1993; Länge and others, 2001). Endocrine disruption 

in surface-water environments is evidenced at biochemical, 

cellular, organismal, and ecological levels of organization. 

Commonly observed endpoints include inappropriate production of vitellogenin, a female egg yolk protein, by males and 

juveniles, the observation of ova in testicular tissue, the induction of female secondary sexual characteristics in putatively 

male fish, and skewed sex ratios in both wild and laboratory 

populations. These effects have been identified in surfacewater environments in North America and Europe and are 

associated with WWTP discharges (Vajda and others, 2008; 

Woodling and others, 2006).

Whereas public and scientific interest in the presence 

and effects of EDCs is substantial, the analytical techniques 

necessary to determine the compositions and concentrations of 

the diverse, heterogeneous suite of EDCs can be complex and 

costly. Bioassay techniques that quantify estrogenic receptor 

response can be a cost-effective means of characterizing the 

estrogenicity both of liquid and solid samples if the relation 

between individual EDC constituents and bioassay estrogenic 

response can be established qualitatively and quantitatively.

Abstract

The ubiquitous presence of pharmaceuticals and other 

emerging contaminants, or trace organic compounds, in 

surface water has resulted in research and monitoring efforts 

to identify contaminant sources to surface waters and to 

better understand loadings from these sources. Wastewater 

treatment plant (WWTP) discharges have been identified 

as an important point source of trace organic compounds to 

surface water and understanding the transport and transformation of these contaminants through wastewater treatment 

processes is essential to controlling their introduction to 

receiving waters.

Whereas the effects of many trace organic compounds 

are not well known, a number are classified as endocrinedisrupting compounds, including natural and synthetic 

estrogens, bisphenol A, alkylphenol ethoxylates, particularly 

nonylphenol ethoxylates, and nonylphenol. This report documents the results from measurements of individual endocrinedisrupting compounds, other trace organic compounds, and 

yeast estrogen screen (YES) bioassay estimates of estrogenic 

activity from a multiple-year, multiple-WWTP study. This 

work was undertaken by scientists of the U.S. Geological 

Survey, the University of Arizona, and AECOM Technology 

Corporation (AECOM) in cooperation with the Water 

Environment Research Foundation (WERF). The purpose of 

the study was to characterize the reduction of estrogenicity 

and endocrine-disrupting compounds (EDCs) through wastewater treatment processes typical of WWTPs in the United 

States, with emphasis on the solids phase. In this study, four 

WWTPs were sampled two to four times annually between 

December 2005 and January 2007. Synoptic liquid samples 

of primary influent, primary effluent, and secondary (treated) 

effluent were collected, and where appropriate, solid samples 

also were collected. Field and laboratory quality-assurance/

quality-control samples also were analyzed in this study. The 

results from this study are published as a U.S. Geological 

Survey Open-File Report to ensure adequate documentation 

of the original results, provide a single citable source of original 

results for subsequent interpretive reports, and ensure continued 

public access to these results through online publication.
2 Pharmaceuticals, Hormones, Waste Indicators, and Estrogenicity in Samples from Sludge Stabilization and Dewatering

Purpose and Scope

This report documents the results from measurements 

of individual EDC and TOC chemicals and yeast estrogen 

screen (YES) bioassays from a multiple-year, multiple-WWTP 

study undertaken by scientists of the U.S. Geological Survey 

(USGS), the University of Arizona, and AECOM Technology 

Corporation (AECOM) in cooperation with the Water 

Environment Research Foundation (WERF). All data tables 

are presented in Microsoft® Excelformat and can be accessed 

through links at their first citation within this report. The purpose of the study was to characterize the reduction of estrogenicity and EDCs through wastewater treatment processes 

typical of WWTPs in the United States, with emphasis on the 

solids phase. Analysis of solid-phase samples was of particular 

importance as the understanding of TOCs in wastewater and 

environmental solid phases is not well documented.

