Determination of estrogenic steroids and microbial and photochemical degradation of...

Environmental ScienceProcesses & Impacts

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Department of Chemistry and Biochemistry,

285 Old Westport Road, North Dartmouth,

edu; Fax: +1 508-999-9167; Tel: +1 508-999

Cite this: Environ. Sci.: ProcessesImpacts, 2013, 15, 1529

Received 13th May 2013Accepted 17th June 2013

DOI: 10.1039/c3em00239j

rsc.li/process-impacts

This journal is ª The Royal Society of

Determination of estrogenic steroids and microbial andphotochemical degradation of 17a-ethinylestradiol(EE2) in lake surface water, a case study

Yuegang Zuo,* Kai Zhang and Si Zhou

In this study, a GC-MS technique was applied to determine 17a-ethinylestradiol (EE2), an active ingredient

of oral contraceptives, and its fate in Lake Quinsigamond, Massachusetts, USA. To the knowledge of the

authors, this is the first study of EE2 and its microbial and photochemical degradation in a lake

ecosystem. EE2 was detected at a concentration up to 11.1 ng L�1. At this concentration EE2 may affect

the reproduction of fish and other aquatic organisms in the lake due to its high estrogenic activity. EE2

was persistent to the biodegradation by the microorganisms in the lake. Under aerobic conditions a

long lag phase (42 days) was observed before the biodegradation of EE2 and a half-life of 108 days was

estimated. Under anaerobic conditions, EE2 experienced even a longer acclimation stage (63 days) and a

slower microbial degradation in the lake water. The photodegradation of EE2 was rapid in the lake

surface water under natural sunlight, with a half-life of less than 2 days in summer sunny days.

Compared to biodegradation, photodegradation may represent a predominant removal mechanism for

EE2 in natural surface waters.

Environmental impact

Natural and synthetic steroid estrogens have become of increasing concern due to their widespread presence in the environment and endocrine disruptionpotential. However, there is still limited knowledge of their occurrence and fate in natural environments, especially in lake ecosystems. This study examined 17a-ethinylestradiol (EE2) and other estrogenic steroids in a freshwater lake in Massachusetts, USA and their microbial and photochemical degradation. EE2 wasdetected at a concentration up to 11.1 ng L�1. At this concentration EE2 may affect the reproduction of sh and other aquatic organisms in the lake due to itshigh estrogenic activity. EE2 was persistent to the biodegradation by the microorganisms in the lake but underwent rapid photodegradation in the surface waterunder natural sunlight. Data obtained in this study are useful for the environmental risk assessment and regulations of the steroid estrogens.

Introduction

Endocrine disrupting chemicals (EDCs) are exogenoussubstances or mixtures that alter the functions of the endocrinesystem and consequently cause adverse health effects in anintact organism, or its progeny, or (sub) populations.1During thepast decades, EDCs have received signicant scientic andpublic attention, because of their widespread occurrence in theenvironment and harmful inuence, particularly on aquaticorganisms. Many studies have shown that EDCs are able tomimic natural hormones or interfere with the action of endog-enous hormones, and thus cause various ecological and healthproblems, such as abnormalities in male reproductive systemsand development in sh and other wildlife species.1–8 Among thevarious EDCs reported in the environment, the natural steroidestrogens estrone (E1), 17b-estradiol (E2), estriol (E3), and

University of Massachusetts Dartmouth,

MA 02747, USA. E-mail: yzuo@umassd.

Chemistry 2013

particularly the synthetic estrogen 17a-ethynylestradiol (EE2) areconsidered to be the most potent estrogenic compounds.2,4–7,9,10

Even at a concentration as low as 0.1 ng L�1, EE2 provokesfeminization in some species of male wild shes.2 EE2 is a mainactive ingredient in some oral contraceptives. It is also widelyused in a number of pharmaceutical formulations as hormonalreplacement therapies, treatments of prostate and breast cancerand hair lotions for contrasting alopecia. EE2 is also applied tocontrol ovulation and treat reproductive disorders in livestock.EE2 and other steroid estrogens are excreted into wastewaterpredominantly as their sulfate and glucuronide conjugates.11–14

These conjugated steroids are not biologically active estrogens.During sewage treatment, the action of microorganisms canconvert the steroidal conjugates to the free active estrogens bycleavage of the conjugate group. Due to incomplete removalduring the wastewater treatment processes, EE2 and naturalestrogens are released to the environment and become majorcontributors to the estrogenic activity in the receiving water.

