Techniques and Methods 5–B4

U.S. Department of the Interior

U.S. Geological Survey

Determination of Wastewater Compounds in Whole Water 

by Continuous Liquid–Liquid Extraction and Capillary-Column 

Gas Chromatography/Mass Spectrometry

Chapter 4

Section B, Methods of the National Water Quality Laboratory

Book 5, Laboratory Analysis

Prepared by the U.S. Geological Survey Office of Water Quality, National Water Quality Laboratory
Determination of Wastewater Compounds 

in Whole Water by Continuous Liquid–

Liquid Extraction and Capillary-Column 

Gas Chromatography/Mass Spectrometry

By Steven D. Zaugg, Steven G. Smith, and Michael P. Schroeder

Chapter 4

Section B, Methods of the National Water Quality Laboratory

Book 5, Laboratory Analysis

Techniques and Methods 5–B4

U.S. Department of the Interior

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

DIRK KEMPTHORNE, Secretary

U.S. Geological Survey

P. Patrick Leahy, Acting Director

U.S. Geological Survey, Reston, Virginia: 2006

Revised: 2007 

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Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Zaugg, S.D., Smith, S.G., and Schroeder, M.P., 2006, Determination of wastewater compounds in whole water by continuous liquid–liquid extraction and capillary-column gas chromatography/mass spectrometry: U.S. Geological Survey 

Techniques and Methods, book 5, chap. B4, 30 p. 
iii

Contents

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

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

Analytical Method..........................................................................................................................................2

 1. Scope and application ..................................................................................................................2

 2. Method summary...........................................................................................................................5

 3. Interferences..................................................................................................................................5

 4. Apparatus and instrumentation...................................................................................................5

 5. Reagents and consumable materials.........................................................................................6

 6. Standards........................................................................................................................................6

 7. Sample preparation.......................................................................................................................8

 8. Safety precautions and waste disposal.....................................................................................9

 9. Gas chromatograph/mass spectrometer performance ........................................................10

10. Instrument Calibration.................................................................................................................10

11. Quality assurance and quality control .....................................................................................14

12. Calculation of results...................................................................................................................15

Results and discussion of method validation..........................................................................................16

Summary and conclusions.........................................................................................................................19

References cited..........................................................................................................................................27

Figures

1. Continuous liquid–liquid extractors with solvent-dispersing frits mounted in a fume 

hood ................................................................................................................................................5

2. Manual integration of the extracted ion profile for the quantitation ion (mass-tocharge ratio 135) of 4-nonylphenol from the 2-nanogram-per-microliter calibration 

solution for the wastewater method .........................................................................................7

3. Boxplot showing results for analysis of 140 environmental whole-water samples ........26

Tables

1. Wastewater method compound names, endocrine-disrupting potential, log Kow, 

parameter/method codes, and possible compound uses......................................................3

2. Wastewater method compound retention time, quantitation ion, confirmation ions, 

surrogate compounds, and internal standard reference compound.................................11

3. Typical analytical sequence suggested for use with this method .....................................15

4. Wastewater method compounds detected in unfortified reagent-water, ground-water, 

and surface-water samples............................................................................................................17

5. Wastewater method mean bias and variability of spike-recovery data for eight 

replicates with compounds spiked at two concentrations that range from 0.5 to 80 

micrograms per liter in reagent-water (including calculated method detection 

limits), ground-water, and surface-water samples...............................................................20

6. Wastewater initial method detection limits calculated from the spike-recovery data 

reported in table 5 using the eight replicate reagent-water samples with compound 

concentrations that range from 0.5 to 80 micrograms per liter ..........................................24
iv

Conversion Factors

ISI to Inch/Pound

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

micrometer (μm) 0.00003937 inch (in.)

millimeter (mm) 0.03937 inch (in.)

meter (m) 39.37 inch (in)

Volume

liter (L) 0.2642 gallon (gal)

microliter (μL) 0.000000264 gallon (gal)

milliliter (mL) 0.000264 gallon (gal)

milliliter per minute (mL/min) 0.0338 ounce per minute

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

kilogram (kg) 2.205 pound avoirdupois (lb)

microgram (μg) 0.00000003527 ounce, avoirdupois (oz)

milligram (mg) 0.00003527 ounce, avoirdupois (oz)

Pressure

kilopascal (kPa) 0.1450 pound-force per inch (lbf/in) 

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

°F=(1.8×°C)+32

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

°C=(°F-32)/1.8

The following units of measurement are used in this report:

in. inch

L/min liters per minute

min minute

mg/mL milligram per milliliter

μg/L microgram per liter

ng/μL nanogram per microliter

The following abbreviations and symbols are used in this report:

AHTN acetyl-hexamethyl-tetrahydronaphthalene

AP alkylphenol

APEC alkylphenol ethoxycarboxylate

APEO alkylphenol polyethoxylate

ASE accelerated solvent extraction

ASR analytical services request

CAS Chemical Abstracts Service
v

CCV continuing calibration verification solution

CLLE continuous liquid–liquid extraction

DCM dichloromethane

DEET N,N-Diethyl-meta-toluamide

DEHP diethylhexyl phthalate

DEP diethyl phthalate

GC gas chromatograph or gas chromatography

GCC glass bottle, amber

GC/MS gas chromatography/mass spectrometry

HHCB hexahydrohexamethyl-cyclopentabenzopyran

IRL interim reporting level

LT–MDL long-term method detection level

MDL method detection limit

MRL minimum reporting level

MS mass spectrometry

m/z mass-to-charge ratio

NP nonylphenol

NPEO nonylphenol ethoxylate

NWQL National Water Quality Laboratory

N/A not applicable

OPEO octylphenol ethoxylate

PAH polycyclic aromatic hydrocarbon

PBDE polybrominated diphenylether

PCB polychlorinated biphenyl

PSDVB polystyrene-divinylbenzene

QA/QC quality assurance and quality control

RRF relative response factor

RRT relative retention time

RSD relative standard deviation

SPE solid-phase extraction

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey ≤ less than or equal to > greater than % percent ± plus or minus
Abstract

A method for the determination of 69 compounds typically found in domestic and industrial wastewater is described. 

The method was developed in response to increasing concern 

over the impact of endocrine-disrupting chemicals on aquatic 

organisms in wastewater. This method also is useful for 

evaluating the effects of combined sanitary and storm-sewer 

overflow on the water quality of urban streams. The method 

focuses on the determination of compounds that are indicators of wastewater or have endocrine-disrupting potential. 