In this study, four WWTPs were sampled two to four 

times annually between December 2005 and January 2007. 

Synoptic liquid samples of primary influent, primary effluent, and secondary (treated) effluent were collected based on 

the typical hydraulic residence time (HRT) of each WWTP, 

as determined by WWTP operations staff. Where appropriate, 

solid samples also were collected on a schedule based on the 

typical HRT of each WWTP. However, due to the long solidsretention times (SRTs) of many solids-treatment unit processes 

(for example, digestion), synoptic sampling was not possible. 

However, the long SRTs also result in concentrations that are 

less variable over time than for liquid samples, so comparisons between sequential unit processes are valid. Upon receipt 

by participating laboratories, solid and liquid samples were 

split; one set of samples was analyzed by chemical methods 

to determine a suite of individual pharmaceuticals, 17 hormones (estrogens, androgens, and progestins), and a suite of 

anthropogenic waste indicator (AWI) compounds. Many of 

the hormones and AWIs are known or suspected EDCs. The 

second set of split samples was assessed for total estrogenicity, 

quantified as ethinyl estradiol equivalents (EEQ) by using the 

YES bioassay. Field and laboratory quality-assurance/qualitycontrol samples also were analyzed in this study and the 

results met acceptance criteria for each method.

Solid and liquid samples from WWTPs are complex samples typically containing many organic and inorganic interferences that were expected to make sample analysis difficult. 

Also, at the time of this study, many of the methods were not 

approved as official methods of the USGS and were classified 

instead as research or custom methods. Data from unapproved 

methods cannot be entered into the USGS National Water 

Information System (Miller, 2004), the standard, publicly 

accessible data repository for results from official USGS methods. Thus, the results from this study were aggregated into this 

USGS Open-File Report to (1) ensure adequate documentation of the original results, (2) provide a clear description 

of the analytical methods used to produce these results, 

(3) provide a single citable source of original results for subsequent interpretive reports, and (4) ensure continued public 

access to the results through online publication as a USGS 

Open-File Report.

Study Sites and Sampling Design

Four WWTPs were sampled as a part of this study. The 

four WWTPs are hereinafter referred to as Plants A, B, C, or D 

in all tables of results. To encourage participation and cooperation for the extensive sample collection this study required, 

WWTPs are not identified by location. All four plants are 

secondary treatment plants, with screening, primary treatment 

(settling), followed by secondary treatment with activated 

sludge, prior to disinfection and discharge. Solids handling 

steps varied among plants and included thickening by gravity, gravity belt, and dissolved air flotation; stabilization using 

lime addition, aerobic digestion, and anaerobic digestion; 

chemical conditioning; dewatering by centrifugation; and 

other processes including composting and pelletization.

Samples were collected from the WWTPs approximately every 3 or 6 months during 1 year. Aqueous samples 

and some high water-content solid samples were collected 

based on the average HRT of each plant, which was provided 

by the operations staff of each plant. The purpose of adjusting sampling time to HRT was to maximize comparability 

among different unit processes when estimating loads of 

TOCs. Sample collection was conducted according to USGS 

water-quality trace-organic compound sample-collection 

protocols (U.S. Geological Survey, variously dated), adapted 

for the specific circumstances present in WWTPs. After 

an initial training period by USGS scientists, AECOM and 

participating WWTP staff assisted the USGS with sample 

collection. After collection, samples were stored on ice in 

insulating coolers and shipped by overnight express to the 

USGS and University of Arizona laboratories for TOC and 

bioassay analyses, respectively.