A great deal of scientic effort has been spent to determinethe sources, distribution, transportation and fate of estrogen

Environ. Sci.: Processes Impacts, 2013, 15, 1529–1535 | 1529

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steroids in the environment. Most of this research has beenfocused on the occurrence of the natural and synthetic estro-gens and their removal in wastewater treatment processes andin receiving waters.9,11–19 For instances, Ternes et al.20 and Silvaet al.14 have shown that in wastewater treatment processes withactivated sludge, E2 was oxidized to E1, which was furthereliminated without any degradation products observed. Thesynthetic EE2 is principally persistent under selected aerobicconditions, where mestranol (MeEE2), another commonsynthetic contraceptive component, was rapidly removed andsmall amount of EE2 were formed by demethylation of MeEE2.Microorganisms in English rivers were capable of transformingE2 to E1 with half-lives of 0.2–9 days at 20 �C, and E1 was thenfurther eliminated at similar rates.21 However, EE2 was muchmore resistant to biodegradation than the natural estrogenicsteroids. Fewer studies have been made to examine the degra-dation of EE2 in natural aquatic environment. In particular,little is known about the fate of synthetic estrogens in lakeecosystems. Lake Quinsigamond is a body of water locatedbetween the city of Worcester and the town of Shrewsbury inWorcester County, Massachusetts, USA, a highly urban area,and supports a variety of recreational uses, including shing,boating, bathing, and water skiing. The entire periphery of thelake is highly developed with many private homes and severalcommercial establishments. It represents a good location tostudy the pollution and fate of steroid estrogens since the lakeand its surrounding tributaries have been used for the disposalof human, animal and industrial wastes for over two centuries.Over the past decades the local municipality has investedmillions of dollars in the construction of sewage treatmentfacilities to process sewage and sewage overows that arereleased into the lake. However, even aer the construction ofthese sewage treatment facilities, sewage overows still occurduring heavy and prolonged rainstorms. In this study, weexamined the concentrations of EE2, MeEE2 and naturalestrogens E1, E2 and E3 in Lake Quinsigamond, and theirmicrobial and photochemical degradation employing a solid-phase extraction (SPE) and GC-MS technique developed in ourlaboratory.16 The chemical structures of the ve estrogenicsteroids studied are presented in Fig. 1.

Materials and methodsChemicals

Methanol, ethyl acetate, and n-hexane were obtained fromPharmco Products (Brookeld, NJ, USA). Derivatization reagentN,O-bis (trimethylsilyl)triuoroacetamide (BSTFA) + trimethyl-chlorosilane (TMCS) was purchased from Supelco (Supelcopark, PA, USA). Newly distilled pyridine was prepared from thepyridine purchased from MC&B (Norwood, OH, USA). Steroidstandards 17b-estradiol (E2), 17a-ethinylestradiol (EE2), andcholesterol (internal standard) used for this study wereobtained from Aldrich Chem. Co. (Milwaukee, WI, USA).Estrone (E1) was from Spectrum Chem. Mtg. Corp. (Gardena,CA, USA). Estriol (E3) was purchased from Sigma-Aldrich (St.Louis, MO, USA). Mestranol (MeEE2) was ordered from TCICo. (Japan).