These compounds include the alkylphenol ethoxylate nonionic 

surfactants, food additives, fragrances, antioxidants, flame 

retardants, plasticizers, industrial solvents, disinfectants, 

fecal sterols, polycyclic aromatic hydrocarbons, and high-use 

domestic pesticides.

 Wastewater compounds in whole-water samples were 

extracted using continuous liquid–liquid extractors and 

methylene chloride solvent, and then determined by capillarycolumn gas chromatography/mass spectrometry. Recoveries 

in reagent-water samples fortified at 0.5 microgram per liter 

averaged 72 percent ± 8 percent relative standard deviation. 

The concentration of 21 compounds is always reported as 

estimated because method recovery was less than 60 percent, 

variability was greater than 25 percent relative standard deviation, or standard reference compounds were prepared from 

technical mixtures. Initial method detection limits averaged 

0.18 microgram per liter. Samples were preserved by adding 

60 grams of sodium chloride and stored at 4 degrees Celsius. 

The laboratory established a sample holding-time limit prior to 

sample extraction of 14 days from the date of collection.

Introduction

The United States faces the difficult challenge of effectively managing wastewater owing to limited water supply, 

increasing population, aging wastewater-treatment facilities and infrastructure, and emerging contaminants in the 

environment. Specific analytical methods are available (U.S. 

Environmental Protection Agency, 1995) to monitor regulated 

compounds in wastewater or the discharge from wastewatertreatment facilities. However, several unregulated compounds 

known to be toxic to aquatic life, such as nonylphenol ethoxylates (NPEOs), that are on the U.S. Environmental Protection 

Agency (USEPA) Toxic Substance Control Act Priority Testing List (U.S. Environmental Protection Agency, 1996), lack 

USEPA-approved analytical methods. To provide an analytical 

method for many unregulated compounds with known toxicity 

or endocrine-disrupting potential (U.S. Environmental Protection Agency, 1997a), the U.S. Geological Survey (USGS) 

National Water Quality Laboratory (NWQL) developed a 

method in 2001 to detect wastewater compounds in filtered 

water. This method uses polystyrene-divinylbenzene (PSDVB) 

solid-phase extraction (SPE) coupled with capillary-column 

gas chromatography/mass spectrometry (GC/MS) (Zaugg and 

others, 2002). This SPE method is appropriate for the analysis 

of wastewater compounds in filtered water, but in some cases 

(particularly surface-water samples) the analysis of wholewater (unfiltered) samples is desirable.

This report describes a method for determining a 

broad range of wastewater compounds in natural wholewater samples that complements the existing NWQL SPE 

method developed for filtered water. Whole-water samples 

are extracted by using continuous liquid–liquid extraction 

(CLLE) with methylene chloride (DCM) solvent, and compounds are determined by capillary-column GC/MS. This 

method requires less solvent and solvent exposure, and has 

far fewer emulsion problems than separatory-funnel methods 

currently (2006) in use at the NWQL. The method is similar 

to other methods that use CLLE and GC/MS for the determination of 4-tert-octylphenol, 4-nonylphenol, and bisphenol A 

in surface water (Jin and others, 2004), volatile compounds 

in wine (Castro and others, 2004), and polychlorinated 

biphenyls in blood plasma lipids (Wingfors and others, 

2005). Other methods of the USGS (that do not use CLLE) 

have been used for the determination of organic substances 

in water and have been described previously by Wershaw 

and others (1987) and by Fishman (1993). However, a CLLE 

Determination of Wastewater Compounds in 

Whole Water by Continuous Liquid–Liquid Extraction 

and Capillary-Column Gas Chromatography/

Mass Spectrometry 

By Steven D. Zaugg, Steven G. Smith, and Michael P. Schroeder
 Determination of Wastewater Compounds in Whole Water

method at the NWQL to extract organophosphate pesticides 

from water has been approved for use since 2003 (Jha and 

Wydoski, 2003). 

The CLLE wastewater method described in this report 

was implemented at the NWQL in October 1998 on a custom 

basis (Brown and others, 1999) as NWQL Laboratory Code 

8033. A comparison of results for more than 30 environmental 

samples between the whole-water CLLE and the filteredwater SPE methods demonstrated that concentrations for some 

of the hydrophobic (low water-soluble) compounds having 

a log octanol-water partition coefficient (log Kow) greater 

than 3 were as much as 400 percent less when determined by 

SPE, presumably because of losses during sample filtration; 

whereas, results for hydrophilic compounds (log Kow less 

than 3) were comparable. The reproducibility of the method 

as evaluated using 47 other environmental samples had correlation coefficients greater than 0.95 for most of the compounds (Wilkison and others, 2002). This CLLE method has 

been used to describe urban sanitary-sewer and storm-sewer 

problems (Wilkison and others, 2002), as well as emerging 

contaminants in U.S. streams and runoff from concentrated 

animal feedlots for the USGS’s Toxic Substances Hydrology 

Program (Kolpin and others, 2002). Results from using the 

method for 8 years (1998–2005) has demonstrated the capability for identifying anthropogenic contaminants over a wide 

range of sample matrices. The method officially was approved 

and implemented at the NWQL July 1, 2006, as analytical 

method O-1433-06.

This report provides a detailed description of all aspects 

of the method, including the equipment, reagents, sampling 

protocol, instrument calibration, and procedure required for 

sample analysis. Method performance (bias and variability) 

and estimated method detection limits for 69 compounds are 

presented.

The scope of this report includes determination of method 

performance in reagent-water, ground-water, and surfacewater samples. Method performance was determined at two 

appropriate concentrations for each compound (0.50 and 4.0 

μg/L for most compounds) in each water type. Method detection limits were determined according to an accepted statistical 

procedure (U.S. Environmental Protection Agency, 1997b).

Analytical Method

Organic Compounds and Parameter Codes: Wastewater 

compounds, whole water, gas chromatography/mass spectrometry, O-4433-06 (see table 1)

1. Scope and Application

This method is suitable for the determination of microgram-per-liter concentrations of compounds in whole wastewater and environmental water samples. The method is applicable to compounds that are efficiently partitioned from the 

water phase into the DCM organic phase, and are sufficiently 

volatile and thermally stable for gas chromatography.

The method includes many compounds that typically are 

associated with industrial and household wastewater (Paxéus 

and others, 1992), as well as some that are known or suspected 

endocrine-disrupting compounds (table 1).

