Analytical Methods Used to Measure Endocrine DisruptingCompounds in Water

S. Snyder1; B. Vanderford2; R. Pearson3; O. Quinones4; and Y. Yoon5

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Abstract: Endocrine-disrupting compounds~EDCs! have been detected in natural waters globally. Additionally, trace concentratiopharmaceuticals and personal care products~PPCPs! have also been detected in various waters, primarily as the result of incomwastewater treatment. Some PPCPs are known to act as EDCs as they can have impacts on the endocrine systems of adetection of EDCs and PPCPs in source water is of great concern since some of these compounds have known physiological rlow concentrations. The majority of EDCs and PPCPs are more polar than traditional contaminants, such as polychlorinated biphorganochlorine pesticides, and several have acidic or basic moieties. These properties, coupled with trace quantities, crechallenges for both removal processes and analytical detection. There are two general approaches for monitoring EDCs andwater: ~1! direct measurement of target compounds via analytical instrumentation and~2! biological assays. These approaches willdiscussed with an emphasis on analytical methods for direct measurements.

DOI: 10.1061/~ASCE!1090-025X~2003!7:4~224!

CE Database subject headings: Analytical techniques; Pollutants; Water pollution; Wastewater treatment; Measurement.

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Introduction

The first difficulty in developing an analytical approach for montoring endocrine disrupting compounds~EDCs! is determiningwhat an EDC actually is, and which compounds are approprtarget analytes. The U.S. Environmental Protection Agency~U.S.EPA! has defined an environmental EDC as an exogenous awhich interferes with the ‘‘synthesis, secretion, transport, bindiaction, or elimination of natural hormones in the body that aresponsible for the maintenance of homeostasis, reproductionvelopment, and/or behavior’’~U.S. EPA 1997!. However, thisdefinition is quite broad and later in the same document the UEPA states, ‘‘ . . . cells within the brain are a potential target foenvironmental chemicals that have no impact on steroid hmones directly, but yet will lead to a disruption of endocrinfunction’’ ~U.S. EPA 1997!. There are numerous other definitionand opinions on what defines an EDC. However, it is generaaccepted that the three major classes of endocrine endpointestrogenic~compounds which mimic or block natural estrogen!,

1Research and Development Project Manager, Southern Nevada WAuthority, 1001 S. Valley View Blvd., Las Vegas, NV 89153. E-maishane.snyder@lvvwd.com

2Research Chemist, Southern Nevada Water Authority, 1001 S. VaView Blvd., Las Vegas, NV 89153.

3Research Chemist, Southern Nevada Water Authority, 1001 S. VaView Blvd., Las Vegas, NV 89153.

4Research Chemist, Southern Nevada Water Authority, 1001 S. VaView Blvd., Las Vegas, NV 89153.

5Research Associate, Arizona State Univ., Department of Civil aEnvironmental Engineering, Tempe, AZ 85287.

Note. Discussion open until March 1, 2004. Separate discussions mbe submitted for individual papers. To extend the closing date bymonth, a written request must be filed with the ASCE Managing EdiThe manuscript for this paper was submitted for review and posspublication on March 24, 2003; approved on June 9, 2003. This papepart of thePractice Periodical of Hazardous, Toxic, and RadioactivWaste Management, Vol. 7, No. 4, October 1, 2003. ©ASCE, ISSN1090-025X/2003/4-224–234/$18.00.

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androgenic~compounds which mimic or block natural testosteone!, and thyroidal~compounds with direct or indirect impacts tthe thyroid gland!. Under amendments to the Safe Drinking WatAct ~Bill Number S.1316! and the Food Quality Protection Ac~Bill Number P.L. 104-170!, all chemicals and formulations musbe screened for potential endocrine activity before they are mafactured or used in certain processes where drinking water anfood could become contaminated. These laws require the UEPA to ‘‘develop a screening program, using appropriate vadated test systems and other scientifically relevant informationdetermine whether certain substances may have an effect inmans that is similar to an effect produced by a naturally occurrestrogen, or other such endocrine effect as the administratordesignate.’’ The outcome of this screening battery is critical towater industry, as it will provide definitive answers to what compounds really are EDCs. However, it is critical to note that tcurrent legislature regulates only the industries producingusing raw chemicals and not the water industry directly.

There are currently no maximum contaminant limits for phamaceuticals and personal care products~PPCPs! in drinking ornatural waters. The Food and Drug Administration requires elogical testing and evaluation of a pharmaceutical only if an evironmental concentration is expected to exceed 1mg/L. The U.S.EPA does not regulate pharmaceuticals in the environment,has taken an active interest in the subject~Daughton and Ternes1999!. The United States Geological Survey recently completenational reconnaissance study that indicates widespread contnation of U.S. surface waters with various pharmaceuticalsother wastewater contaminants~Kolpin et al. 2002!.

