Transformation of 17Î±-Estradiol, 17Î²-Estradiol, and Estrone in

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Transformation of 17#-Estradiol, 17#-Estradiol, and Estrone in Sediments Under Nitrate- and Sulfate-reducing Conditions Michael L Mashtare, Linda S Lee, Loring Nies, and Ronald F Turco Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es4008382 • Publication Date (Web): 24 May 2013 Downloaded from http://pubs.acs.org on June 7, 2013

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Environmental Science & Technology

1 2 3 4

Transformation of 17α-Estradiol, 17β-Estradiol, and Estrone in Sediments Under Nitrate- and Sulfate-reducing Conditions

5 6 7 8 9 10 11 12 13 14

Michael L. Mashtarea,b, Linda S. Leea,b,*, Loring F. Niesc, and Ronald F. Turcod a Ecological

Sciences and Engineering Interdisciplinary Graduate Program, Purdue University, West Lafayette, IN 47907, USA b College of Agriculture, Department of Agronomy, Purdue University, West Lafayette, IN 47907, USA c School of Civil Engineering, Purdue University, West Lafayette, IN 47907, USA d College of Agriculture, Laboratory for Soil Microbiology, Purdue University, West Lafayette, IN 47907, USA

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*

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.

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Corresponding author: Linda S. Lee, Purdue University, 915 W State St., West Lafayette, IN 47907, USA. Tel.: +1 765 494 8612; fax: +1 765 0496 2926, [email protected]

Prepared for ES&T Revision of es-2013-008382 May 11, 2013

ACS Paragon Plus Environment

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ABSTRACT

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The natural manure-borne hormones, 17α-estradiol (17α-E2), 17β-estradiol (17β-E2), and

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estrone (E1), are routinely detected in surface water near agricultural land and wastewater

29

treatment facilities. Once in the stream network, hormones may enter the sediment bed

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where they are subject to anaerobic conditions. This study focuses on the difference in

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anaerobic transformation rates and formation of metabolites from 17α-E2, 17β-E2, and E1

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(applied at ~3.66 mol kg-1 of sediment on a dry weight basis) under nitrate- and sulfate-

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reducing conditions. Sediment extracts were analyzed using negative electrospray

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ionization tandem mass spectrometry. Under both redox conditions, degradation was

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stereospecific and followed similar trends in half-lives: 17β-E2 < 17α-E2 < E1, with

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degradation considerably slower under sulfate-reducing conditions. Both E2 isomers were

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predominantly converted to E1; however, isomeric conversion also occurred with peak

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concentrations of ~1.7 mol% of 17β-E2 formed in 17α-E2 amended soils and peak

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concentrations of ~2.4 mol% of 17α-E2 formed from 17β-E2. In E1-amended systems, E1

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transformed to E2 with preferential formation of the more potent 17β isomer up to ~30

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mol% suggesting that isomer interconversion is through E1. Sediments, therefore, may

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serve as both a sink and a source of the more estrogenic compound E2. Transformation of

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amended hormones in autoclaved sediments was markedly slower than in non-autoclaved

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sediments. Results support the inclusion of isomer-specific behavior and the potential for

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reversible transformation and interconversion in anaerobic sediments in modeling fate in

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stream networks and developing risk management strategies.

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Keywords: 17α-estradiol, 17β-estradiol, estrone, reversible transformation, anaerobic

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degradation, stereoisomers, nitrate-reducing, sulfate-reducing, sediments


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INTRODUCTION

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The persistence and subsequent transport of estrogenic compounds from the agronomic

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land application of manure, effluent, and biosolids have contributed to increased estrogen

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detection in drainage ditches and surface water.1,2 Once in the water, these compounds

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including 17α-estradiol (17α-E2), 17β-estradiol (17β-E2) and their primary metabolite,

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estrone (E1) (Fig. S-2) have the potential to alter secondary sex characteristics, disrupt

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endocrine function, and activate hormone responses in sensitive aquatic species at low

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ng/L concentrations, which are common in environmental samples.1,3,4

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Most of the studies addressing the degradation of the natural estrogens have

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focused on 17β-E2 including aerobic stream sediments;5 aerobic soils;5-10 anaerobic or

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saturated soils;11-13 sediments impacted by wastewater treatment plant discharge;5 marine

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sediments,13 river water and sediments;14 activated sludge in membrane reactors;15

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alternating anoxic and aerobic conditions,16 and anaerobic lake sediments.17 The latter two

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studies16, 17 observed the conversion of 17β-E2 to 17α-E2 under reducing conditions, which

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Hutchins et al.18 hypothesized as a reasonable explanation for the unexpected elevated

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concentrations of 17α-E2 in swine and poultry lagoons.

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Much less is known on the environmental fate of 17α -E2 and E1, although these

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compounds are more frequently detected in beef cattle and dairy lagoons, stream and ditch

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water, and stream sediments than 17β-E2.3, 19, 42, 43 To date, laboratory degradation

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studies of 17α-E2 have been limited to soil bacteria cultures,20, 21 dairy lagoon water,22 a

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sterile/unsterile soil mixture,9 and aerobic soils.10 Laboratory degradation studies of E1

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have focused on aerobic soils;6, 8 aerobic stream sediments;5 anaerobic soils;8 anaerobic

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river water and sediments;14 activated sludge;15 and dairy lagoon water.22 Limited field


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studies have reported the apparent conversion 17α-E2 to 17β-E2 and E1 in a simulated

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feedlot under saturated conditions23 and in dairy manure and waste lagoons24 based on

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changes in relative concentrations in environmental samples. However, a direct

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assessment of this conversion potential has not been well explored and little is known

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about how 17α-E2 and E1 will behave in anaerobic sediment systems.