The alkylphenol polyethoxylates (APEOs) are branchedchain nonionic surfactants that consist of the nonylphenol 

polyethoxylates (NPEOs) and the octylphenol polyethoxylates 

(OPEOs). The APEO surfactants generally contain up to about 

20 ethoxy-units (Barber and others, 2000). The alkylphenol 

mono-ethoxylates (APEO1) are represented in the method by 

OPEO1 and NPEO1 (total, resulting from addition of several 

isomers). The alkylphenol di-ethoxylates are represented in the 

method by OPEO2 and NPEO2 (total) (see table 1). The degradation of APEOs in the environment can result in the formation of alkylphenols (APs) and alkylphenol ethoxycarboxylates (APEC). The APs are represented in the method (see table 

1) by 4-cumylphenol, 4-n-octylphenol, 4-nonylphenol (total), 

and 4-tert-octylphenol. The APEOs also are degraded during 

sewage treatment (Geiger and others, 1984), and sewagetreatment-plant effluents can be major point sources for the 

APs and APECs. The APECs are too polar to be determined 

by this method because they thermally degrade on the GC. The 

volatility and availability of reference standard APEOs limit 

the method to the determination of OPEO1, OPEO2, NPEO1 

(total), and NPEO2 (total), even though APEOs typically 

might contain up to about 20 ethoxy-units. However, most 

of the APEOs present in the environment after degradation 

contain less than four ethoxy-units. All of these compounds 

are known for their estrogenic activity in aquatic organisms 

(Jobling and Sumpter, 1993). A comprehensive analysis (Barber and others, 2000) of this family of compounds (APEOs 

and their degradates) is needed to understand their fate and 

transport in the environment. 

Caffeine is an important indicator for surface-water contamination; however, it is not conservative in the environment 

because of rapid degradation by bacteria. Coprostanol has long 

been a traditional indicator of sewage contamination and is 

persistent in the environment under anaerobic conditions but is 

degraded by aerobic bacteria (Shigenaka and Price, 1988). Triclosan, an antimicrobial agent used in several common household products, and the synthetic polycyclic musk fragrance 

compounds acetyl-hexamethyl-tetrahydro-naphthalene (AHTN) 

and hexahydrohexamethyl-cyclopentabenzopyran (HHCB) are 

persistent in the environment and have been frequently detected 

during the custom implementation of this method. The presence 

and fate, as well as uses for the APEOs and other wastewater 

indicator compounds (caffeine, sterols, flame retardants, triclosan, AHTN, and HHCB), are described in more detail in Zaugg 

and others (2002). 

The widespread detection of DEET by this method prior 

to method approval (1998–2002) also has been documented 

(Sandstrom and others, 2005). The polybrominated diphenylethers (PBDE), as well as other polybrominated flame-retarding 

chemicals, have the potential to become a widespread, persistent 
Table 1. Wastewater method compound names, endocrine-disrupting potential, log Kow, parameter/method codes, and possible 

compound uses. 

[EDP, endocrine-disrupting potential; K, known; S, suspected; CAS, Chemical Abstract Service; F, fungicide; H, herbicide; I, insecticide; GUP, general use 

pesticide; FR, flame retardant; PAH, polycyclic aromatic hydrocarbon; Kow, octanol-water partition coefficient; WW, wastewater; Manuf, manufacturing; %, 

percent; >, greater than; CP, combustion product; USEPA, U.S. Environmental Protection Agency; UV, ultraviolet; --, no data]

Compound name EDP1 Log 

Kow2

CAS 

number3

Parameter/

method 

codes4

Possible compound uses or sources5

1,4-Dichlorobenzene6 S 3.28 106-46-7 34571Y Moth repellent, fumigant, deodorant

1-Methylnaphthalene -- 3.72 90-12-0 81696Z 2–5% of gasoline, diesel fuel, or crude oil

2,2',4,4'-Tetrabromodiphenyl ether -- 6.77 5436-43-1 63147A Widely used brominated flame retardant

2,6-Dimethylnaphthalene -- 4.26 581-42-0 62805Z Present in diesel/kerosene (trace in gasoline)

2-Methylnaphthalene -- 3.72 91-57-6 30194Z 2–5% of gasoline, diesel fuel, or crude oil

3,4-Dichlorophenyl isocyanate -- 3.88 102-36-3 63145A Degradate of diuron, a noncrop herbicide

3beta-Coprostanol -- 8.82 360-68-9 62806Z Carnivore fecal indicator

3-Methyl-1H-indole (skatol) -- 2.60 83-34-1 62807Z Fragrance, stench in feces and coal tar

3-tert-Butyl-4-hydroxyanisole (BHA) K 3.50 25013-16-5 61702Z Antioxidant, general preservative

4-Cumylphenol K 4.12 599-64-4 62808Z Nonionic detergent or metabolite

4-n-Octylphenol K 5.50 1806-26-4 62809Z Nonionic detergent or metabolite

4-Nonylphenol (total, NP) K 5.92 84852-15-3 62829Z Nonionic detergent or metabolite

4-tert-Octylphenol K 5.28 140-66-9 62810Z Nonionic detergent or metabolite

5-Methyl-1H-benzotriazole -- 1.71 136-85-6 61944Z Antioxidant in antifreeze and deicers

Acetophenone -- 1.67 98-86-2 62811Z Fragrance in detergent and tobacco, flavor in beverages

Acetyl-hexamethyl-tetrahydro-naphthalene 

(AHTN, Tonalide) -- 6.35 21145-77-7 62812Z Musk fragrance, persistent, widespread in ground 

water, concern for bioaccumulation and toxicity

Anthracene6 -- 4.35 120-12-7 34220Z Component of tar, diesel, or crude oil, CP

Anthraquinone -- 3.34 84-65-1 62813Z Manuf dye/textiles, seed treatment, bird repellent

Atrazine6 K 2.82 1912-24-9 39630C Selective triazine herbicide

Benzo[a]pyrene6

 K 6.11 50-32-8 34247Z Regulated PAH, used in cancer research, CP

Benzophenone S 3.15 119-61-9 62814Z Fixative for perfumes and soaps

beta-Sitosterol -- 9.65 83-46-5 62815Z Plant sterol

beta-Stigmastanol -- 9.73 19466-47-8 61948Z Herbivore fecal indicator (digestion of sitosterol)

Bisphenol A K 3.64 80-05-7 62816Z Manuf polycarbonate resins, antioxidant, FR

Bromacil -- 1.68 314-40-9 30234Z H (GUP), >80% noncrop usage on grass/brush

Bromoform -- 1.79 75-25-2 32104Y WW ozination byproduct, military/explosives

Caffeine -- 0.16 58-08-2 81436Z Beverages, diuretic, very mobile/biodegradable

Camphor -- 3.04 76-22-2 62817Z Flavor, odorant, ointments

Carbaryl K 2.35 63-25-2 39750Z I, crop and garden uses, low persistence

Carbazole -- 3.23 86-74-8 77571Z I, Manuf dyes, explosives, and lubricants

Chlorpyrifos K 4.66 2921-88-2 38932Z I, domestic pest and termite control (domestic use 

restricted as of 2001)