1530 | Environ. Sci.: Processes Impacts, 2013, 15, 1529–1535

Sample collection and preparation

Lake water samples were collected in summer 2004, 2005 and2012 from Lake Quinsigamond, near the Hospital of theUniversity of Massachusetts Medical School, Worcester, MA,USA, stored at 4 �C and used within 24 h aer collection. Todetermine steroid estrogens in the lake water, the watersamples were ltered through 0.45 mm Nylon membrane lters(Whatman International Ltd., Maidstone, England). Aerltration, the used lter membranes were collected, dried atroom temperature, and then immersed in 15mL of ethyl acetatein a 30 mL glass vial for 4 h. The ethyl acetate extract solutionwas then transferred to another glass vial. Meanwhile, 7.0 L ofthe ltered water sample was extracted with Supelco SPE C18

cartridge equipped with a vacuum system. Prior to extraction,the cartridge was conditioned sequentially with 5.0 mL ofhexane, 5.0 mL of ethyl acetate, 5.0 mL of methanol, and 5.0 mLof distilled water. Aer extraction nished, the C18 cartridge wastreated with 5.0 mL of a mixture of methanol and ethyl acetate(1 : 4, v/v) to wash down the absorbed EE2. The SPE extract wascombined with the ethyl acetate extract of lter membrane inthe 30 mL glass vial, and dried under nitrogen to completedryness for the derivatization and GC-MS analysis.

Aerobic and anaerobic microbial degradation of estrogens inthe lake water

The degradation of EE2 was investigated at a concentration of2.0 mM in the lake water. Two 0.5 L samples of freshly collectedlake waters were prepared in two Nalgene bottles (NalgeCompany, Rochester, NY, USA), respectively. One of the bottleswas sterilized by autoclaving for 60 min at 120 �C and used as asterile control. To avoid the potential interference of methanolas a co-solvent, 100 mL of prepared stock solution with aconcentration of 10 mM of the estrogens in methanol wasadded to each bottle andmethanol was evaporated under gentlenitrogen stream prior to the addition of water. Then 0.5 L of lakewater was added to make the concentration of EE2 to 2.0 mM.The bottles were incubated in dark at room temperature (20 �3 �C), which is a common temperature in surface waters duringsummer in Massachusetts. All the bottles were shaken for oneminute before sampling. The concentrations of EE2 weremonitored on days 0, 1, 2, 4, 7, and then weekly until 70 days.

Lake water used for anaerobic study was prepared usingnitrogen to strip out dissolved oxygen. The sample bottles weresealed with rubber lids tightly aerwards. To maintain theestablished anaerobic conditions, syringes equipped with sharpneedles were used to collected water samples out of the bottlesby piercing through the rubble lids. Operations such as incu-bation temperature, sterile controls, and sampling time inter-vals were the same as the above aerobic experiments.

Photodegradation of EE2 in lake water

All photodegradation experiments were conducted on clearsummer sunny days on the top of the Chemistry DepartmentBuilding at University of Massachusetts, Dartmouth Campus(North Dartmouth, MA; 41.63�N). The ambient temperature was

This journal is ª The Royal Society of Chemistry 2013

Fig. 1 Chemical structures of the five estrogenic steroids studied and cholesterol, the internal standard.

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25 �C. The sunlight intensity was recorded using a quantumsensor (Li-Cor, Q-27003) that responds to light of 400–700 nm.The photodegradation was performed using quartz photolysistubes (1.4 cm i.d.� 20 cm) held in a rack at a 45� angle from thehorizontal and about 0.5 m above a black pavement. Darkcontrol was performed in same quartz tubes covered byaluminum foil. The initial concentration of EE2 was 2.0 mM inlake water. Aer irradiation started, at different time intervals(0, 1, 2, 3, 4, and 5 h) 5 mL of the irradiated solution waspipetted out and used to monitor the concentration change ofthe starting estrogenic compound.