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The objectives of this study were to unequivocally assess: (1) differences in

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transformation rates of 17α-E2, 17β-E2, and E2 in sediments under nitrate-reducing and

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sulfate-reducing conditions; (2) if interconversion between the E2 isomers occurs; (3) if E1,

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the primary metabolite from E2 degradation, is transformed back to E2; and (4) if a

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particular isomer is preferentially formed from E1. To achieve these objectives, separate

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sediment microcosm treatments were prepared for 17α-E2, 17β-E2, and E1.

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MATERIALS AND METHODS

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Sediment. Sediment samples were collected from the ditch and stream network at the

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Purdue University Animal Science Research and Education Center (ASREC) and Little Pine

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Creek (West Lafayette, IN) in July and October 2010, and February and April 2011. Sample

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collection times and locations were selected to achieve a representative composite of

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sediments in the network. After each collection, stream water-saturated sediments were

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stored in closed containers at 4 °C in the dark. Immediately prior to initiating the anaerobic

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microcosms (May 2011), all collected sediments were passed through a 2 mm sieve and

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thoroughly mixed to create a homogenous sediment sample. Homogenized sediment

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properties are summarized in Table 1.

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Table 1. Selected sediment properties OMa (%) CECbcmolc kg-1) 4.0

25

pHc 7.07

Clayd (%) Sandd (%) Siltd (%) 20

42

38

Clayse RIS >> I > K

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a

% organic matter determined by loss on ignition at 360 °C 25; b cation exchange capacity

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determined using the Mehlich III Extraction method (1 M NH4OAc buffered at pH 7.0)25; cpH

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of a 1:1 soil (g): water (mL) slurry25; d determined by hydrometer method26;

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eRIS=randomly

interstratified illite-smectite, I=illite, K=kaolinite, as identified by XRD.

99 100

Chemicals. All estrogens (17α-E2, 17β-E2, E1) and the internal standard 17β-estradiol-D3

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(17β-E2-D3) were obtained from Sigma Aldrich, St. Louis MO, USA. Acetonitrile (ACN),

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ethyl ether (Et2O), and methanol (MeOH) were purchased from Mallinckrodt, Phillipsburg,

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NJ, USA. Food grade protein gelatin used as an electron donor was purchased from Kroger

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Co., Cincinnati, Ohio, USA. All chemicals were analytical-reagent grade or higher purity

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(>99%) except for 17α-E2, 17β-E2, 17β -E2-D3, which were >98% purity, and used as

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received. Ultrapure water was prepared using a Mega-Pure System, MP-3A from Barnstead,

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Dubuque, IA, USA. Hormone stock solutions were prepared in pure methanol and stored at

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4 °C in the dark.

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A synthetic freshwater medium (pH adjusted to 7 with HCL) was prepared as

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described by Homklin et al.27 by dissolving 1.0 g of NaCl, 0.4 g of MgCl2×2H2O, 0.1 g of

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CaCl2×2H2O, 0.25 g NH4Cl, 0.2 g of KH2PO4, 0.5 g of KCl, 1 mL of trace element mixture, 2.52

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g of NaHCO3, 0.36 g Na2S nonhydrate into a total volume of 1 liter of Milli-Q water. The

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trace element mixture was prepared by mixing 12.5 mL HCl (7.7 M), 2.1 g FeSO4×7H2O, 30

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mg H3BO3, 100 mg MnCl2×4H2O, 190 mg CoCl2×6H2O, 29.36 mg Ni(NO3)2, 2.9 mg

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CuSO4×5H2O, 144 mg ZnSO4×7H2O and 36 mg Na2MoO4×2H2O into 1 liter of Milli-Q water.


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The freshwater medium was autoclaved and cooled under nitrogen. Sodium nitrate or

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sodium sulfate was added to each bottle to achieve an initial 20 mM electron acceptor

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solution. The headspace was then purged with nitrogen, bottles capped, sealed with

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parafilm, and transferred into a large 4-glove vinyl anaerobic chamber (Fig. S-2). The

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chamber is equipped with an automated airlock pass-through chamber, oxygen and

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hydrogen analyzer, 3 fan boxes equipped with palladium catalysts, and a dark storage

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incubator. At the start of the experiment, 0.5 mL of a 500 mg/L methanol hormone stock

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solution was transferred into a glass flask, the methanol evaporated, and hormones re-

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suspended in 500 mL of a 20 mM electron acceptor-solution and mixed on a magnetic stir

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plate to achieve ~0.5 mg hormone/L. Each hormone solution (17α-E2, 17β-E2, and E1) was

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prepared separately as well as a no-hormone blank. A sample from each solution was

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saved and extracted using diethyl ether (Et2O) at a 4:4.4 water:Et2O liquid-liquid exchange

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for analysis on the LC/MS/MS.

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Batch Sediment Pre-incubation. Sediments were pre-incubated to ensure the onset of

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nitrate-nitrate-reducing or sulfate-reducing conditions prior to hormone addition (defined

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as time t=0). Homogenized wet sediment was transferred to a plastic container and

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covered with 500 mL of 20 mM nitrate or 20 mM sulfate solution. The protein gelatin

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electron donor (3 g) was dissolved in the freshwater medium prior to amendment to

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provide a complex food source and promote diverse anaerobic microbial community

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development as described by Kourtev et al.28, 29 The container was tightly sealed by

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wrapping electrical tape and parafilm around the lid edges followed by purging the

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headspace with nitrogen for 5 minutes using two 16-gauge syringe needles (one for N2

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input and one for exhaust) after which the syringe holes were sealed with electrical tape.