Analyses of Pharmaceuticals, 

Hormones, and Anthropogenic 

Waste Indicators

Three separate trace organic chemical analyses were 

done on separate subsamples of liquid and solid samples. The 

three classes of chemicals measured by each analysis were 

(1) pharmaceuticals; (2) hormones, which included estrogens, 

androgens, progestins, and the fecal sterols cholesterol and 

coprostanol; and (3) anthropogenic waste indicators (AWIs), 

which included the estrogenic APEs, bisphenol A, NPEOs, 

and NP. Table 1 lists the chemical name, Chemical Abstracts 

Service (CAS) number, and typical use of each chemical.
Analyses of Pharmaceuticals, Hormones, and Anthropogenic Waste Indicators 3

Pharmaceutical Analysis

Pharmaceuticals were determined in liquid samples 

by using the procedure of Cahill and others (2004). In this 

method, the target pharmaceuticals and an added performance 

surrogate first were removed from filtered 1-liter (L) water 

samples by passing the water through an Oasis® hydrophiliclipophilic balanced reversed-phase sorbent for acids, bases, 

and neutrals (HLB) solid-phase extraction (SPE) cartridge 

(500-milligram [mg] bed mass; Waters Corporation, Milford, 

Massachusetts). The pharmaceuticals then were eluted from 

the SPE cartridge by using 6 milliliters (mL) of methanol 

followed by 4 mL of methanol acidified with trifluoroacetic 

acid (0.1 percent). The two eluents were combined, concentrated to near dryness, reconstituted into formate buffer 

with an internal standard, and filtered. Pharmaceuticals in 

the concentrated extracts were separated using reversedphase, high-performance liquid chromatography (HPLC), 

which was interfaced with a quadrupole mass spectrometer 

(MS) equipped with an electrospray ionization source 

(HPLC–ESI/MS) for identification and quantitative analysis. 

The electrospray ionization source was operated in the 

positive-ion mode. Selected-ion monitoring MS was used to 

improve sensitivity and specificity. Qualitative identification 

was based on HPLC retention and at least two compoundspecific fragment ions for each pharmaceutical. Quantitation 

was by the injection internal standard method.

For solid samples, the approach of Kinney and others 

(2006) was used. Approximately 1 gram (g) of wet solids was 

weighed into an 11-mL pressurized liquid extraction vessel, 

an aliquot of a cocktail of performance surrogates was added, 

and the sample was extracted using pressurized liquid extraction (ASE 200; Dionex Corporation, Sunnyvale, California), 

using five static cycles (10 minutes each at 130°C and 

10,300-kilopascals [kPa]) with an acetonitrile/water mixture 

(70:30, volume to volume [v/v]). One mL of the total extract 

was filtered using a 0.20-μm (micrometer) syringe filter into 

an HPLC vial, and the acetonitrile evaporated under nitrogen. The sample was reconstituted to about 0.95 mL using a 

10-millimolar aqueous ammonium formate buffer; an aliquot 

of a nicotinamide-2,4,5,6-d4

 solution was added as an injection internal standard and the final volume of the extract was 

brought to about 1 mL. Analysis by HPLC–ESI/MS followed 

the procedure of Cahill and others (2004) outlined in the 

preceding paragraph.

Hormone Analysis

Estrogens, androgens, progestins, and fecal sterols 

were determined in whole-water (liquid + solids) samples 

using C–18 SPE followed by isotope-dilution capillary gas 

chromatography/tandem mass spectrometry (GC/MS/MS). 

Briefly, 1-L whole-water samples were poured into stainlesssteel extraction tubes equipped with a multigrade glass-fiber 

filter (GFF) over a 47-millimeter (mm) C–18 SPE (Supelco 

ENVI+; Sigma-Aldrich Corporation, St. Louis, Missouri) disk. 