Since EDCs and PPCPs represent an extremely broad strum of compounds, the development of analytical techniquequite challenging. This manuscript outlines several biological ainstrumental methods for the detection and quantitation of themerging contaminants in water. Screening for contaminabased on biological activity is useful for determining the presenor absence of a particular class of compounds~e.g., estrogenic orandrogenic!, while the majority of instrumental techniques a

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Fig. 1. MVLN cellular bioassay for estrogenic compounds

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used to identify and quantify specific target compounds. No sinmethod alone can predict or detect all contaminants present ienvironmental sample, nor can all biological mechanisms oftion be accounted for in one simple test. Therefore, it is critithat the toxicological relevance of EDCs and PPCPs be demined in order to develop an appropriate target compound listto establish meaningful method detection limits~MDLs!.

Biological Screening Tools

In Vitro Bioassays

Several types of in vitro assays are available for measuringestrogenic or androgenic activity of single compounds or compmixtures~Soto et al. 1992; Jobling and Sumpter 1993; Anderset al. 1996; Sanderson et al. 1996; Zacharewski 1997; Desbet al. 1998; Snyder et al. 2000b; Giesy et al. 2002!. In vitro bio-assays utilize cells or tissues as compared to in vivo tests thatentire organisms. Common in vitro assays measure end posuch as receptor binding, expression of endogenous or exogegenes, and cell proliferation. In vitro systems are attractivescreening tools because they are rapid, inexpensive, and genereproducible. For these reasons, precise estimates of the relpotency of many samples or compounds can be obtainedrather short period of time. Expression assays examine inducor suppression of proteins encoded by genes whose transcripis modulated through an estrogen-~ER! or androgen-~AR! recep-tor mediated mechanism. Increases or decreases in the activithe protein of interest upon exposure to a single compoundcomplex mixture, such as an environmental extract, suggestpresence of one or more ligands with the potential to modulabroad range of genomically controlled estrogenic or androgeresponses.

The most widely studied EDCs have been estrogenic andtiestrogenic. This is due in part to the role of estrogens in brecancer. In fact, some of the most widely used cellular bioassfor estrogens were developed for breast cancer research~e.g.,MCF-7 cell proliferation assay! ~Zacharewski 1997!. In past re-search efforts, we have used the MVLN~MCF-7-luc! in vitrogene expression assay. MVLN cells are human breast carcincells transfected with a luciferase reporter gene under controestrogen responsive elements~EREs! of theXenopusvitellogeninA2 gene~Pons et al. 1990!. When cells are exposed to an envronmental mixture, ER ligands can enter cells and bind theand upregulate expression of an exogenous luciferase repgene~Fig. 1!. Upon the addition of the appropriate substrate,

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ciferase catalyzes a light producing reaction that can be measuconveniently and with great sensitivity using a 96 well platereading luminometer. The measured luciferase activity is proptional to the sample’s ability to modulate ERE-mediated genexpression. By combining analytical techniques for water extration and fractionation with the MVLN assay, several estrogencompounds were detected in Lake Mead, Nevada~Snyder et al.1999; Snyder et al. 2001c!. However, several other PPCPs werlater detected in Lake Mead using instrumental analyses that wnot detectable using the MVLN assay~Snyder et al. 2001b!. Al-though ER and AR cell lines are the most widely used in EDtesting, cell lines capable of detecting impacts to the thyroid habeen developed and are needed for more comprehensive Escreening~Hohenwarter et al. 1996; Gray et al. 2002!.

Receptor binding assays are also useful for rapid screeningcertain classes of EDCs~U.S. EPA 1998; Zacharewski 1997!. Inthese assays, purified receptors~i.e., ER or AR! are used to assessthe relative binding affinities~RBAs! of various chemicals. Al-though one aspect of the receptor binding assays would becomparison of RBAs to biological potencies, this can be quidifficult as nonreceptor mediated endocrine activity is also posible ~i.e., aromatase activity!. RBAs are often determined usingcompetition assays where a fixed concentration of a labeligand ~i.e., radioactive or fluorescent! and various concentrationsof unlabeled competitor compete for binding to the particular rceptor~Fig. 2! ~Folmar et al. 2002; Inoue et al. 2002; Ohno et a2002!. The more unlabeled competitor that is present, the greathe labeled ligand is displaced from the receptor. Thus individucompounds or complex mixtures can be tested at various conctrations until the concentration required to displace 50% of thlabeled ligand (EC50) is determined. From the EC50 value, theRBAs can be calculated from the ratio of the concentrationchemical or mixture to the concentration of labeled ligand useRBAs then can be compared to endogenous steroids and checals or mixtures can be assigned to general potencies, suchweak, moderate, or strong receptor binding compounds. Additioally, quantitative structural-activity relationship~QSAR! modelsare being developed to predict receptor binding affinities basedstructural properties~U.S. EPA 1998; Mekenyan et al. 2002!.These QSAR models may be used for high-throughput prescreing as part of the U.S. EPA’s Endocrine Disruptor Screening Prgram ~EDSP!.