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The sediment was mixed by gently rocking the container and then placed into a small foil-

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covered plexi-glass anaerobic chamber to pre-incubate (Fig. S-2). The chamber was

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maintained under a positive N2 pressure to minimize the risk of O2 contamination and a

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methylene blue indicator solution was used to confirm anaerobic conditions. After a week

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in the small anaerobic chamber, the sediment-water mixtures were covered in foil and

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transferred to a larger vinyl chamber as previously described (Fig. S-2) for which an

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atmosphere of N2 with ~3-5% H2 was maintained.

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Individual Anaerobic Microcosm Preparation. Glass centrifuge tubes (40 mL) and

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Teflon-lined screw caps were rinsed with acetone and wet autoclaved for 30 minutes,

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transferred to a 105 °C oven until dry and cooled to room temperature. Water and solvent-

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resistant labels were applied and the microcosm tubes were transferred into the large

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anaerobic chamber for at least 24 hours prior to the start of the experiment to allow

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sufficient time to degas.

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The water above the sediment surface in the pre-incubation batch container was

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siphoned off followed by mixing the sediment with a metal spatula. Approximately 8.5 g of

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wet sediment (~5 g dry wt basis) was transferred into each centrifuge tube and lightly

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capped until time of amendment. Sediment slurry was sampled in triplicate for gravimetric

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moisture content determination. Pore water was extracted by centrifuging wet sediments

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and saved along with the siphoned off water for nitrate-nitrite or sulfate analysis to

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monitor nitrate-nitrate and sulfate reduction activity. A hormone solution (10 mL) or blank

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solution was added to each microcosm, tightly capped, gently shaken to suspend the

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sediments in the solution, and the time of amendment for each microcosm recorded. For


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hormone amended microcosms, the initial hormone concentration was ~3.66 μmol kg-1 dry

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wt. basis. Microcosms were placed in the dark anaerobic storage incubation chamber until

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time of sacrifice.

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In addition, a separate set of sediment microcosms were prepared using autoclaved

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sediment. Wet sediment (~8.5 g of sediment) was transferred into 40 mL centrifuge tubes

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and autoclaved for 1 h on each of 3 consecutive days as described by Wolf et al.30 These

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microcosms were transferred to the anaerobic chamber 24 hours prior to the start of the

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experiment and amended with hormones as described above.

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For each hormone and reducing condition, five hormone-amended microcosms

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were analyzed at each sampling time: four for hormone analysis (including 1 autoclaved

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microcosm), and 1 for electron acceptor analysis. Triplicate soil blanks (no hormones)

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were also analyzed at each sampling time. Microcosm sets were sacrificed after 0, 1, 3, 7,

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14, and ~21 days and at two later times between 45 – 80 days.

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Electron Acceptor Analysis. Microcosms for electron acceptor analysis were gently

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shaken and centrifuged at 1600 rpm for at least 20 min. Supernatant (1 mL) was

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transferred into a micro-centrifuge tube and centrifuged at 13000 rpm for 30 min to

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remove fine particulates followed by analysis on a Seal AQ2. AQ2 Methods No: EPA-114-A

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Rev. 6 and EPA-123-A Rev. 4 were used for nitrate-nitrite and sulfate analyses,

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respectively. If electron acceptor concentrations were determined to be less than 10% of

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the amended concentration, the remaining microcosms in the anaerobic chamber were re-

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amended with a sterilized and degassed concentrated electron acceptor solution until


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concentrations approached ~20 mM, which was only required for the nitrate-reducing

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systems.

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Hormone Extraction and Analysis. At each sampling time, microcosms for hormone

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analysis were transferred into the small anaerobic chamber for liquid extraction. Diethyl

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ether (Et2O) was added to each microcosm to minimal headspace and capped tightly. Each

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bottle-cap was wrapped with parafilm, tubes covered with foil to minimize

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photodegradation potential, equilibrated end-over-end at 35 rpm for ~24 h at room

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temperature (22 ± 2 °C) and centrifuged at 1600 rpm for 20 min. Approximately 1.2 mL

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(by weight) of Et2O was transferred into an HPLC vial, evaporated, and residues re-

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dissolved in 0.5 mL of MeOH containing an internal standard (17β-E2-D3). For the sulfate-

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reduction experiment, a second extraction was carried out by removing the excess Et2O

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from the first extraction and repeating the extraction steps above.

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Estrogen analysis was performed on a Shimadzu high performance reverse-phase liquid

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chromatography coupled to a Sciex API3000 mass spectrometer operated in positive

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electrospray ionization (ESI) mode with multiple reaction monitoring. Separation was

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performed using 20-25 μL injections on a Phenomenex Gemini C-18 column (150 mm x 2

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mm, dp= 5 μm) with a gradient elution using water:methanol (90:10) containing 2 mM

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ethanolamine [solvent A] and acetonitrile containing 2 mM ethanolamine [solvent B] at

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0.35 mL min-1. Initial mobile phase composition was 30% solvent B followed by a linear

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gradient to 50% solvent B from 0 to 8.5 min after which solvent B was ramped to 100% for

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2 min to wash the column and then re-equilibrated at 30% solvent B for 2 min prior to the

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next injection. The chromatographic retention times for E3, 17β-E2, 17β-E2-D3, 17α-E2,


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and E1 were 3.8 min, 8.5 min, 7.9 min, 7.9 min, and 9.2 min, respectively. E3 (m/z 287

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145), 17α-E2 and 17β-E2 (m/z 271 145), and E1 (m/z 269 145) were quantified

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using independent external calibration curves with check standards run approximately

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every 12 samples. The internal standard, 17β-E2-D3 (m/z 274 145), was used to assess

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matrix effects in the MS, which were found to be negligible. A deuterated 17α-E2 internal

209

standard was not available. For a 25 uL injection, the limit of detection for all estrogens was

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0.015 μg L-1 and the method limit of quantitation (MLOQ) was 0.03 μg L-1.