Cholesterol -- 8.74 57-88-5 62818Z Often a fecal indicator, also a plant sterol

Cotinine -- 0.34 486-56-6 61945Z Primary nicotine metabolite

Diazinon6

 K 3.86 333-41-5 39570Y I, > 40% nonagricultural usage, ants, flies

Dichlorvos S 0.60 62-73-7 30218Z I, pet collars; naled or trichlofon degradate 

Diethyl phthalate (DEP) 6 K 2.82 84-66-2 34336B Plasticizer for polymers and resins

Diethylhexyl phthalate (DEHP)6 K 8.39 117-81-7 39100C Plasticizer for polymers and resins, pesticide inert

d-Limonene -- 4.83 5989-27-5 62819Z F, antimicrobial, antiviral, fragrance in aerosols

Analytical Method   
   Determination of Wastewater Compounds in Whole Water

Table 1. Wastewater method compound names, endocrine-disrupting potential, log Kow, parameter/method codes, and possible 

compound uses—Continued.

[EDP, endocrine-disrupting potential; K, known; S, suspected; CAS, Chemical Abstract Service; F, fungicide; H, herbicide; I, insecticide; GUP, general use 

pesticide; FR, flame retardant; PAH, polycyclic aromatic hydrocarbon; Kow, octanol-water partition coefficient; WW, wastewater; Manuf, manufacturing; %, 

percent; >, greater than; CP, combustion product; USEPA, U.S. Environmental Protection Agency; UV, ultraviolet; --, no data]

Compound name EDP1 Log 

Kow2

CAS 

 number3

Parameter/

method 

codes4

Possible compound uses or sources5

Fluoranthene6

-- 4.93 206-44-0 34376Z Component of coal tar and asphalt (only traces in 

gasoline or diesel fuel), CP

Hexahydrohexamethyl-cyclopentabenzopyran (HHCB, Galaxolide) -- 6.26 1222-05-5 62823Z Musk fragrance, persistent, widespread in ground 

water, concern for bioaccumulation and toxicity

Indole -- 2.05 120-72-9 62824Z Pesticide inert ingredient, fragrance in coffee

Isoborneol -- 2.85 124-76-5 62825Z Fragrance in perfumery, in disinfectants

Isophorone6 -- 2.62 78-59-1 34408Z Solvent for lacquer, plastic, oil, silicon, resin

Isopropylbenzene (cumene) -- 3.45 98-82-8 77223Y Manuf phenol/acetone, fuels and paint thinner

Isoquinoline -- 2.14 119-65-3 62826Z Flavors and fragrances

Menthol -- 3.38 89-78-1 62827Z Cigarettes, cough drops, liniment, mouthwash

Metalaxyl -- 1.70 57837-19-1 04254Z H, F (GUP), mildew, blight, pathogens, golf/turf

Methyl salicylate -- 2.60 119-36-8 62828Z Liniment, food, beverage, UV-absorbing lotion

Metolachlor6 -- 3.24 51218-45-2 82612Z H (GUP), indicator of agricultural drainage

N,N-Diethyl-meta-toluamide (DEET) -- 2.26 134-62-3 61947Z I, urban uses, mosquito repellent

Naphthalene6

 -- 3.17 91-20-3 34696Y Fumigant, moth repellent, major component (about 

10%) of gasoline

Nonylphenol, diethoxy- (total, NPEO2) K -- 26027-38-2 61703Z Nonionic detergent

Nonylphenol, monoethoxy- (total, NPEO1) K -- 104-35-8 61704A Nonionic detergent

Octylphenol, diethoxy-(OPEO2) K -- 26636-32-8 61705Z Nonionic detergent

Octylphenol, monoethoxy-(OPEO1) K -- 26636-32-8 61706Z Nonionic detergent

p-Cresol -- 2.06 106-44-5 77146Z Wood preservative

Pentachlorophenol6 S 4.74 87-86-5 39032Z H, F, wood preservative, termite control

Phenanthrene6

 -- 4.35 85-01-8 34461Z Manuf explosives, component of tar, diesel fuel,

or crude oil, CP

Phenol6

 -- 1.51 108-95-2 34694Z Disinfectant, Manuf several products, leachate

Prometon6 -- 3.57 1610-18-0 39056Z H (noncrop only), applied prior to blacktop

Pyrene6

 -- 4.93 129-00-0 34469Z Component of coal tar and asphalt (only traces in 

gasoline or diesel fuel), CP

Tetrachloroethylene -- 2.97 127-18-4 34475Y Solvent, degreaser, veterinary anthelmintic

Tri(2-butoxyethyl) phosphate -- 3.00 78-51-3 62830Z Flame retardant

Tri(2-chloroethyl) phosphate S 1.63 115-96-8 62831Z Plasticizer, flame retardant

Tri(dichloroisopropyl) phosphate S 3.65 13674-87-8 61707Z Flame retardant

Tributyl phosphate -- 3.82 126-73-8 62832Z Antifoaming agent, flame retardant

Triclosan S 4.66 3380-34-5 61708Z Disinfectant, antimicrobial (concern for acquired 

microbial resistance)

Triethyl citrate (ethyl citrate) -- 0.33 77-93-0 62833Z Cosmetics, pharmaceuticals

Triphenyl phosphate -- 4.70 115-86-6 62834Z Plasticizer, resin, wax, finish, roofing paper, FR

1

Colburn and others (2000), Our Stolen Future website (http://www.ourstolenfuture.org/Basics/chemlist.htm). 

2

Log Kow calculated using USEPA’s exposure assessment tools and models (EPI-suite software, WSKOWWINTM version 1.40; U.S. Environmental Protection 

Agency, 2005).

3

CAS Registry Number® is a Registered Trademark of the American Chemical Society. CAS recommends the verification of the CASRNs through CAS Client 

Services. See www.cas.org

 4Parameter codes define sample constituent variables linked to compound analytical results stored in the National Water Information System data base.

5

ChemFinder Webserver (2006); National Toxicology Program (2006); National Institute of Standards and Technology (2006); Spectrum Laboratories, Inc. 

(2006); HealthCentral.com (2006); EXtension TOXicology NETwork (2006).

6

Compound determined by at least one other whole-water method at the National Water Quality Laboratory.
environmental problem similar to the PCBs (de Boer and others, 

1998) because of their toxicity and extensive use in many common 

products. These compounds are present at low concentrations in 

the environment (nearly less than the detection capabilities of this 

method) because of their low water solubility. However, the most 

common PBDE detected in the environment (2,2', 4,4'-tetrabromodiphenyl ether) has been detected in a few samples using this 

method.