The samples were passed through the GFF/C–18 disk under 

pressure, as needed. Just prior to extraction, an aliquot of a 

cocktail of 12 stable-isotope-labeled analogues of estrogens, 

androgens, progestins, and fecal sterols was added to each 

sample to quantify the hormones by the stable isotope dilution 

method (De Hoffman and others, 1996). Following compound 

isolation, the GFF/C–18 disk was rinsed with 25 percent 

methanol (MeOH) in water, dried with nitrogen, and the compounds eluted with MeOH. The MeOH eluent was evaporated 

to dryness and reconstituted in a mixture of 20 percent diethyl 

ether in dichloromethane (DCM/DEE). The extract was then 

passed through a 1-g Florisil SPE column and eluted with an 

additional aliquot of the DCM/DEE solution. The eluent was 

reduced in volume with nitrogen, quantitatively transferred to 

a 5-mL reaction vial, and evaporated to dryness. Ketone and 

alcohol groups on the analytes and isotopically labeled surrogates were derivatized to trimethylsilyl (TMS)-substituted 

analogs to make them stable for separation by gas chromatography. Derivatization was accomplished using N-methylN-(trimethylsilyl)trifluoroacetamide (MSTFA) activated with 

2-(trimethylsilyl)ethanethiol. The analytes were separated by 

capillary gas chromatography and quantified by tandem quadrupole mass spectrometry using an isotope dilution procedure.

For solid samples, the derivatization, chromatographic 

separation, and mass spectrometric quantitation steps used for 

water samples remained the same, whereas sample extraction 

and isolation steps were substantially different.1

 Wet solids 

were loaded into a 33-mL stainless-steel extraction vessel. 

Approximately 1 g of solid material (on a dry mass basis) 

was used. Any remaining cell volume was filled with ashed 

quartz sand. Just prior to extraction, an aliquot of a cocktail of 

12 stable-isotope-labeled analogues of estrogens, androgens, 

progestins, and fecal sterols was added to each sample to 

quantify the hormones by the stable-isotope dilution method 

(De Hoffman and others, 1996). The wet sample was then 

extracted using an automated pressurized liquid extraction 

system (ASE 200; Dionex Corp., Sunnyvale, California), 

using a programmed multiple-cycle extraction procedure. In 

the procedure, the samples are extracted three times using a 

mixture of water:isopropyl alcohol (50:50, v/v) at 120°C and 

then extracted three times with water:isopropyl alcohol (20:80, 

v/v) at 200°C. The entire extraction sequence is carried out at 

a pressure of 13.8×103

 kPa.

1

Subsequent to this study, some changes were made to the hormone analysis 

procedures that could result in unknown data bias. Specifically, for both 

water and sediment methods, a few different isotopically labeled compounds 

were added and others subtracted to address intermittent performance issues 

not apparent during this study. Also, Florisil elution solvent was changed 

from 80 percent DCM/20 percent DEE to 95 percent DCM/5 percent MeOH 

(waters) or 90 percent DCM/10 percent MeOH (solids) to improve polar 

compound recovery, address safety concerns associated with formation of peroxides in oxygen-exposed DEE, and to address intermittent losses of phenolic 

compounds due to reaction with peroxides not apparent in this study.
4 Pharmaceuticals, Hormones, Waste Indicators, and Estrogenicity in Samples from Sludge Stabilization and Dewatering

The extraction aliquots were automatically transferred 

by nitrogen pressure to a glass vessel sealed with a Teflonfaced silicone septum. The three extraction aliquots processed 

at 120°C were automatically combined in a single glass 

vessel. The three extract aliquots processed at 200°C were 

automatically combined in a separate sealed glass vessel. 

The 200°C extract was passed through an SPE cartridge 

(Oasis® HLB, 500-mg bed mass; Waters Corporation, Milford, 

Massachusetts), followed by the 120°C extract. The Oasis®

cartridge was dried with nitrogen, connected to a second 

cartridge containing 1.5 g anhydrous sodium sulfate over 2 g 

of Florisil bed mass, and the hormones were eluted and purified in a single step with three successive 10-mL aliquots of 

DCM/DEE (80:20, v/v). The combined DCM/DEE aliquots 

were reduced under nitrogen to dryness. The dried sample was 

then derivatized, separated, and quantified as described for the 

whole-water samples.