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Estimating Degradation Rates. A first-order exponential decay model was fit to the data

212

to estimate net (apparent) degradation rates (ka and kb, d-1) of the hormones following the

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assumption of a simple set of consecutive reactions (Eq. 1) and using solutions defined in

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Eqs. 2 and 3 (derivation detailed in the supplemental information).

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→

→

(1)

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[A]t = [A]0 e-kat

(2)

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[B]t = {(ka[A]0)(e-kat – e-kbt)}(kb-ka)-1

(3)

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where [A]0 and [A]t represent the extracted applied hormone concentrations on a mole

219

basis at time t=0 and time t (d), respectively, and [B]t represents the mole concentration of

220

the extracted primary daughter metabolite at time t (d). At time=0, all compounds except

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the applied hormone is assume to be zero (i.e., [B] 0 = [C]0 =0). This model assumes a

222

steady-state biomass, and does not account for irreversible transformations or effects of

223

sorption on bioavailability.


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Statistical Analysis. Minitab v16 (State College, PA: Minitab, Inc.) was used for statistical

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analysis. An ANOVA was used to determine the significance of differences between the

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observed temporal changes in transformation of 17α-E2 and 17β-E2 as well as between the

227

rate of E1 metabolite production and subsequent loss from 17α-E2 and 17β-E2 within and

228

between redox conditions. Statistical tests used α = 0.05 as the level of significance.

229 230

RESULTS AND DISCUSSION

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All hormone concentrations are presented and discussed on a mol % basis relative to the

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parent hormone applied. Electron acceptor concentrations are presented in mM. Applied

233

hormone concentrations over time for nitrate-reducing and sulfate-reducing conditions are

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shown in Figs. 1A and 1B, respectively, with the corresponding electron acceptor trends

235

shown in Figs. 1C and 1D. Metabolite formation and isomeric conversion for each E2-

236

isomer are summarized in Fig. 2 with reversible transformation of E1 to 17α-E2 and 17β-

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E2 under both reducing conditions shown in Fig. 3. The pseudo first-order exponential

238

decay model fits to the hormone data are summarized in Table 2 along with select apparent

239

degradation rates (ka and kb), modeled t½ values, and coefficients of determination (R2)

240

from fitting Eqs. 2 and 3.

241 242

17α-E2 and 17β-E2 Transformation Under nitrate- and sulfate-reducing conditions.

243

The loss 17β-E2 was rapid and significantly faster than 17α-E2 under both nitrate- and

244

sulfate-reducing conditions with faster dissipation of both isomers under nitrate-reducing

245

conditions (Table 2). The continuous reduction over time in electron acceptor


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concentrations indicates that the targeted redox activity was occurring and that the

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microbial communities in the live microcosms remained active during incubation (Figs. 1C

248

and 1D). However, 17α-E2 and 17β-E2 t½ values were ~16 and ~6 times longer,

249

respectively, under sulfate-reducing conditions than under nitrate-reducing conditions.

250

Sulfate utilization (Fig. 1D) was slower relative to nitrate (Fig. 1C) with nitrate

251

concentrations falling to below 20% of amended concentrations by day 7 while it took 80 d

252

to achieve a similar loss in the sulfate-reducing systems suggesting lower microbial activity

253

in the latter. E3 was not detected in either system. The mass balance of total estrogens

254

(Fig S-3) decreased with time under both redox conditions suggesting that either E1 or the

255

E2 isomers were mineralized, degraded to unknown metabolites, or irreversibly sorbed.

256

For 17β-E2 under nitrate-reducing conditions, the t½ of < 0.3 d is similar to values of
80 * E1 concentrations dropped to ~42 mol % but then rose above 60 mol% for the rest of the 80-d incubation period.

303 304


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305 306

Figure 1. Composite of single hormone-amended experiments showing the loss of 17α-E2 (

307

), 17β-E2 ( ), and E1 ( ) in mol % over time under (A) nitrate-reducing and (B) sulfate-

308

reducing conditions; and associated changes in electron acceptor concentrations (mM)

309

over time shown for (C) nitrate and (D) sulfate. Re-amendment of nitrate (+) is shown for

310

8 and 15 d and re-amendment with both nitrate and protein (*) at 48 d. Solid lines (______)

311

represent first-order decay model fits (Eq. 2) over the first 14 d with extrapolation after 14

312

d represented by dotted lines (…….). Dashed lines (----) represent the model fits to the entire

313

incubation period. Error bars represent the standard deviation (n=3).


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E1 Formation from 17α-E2 and 17β-E2 and Interconversion. Under nitrate-reducing

315

conditions, the first-order exponential decay model fits to the formation of E1 from each

316

isomer and E1’s subsequent loss were good (R2 of 0.66-0.98) (Table 2, Figs. 2A and 2C). The

317

estimated t½ of E1 that formed from 17α-E2 is approximately a factor of two slower than

318

the subsequent degradation of E1 formed from 17β-E2. Under aerobic conditions,

319

Mashtare et al. 10 also observed differences in E1 formation and loss between the E2

320

isomers. Half-lives estimated for fits to the first 14 d under nitrate-reducing conditions

321

compared to the entire incubation period are 25 to 50 % shorter, suggesting the decrease

322

in nitrate-reduction may have slowed down the apparent loss of E1 formed from E2 in the

323

microcosms.