In Vivo Bioassays

Although in vitro assays are an attractive option for screening amechanistic studies, they may miss effects that would take pla

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Table 1. End Points Used to Assess Reproductive Impacts of Endocrine Disrupting Compounds in Fish

End point Selected references

Vitellogenin ~alkaline labile phosphorus, immunoassay, liver mRNA! Folmar et al. 1996; Harries et al. 1997Gonadosomatric index~GSI! Pereira et al. 1993; Monosson et al. 1994; Singh et al. 1994

Jobling et al. 1996Plasma sex hormone levels~T, 11-KT, E2 and other estrogens! MacLatchy and Van Der Kraak 1995; Van Der Kraak et al. 1992Steroidogenesis enzyme activities and intermediates MacLatchy et al. 1997Production of hormones during in vitro organ incubation~gonad, pituitary! Singh et al. 1994; MacLatchy and Van Der Kraak 1995Gonadotropins and gonadotropin releasing hormone Van Der Kraak et al. 1992; Singh et al. 1994Gonad structure and histology~intersex, gonad duct development! Gimeno et al. 1997; Gray andMetcalfe 1997; Jobling et al. 1998Activity of liver enzymes involved in steroid metabolism~mixed functionoxygenase, or MFO, activity!

Johnson et al. 1993; McMaster et al. 1991

Fertility and fecundity Giesy et al. 2000; Monosson et al. 1994Secondary sex characteristics Bortone and Davis 1994; Miles-Richardson et al. 1999Reproductive behavior Kindler et al. 1991

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only in whole organisms. Some toxicants require metabolic acti-vation through one or more pathways that occur in vivo, but notin vitro. Homeostatic controls and bioaccumulation generally arenot simulated by in vitro testing systems. Toxicant effects canoccur by different mechanisms in multiple tissues simultaneously.In addition, the same chemical exposure can result in very differ-ent responses in an animal depending upon its life stage, sex, andreproductive state. Fish are useful in vivo models for determiningthe aquatic impact of EDCs in water. Some species are extremelysensitive to such effects, and fish can act as integrators of re-sponses to mixtures of toxicants that occur in the environment.Furthermore, effects related to growth and reproduction in fish aremore easily related to population-level and ecological effects thanare effects measured by in vitro systems. Table 1 provides someof the more common bioindicators used for EDC testing in fishassays. Although fish assays are most commonly used for EDCtesting for surface water contamination issues~i.e., wastewatereffluents!, mammalian studies are generally used for drinkingwater testing where human toxicology endpoints need to be mea-sured. As part of the U.S. EPA’s EDSP, several mammalian testsare being evaluated~U.S. EPA 1998!.

Direct Measurements

Generally, analytical methods to detect EDCs/PPCPs are based onpreselected target compounds. Although some reports have shownthat novel or potential EDCs/PPCPs can be detected using ad-vanced mass spectrometric techniques, these techniques are stilllimited to identification and cannot determine whether the com-pounds will have EDC toxicological impacts~Grange et al. 1996;Grange and Brumley 1997; Snyder et al. 2001b!. Since EDCs andPPCPs represent broad classes of chemical compounds, the meth-ods employed for analyses are also quite varied. Table 2 presentsseveral analytical approaches that have been used for EDC/PPCPanalysis with corresponding detection limits and instrumentationutilized. We will describe some of the most common and modernanalytical tools used for the analysis of EDCs/PPCPs in water.

Extractions

Detection of EDC/PPCP compounds in water is often desired attrace levels~sub-mg/L! since some compounds have been foundto have aquatic impacts at these concentrations~Routledge et al.1998; Segner et al. 2003; van Aerle et al. 2002!. However,