Analytical method

The microbial and photochemical degradation was followed byGC-MS measurements.16 To prepare for GC-MS analysis, solidphase extraction using Supelco Discovery SPE C18 cartridgescoupled with a vacuum system (Supelco, Bellefonte, PA, USA)was applied to all samples as described in Section 2.2. Prior toextraction, the cartridges were conditioned sequentially with5.0 mL of hexane, 5.0 mL of ethyl acetate, 5.0 mL of methanol,

and 5.0 mL of distilled water. Aer loading 5 mL of watersample, the cartridge was treated with 5.0 mL of a mixture ofethyl acetate–methanol (4 : 1, v/v) to elute the absorbed chem-icals. Aer being dried under nitrogen, eluted extracts werederivatized using 50.0 mL of BSTFA + TMCS in 50.0 mL of pyri-dine, and heated at 80 �C for 2 h in a sand bath. To examine theextraction efficiency, recovery experiments were carried outusing standard spiked lake water samples at concentration of2 mM of estrogens.

A Hewlett–Packard (HP) Model GC 5890 Series II gas chro-matograph coupled with an HP 5971 Series mass selectivedetector and an HP 7673 GC auto-sampler was employed for allanalyses. Samples were separated on a 30m� 0.32mm, 0.25 mm,DB-5 fused silica capillary column (J &W Scientic, Folsom, CA).The column temperature was programmed as follows: the initialtemperature was 80 �C for 4min and increased to 200 �C at 20 �Cmin�1, then itwas increased to300 �Cat 10 �Cmin�1 andheld for4 min. The total run time was 24 min. Ultra high purity heliumwith an inline Alltech oxygen trap was used as carrier gas. Thecarrier gas was set at 40 psi, column head pressure at 8 psi.Injector temperaturewasmaintained at 280 �C, and the injection

Table 1 Extraction recoveries,a RSD and concentrations of identified estrogeniccompounds in Lake Quinsigamond surface water samples

Spiked water samplesAverage recoveryrate (%)

RSD(%)

Concentration(ng L�1)

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volume was 1.0 mL in the splitless mode. The interface temper-ature was held at 280 �C.Mass spectra were scanned fromm/z 50to 650 at a rate of 1.5 scans per second. Electron impact ioniza-tion energy was 70 eV.

Estrone (E1) 105 6.49 n.d.17b-Estradiol (E2) 107 8.41 n.d.Estriol (E3) 103 10.5 n.d.17a-Ethinylestradiol (EE2) 92.6 11.7 8.72–11.1Mestranol (MeEE2) 80.1 7.26 n.d.

a Average of triplicate analysis; n.d.: not detected.

Fig. 3 (A) GC-MS chromatogram of water sample collected in Lake Quinsiga-mond. (B) Mass spectra of the EE2 detected in the sample.

Results and discussionDetermination of steroid estrogens in Lake Quinsigamondwater samples

Determination of steroid estrogens in natural water has been achallenge for environmental scientists because of the extremelylow analyte concentration levels and complex matrix composi-tion. Consequently, it is necessary to preconcentrate theselected analytes and clean-up the samples from interferingcomponents, especially in the presence of humic substances orsuspended particles in surface waters. Analytical and technicalproblems caused by the suspended particles affect both preci-sion and accuracy of estrogen determination in the water.Filtration with various lter materials, such as glass ber,acetate cellulose, Teon, nylon and polycarbonate has beencommonly employed to remove suspended particles in watersamples.11 Some of these lter materials may adsorb steroidestrogens because of their low volatility and hydrophobiccharacter, and thus lower the analytical results if not properlytreated. To test this potential problem, 10 mL of 5.0 mg L�1 ofEE2 solution was ltered through one of commercial membranelters [cellulose acetate: 0.45 mm pore size (Millipore Corpora-tion, Billerica, MA, USA), polycarbonate: 0.2 mm pore size(Whatman International Ltd.), or nylon: 0.2 mm pore size, all in13 mm diameter]. As shown in Fig. 2, approximately 64% of EE2was retained on the nylon membrane lter. Therefore, aer theltration, the ltration system should be washed with anorganic solvent to remove any possible adsorbed analytes, andadd this organic solvent extract into the sample. Analyticalrecoveries for all steroids tested were good (Table 1). Repro-ducibility between triplicate extractions was also good with arelative standard deviation ranged from 6.49 to 11.7%. A typicalGC-MS chromatogram of lake water samples is presented inFig. 3(A), and the mass spectrum of EE2 derivatives identiedfrom the extract in Fig. 3(B). Although no natural estrogenic