324

In addition to E1 formation, apparent interconversion between E2 isomers occurred by

325

day 1 sampling under both redox conditions. 17α- and 17β-E2 concentrations formed from

326

isomeric conversion peaked and then decreased over time under nitrate-reducing

327

conditions, but generally continued to accumulate over time under sulfate-reducing

328

conditions (Fig. 2). In 17α-E2 amended nitrate-reducing microcosms, 17β-E2 peaked at

329

0.62 mol% on day 21 (Fig. 2C) while under sulfate-reducing conditions, 17β-E2 peaked

330

earlier (14 d) and at 1.72 mol% (Fig. 2D). Likewise, in the 17β-E2 amended microcosms ,

331

greater isomeric conversion occurred with 17α-E2 concentrations reaching 2.4 mol% (Fig.

332

2A), whereas in nitrate-reducing conditions, only 0.24 mol% of the isomer was formed (Fig.

333

2D). Formation of 17α-E2 from 17β-E2 in laboratory studies has previously been reported

334

for anaerobic lake sediments under methanogenic, sulfate-reducing, and iron-reducing

335

conditions (not observed in nitrate-reducing)17 and in anoxic activated sludge.16 Isomeric

336

conversion from 17α-E2 to 17β-E2 in sediments has not been previously reported;


16

Page 17 of 29

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337

however, it was observed in blended dairy lagoon water22 and simulated feedlot runoff.23

338

The greater accumulation of E2 isomers in sediments under sulfate-reducing versus nitrate

339

reducing conditions is likely due to both the slower degradation rates of E2 and reversible

340

transformation of E1 back to E2 as discussed below.

341 342

Figure 2. Mol % of E1 ( ) (right axis), 17α-E2 ( ) (left axis), and 17β-E2 ( ) (left axis) in

343

soil amended with either 17β-E2 (upper graphs A and B) or 17α-E2 (lower graphs C and D)

344

microcosms under nitrate-reducing (left graphs A and C) and sulfate-reducing conditions

345

(right graphs C and D). Solid lines (______) represent first-order decay model fits (Eq. 3) over

346

the first 14 d with extrapolation after 14 d shown with dotted lines (…….). Dashed lines (----)

347

represent the model fits to the data over the entire incubation period. Error bars represent

348

the standard deviation (n=3).


17


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

Page 18 of 29

349

E1 Fate in E1-Amended Sediments. In E1-amended nitrate-reducing microcosms, E1 half-

350

life was 35.9 d (Table 2, Fig. 3A), which is similar to the subsequent loss of E1 (t½ ranged

351

between 22 and 43 d) after forming from either E2 isomer. In the E1-amended sulfate-

352

reducing microcosms, E1 dropped to ~42 mol % within 3 days. However, E1

353

concentrations subsequently rose to >60 mol% by 14 d presumably due to the reversible

354

transformation between E1 and E2 (Fig. 3B) and remained above  60% through the 80-d

355

incubation period. In anaerobic sludge (without nitrate) membrane reactors spiked with

356

E1, a constant E1-E2 ratio was observed within hours, supporting that the reversible

357

transformation of E1 and E2 results in an apparent increase in persistence. Czajka and

358

Londry17 also reported that the E1 formed from 17β-E2 in their sandy lake sediments

359

under nitrate-, iron-, and sulfate-reducing conditions as well as methanogenesis did not

360

appear to dissipate substantially within their 383 d incubation period. The slow or

361

apparent non-loss of E1 in these studies relative to E2 suggests that the processes and

362

microbial populations responsible for degrading 17β-E2 are different than those capable of

363

E1 degradation. While denitrifying bacterium has been isolated from activated sludge that

364

can degrade E2 as a sole source of carbon,44 only a limited number of bacteria are known to

365

be able to degrade both E1 and E2.34 The current study is the first to investigate E1-

366

amended anaerobic sediments; however, E1 fate as the starting compound was assessed in

367

anaerobic sludge15 and in lagoon water22 for which t½ of E1 was reported to be >52 d.

368

E1 is presumed to be the intermediate in the isomeric conversion of the parent

369

hormones observed in the E2-amended microcosms. Reversible transformation from E1

370

was observed under each redox condition with both precursors formed in the E1-amended

371

microcosms (Figs. 3A and 3B). Under nitrate-reducing conditions, formation of 17α-E2


18

Page 19 of 29

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372

peaked at 0.19 mol% while 17β-E2 peaked at 2.14 mol%. This reversible transformation

373

was more pronounced under sulfate-reducing conditions with E1 to 17α-E2 conversion

374

steadily increasing to 2.6 mol% by 81 d, while 17β-E2 peaked at 28.9 mol% within 3 d,

375

before declining to a pseudo steady state concentration of ~7 mol%. The formation and

376

loss of 17β-E2 under sulfate-reducing condition occurred with a near stoichiometric loss

377

and gain of E1 (Fig. 3B). While a preference for 17β-E2 formation from E1 was observed

378

under both redox conditions, the temporal accumulation of these compounds appears to be

379

influenced by the decay rate of the isomer. This reversible transformation may also help

380

explain the sizeable remaining mass of E1 and the E2 isomers observed under sulfate-

381

reducing conditions. Zheng et al.22 also observed reversible transformations from E1 to

382

both E2 isomers in blended dairy lagoon water with preferential formation of 17β-E2. This

383

preference for 17β-E2 formation is also consistent with the relative activities and stability

384

of 17α-E2 and 17β-E2 dehydrogenases.33

385


19


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Page 20 of 29

386 387

Figure 3. Mol % of 17α-E2 ( ) and 17β-E2 ( ) (right axes) in E1-amended systems under

388

(A) nitrate-reducing and (B) sulfate-reducing conditions. Lines represent E1 decay patterns

389

(left axis). Error bars represent the standard deviation (n=3).