most analytical instruments are not able to directly detect compounds at these trace levels. Therefore, an extraction step is uto concentrate the target compounds to a detectable level. Cventional extraction techniques such as liquid-liquid~Yook et al.1994; Holm et al. 1995; Romero et al. 2002!, Soxhlet ~Bennieet al. 1997; Pryor et al. 2002; Fatoki and Awofolu 2003!, andsteam distillation~Kubeck and Naylor 1990; Snyder et al. 2001a!,have been used to extract EDC/PPCP compounds from water asolids. However, solid-phase extraction~SPE! is by far the mostcommon technique employed for extraciton of trace contaminanfrom water. Generally, the stationary phase is packed into a ctridge or column and water is pumped or pulled through. Frequently, after the water sample has passed through the stationphase, the stationary phase is dried by passing nitrogen orthrough the SPE column/cartridge. This drying step removes mowater from the resulting extract, which can be detrimental focertain types of instrumental analyses. Solvents with a greaaffinity for the target compounds than for the stationary phase aused to elute the compounds from the SPE column/cartridgOften a multisolvent system is required to remove all target compounds from the SPE. The resulting extract volume is dependeupon how much solvent was used in the elution process. Thextract is generally concentrated further by evaporation withgentle stream of nitrogen to one mL or less. For certain analysa cleanup step may be required, such as gel permeation chrotography to remove large molecular weight compounds or silicgel or alumina columns that can fractionate the extract basedpolarity ~Khim et al. 1999a,b,c; Snyder et al. 1999; Snyder et a2001b,c!. More modern variants of SPE include solid-phase mcroextraction~SPME! and various on-line and automated SPEtechniques. SPME involves a fine silica fiber that is coated extenally with a stationary phase. This small~1–2 cm! fiber is im-mersed into the aqueous sample~generally 20 mL or less! andremains in contact with a stirred solution until a predetermineequilibrium is established. The SPME fiber is introduced directinto the analytical instrumentation through a sample injection sytem and compounds desorbed from the fiber using heat in gchromatography or a solvent in liquid chromatography. Passisamplers are also of great use in the monitoring of water fvarious EDCs. Semipermeable membranes devices~SPMDs! arepassive samplers often used for an integrated assessment of wcontaminants. These devices generally consist of a low-denspolyethylene tube filled with triolene or other lipophillic materialand remain in contact with water for an extended

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Table 2. Summary of Analytical Methods for Determination of Endocrine-Disrupting Compounds and Pharmaceuticals and Personal Care Products

Class Compound Limit of detection Method Water type Reference

Estrogens/surfactants

Estrone, mestranol, estradiol,ethynylestradiol, norethistrone,ethistrone, d3-estradiol,stigmasterol, cholesterylbutyrate,cholesterol methylether

1–5 ng/L Gas chromatography/massspectrometers w/silicacapillary column

Drinking water Carlile et al. 1996

17b-estradiol~E2!,17a-ethynylestradiol~EE2!/nonylphenol~NP!, octylphenol~OP!,nonylphenol polyethoxylates~NPE!

11, 2, and 52 ng/L~NP,OP,NPE!

HPLC w/fluorescence Wastewater effluent/surface water

Snyder et al. 2000a

107 and 53 pg/L~E2and EE2!

competitive radioimmunoassay

Estrogens/phthalates/surfactants/plasticizers

E2/diethyl phthalate, bisoctylphthalate/nonylphenol,nonylphenolethoxylate/bisphenol A

0.3 ng/L~E2!, 0.2mg/L ~phthalates!, 0.2to 4 mg/L ~surfactant!

Gas chromatography/massspectrometers/massspectrometers

Sewage effluent/surface water

Fawell et al. 2001

5.1 mg/L ~bisphenolA!

HPLC

E2 ;2.75 ng/L in vitro recombinant assay with yeastcells

Surface water Witters et al. 2001

Estrogens/phenols Estrone, E2, EE2, NP, OP ;50 ng/L High-resolution gas chromatographywith negative chemical ionization-mass spectrometers

Surface water/drinking water

Kuch and

Ballschmiter 2001

Bisphenol A~BPA!, NP,butylbenzylpthalate, EE2

,2.8 ng/L EE2BPANP

E-screen~in vitro assay!Gas chromatography/mass spectrometersEnzyme linked immunosorbentassays

Leachate effluent Behnisch et al.

2001

Estrogens/testosterone

TestosteroneE2EE2

1.56 nM Yeast estrogenic assay Municipal/industrial wastewater

Layton et al. 2000

Flame retardant/phenols

Polybrominated diphenyl ether Various Gas chromatography/mass spectrometer

Synthetic water Rahman et al.

2001

Tetrabromo-bisphenol A,tetrachlorobisphenol A

HPLC w/ultraviolet-visibledetector

Sediment Voordeckers et al.

2002

Musk Galaxolide 5mg/L HPLC w/ultraviolet Synthetic water Rimkus 1999

Musk xylene, musk ketone 0.001mg/L ~water!0.5 mg/L ~fish!

Gas chromatography/endocrine-disruptingcompounds

Surface water/fish

Yamagishi et al. 1983

Organotins Butyltins, phenyltin 5 ng/g as Sn Gas chromatography/flame-photometric

Sediment Byrns 2001

Polyaromatichydrocarbons~PAHs!

Phenanthrene, acenaphthene,fluoranthene, pyrene

10 ng/L Gas chromatography/mass spectrometers

Sediment Schults et al. 1992

Anthracene 0.6mM HPLC at 250 nm Synthetic water Fischer et al. 2002

Phenanthrene, fluoranthene,pyrene, benz~a!anthracene,benzp~a!pyrene

60–130 pg Gas chromatography/mass spectrometer

Harbor water Leppard et al. 1998

Fluoranthene, benzo~a!pyrene,benzo~a!pyrene

0.3–1.0mg/L HPLC Sewage effluent Blanchard et al. 2001

Pyrene, fluoranthene,anthracene

11 ng/L ~pyrene,fluoranthene!, 0.8ng/L ~anthracene!