A complementary method to detect wastewater compounds in sediments and suspended particles using accelerated 

solvent extraction (ASE) also has been developed (Burkhardt 

and others, 2006). The ASE sediment method has been developed primarily to detect all the method compounds except 

5-methyl-1H-benzotriazole, caffeine, cotinine, dichlorvos, and 

triethyl citrate (log Kow less than about 2, see table 1).

2. Method Summary

Whole-water samples are collected in 

1-L amber glass pesticide bottles according 

to standard USGS sampling procedures for 

the determination of trace organic compounds 

(U.S. Geological Survey, variously dated) and 

shipped at 4°C to the NWQL. The whole-water 

samples (1-L) are immediately treated with the 

addition of 60 ± 10 g NaCl and stored at 4oC. 

Samples are transferred to CLLE vessels before 

the expiration of the 2-week holding time and 

extracted with DCM at ambient pH for 3 hours 

and initially at pH 2 for an additional 3 hours. 

Next, the DCM extract is evaporated by using 

a gentle stream of nitrogen to a final volume 

of 400 μL. The extracts are stored in a freezer 

at ≤ −10oC until compounds are determined by 

capillary-column GC/MS.

3. Interferences

Organic compounds that have gas chromatographic retention times and characteristic 

ions with a mass identical to those of the compounds of interest might interfere. There might 

be unknown compounds that interfere because 

of the complex nature of wastewater.

Sample-collection protocols and cleaning 

procedures for field equipment (U.S. Geological Survey, variously dated) must be followed 

to reduce the possibility of interferences and to 

minimize potential contamination. Many method 

compounds are widespread in the environment 

and have been detected in laboratory reagentwater-blank samples. For example, diethylhexyl 

phthalate might contribute to environmental contamination. Samples and collection equipment 

that are handled improperly also might become 

contaminated with soaps, caffeine, and fragrances. Precautions 

are necessary to avoid and minimize the potential for contamination during sample collection (see section 7.1) because some 

method compounds are contained in commonly used products. 

4. Apparatus and Instrumentation

4.1 Continuous liquid–liquid extractors (custom made by 

Allen Scientific Glass, Inc. and not commercially available) 

include a condenser, a coarse frit at the head of the water 

sample for microdispersion of methylene chloride, and a 1-L 

extraction chamber to hold the sample (see fig. 1). A heating mantle is placed over a 25-mL receiver to gently boil the 

methylene chloride solvent. The solvent vapor is condensed 

and returns to the frit to continuously extract the sample. During the CLLE process the extracted sample is concentrated in 

CONDENSER

SOLVENT

RETURN

ARM

FRIT


MILLILITER

RECEIVER L
LITER

SAMPLE

CHAMBER

Figure 1. Continuous liquid–liquid extractors with solvent-dispersing frits mounted 

in a fume hood.

Analytical Method   
 Determination of Wastewater Compounds in Whole Water

the 25-mL receiver (see Section 7, Sample Preparation) for 

details.

4.2 Variac variable transformer; 0 to 140 volts or equivalent.

4.3 Heating mantle; tube or teardrop-shaped.

4.4 Carboy or equivalent waste container resistant to chlorinated solvents.

4.5 Kuderna-Danish receivers (concentrator tubes); 25-mL 

graduated, Kontes part number 570081-2526 or equivalent.

4.6 Analytical balances, capable of accurately weighing 1,500 

± 1 g for samples and 50 ± 0.01 mg for standard preparation.

4.7 Nitrogen evaporative concentrator; Organomation 

N-Evap or equivalent.

4.8 Micropipettes; 50-, 100-, and 200-μL fixed-volume and 

variable-volume micropipettes with disposable glass bores; 

VWR Scientific or equivalent.

4.9 Glass syringes; 10-, 50-, 100-, 250-, and 500-μL volume.

4.10 Funnels; stainless steel, baked 8 hours at 450°C.

4.11 Fused-silica capillary column; any column that provides adequate resolution, capacity, accuracy, and precision. 

A 30-m by 0.25-mm inside diameter fused-silica capillary 

column coated with a 0.50-μm bonded film of 5-percent 

polyphenylmethylsilicone; J&W Scientific DB-5MS column 

or equivalent.

4.12 GC/MS bench-top system; Agilent Technologies, 

Model 5973 or equivalent.

4.12.1 Recommended GC conditions; oven, 40°C (hold 3 

minutes), then ramp at 4°C/min to 100°C, and 9°C/min to 

320°C; injection port, 290°C with electronic pressure control 

set for a constant flow of helium carrier gas of 1 mL/min; 

injection volume, 2 μL, splitless injection.

4.12.2 Recommended MS conditions; source, 200°C; 

quadrupole analyzer, 100°C; interface, held at 250°C and 

programmed at 9°C/min to 290°C when the oven temperature 

surpasses 250°C; electron-impact ionization mode (70 electron 

volts), full-scan mode from 45 to 450 atomic mass units in 0.5 

second. 

5. Reagents and Consumable Materials

5.1 Carrier gas; helium (99.999 percent).

5.2 Nitrogen gas; for evaporation, ultrapure.

5.3 Sodium chloride; reagent grade, baked 8 hours at 450°C, 

VWR or equivalent.

5.4 Glass bottles; amber, 1,000-mL, 33-mm neck, baked at 

450°C for 2 hours, fitted with Teflon-lined screw caps; NWQL 

GCC or equivalent.

5.5 Solvents; isopropyl alcohol and acetone, B&J Brand, 

pesticide grade or equivalent.

5.5.1 Dichloromethane, 20-L pressurized solvent keg, B&J, 

catalog number NS300-20.

5.6 Organic contaminant-free water; Solution 2000 reagent 

water; prepared by Solution 2000 purification system or 

equivalent.

5.7 Pasteur pipettes; disposable, baked 8 hours at 450°C.

5.8 Vial; 2-mL, amber glass, with Teflon-lined screw caps, 

Supelco Inc. or equivalent.

5.9 Boiling chips; four-mesh granule sizes, baked 8 hours at 

450°C.

5.10 Sulfuric acid; reagent-grade, 25 percent, diluted in 

Solution 2000 water.