390 391

Autoclaved Sediments. In an attempt to discern differences between biotic and abiotic

392

transformations, we employed autoclaved sediments in a single microcosm set. Autoclaving

393

as a sterilization procedure is commonly used in our lab and traditionally used for aerobic

394

soils,30 but which may have been inadequate for effective sterilization of anaerobic

395

systems. Bradley and Chapelle35 in regards to biodegradation of chlorinated solvents

396

comment that while heat-sterilization under high pressure (e.g., autoclaving), greatly

397

suppresses biological activity, it may not completely inhibit biological activity in sediments.

398

Both Slepova et al.36 and Hyun et al.37 found thermophilic bacteria and their spores to be

399

extremely heat resistant. In addition, Carter et al.38 found that although autoclaving

400

appears to kill aerobic soil microbes, microbial enzymes remained active.


20

Page 21 of 29

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401


Sterility was not confirmed in our experiments and only a single microcosm was

402

sacrificed at each sampling point, thus providing no measure of variability. Nevertheless,

403

some interesting trends were observed in the autoclaved sediments. In general,

404

transformation of the applied hormones was slower than the non-autoclaved (live)

405

sediments under both nitrate-reducing (Fig. S-5) and sulfate-reducing (Fig. S-6) conditions.

406

One exception is that the apparent loss of amended E1 under sulfate-reducing conditions

407

was similar to the live sediments, but no formation of 17β-E2 was observed until day 14

408

resulting in an apparent pseudo steady state of 17β-E2 (~16 mol %) and E1 (~30 mol %).

409

Metabolite formation was generally slower across all autoclaved microcosms. With the

410

exception of 17α-E2 formed from E1 under nitrate-reducing conditions (~1.2 mol %), peak

411

E2 isomeric conversion and E1 to E2 conversion was generally smaller across the

412

incubation period under both redox conditions. Mass balances in all autoclaved

413

microcosms (Fig. S-4) are higher than in the live microcosms under nitrate-reducing

414

conditions. In autoclaved sulfate-reducing conditions, E2 mass balance in E2 amended

415

microcosms was similar to the live soils, but lower for E1. The latter could be due to

416

transformation of E1 to an unknown metabolite or loss to irreversible sorption, which can

417

increase with increasing residence time. Van Emmerik et al. 39 reported irreversible

418

sorption of 17β-E2 in a smectite clay, which is the dominant clay type in the sediment used

419

in our study. Although abiotic transformation of 17β-E2 to E1 in aerobic soils was reported

420

by Collucci et al.6, we are unable to definitively differentiate between biotic and abiotic

421

transformation in our study. However, if the slower transformation patterns in the

422

autoclaved sediments are due to abiotic transformation, which is not unexpected, much of

423

the observed transformations in the live systems appear microbially-mediated.


21


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Page 22 of 29

424

Nevertheless, further work is needed to elucidate the difference between biotic and abiotic

425

transformation of these compounds.

426 427

Environmental Implications. In soils under aerobic conditions, the rapid dissipation (t½

428

generally < 3 d) of 17α-E2, 17β-E2, and E1 has been observed.6-10 For hormones entering

429

the surface water via discharge or run off, sediments may serve as a sink or source for

430

hormones.40 Once in the sediment bed, this study suggests that the anaerobic degradation

431

rates of 17α-E2 and 17β-E2 cannot be assumed to be the same. For our study, t½ of 17α-E2

432

in stream sediment is 16-46 times longer than 17β-E2 depending on the extant redox

433

condition, with higher persistence under sulfate-reducing conditions. Interconversion was

434

observed between 17α-E2 and 17β-E2, presumably with E1 as the intermediate, which was

435

observed to reversibly transform back to its precursors. Of particular concern is the

436

apparent preferential formation of 17β-E2, the more potent of the estradiol isomers,

437

although the loss rate of 17β-E2 is more rapid. The slower degradation rate of 17α-E2, to

438

which some aquatic species have been shown to be more sensitive to than their

439

mammalian counterparts, suggests that both isomers, and their primary metabolite, E1,

440

which exhibited even slower degradation than E2, have the potential for prolonged

441

environmental persistence under highly reduced conditions.

442

The potential for isomeric conversion and reversible transformations from E1, suggest

443

the risk to aquatic species may not be adequately predicted by looking at inputs/discharge

444

into the water column alone. For example, E1, often the dominant hormone detected in

445

impacted surface water,42, 43 may transform back to the more potent 17β-E2 and17α-E2

446

once partitioned into the sediment bed. The lower partition coefficients of E241 relative to


22

Page 23 of 29

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447

E1 suggest that sediments may then serve as a long-term source of the E2 isomers re-

448

entering the water column via diffusion and under turbulent conditions where they may

449

come into contact with sensitive species. Thus, understanding the transformation potential

450

of these compounds once in the sediment bed is paramount in developing an effective risk

451

management strategy.

452 453

ACKNOWLEDGEMENTS

454

This work was funded in part by a U.S.D.A. AFRI Water and Watersheds Award No. 104117.

455

We also wish to acknowledge Stephen Sassman for his analytical chemistry support,

456

Chandeepa T. Cooray Bulathsinhala for his help in sediment collection and preparation,

457

Leila Nyberg for her assistance with the anaerobic chamber, and Nicole De Armond for her

458

help with the Seal AQ2.

459 460

SUPPORTING INFORMATION

461

Structures of hormones, photos of experiment, extraction efficiencies, and mass balance in

462

microcosms and autoclaved microcosms under nitrate-reductucing and sulfate-reducing

463

conditions are available. Thiis material is available free of charge via the Internet at

464

http://pubs.acs.org.

465 466


23


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Page 24 of 29

467

REFERENCES

468

(1) Aga, D. S. Fate of pharmaceuticals in the environment and in water treatment systems.