Liquid chromatographyw/ultraviolet at 254 nm

Sediment Ravelet et al. 2001

Pesticides/polyaromatichydrocarbons

Fluoranthene, pyrene,anthrancene, organochlorinepesticides,hexachlorobenzene

0.01–10 ng/g dry wt Revere-phase HPLC w/fluorescence

Lake water Khim et al. 1999c

Phenanthrene, acenaphthene,fluoranthene, pyrene, etc.

10 ng/L Gas chromatography/mass spectrometer

Sediment Schults et al. 1992

Pesticides/herbicides

Atrazine 10mg/L HPLC w/ultraviolet at 225 nm Wetland/agriculturalwastewater effluent

McKinlay and

Kasperek 1999

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Table 2. ~Continued!

Class Compound Limit of detection Method Water type Reference

Triazines, organochlorines~OCs!, organophosphorus~OPs!, amide

0.05mg/L: triazines,organochlorines,organophosphorus, amide

Gas chromatography/mass spectrometer

Synthetic water Mouvet and Jucker

1997

0.05mg/L: dinoseb,dinoterb, bentazone

HPLC/ultraviolet at 244 nm

Pesticides/pharmaceuticals/nonphenylicpesticides

Terbutylazine, metolachlor,dichlobenil/carbamazepine,caffeine

0.3–2 ng/L Gas chromatography/massspectrometer

North Sea water Weigel et al. 2001

Atrazine, simazine, diuron,isoproturon

5 mg/L0.1–0.5mg/L

HPLC w/o pretreatmentHPLC w/pretreatment

Ground water Van der Bruggen et al.

2001

Simazine, atrazine, pyridine, etc. Sub-mg/L HPLC w/ultraviolet~220 nm! Synthetic water Kiso et al. 2000, 2001Pharmaceuticals~stimulants!

Caffeine, propyphenazone, 4-aminoantipyrine, diazepam,clibenclamide, nifedipine,omeprazole, oxyphenbutazone

25–50 ng/L HPLC/mass spectrometer/mass spectrometer

Wastewater/river water

Ternes et al. 2001

Caffeine 5mg/L HPLC Groundwatercontaminate withwastewater

Seiler et al. 1999

0.04mg/L Gas chromatography/massspectrometer

Bezifibrate, clofibric acid,gemfibrozil, diclofenac,ibuprofen, carbamazepine

Lower ng/L Gas chromatography/massspectrometer/mass spectrometerLC-electrospray/mass spectrometer/mass spectrometer

Sewage effluent/river water

Ternes 1998

Diclofenac ,1 ng/L Solid phase extraction/gaschromatography/mass spectrometer

Surface water Buser et al. 1998b

Ibuprofen 0.1–1.0 ng/L Solid phase extraction/gas chromatography/mass spectrometer

Surface water Buser et al. 1999

Wastewater

Clofibric acid, ibuprofen,diclofenc, bezafibrate,naproxen

50–150 ng/L~sewage!,10–25 ng/L~surfacewater!,1–25 ng/L~drinkingwater!

Solid phase extraction/gas chromatography/mass spectrometer

Sewage water/surface water/drinking water

Stumpf et al. 1999

Clofibric acid 0.2–1.0 ng/L Solid phase extractiongas chromatography/mass spectrometer

Lake/North Sea waterNorth Sea water

Buser et al. 1998a

Amoxicillin 50 mg/L HPLC w/ultraviolet~250 nm! and

fluorescence 1990!

Synthetic water Mascher and Kikuta 1990

Carbamazepine and metabolites 2–4mg/L HPLC w/ultraviolet~237 nm! Epileptic patient Mandrioli et al. 2001

Polyaromatichydrocarbon

Anthracene 0.6mM HPLC at 250 nm Synthetic water David and Riguier 2002

Phenanthrene, fluoranthene,pyrene, benz~a!anthracene,benzp~a!pyrene

60–130 pg Gas chromatography/mass spectrometer

Harbor water Leppard et al. 1998

Fluoranthene, benzo~a!pyrene,benzo~a!pyrene

0.3–1.0mg/L HPLC Sewage effluent Blanchard et al. 2001

Pyrene, fluoranthene,anthracene

11 ng/L ~pyrene,fluoranthene!, 0.8 ng/L~anthracene!