6. Standards

6.1 Stock standard solutions at 10,000 ng/μL. Obtain method 

compounds and surrogate compounds at greater than 99-

percent purity from commercial vendors. Prepare stock standard solutions of each individual compound at about 10,000 

ng/μL (10 mg/mL) by accurately weighing, to the nearest 

0.002 mg, 20 mg of the neat material in a 2-mL volumetric 

flask and dilute to volume with DCM. Three of the method 

compounds (4-NP, OPEO1, and OPEO2) are only available 

in technical mixtures. For the technical grade nonylphenol 

(NP) mixture (1-NP and 4-NP) and the Igepal 210 (Dupont, 

Inc.) mixture (OPEO1 and OPEO2), the final concentration 

of the stock standard solutions is calculated on the basis of 

the percentage contribution of each compound to the total ion 

chromatograms of the technical mixtures. These compounds 

are identified in the total ion chromatogram by referring to 

their characteristic ions and relative retention times (see table 

2 in Section 10, Instrument Calibration).

The contribution of the 4-nonylphenols (total) in the 

NP technical mixture is determined by manually integrating the sum of the peaks within the expected retention time 

window (fig. 2) for the quanitation ion (m/z 135, see table 

2, section 10). Also, note how the confirmation ion profiles 

(m/z 220 and 107) must coincide in a similar pattern with 

ion 135. The ortho-NPs elute prior to the 4-NPs and are not 

determined in this method because their contribution to the 
total ion chromatogram is minimal (less than 7 percent). In 

general, it is desirable, for the purposes of making dilutions 

of the mixed standard solution, to prepare a stock standard 

solution of the 4-NP isomers (total), which is 16 times the 

concentration of the stock standard solutions of the singlecomponent compounds in the method. To prepare this stock 

standard solution, calculate the necessary amount of the 

technical mixture needed (about 180 mg/mL). 

The Igepal 210 technical mixture is mainly composed of 

single components of OPEO1 and OPEO2 in a ratio of about 

10 to 1, respectively. A convenient concentration of a stock 

standard solution for OPEO1 is prepared at 4 times the concentration of the single-component compounds, or 40 mg/mL. 

This concentration also provides enough material for calibrating OPEO2 (about 4 mg/mL) from the same stock standard 

solution. The preparation of the OPEO1 and OPEO2 stock 

standard solution, thus, generally requires about 45 mg/mL 

(as calculated) of the Igepal 210 technical mixture. Reference standards for the NPEO1s and NPEO2s were obtained 

as separate mixtures from the Aldrich chemical company by a 

custom synthesis requested by the U.S. Environmental Protection Agency.

6.2 Intermediate method compound standard solution at 100 

ng/μL. Prepare a mixed stock standard solution that contains 

each intermediate method compound standard solution at 100 

ng/μL (not including surrogate compounds). Use an adjustable 

100-μL dispenser and a 10-mL volumetric flask to prepare this 

intermediate method compound standard solution and dilute 

with DCM. 

6.3 Surrogate spiking solution at 20 ng/μL. Combine 100 μL 

of stock standard solution at 10,000 ng/μL for each surrogate 

compound listed in table 2 (see Section 10, Instrument Calibration) in a 50-mL volumetric flask and dilute with isopropyl 

alcohol. Add 100 μL of the 20-ng/μL intermediate method 

compound standard solution to a 1-L sample to obtain a surrogate spiking solution of 2.0 μg/L. A surrogate concentration 

of 5.0 ng/μL is expected in a 400-μL extract if 100 percent 

of the surrogate is recovered through the sample-preparation 

procedure.

6.4 Mixed surrogate and method compounds solution at 50 

ng/μL. Add 5.0 mL of the 100-ng/μL intermediate method 

compound standard solution (see section 6.2) to a 10-mL 

flask. Add 50 μL of each of the stock standard solutions and 

surrogate standard solutions at 10,000 ng/μL (see section 6.1) 

and dilute with DCM. This mixture is used to prepare the 

calibration solutions (section 6.7).

6.5 Spike solution at 20 ng/μL. Dilute 2.0 mL of the intermediate method compound standard solution at 100 ng/μL 

(see section 6.2) in a 10-mL volumetric flask with isopropyl 

alcohol. Add 100 μL to a 1-L sample to obtain a compound 

concentration of 2.0 μg/L. A concentration of 5.0 ng/μL is 

expected in a 400-μL extract if 100 percent of the spike is 

recovered.

6.6 PAH procedural internal standard solution at 100 ng/μL. 

The internal standards (see table 2, Section 10, Instrument 

Calibration) are obtained from Supelco (or equivalent) in a 

mixture at 2,000 ng/μL. Add 2.5 mL of this mixture to a 50-

mL flask and dilute with DCM. Note that 20 μL of the polycyclic aromatic hydrocarbon (PAH) procedural internal standard 

solution at 100 ng/μL in a 400-μL extract is equivalent to a 

concentration of 5 ng/μL.

6.7 Calibration solutions. Prepare a series of calibration 

solutions in DCM that contain all of the method and surrogate 

compounds at concentrations for most compounds ranging 

from 0.05 to 40.0 ng/μL (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 

10, 20, 40 ng/μL). The concentration of single-component 

compounds in the calibration mixture that respond poorly by 

GC/MS (bromacil, cholesterol, cotinine, 3beta-coprostanol, 

beta-stigmastanol, beta-stitosterol, 5-methyl-1H-benzotriazole, and pentachlorophenol) is 4 times that of the other singlecomponent compounds. The concentration of the multicomponent compounds in the calibration mixture, NP (total), NPEO1 

(total), and NPEO2 (total), is 18 times, 16 times, and 16 times, 

respectively, that of the single-component compounds in the 

calibration mixture. The concentration of the PAH procedural 

internal standard solution in the calibration solutions is kept 

constant at 5.0 ng/μL. Prepare these calibration solutions by 

adding the appropriate volumes of the mixed surrogate and 

method compounds solution at 50 ng/μL and the PAH procedural internal standard solution at 100 ng/μL into volumetric 

flasks and diluting to volume with DCM.

Ion 135.00

Ion 220.00

Ion 107.00

21.8 22 22.2 22.4 22.6 22.8 23 23.2 23.4 23.6

TIME, IN MINUTES

ION ABUNDANCE, COUNTS

30,000

20,000

10,000

0

3,000

2,000

1,000

0

15,000

10,000

5,000

0

Figure 2. Manual integration of the extracted ion profile for 

the quantitation ion (mass-to-charge ratio 135) of 4-nonylphenol 

from the 2- nanogram-per-microliter calibration solution for the 

wastewater method (from Zaugg and others, 2002).

Analytical Method 
 Determination of Wastewater Compounds in Whole Water

7. Sample Preparation

7.1 Collect whole-water samples in 1-L amber glass pesticide bottles (NWQL type GCC) before shipping at 4o

C to the 

NWQL, preferably by express overnight mail. Fill the bottle 

to the neck, and leave sufficient headspace (about 30 – 40 

mL) for the addition of 60 g NaCl upon arrival at the NWQL. 