Anthropogenic Waste Indicator Analysis

The AWIs, which included the nonhormonal, synthetic 

organic EDCs, were determined in filtered liquid samples 

by the method of Zaugg and others (2007). Briefly, liquid 

samples were filtered through a 0.7-μm ashed glass-fiber filter, 

and approximately 1 L of filtered sample was passed through 

an Oasis® HLB SPE cartridge under negative pressure. Just 

prior to isolation by SPE, an aliquot of a cocktail of surrogate 

compounds was added to the sample. The recovery of these 

surrogates was determined as part of the analysis to assess 

method performance.

The SPE cartridge was dried with nitrogen and the AWIs 

were then eluted with two successive aliquots of DCM/DEE 

(4:1, v/v). The first 15-mL aliquot is used to rinse the sample 

bottle prior to elution through the SPE cartridge to extract any 

AWIs adsorbed to the bottle surface. The second 3-mL elution is applied directly to the SPE cartridge to elute any AWIs 

remaining on the cartridge. A final elution with 3 mL of DEE 

is then applied to the SPE cartridge to maximize compound 

recovery. An aliquot of an injection internal standard cocktail of 

perdeuterated polycyclic aromatic hydrocarbons was added to 

the combined eluent fractions of each sample, and the sample 

extract was reduced to 0.4 mL final volume. The sample final 

extract was analyzed for the 67 AWI compounds by capillary 

gas chromatography coupled to a quadrupole mass spectrometer 

operated under full-scan, electron-impact ionization conditions. Sample AWI detections were verified by comparison to 

an in-house mass spectral library developed from analysis of 

authentic standards. Multiple-point calibration using the injection internal standard method was used to determine sample 

concentrations for each compound.

The AWIs were determined in solid samples by using 

the method of Burkhardt and others (2006). The extraction 

procedure is identical to the hormone extraction procedure 

except as noted. Just prior to extraction, an aliquot of a cocktail of surrogates was added to each sample; the surrogates 

were quantified as part of the analysis to evaluate method 

performance. After extraction, the combined DCM/DEE 

aliquots were reduced under nitrogen to between 2 and 4 mL, 

an aliquot of an injection internal standard cocktail of perdeuterated polycyclic aromatic hydrocarbons was added, and 

the sample extract was reduced to a volume of 1 mL. The 

sample final extract was analyzed for the 67 AWI compounds 

by capillary gas chromatography coupled to a quadrupole 

mass spectrometer operated under full-scan, electron-impact 

ionization conditions. Sample AWI detections were verified 

by comparison to an in-house mass spectral library developed 

from analysis of authentic standards. Multiple-point calibration using the injection internal standard method was used to 

determine sample concentrations for each compound.

Compounds that were not detected were typically reported 

as “less than” values; that is, “<20 μg/L” for liquid samples and 

“ND” for solid samples. These values are based on the reporting level of the method. The reporting level is provided as a 

concentration that is based upon a standard sample size. For 

liquid samples in this study, the standard sample size is one liter. 

Standard solid sample sizes varied: for hormones the standard 

sample size used to determine reporting levels was 0.2 g dry 

mass, for AWIs 25 g dry mass, and for pharmaceuticals 35 g dry 

mass. If the dry mass of the sample is less than the standard dry 

mass for the analysis, the reporting levels are raised to reflect 

the lower dry mass of sample analyzed.

Determination of Total Estrogenicity

Centrifugation and Filtration

Liquid-phase samples (raw influent, primary clarifier 

effluent, secondary clarifier effluent, effluent from dewatering 

of thickened sludge, and liquid from the dewatering process 

after anaerobic digestion [centrate]) were separated into liquid 

and solid fractions using a Beckman J–10 centrifuge (Beckman 

Coulter, Incorporated, Brea, California) with a JA–10 rotor 

(20 minutes, relative centrifugal force [RCF]=17,000). Liquid 

portions were decanted and filtered using 3.1-μm and 0.7-μm 

Pall glass-fiber filters (Pall Corporation, Port Washington, 

New York).