HPLC w/ultraviolet~254 nm! Sediment Ravelet et al. 2001

Surfactant/polyaromatichydrocarbon/estrogen

NPE, NP, BPA,benzo~a!pyrene,E2

0.1 mg/L NP & NPE Gas chromatography/mass spectrometer

Wastewater Nasu et al. 2001

0.01mg/L BPA &benzo~a!pylene

0.2 ng/L E2 Enzyme linked immunosorbentassays

Surfactants Nonylphenol polyethoxycarboxylate

0.2–2 ng/L Solid phase extraction/gaschromatography/mass spectrometer

Sewage effluent/river water

Field and Reed 1996

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Table 2. ~Continued!

Class Compound Limit of detection Method Water type Reference

Polyethoxylated nonylphenol,nonylphenol

50–500 ng/L HPLC-diode array detection-massspectrometer

Sewage water/surface water

~Sole et al.

2000!4-nonylphenol4-nonylphenol, 4-tert-octylphenol

,81 ng/L10 ng/L ~NP!

HPLC w/fluorescenceGas chromatography/mass spectrometer

Fathead minnow ~Giesy et al.

2000!

Fish in river water ~Tsuda et al.

2001!1 ng/L ~OP!

Nonylphenol ethoxylate ;1 mg/L HPLC w/fluorescence Synthetic water ~Fielding et al.

1998!X-ray contrastmedia

Diatrizoate, iopamidol,iopromide, iothalamic acid,iomeprol, ioxithalamic acid

5 ng/L–10mg/L Liquid chromatography/massspectrometer

Sewage effluent/river/groundwatersewage

~Ternes and

Hirsch 2000!

Diatrizoate and iopromid 50 ng/L Liquid chromatography/massspectrometer

Wastewater/river water

~Putschew et al.

2000!Iopromide 10 ng/L HPLC at 254 nm Wastewater/

surface water~Steger-

Hartmann et al. 2002!

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period of time~days–months!. The SPMD can mimic the bioac-cumulation of chemicals into aquatic organisms and provide esti-mated water concentration data~Bevans et al. 1996; Bennett andMetcalfe 2000; Echols et al. 2000; Petty 2000; Lindstrom et al.2002!. Typical SPMDs using nonpolar liquids would be unlikelyto accumulate polar compounds, like many EDCs and PPCPs;however, efforts are underway to refine passive samplers for ef-ficient uptake of polar compounds.

Gas Chromatography

Gas chromatography~GC! has been used for many years forchemical analysis. Traditionally, GC works by injecting a liquidsample into a heated injection chamber, where the compounds arequickly vaporized and swept by a carrier gas, usually helium,onto a long, thin coated silica chromatography column~i.e., 30 mlong by 0.25mm diameter! internally coated with a stationaryphase. The chromatography column is positioned in an electroni-cally controlled oven. The individual compounds will separate asthey flow through the column based on their boiling point, affinityfor the stationary phase, and temperature profile of the GC oven.A detector then monitors the compounds as they leave the col-umn. There are many types of detectors including flame-ionization, electron capture, nitrogen-phosphorus, flame-photometric, and mass spectrometric. Each detector has its ownadvantages and disadvantages with regard to sensitivity, selectiv-ity, and cost.

Mass spectrometry~MS! is one of the most useful and com-mon techniques because of its superior sensitivity and selectivity.Since MS separates and detects an analyte based on mass tocharge ratio~m/z!, the compound must be ionized before it entersthe MS. The interface where this takes place is known as theionization source. There are two major techniques to induce ion-ization in the source: electron ionization~EI! and chemical ion-ization ~CI!. In EI, the GC column effluent is directed through abeam of electrons created by a filament. This results in the loss orgain of an electron by the analyte creating a positively or nega-tively charged molecule, respectively. This type of ionization re-sults in molecular fragmentation, which is related to the structuralproperties of the compound. Thus, each compound has a uniqueMS ‘‘fingerprint’’ that allows for the identification of the com-pound based on its fragmentation pattern. In CI, a gas~typicallymethane! is first ionized, creating a radical that then collides with

the analyte, resulting in the gain or loss of a proton. CI is consered a ‘‘soft’’ ionization process because it generally resultsless fragmentation than EI. Therefore, even though CI cancrease analyte sensitivity, it often provides less structural inmation.

There are several types of mass analyzers including ion trmagnetic/electric sectors, single and triple quadrupoles, timeflight, Fourier-transform systems, and hybrid mass spectromeSome mass analyzers~i.e., ion trap, triple quadrupole! can per-form tandem mass spectrometry~MS/MS!. In MS/MS, an iontransition is monitored, where a specific ion is isolated~precursorion! and fragmented, followed by an isolation and monitoring oresulting fragment ion~product ion!. MS/MS transitions areunique for a given compound and result in lower backgrounoise and higher signal to noise ratios. Thus MS/MS techniqare favored in the analysis of complex matrices, such as wawater effluents.