Field-sampling procedures need to follow those typically used 

to collect samples for trace organic compound determinations 

(Ward and Harr, 1990; Wilde, April 2004; Wilde and others, 

April 2004; U.S. Geological Survey, variously dated) with specific instructions for the collection of wastewater compounds 

(Wilde and others, section 5.6.1.F, April 2004). 

CAUTION: Some of the compounds that are determined by 

this method are found in commonly used products, such as coffee, tea, cola, soap, and insecticide repellent. Project personnel 

need to be careful to avoid potential contamination of samples 

from such sources by avoiding consumption or contact with 

these materials immediately prior to and during sampling 

procedures. Limit or avoid contact with any fragranced materials, including colognes, scented-detergents, shampoos, and 

conditioners. If samples are suspected of containing untreated 

sewage or biohazards, special shipping, handling, and processing requirements need to be followed (U.S. Geological Survey, 

variously dated) prior to shipping. Clean the outside of the 

bottle thoroughly with isopropyl alcohol and secure the lid 

tightly before wrapping in four high-capacity 3M sorbent pads 

(Lab Safety and Supply, Janesville, Wis., catalog number 2BD26577). Finally, enclose the wrapped bottle in three resealable 

plastic bags (two bags are necessary and one bag is precautionary).

The probability of sample contamination with compounds 

determined by this method is higher than for other NWQL 

methods. For this reason, routinely prepare field blanks for 

analysis by using EMD Chemicals, Inc. Omni Solv® water or 

NWQL blank water (OneStop Shopping number N1570) to 

monitor for potential sample contamination.

7.2 When the sample is received, if the sample bottle has less 

than about 30 mL of headspace, discard a minimum amount 

of water to provide enough headspace for later addition of 

60 g NaCl. Weigh the sample plus bottle and record the gross 

weight (± 1 g). After the gross weight has been recorded, add 

60 ± 10 g NaCl (baked) to make the sample about 1 molar 

NaCl. Mix well to ensure the NaCl has dissolved completely. 

Store samples in a refrigerator at 4°C for up to 14 days from 

the time of sample collection.

NOTE: Addition of NaCl to samples increases the ionic 

strength and improves recovery of polar compounds during 

extraction.

7.3 The CLLE vessels must be mounted in a fume hood (see 

fig. 1) because of the organic solvents that are used in this 

method. Prior to loading samples in the CLLE vessels (usually the day before extraction), rinse each vessel, stopper, and 

receiver (first with reagent water, second with acetone, and 

third with DCM). Close the Teflon stopcock at the base of the 

CLLE vessels. Place two to five boiling chips in the bottom of 

each 25-mL receiver and attach firmly to the extractor with a 

connector clip.

7.4 Prepare two additional 1-L bottles for each set of up to 

18 samples for the laboratory reagent set blank and the reagent 

set spike containing about 900 mL of Solution 2000 water and 

60 ± 10 g of NaCl. Add 100 μL of surrogate spiking solution 

(20 ng/μL; see section 6.3) to each sample, including the set 

blank and spike. Add 100 μL of primary fortification spike 

solution (20 ng/μL; see section 6.5) into the reagent water 

spike sample. (This step results in a concentration of 2.0 μg/L 

for the fortified spike compounds and surrogate compounds in 

a 1-L sample.) Cap each sample bottle and shake well to mix. 

Ensure all the NaCl has dissolved.

7.5 Add 50 mL of DCM to each CLLE vessel using the pressurized solvent keg. Shake each sample and carefully transfer 

into a CLLE vessel through the side access arm. Use a clean 

stainless-steel funnel to facilitate this transfer. Transfer the 

water and any particulate matter to the CLLE vessel. Rinse the 

sample bottle well with about 10 to 15 mL of Solution 2000 

water and pour the rinse into the CLLE vessel. Then, rinse 

the sample bottle with about 10 to 15 mL DCM, and swirl or 

rotate the bottle to ensure the entire inside glass surface has 

contact with solvent, and pour the solvent rinse into the CLLE 

vessel. Rinse with the solvent a second time, following the 

same procedure. Finally, rinse the sample bottle with Solution 

2000 reagent water to remove residual DCM and transfer to 

the CLLE vessel.

NOTE: Thoroughly rinsing sample bottles is important 

because more than 30 percent of some hydrophobic compounds (particularly PAHs, sterols, and organochlorine compounds) might adhere to glass walls.

7.6 With the frit assembly raised out of the way, add Solution 2000 water to the extractor until DCM just begins to 

drip from the CLLE vessel solvent return arm into the 25-mL 

receiver. Lower the frit assembly until the bottom of the frit 

just touches the surface of the water. 

7.7 Add DCM with a Teflon® squirt bottle (or preferably 

directly from a pressurized solvent dispensing drum) into the 

spout at the top of the condenser until the reservoir above the 

frit is filled. DCM will begin to disperse from the microdroplet 

dispersing frit. Continue adding DCM until there is about 15 

mL of DCM in the receiver. 

NOTE: Sometimes the frit will need to be rattled to remove 

trapped air pockets and start DCM “raining.”
7.8 Turn on the water to the condensers and close the side 

access arm with a no. 22 glass stopper. Hook the heating 

mantle onto the receiver. Turn on the Variac voltage controllers 

and set at 70 volts. Watch the receiver to ensure that the DCM 

is boiling vigorously and extract for 3 hours at ambient pH. 

NOTE: Ambient pH is used because recoveries of a few compounds subject to hydrolysis (particularly phthalate esters) are 

somewhat better than at basic pH.

After 3 hours, reduce the pH with the addition of 3 mL H2SO4

(25 percent, pH 2) through the side arm of the CLLE vessel. 

NOTE: Check the volume of DCM in the receiver every 15 

to 30 minutes to maintain a volume of about 15 to 20 mL. Add 

DCM through the spout at the top of the condenser to make 

this volume adjustment during the first 3 hours of extraction. 

After 3 hours, if the volume of DCM in the CLLE vessel is 

greater than 50 mL, add Solution 2000 reagent water through 

the side access arm to make this volume adjustment. If at any 

time the volume of DCM in the CLLE vessel is less than 50 

mL, add DCM through the spout at the top of the condenser.

7.9 After 6 hours of sample extraction, drain the CLLE vessel 

into a designated waste carboy. This step also will cut off the 

return of DCM to the receiver. Continue to boil the DCM in the 

receivers until the level reaches 4 to 8 mL. Remove the heating 

mantle and allow the receivers to cool before removing them. 