The liquid and solid fraction of each sample was analyzed 

for estrogenic activity. Solid-phase samples (sludges/biosolids) 

were separated using centrifugation and the solid fractions 

were analyzed. Liquid centrates were not tested because it was 

assumed that the EDCs of interest, which typically are hydrophobic, would be found in the solid fraction.

Microwave-Assisted Extraction

After centrifugation/filtration, all solid samples (sludges/

biosolids) were extracted in methanol using a microwaveassisted extraction (MAE) procedure. About 1 g (dry 

weight) of solid was suspended in 20 mL of methanol and 

extracted at constant pressure (138 kPa for 30 minutes) 

using a CEM–MDS 2100 Microwave Digestion System 

(CEM Corporation, Matthews, North Carolina). Reactor 

contents were cooled for 45 minutes inside the microwave unit 

before liquids were decanted into ashed glass vials. Methanol 

extracts were evaporated to 1 mL under nitrogen gas.
Determination of Total Estrogenicity 5

Separation on C–18 Resin

Solid-phase microwave extracts were diluted to 1-percent 

methanol (v/v) in Nanopure water (Nanopure Infinity, Barnstead 

Thermolyne, Dubuque, Iowa) water and passed through reversephase (C–18 octadecyl) resin disk (Empore, 3M Purification, 

Incorporated, Eagan, Minnesota). The 47-mm C–18 disks were 

preconditioned with two 10-mL volumes of 100 percent ethyl 

alcohol (Pharmco-AAPER, a subsidiary of Greenfield Ethanol, 

Chatham, Ontario, Canada) and 10 mL of Nanopure water as 

prescribed by the resin manufacturer. Retained organics were 

sequentially eluted off C–18 disks using 10 mL of a 20-percent 

methanol in water solution, followed by 10 mL of a 50-percent 

methanol in water solution, and then 10 mL of an 80-percent 

methanol in water solution.

For liquid-phase 0.7-μm filtrates, whole samples (undiluted) were applied and sequentially eluted (as described above) 

from C–18 disks. Eluates were dried under nitrogen gas and 

redissolved in autoclaved Nanopure water to yield final concentration factors of 200–500 for estrogenic activity analysis. 

Solid-phase eluates were similarly dried under nitrogen gas, 

resuspended with 1–2 mL of autoclaved Nanopure water, then 

filtered through a 0.7-μm glass-fiber filter prior to analysis.

Yeast Estrogen Screen (YES) Bioassay

Total estrogenic activity was measured using the YES 

bioassay of Routledge and Sumpter (1996) as amended by 

DeBoever and others (2001). The Saccharomyces cerevisiae

strain was provided by John Sumpter of Brunel University, 

Oxbridge, U.K. The YES is a yeast-based in vitro bioassay utilizing a human estrogen receptor recombinant engineered with 

a beta-galactosidase reporter gene (lacZ) downstream from the 

estrogen response element. Resultant total estrogenic activity is expressed as an equivalent concentration of a known 

estrogenic compound—here, 17-alpha-ethnylestradiol (EE2), 

an oral contraceptive.

Each sample concentrate was serially diluted across 

10 wells of a 96-well micro-titer plate (Corning, Incorporated, 

Lowell, Massachusetts). Each dilution series was initiated by 

placing 100 microliters (μL) of sample concentrate in the first 

well of a single row. Fifty μL were transferred to the second 

column and mixed with 50 μL of Nanopure water (twofold 

dilution per step). The process was repeated across each row 

to produce a maximum dilution factor of 29. Fifty μL of Nanopure water that was pretreated by passage through the C–18 

resin were added to wells 11 and 12 of each row to serve as 

(negative) process controls. The eight rows of each 96-well 

plate provided replicate data (n=8) for estimation of experimental error. A standard series was developed in a similar 

manner with each set of measurements using concentrations of 

EE2 from 1.0×10–7 to 5.0×10–12 molar.