GC/MS has been used for a variety of applications, includthe analysis of EDCs. GC/MS amenable EDCs include traditiocontaminants such as polycyclic aromatic hydrocarbons~i.e., ben-zo@a#pyrene! and pesticides~i.e., lindane!, as well as semivolatilenonpolar EDCs and PPCPs. While some EDCs and PPCPs caanalyzed directly, many require structural modification to mathem amenable to GC/MS. Compounds that are very polar, tmally labile, are acidic or basic, and/or have large molecuweights are not suited to the conditions present in a GC. Toprove performance, some compounds can be chemically modby derivatization. Derivatization alters the structure of the tarmolecule such that the molecule has lower polarity and/or grevolatility. However, these procedures can be time-consuminglabor intensive, and many of the chemical agents involvedtoxic and/or explosive.

Although GC/MS has many advantages over other analytmethods, the instrumentation is costly and complex to operatemaintain. Techniques such as SPME and derivatization have bdeveloped to reduce the amount of sample preparation requand increase the applicability of GC/MS, respectively. HowevGC/MS still needs a considerable amount of sample clean uppreparation in order to prevent costly maintenance and dimished results. These disadvantages have led to the increasedlarity of other types of analytical methods such as liquchromatography/mass spectrometry~LC/MS!.

PRACTICE PERIODICAL OF HAZARDOUS, TOXIC, AND RADIOACTIVE WASTE MANAGEMENT © ASCE / OCTOBER 2003 / 229

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Liquid chromatography~LC! has long been used for the analysof organic compounds in water. Typical LC systems consist osolvent pump, sample injector, analytical column, and a deteAn LC system allows for the separation of analytes in the liqphase such that time-resolved peaks relate to a compounds’tive affinity for the stationary and mobile phases. Detectionmost commonly accomplished using a non-specific detector,as ultraviolet or visible~UV/VIS! transmittance or absorbancLC methodology provides for the analysis of nearly all EDPPCP compounds including those that have extreme pola~e.g., acidic compounds!, are thermally labile~e.g., steroids!,and/or have relatively large molecular weights~e.g., macrolideantibiotics!. These types of compounds have generally beenyond the capabilities of GC based systems or required ticonsuming and labor intensive derivatizations. However, thLC systems~i.e., UV/VIS! lack the sensitivity and selectivity oGC/MS and therefore cannot be used for the detection of mEDCs/PPCPs at environmentally relevant concentrations. Mrecently, LC coupled with mass spectrometry~LC/MS! has be-come a powerful tool for the analysis of these contaminantwater. This combination allows for the determination of copounds that are not amenable to GC while providing the sensity and selectivity of a mass spectrometer.

Unlike GC/MS, the separation of analytes in LC/MS occursthe liquid phase. Thus, analytes reach the mass spectrometeliquid rather than as a gas. Since ionization in a GC/MS occvia the use of a filament at well below atmospheric pressursimilar type of ionization for LC/MS is not practical. Thereforionization in an LC/MS occurs in an entirely different mannThere are three main types of ionization used in LC/MS: elecspray ionization~ESI!, atmospheric pressure chemical ionizati~APCI!, and atmospheric pressure photoionization~APPI!.

ESI is accomplished by guiding the eluent from a LC systinto a capillary tube upon which a potential has been applied.eluent from the capillary is composed of fine droplets and, duthe electric potential applied to the capillary, the emerging drlets are charged. These droplets then encounter a heat sourcrapidly evaporates the mobile phase, thereby transferringcharge from the droplet to the analyte. In APCI, atmosphgases are ionized by a corona discharge and react with somolecules to form reagent ions. These reagent ions thenwith analyte molecules to form charged species. SimilarlyAPCI, an APPI source sprays the analyte into a heated regiovolatilization then analytes are bombarded with photons of atain energy that are emitted by an APPI lamp. This createslecular ions of the analytes while leaving the solvent unchargLike CI GC/MS all of these ionization processes are ‘‘soft’’ aresult in little fragmentation of the original compound.

As with GC/MS, there are a number of different mass sptrometers that can be coupled to a LC. Ion traps~Jeannot 2000!,single and triple quadrupoles~Snyder et al. 2001b; Ternes 2001!,TOF ~Hirsch et al. 2001!, and sector mass spectrometers~Snyderet al. 2001b! have all been used for the analysis of endocrdisruptors in water.

Although LC/MS has made the analysis of many compoueasier, it has several limitations. LC/MS systems are relativexpensive and analysts often need specialized training and erience to become proficient in their use. Perhaps the most trousome limitation is due to a phenomenon known as matrix spression. Due to the nature of ESI, compounds that elute togehave to compete for charge in the source. Thus, backgroun

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terferences, if present in large enough quantities, can alterresponse of target compounds. To compensate for this, reseaers have used the method of standard addition and/or isotopiclabeled surrogate and internal standards. However, few isotocally labeled EDC/PPCP standards are readily available andbe very expensive. APCI is generally not susceptible to matsuppression, but many compounds that ionize by ESI do notficiently ionize using APCI. APPI is a relatively new techniquthat has shown promise in ionizing a broad spectrum of copounds without matrix suppression and research efforts usingsource are ongoing.