469

2008. CRC Press: Taylor & Francis Group.

470

(2) Khanal, S. K.;Xie, B.; Thompson, M. L.; Sung, S.; Ong, S. K. ; Leeuwen, J. Fate, transport and

471

biodegradation of natural estrogens in the environment and engineered systems. Environ.

472

Sci. Technol. 2006, 40, 6537-6546.

473

(3) Hanselman, T. A., Graetz, D.A; Wilkie, A.C. Manure-borne estrogens as potential

474

environmental contaminants, a review. Environ. Sci. Technol. 2003, 37, 5471-5478.

475

(4) Young R. B.; Borch, T. Sources, presence, analysis, and fate of steroid sex hormones in

476

freshwater ecosystems – a review. Aquatic Ecosystem Research Trends, 2009 Nova Science

477

Publishers.

478

(5) Bradley, P. M.; Barber, L. B.; Chapelle, F. H. ; Gray, J. L.; Kolpin, D. W.;. B. McMahon, P. B.

479

Biodegradation of 17β-estradiol, estrone, and testosterone in stream sediments. Environ.

480

Sci. Technol. 2009, 43, 1902-1910.

481

(6) Colucci, M. S.; Bork, H.; Topp, E. Persistence of estrogenic hormones in agricultural

482

soils: I. 17β-estradiol and estrone. J. Environ. Qual. 2001, 30, 2070-2076.

483

(7) Das, B. S.; Lee, L. S.; Rao, P. S. C;. P. Hultgren, R. P. Sorption and degradation of steroid

484

hormones in soils during transport, Column studies and model evaluation. Environ. Sci.

485

Technol. 2004, 38, 1460-1470.

486

(8) Ying, G. G.; Kookana, R. S. Sorption and degradation of estrogen-like-endocrine

487

disrupting chemicals in soil. Env. Tox. Chem. 2005, 24, 2640-2645.

488

(9) Xuan, R.; Blassengale, A.A.; Wang, Q. Degradation of estrogenic hormones in a silt loam

489

soil. J. Agr. Food Chem. 2008, 56, 152–9158.

490

(10)

491

in aerobic soils. Chemosphere 2013, 90, 647-652.

492

(11)

493

of common pharmaceuticals and personal care products in soil under aerobic and reduced

494

oxygen conditions. Water Air Soil Pollut. 2011, 216, 633-642.

Mashtare, M. L.; Green, D. A.; Lee, L. S. Biotransformation of 17α- and 17β-estradiol

Carr, D. L.; Morse, A. N.; Zak, J. C.; Anderson, T. A. Microbially mediated degradation


24

Page 25 of 29

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


495

(12)

Fan, Z.; Casey, F. X. M.; Hakk, H.; Larsen, G. L. Persistence and fate of 17β-estradiol

496

and testosterone in agricultural soils. Chemosphere 2007, 67, 886-895.

497

(13)

498

chemicals in seawater and marine sediment. Environ. Sci. Technol. 2003, 37, 1256-1260.

499

(14)

500

Williams, R. J. The potential for estradiol and ethinylestradiol degradation in English rivers.

501

Environ. Tox. and Chem. 2002, 21, 480-488.

502

(15)

503

municipal wastewater treatment under aerobic and anerobic conditions: consequences for

504

plant optimization. Environ. Sci. Technol. 2004, 38, 3047-3055.

505

(16)

506

nitrifying activated sludge under aerobic and alternating anoxic/aerobic conditions. Water

507

Environ Res. 2008, 80, 47-52.

508

(17)

509

Environ. 2006, 367, 932-941.

510

(18)

511

different concentrated feeding operations for estrogens and estrogen conjugates. Environ.

512

Sci. Technol. 2007, 41, 738–744.

513

(19)

514

occurrence and fate of pharmaceuticals and steroid hormones in agricultural

515

environments. Water Environ. Res. 2009, 83, 869-82.

516

(20)

517

by the microflora of soils. J. Bacteriol. 1947, 54, 557-562.

518

(21)

519

molecule. J. Biochem. 1947, 42, 376-83.

520

(22)

521

kinetics and mechanism of steroid estrogenic hormones in dairy lagoon water. Environ. Sci.

522

Technol. 2012, 46, 5471-5478.

Ying, G. G.; Kookana, R. S. Degradation of five selected endocrine-disrupting

Jürgens, M. D.; Holthaus, K. I. E.; Johnson, A. C.; Smith, J. J. L.; Hetheridge, M.;

Joss, A.; Andersen, H.; Ternes, T.; Richle, P. R.; Siegrist, H. Removal of estrogens in

Dytczak, M. A.; Londry, K. L.; Oleszkiewicz, J. A. Biotransformation of estrogens in

Czajka C. P.; Londry, K. L. 2006. Anaerobic biotransformation of estrogens. Sci. Tot.

Hutchins, S.R.; White, M.V.; Hudson, F.M.; Fine, D.D. Analysis of lagoon samples from

Snow, D. D.; Bartelt-Hunt, S. L.; Brown, D. L.; Sangster, J.; Cassada, D. A. Detection,

Turfitt, G. E. Microbial agencies in the degradation of steroids. II. Steroid utilization

Turfitt, G. E. The microbial degradation of steroids. 4. Fission of the steroid

Zheng, W.; Li, X.; Yates, S. R.; Bradford, S. A. 2012. Anaerobic transformation


25


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

Page 26 of 29

523

(23)

Mansell, D. S.; Bryson, R. J.; Harter, T.; Webster, J. P.; Kolodziej, E. P.; Sedlak, D. L.