Yeast cells were grown in the Routledge/Sumpter 

medium to an absorbance (A630) of 1.0 cm–1. The culture was 

then diluted in the same medium to an absorbance (A630) of 

0.133 cm–1, and 150 μL of the diluted suspension was added 

to each well of the 96-well plate (total volume 200 μL per 

well). The resultant A630 value in each well was then about 

0.10 cm–1. Plates were incubated for 24 hours at 32°C for 

growth of S. cerevisiae and estrogen-dependent expression of 

lacZ. At that point, 50 μL of cycloheximide/CPRG (chlorophenol red β-D-galactopyranoside) solution consisting of 3 mL 

of autoclaved Nanopure water, 2 mL of 10 mg/mL cycloheximide, and 200 μL of 10 mg/mL CPRG was added to each test 

well. Following an additional 24-hour incubation at 32°C for 

β-galactosidase-dependent color development, absorbance was 

measured at 570 nm (β-galactosidase activity) and 630 nm 

(turbidity). The contribution of cell-dependent light scattering to A570 measurements was determined by measuring 

the ratio of A570/A630 (here defined as R) in the negative 

control wells. The β-galactosidase activity was then corrected 

to A570 – R × A630. Dose-response curves were plotted for 

environmental samples and the positive (EE2) control.

KBluc Bioassay Summary

Due to its high operational costs, the T47D–KBluc cell 

line bioassay developed by Wilson and others (2004) was used 

on a subset of samples as a second in vitro technique to measure estrogenic activity. Cells were maintained in RPMI–1640 

Medium with 10 percent fetal bovine serum (FBS) (HyClone 

Laboratories, Incorporated, Logan, Utah); no antibiotics were 

added to the medium. The bioassay was conducted in 24-well 

plates, and wells were rinsed with estrogen-free medium 

containing 3 percent charcoal dextran-treated FBS (Atlanta 

Biologicals, Incorporated, Lawrenceville, Georgia). Samples 

were serially diluted in triplicate across plates in estrogenfree growth medium, and 50,000 T47D cells were seeded per 

well. Plates were incubated in 5 percent carbon dioxide for 

48 hours at 35°C. Subsequently, cells were harvested using 

lysis buffer of which 100 μL from each well of the lysed cell 

solution was collected and transferred to a 96-well luminometer plate. Luciferase activity was quantified using an Analyst 

AD Plate Reader (Molecular Devices, Sunnyvale, California). 

The positive estrogen control consisted of decline dilutions of 

EE2 (Sigma-Aldrich Corporation, St. Louis, Missouri) from 

10 nanomole (nM) to 1 femptomole (fM). Data were plotted 

as relative light units (RLU) relative to EE2 concentration. A 

negative control plate consisting of medium and cells was run 

concurrently with each set of environmental samples.

Standard Data Reduction Method for Bioassays

The traditional technique to quantify estrogenic activity 

in environmental samples relies upon identifying the midpoint 

(50 percent, EC50) level of response in both the environmental sample and positive control (EE2) dose response curves. 

In this approach, the estrogenic response of an environmental 

sample is converted to an equivalent concentration (EEQ) of 

the known estrogen concentration (EE2, used in this project) using: EEQ = EC50EE2/(EC50sample × CF), where 

EC50sample is the volume fraction of the sample producing a 
6 Pharmaceuticals, Hormones, Waste Indicators, and Estrogenicity in Samples from Sludge Stabilization and Dewatering

50 percent maximal response, EC50EE2 is the concentration 

of EE2 that produces a 50 percent maximal response in the 

positive control dose response curve, and CF is the concentration factor of the sample extract (typically 200–500 for 

liquid-phase samples). In this project estrogenic activity in 

several samples could not be determined using the traditional 

EC50 method due to sample toxicity inhibiting the estrogenic 

response. A new data reduction method was devised and was 

deemed the “First Response” method. The method relies 

upon identifying the lowest concentration of sample in the 

assay plate dilution series that exhibits an estrogenic response 

significantly above background. A statistical approach using 

Student’s t-tests was used to determine when significant 

departure from baseline occurred. Using this information, the 

method then follows in a similar fashion to the EEQ equation 

above. The new method is described in Teske (2009).