Inductively Coupled Plasma Mass Spectrometry

The inductively coupled plasma mass spectrometer~ICP/MS! hastypically been used for elemental analysis of metals. ICP/MS ulizes a plasma, generally argon, as the ionization source anmass spectrometer to detect the ions produced. It can simuneously measure most elements in the periodic table and demine the concentrations of some elements at sub-ng/L levels.cause it employs a mass analyzer, it also has the abilitymeasuring isotopic ratios. This makes ICP/MS an excellent alytical tool for certain organic compounds after GC, LC, or iochromatography~IC! separation. Some modern ICP/MS systemare equipped with reaction or collision chambers that declusions that often cause interferences. For instance, isotopes of ar(40Ar) can form a dimer cluster to have an apparent molecuweight of 80 Daltons, which interferes with the analysis of thmost abundant selenium isotope (80Se). Using modern reaction orcollision chamber equipped ICP/MS systems, these clustersbe separated and interferences minimized. Likewise, this technogy provides for more sensitive analysis of previously difficuelements due to high background interferences, such as carnitrogen, and phosphorus. Future applications of ICP/MS systewill likely include the analysis of several EDC/PPCP compound

LC- and IC-ICP/MS applications are routinely used for detemining metal speciation~i.e., arsenic and chromium!. The metalspecies present is critical to both toxicity and water treatmestrategies~Edwards 1994; Manning et al. 2002!. LC-ICP/MS hasalso shown good sensitivity in the analysis of halogenated copounds such as bromate, at low detection levels and with hselectivity ~Salov et al. 1992; Diemer and Heumann 1997; Daet al. 2001!. By using isotopically labeled standards, it is possibto monitor and correct the rate of change and/or degradationtarget compounds. This methodology has been exploited inspeciation of Cr, Hg, and Sn compounds. Early experiments frour research group have shown that perchlorate and iodinacontrast media can be analyzed using LC-ICP/MS with detectlimits similar or superior to conventional methods. Further rsearch is underway to determine the applicability of LC- anIC-ICP/MS for various organohalides and organometallic compounds, many of which are classified as EDCs and PPCPs.

Of increasing interest in organometallic analyses are the ornotin compounds known to cause reproductive abnormalitiessome aquatic organisms~Bryan et al. 1988; Snyder et al. 2000b!.GC-ICP/MS has recently been shown to be an extremely sensiand selective approach to monitor various organotin compounin water ~Ruiz Encinar et al. 2002!. Likewise, other organomet-alic compounds would be amenable to GC-ICP/MS analygiven sufficient volatility or derivatization. GC-ICP/MS alsoholds promise in the analysis of halogenated disinfection byproucts from water treatment~Magnuson 1998!.

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Immunosorbent Techniques

Immunosorbent techniques have been used to monitor vaEDCs and PPCPs~Aherne et al. 1985; Snyder et al. 1999; Huaand Sedlak 2001; Fallon et al. 2002!. Enzyme linked immunosorbent assays~ELISAs! and radioimmunoassays~RIAs! have beenwidely used because they are inexpensive and very sensThese methods utilize antibodies that bind with high-specificitan analyte. In those techniques, sensitivity increases becausdioactivity or products of certain enzymatic reactions can be msured in very small amounts. This increased sensitivity has bextremely helpful to determine EDCs and PPCPs at ng/Llower levels in wastewater effluents and surface waters~Aherneet al. 1985; Fallon et al. 2002; Huang and Sedlak 2001!. Thereare two basic ELISA methodologies, one for detecting anti~direct ELISA! and the other for antibodies~indirect ELISA!.Radioisotopes are employed for the RIA technique instead ofzymes as antibody conjugates~Snyder et al. 1999!. Iodine-125 isthe most commonly used radioisotope used in RIAs, as mcompounds~such as hormones! can be readily iodinated withoudisrupting their specificity. Generally, RIA is a more sensittechnique than ELISA, yet is more costly and requires the hdling of radioactive materials.

Conclusions

It is apparent that EDCs and PPCPs have a broad range of piochemical characteristics. Therefore, no single analytical mewill be sufficient for comprehensive EDC/PPCP monitoring.integrated approach combining bioassays and instrumental ases allows for screening of many classes of EDCs/PPCPs. Mern mass spectrometers and genetically engineered cell assaexamples of new tools for the screening of these emergingtaminants. As analytical and bioanalytical technologies advathe ability to detect more xenobiotics at even lower concentions will certainly improve as well.

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