524

Fate of endogenous steroid hormones in steer feedlots under simulated rainfall-induced

525

runoff. Environ. Sci. Technol. 2011, 45, 8811-8818.

526

(24)

527

typical dairy waste disposal system. Environ. Sci. Technol. 2008, 42, 530-535.

528

(25)

529

Central Region, North Central Region Research Publication No. 221. (Revised Jan. 1998),

530

Missouri Agricultural Experiment Station SB 1001.

531

(26)

532

Physical and Mineralogical Methods. 1986. American Society of Agronomy, Inc., Madison,

533

WI.

534

(27)

535

methyltestosterone in sediment under different electron acceptor conditions. Chemosphere

536

2011, 82, 1401-1407.

537

(28)

538

community to addition of organic C in chromium(vi)- and iron(iii)-amended microcosms.

539

Appl. Environ. Microb. 2006, 72, 628-637.

540

(29)

541

chromium(vi) in anaerobic soil microcosms. Appl. Environ. Microb. 2009, 75, 6249-6257.

542

(30)

543

methods on selected microbiological, physical, and chemical properties. J. Environ. Qual.

544

1989, 18, 39–44.

545

(31)

546

Phytopathol. 1976, 13, 357-373.

547

(32)

548

17β-estradiol in soils receiving swine manure or municipal biosolids. J. Environ. Qual.

549

2005, 34, 861-871.

Zheng, W.; Yates, S. R.; Bradford, S. A. 2008. Analysis of steroid hormones in a

Brown, J. R. (Ed). Recommended Chemical Soil Test Procedures for the North

Gee, G. W.; Bauder, J. W. Particle-size analysis. Methods of Soil Analysis, Part 1 –

Homklin, S.; Ong, S. K.; Limpiyakorn, T. Biotransformation of 17-

Kourtev, P. S.; Nakatsu, C. H.; Konopka, A. Responses of the anaerobic bacterial

Kourtev, P. S.; Nakatsu, C. H.; Konopka, A. Inhibition of nitrate reduction by

Wolf, D.C.; Dao, T.H.; Scott, H.D.; Lavy, T.L. 1989. Influence of soil sterilization

Marshall, K. C. Clay mineralogy in relation to survival of soil bacteria. Annu. Rev.

Jacobsen, A.; Lorenzen, A.; Chapman, R. ; Topp, E. Persistence of testosterone and


26

Page 27 of 29

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


550

(33)

Renwick, A. G. C.; Engel, L. L. The partial purification of 17α- and 17β-estradiol

551

dehydrogenase activities from chicken liver. Biochimica et Biophysica Acta 1967, 146, 336-

552

348.

553

(34)

554

degradation by stenotrophomonas maltophilia. Environ Sci. Technol. 2012, 46, 5947-5955.

555

(35)

556

In Situ Remediation of Chlorinated Solvent Plumes. Edited by H. F. Stro and C. H.

557

Ward. Springer Science. 2012, pp 39-67.

558

(36)

559

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic

560

prokaryotes. Microbiol. 2007, 76, 523-529.

561

(37)

562

heat resistance of spores from thermophilic Clostridium species. J. Bacteriol. 1983, 156,

563

1332.

564

(38)

565

enzymes remain active. Pedobiologia 2007, 51, 295-299.

566

(39)

567

of 17[beta]-estradiol onto selected soil minerals. J. Colloid and Interface Sci. 2003, 266, 33-

568

39.

569

(40)

570

bioavailability of estrogenic chemicals from sediments. Chemosphere 2009, 76, 395-401.

571

(41)

572

17α-estradiol and 17β-estradiol. Chemosphere 2011, 82, 847–852.

573

(42)

574

midwest tile-drained agroecosystem receiving animal wastes. Environ. Sci. Technol. 2011,

575

45, 8755-8764.

576

(43)

577

Buxton, H. T. Pharmaceuticals, hormones, and other organic wastewater contaminants in

Li, Z.; Nandakumar, R.; Madayiputhiya, N.; Li, X. Proteomic analysis of 17β-estradiol

Bradley, P. M.; Chapelle, F. H. Biodegradation of chlorinated ethenes (Chap 3). IN:

Slepova, T. V.; Rusanov, I. I.; Sokolova, T. G.; Bonch-Osolovskaya, E. A.; Pimenov, N. V.

Hyun, H. H.; Zeikus, J. G.; Longin, R.; Millet, J.; Ryter, A. Ultrastructure and extreme

Carter, D. O.; Yellowlees, D.; Tibbett, M. Autoclaving kills soil microbes yet soil

Van Emmerik, T.; Angove, M. J.; Johnson, B.B.; Wells, J. D.; Fernandes, M.B. Sorption

Duong C. N.; Schlenk, D; Chang, N.I.; Kim, S.D. The effect of particle size on the

Mashtare, M. L.; Khan, B.; Lee, L. S. Evaluating stereoselective sorption by soils of

Gall, H.E.; Sassman, S. A.; Lee, L. S.; Jafvert, C. T. Hormone discharges from a

Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.;


27


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Page 28 of 29

578

U.S. streams, 1990-2000: a national reconnaissance. Environ. Sci. Technol. 2002, 36, 1202-

579

1211.

580

(44)

581

oestradiolicum gen. nov., sp. Nov., a 17B-oestradiol-degrading, denitrying

582

betaproteobacterium. Intern. J. of Systematic and Evolutionary Microbiology. 2006, 56,

583

1547-1552.

Fahrbach, M.; Kuever, J.; Meinke, R.; Kampfer, P.; Hollender, J. Denitratisoma

584


28

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585 586


Graphical Abstract:

587


29

Transformation of 17Î±-Estradiol, 17Î²-Estradiol, and Estrone in

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