Fig. 2 Concentration loss of EE2 in filtrate after 10 mL of 5.0 mg L�1 of EE2 wasfiltered through nylon (0.2 mmpore size, 13 mm diameter), polycarbonate (0.2 mmpore size, 13 mm diameter) and cellulose acetate (0.45 mm pore size, 13 mmdiameter) membrane filters at pH 6.0.

hormones [above the limit of detection (LOD) of 0.1 ng L�1]were detected in any of the samples collected, the syntheticestrogen, EE2, was found in Lake Quinsigamond surface waterwith a concentration of 11.1 ng L�1 in the summer, which couldcertainly affect the development and reproduction of sh andaquatic organisms in the water body. The occurrence of EE2 inLake Quinsigamond surface water at such high concentrationwas probably due to the pollution from the Hospital, and otherunknown sources such as combined sewage and sewage over-ows, livestock wastes, and the resistance of EE2 to the degra-dation. It is important to examine the possible fate of EE2detected in the lake water.

Aerobic and anaerobic microbial degradation of EE2 in LakeQuinsigamond surface water

EE2 experienced a long microbial acclimation stage underaerobic conditions before a slow degradation (Fig. 4). Aer 70days of incubation in the aerated lake water, approximately 38%of EE2 was degraded with an estimated half-life of 108 days.

Fig. 4 Aerobic and anaerobic microbial degradation of EE2 in lake water. Opencircles, aerobic; open squares, anaerobic; closed triangles, sterilized lake water. Fig. 5 Photodegradation of EE2 in Quinsigamond lake water under natural

sunlight irradiation.

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Under anaerobic conditions, the lag phase was even longer.Little degradation was observed within 63 days incubation whencompared with the sterile control. A slow degradation of EE2occurred aer 63 days acclimation under anaerobic conditions.It is not surprising that EE2 was persistent in either the oxic oranoxic lake waters. EE2, like other anthropogenic compounds,has substituents and structural features that differ from thosein natural steroid estrogens (Fig. 1), and indigenous microor-ganisms may not have evolved the enzymes to deal with thesechemical structures.22–24

Microbial degradation of natural steroid estrogens has beenreported to play a major role in the elimination of these EDCsfrom aquatic environment. Jurgens et al. observed that inEnglish rivers EE2 had a half-life for 17 days. Although thisbiodegradation of EE2 is very slow, it is still signicantly fasterthan that observed in this study perhaps because the microor-ganisms in the receiving waters had gradually adapted to thesynthetic estrogens in thewastewater effluent. As shown inFig. 1,E2 has twoOH functional groups on its structure, whichmakes itamenable to microbial transformation. However, EE2 has twoadjacent quaternary carbon atoms with an ethinyl group, whichmake it more resistant to microbial attack. Furthermore, it isspeculated that microorganisms can use natural estrogens thatexist in soils, natural waters, and sewage sludge for growth; whilefor synthetic estrogens,microorganisms onlymodify but not usethem for growth via a cometabolic process.25,26 These differencesin the degradation mechanisms might also contribute to theslow biodegradation of EE2 in natural water.