Data for Liquid and Solid Samples

The analytical results from this study are contained in 

tables 2–18. The concentrations of hormones in liquid and 

solid samples are reported for plants A (table 2), B (table 3), 

C (table 4), and D (table 5). Individual hormone concentrations 

in liquid samples are reported in nanograms per liter, and individual hormone concentrations for solid samples are reported in 

micrograms per kilogram of dry solid. Similarly, AWI concentrations (including nonhormonal synthetic EDC trace organic 

compounds) for liquid and solid samples are reported for 

plants A (table 6), B (table 7), C (table 8), and D (table 9). The 

concentrations of individual AWIs in liquid samples are reported 

in micrograms per liter, and the concentrations of individual

AWIs in solid samples are reported in micrograms per kilogram 

of dry solid. These two classes of TOCs follow each other in the 

table order of this report to aid comparison with the estrogenicity assay results because individual hormones and AWIs have 

been identified as contributors to estrogenic response in assays 

of environmental samples.

The YES bioassay results are reported for plants A 

(table 10), B (table 11), C (table 12), and D (table 13). The 

KBluc estrogen receptor bioassay was used for a limited 

set of samples from plant D and is reported in table 14. The 

estrogenicity results for both the YES and KBluc assays 

are reported as molar equivalents of EE2. The molar concentrations in tables 10 to 14 are reported in exponential 

notation, a standard format for this assay, in the commonly 

used engineering notation of X.X E–YY; that is, 9.1 E–13 

moles as EE2 equivalents corresponds to 9.1×10–13 moles as 

EE2 equivalents.

Concentrations of individual pharmaceuticals in liquid and solid samples are reported for plants A (table 15), B 

(table 16), C (table 17), and D (table 18). The concentrations 

of individual pharmaceuticals in liquid samples are reported 

in micrograms per liter, and the concentrations of individual 

pharmaceuticals in solid samples are in reported micrograms 

per kilogram of dry solid.

Quality-assurance/quality-control results are included in 

the tables for hormones, AWIs, and pharmaceuticals. Typically, surrogate recovery results are listed for each sample or 

replicate analysis results from two subsamples of the same 

sample. The surrogates are isotopically labeled versions of 

compounds determined in this study or are synthetic compounds not found in environmental samples. Results from 

these surrogates are reported as a percentage of the amount 

added and are used to assess method performance for each 

sample. Replicate sample results are in the concentration 

units appropriate for hormones or AWIs in solid or liquid 

samples. Reagent blank samples were processed with each set 

of environmental samples, and if blank contamination of any 

compound was detected, the environmental results for that 

sample set were censored if the concentration of the compound in the blank was greater than 5 percent of the environmental sample.

Acknowledgments

A substantial portion of the results of this study was 

supported by Water Environment Research Foundation 

(WERF) Grant 04–HHE–6 “Fate of Estrogenic Compounds 

during Municipal Sludge Stabilization and Dewatering.” The 

authors of this report also gratefully acknowledge extensive support from Dana W. Kolpin, USGS Toxic Substances 

Hydrology Program Emerging Contaminants Project, and 

Daniel Edwards, New York Water Science Center, USGS, for 

critical assistance in training and collection of liquid samples. 

We also thank the technical staff of the four WWTPs sampled 

in this study, without whose cooperation this research could 

not have been completed.

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8 Pharmaceuticals, Hormones, Waste Indicators, and Estrogenicity in Samples from Sludge Stabilization and Dewatering

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Furlong and others—Pharmaceuticals, Hormones, Waste Indicators, and Estrogenicity in Samples from Sludge Stabilization and Dewatering—OFR 2011–1132