Photochemical degradation of EE2 in Lake Quinsigamondsurface water

Although EE2 is resistant to microbial degradation, it possessesan aromatic functional group, which is susceptible to sunlight-inducedphotodegradation (Fig. 5). Nodegradation of the steroidcompound was observed in the dark controls. It is assumed that

sunlight induced photodegradation of the estrogen in naturalwater system follows pseudo rst-order kinetics.

ln(C/C0) ¼ �kt (1)

where C0 and C are the concentrations of EE2 at time zero andreaction time t (h), respectively; k is the pseudo-rst-orderdegradation rate constants (h�1). The rate constants were esti-mated from the slope of a plot of ln(C/C0) versus time, and thehalf-life (t1/2) was calculated using equation:

t1/2 ¼ ln 2/k (2)

Under sunlight irradiation the half-life of EE2 in the lakewater was 23 hours which is much faster than biodegradation(half-life of approximately 108 days in the same lake water).Photochemical transformation may be a signicant removalmechanism in the aquatic environment in the northernsummer considering the long sunlight irradiation time insummer seasons in Massachusetts. However, factors such asturbidity and coloring of the water would reduce the intensity ofsunlight. Since the water samples used in this study wereltered prior to use, making sunlight absorption more efficient,the corresponding photodegradation in the environment mightbe slower.

The photodegradation of the steroid estrogens may occurthrough direct and indirect photochemical processes. Thedirect photolysis is due to the absorption of energy fromsunlight by estrogenic compounds themselves (steroid estro-gens absorb light from 280–390 nm, overlapping with solarspectrum). Many studies have been conducted to investigateindirect photodegradation reactions in the environment.9,27–36

Indirect photolysis is driven by oxidative species such as OHradicals generated by other light absorbing species, such ashumic substances, transition metals, nitrate and nitrite in

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natural water under sunlight irradiation. The reactions (3) to(11) demonstrate these indirect photodegradation of EE2 innatural surface water.

DOM + hn / DOM_� (3)

DOM_� + O2 / oxidized DOM + O2_� (4)

Fe(III)–organic complex + hn / Fe(II) + Org_ (5)

Org_+ O2 / oxidized org + O2_� (6)

2O2_� + 2H+ / H2O2 + O2 (7)

Fe(II) + H2O2 / Fe(III) + OH_+ OH� (8)

H2O2 + hn / 2OH_ (9)

NO2�/NO3

� + H2O + hn / NO/NO2 + OH_+ OH� (10)

EE2 + OH_/ products (11)

Thus, the photodegradation of steroid estrogens iscontrolled by factors such as irradiation time, light intensity,and concentration of the photochemically reactive species, andtotal organic content.

Conclusions

In this study, 17a-ethynylestradiol (EE2) has been determinedin Lake Quinsigamond surface water for the rst time atconcentrations up to 11.1 ng L�1. Neither natural steroidestrogens nor mestranol have been detected in the lake. Thepresence of EE2 at this concentration level may adversely affectthe reproduction of shes and other aquatic organisms in thewater body. EE2 was persistent to the microbial degradationwith an estimated half-life of 108 days in the surface lake waterunder aerobic conditions. The anaerobic biodegradation waseven much slower. But EE2 can undergo a rapid photo-degradation in the surface lake water under sunlight irradiationwith a half-life of 23 h. Further study is needed to examine thephotodegradation products of EE2 and other estrogenicpollutants, and to investigate their effects on sh and otheraquatic organisms in the lake ecosystems.

Acknowledgements

The authors would like to thank Dr D. Ryan and E. Ojadi fortheir contributions to this work. This research project waspartial supported by the US National Science Foundation underGrant OCE 0752033, the US National Oceanic and AtmosphericAdministration (NOAA), and the US Geological Survey (USGS).

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The Management of Estrogens, Estrogen Receptors and ...bioidenticalhormonesny.com/.../uploads/2014/08/Managements-of-E… · anti-estrogenic 2. Estrogens are absorbed in the liver

Photochemical Alteration of Dissolved Organic Nitrogen in the Surface Ocean of the North Atlantic

Physical Organic Photochemistry and Basic Photochemical ... · PDF filePhysical Organic Photochemistry and Basic Photochemical Transformations Group Meeting Jan 26th 2011 Scott Simonovich

Physical Organic Photochemistry and Basic Photochemical ...chemlabs.princeton.edu/macmillan/wp-content/uploads/sites/6/unnamed-file.pdf · Photochemistry occurs from S 1 (infrequent)