Turn off the Variac heater and the water to the condensers.

NOTE: This boiling concentration step follows quickly and 

requires constant monitoring to remove the heating mantle 

when the DCM level is between 4 and 8 mL. At no time 

should the solvent be allowed to boil dry because of volatile 

compound losses.

7.10 After the sample has drained from the CLLE vessel, 

rinse all CLLE surfaces immediately with copious amounts of 

Solution 2000 reagent water to ensure all surfaces are thoroughly wetted. Make sure all suspended material has been 

rinsed out of the CLLE vessel. While the walls of the CLLE 

vessel are still wet, rinse with 60 mL of acetone followed by 

60 mL of DCM.

NOTE: The NWQL needs to be notified if a sample might 

contain raw sewage (section 8.1). If this is the case, double or 

triple the amount of water and solvents in the rinse to ensure 

that the CLLE vessel is clean.

7.11 Record the empty sample bottle weight (± 1 g) to obtain 

the weight of the extracted sample. 

7.12 Add 20 μL of the PAH procedural internal standard 

solution (100 ng/μL; see section 6.6) to each sample extract 

by using a 1.0-mL stepper syringe. Prepare the 24-position 

N-Evap nitrogen evaporator by attaching cleaned and burned 

stainless-steel needles in each position. Set the nitrogen flow 

rate to about 3.5 L/min and adjust the flow visually so that 

a slightly detectable ripple can be seen on the surface of the 

extracts. Remove the extracts when the final volume is 400 μL. 

To maintain a more consistent flow rate for remaining extracts, 

leave the needles attached to the N-Evap with the flow of 

nitrogen remaining unaltered. Concentrate the DCM extract at 

ambient temperature and periodically check the extracts. Do 

not allow the extract to evaporate to less than 300 μL.

7.13 Vortex the extract so that the solvent rinses the glass 

walls of the receiver. Then use a baked, disposable glass 

Pasteur pipette to transfer concentrated (400 μL) extracts to 

appropriately labeled, screw-cap GC vials that contain 400-μL 

glass inserts. Ensure that the screw cap forms a tight seal. 

Store extracts in a freezer at ≤ −10oC for up to 45 days prior to 

GC/MS analysis.

8. Safety Precautions and Waste Disposal

8.1 Sample Handling. All field and laboratory staff who 

handle samples are required to have tetanus/diphtheria, hepatitis A, and hepatitis B immunizations. Nitrile or latex gloves, 

protective glasses, and laboratory coats also are necessary. If 

samples might contain untreated sewage or biohazards, field 

personnel are instructed to clean the outside surfaces of the 

bottle thoroughly with isopropyl alcohol and water to prepare for shipment to the NWQL according to the USGS field 

manual (U.S. Geological Survey, variously dated). It is necessary for all NWQL personnel to wear disposable gloves when 

handling sample bottles. Change gloves frequently.

NOTE: As a warning to NWQL personnel, the accompanying 

analytical services request (ASR) form for samples that possibly contain untreated sewage or biohazards must clearly indicate in capital letters in the hazards comment field: “SAMPLE 

MIGHT CONTAIN SEWAGE. HANDLE WITH CAUTION.”

8.2 Conduct the CLLE extraction and DCM concentration 

in a fume hood. Eye protection, gloves, and protective clothing must be worn in the laboratory area and when handling 

reagents or solvents. Avoid contact with DCM because typical 

laboratory disposable, nitrile gloves do not provide adequate 

protection from DCM.

8.3 The sample waste stream produced during sample 

preparation is about 1 molar NaCl and pH 2. It is collected in 

carboys, and must be disposed of according to local regulations. Solvent and DCM used to clean or rinse glassware, SPE 

cartridges, or equipment also must be disposed of in the appropriate waste containers. All bags, bottles, and consumable 

items associated with sample processing must be segregated 

and disposed of properly.

Analytical Method 
10 Determination of Wastewater Compounds in Whole Water

9. Gas chromatograph/mass spectrometer 

performance

The performance of the analytical instrumentation is 

checked at least every 24 hours to ensure that it meets qualityassurance guidelines of sensitivity and accuracy necessary to 

obtain reproducible sample results.

9.1 Gas chromatograph performance 

evaluation

Gas chromatograph performance is indicated by peak 

shape, compound resolution, and verification of selectedcompound response factors of a low-level standard. An example of the separation and peak shape for the complex mixture 

of NP compounds is shown in the selected ion chromatogram 

(fig. 2) of a 2.0-ng/μL calibration solution. If peak shape and 

resolution deteriorates (indicated by a loss in the number of 

resolved NP isomers, fig. 2) or if compounds fail to meet 

the calibration criteria (see Section 10, Instrument Calibration), change the injection port liner or maintain the capillary 

column to bring the gas chromatograph into compliance. 

About 0.6 m (one column loop) of the capillary-column inlet 

end often can be removed to restore GC performance. Specifically, a loss in response greater than 30 percent for cholesterol 

indicates the need for replacement of the GC inlet liner, or 

maintenance of the column, or both. Instrument maintenance 

requires recalibrating the method compounds and reanalysis of 

samples.

9.2 Mass spectrometer performance 

evaluation

9.2.1 Check for air (m/z 28 and 32) and water (m/z 18) leaks 

in the GC/MS prior to analysis. If air leaks are detected, as 

indicated by the presence of nitrogen (m/z 28) greater than 10 

percent of the m/z 69 peak area of the perfluorotributylamine 

tuning compound, then locate and fix the leaks. Also, check the 

instrument before each sequence of analyses to ensure that mass 

spectrometer performance is in accordance with the perfluorotributylamine tuning criteria outlined below (section 9.2.2). In 

addition, adjust the mass spectrometer response (also outlined 

in section 9.2.2) to ensure that the established interim reporting 

level (IRL) for each selected compound can be achieved.

9.2.2 Check the mass spectrometer tune prior to sample 

analysis. 

NOTE: The following guidance applies to the Agilent Technologies model 5973 GC/MS system. Other GC/MS systems 

might require different adjustments to achieve the method 

performance criteria.

The mass axis and MS peak-width adjustment characteristics must be set to give ± 0.15-atomic mass unit accuracy at 

masses 69, 219, and 502 in the spectrum of perfluorotributylamine. Adjust the electron multiplier voltage to achieve about 

1,000,000 counts for the mass 69 ion. This setting generally 

will provide sufficient signal to meet detection requirements 

for method compounds at method detection limit (MDL) 

concentrations in samples, provided that the GC is performing properly. Manually adjust the resolution so that m/z ion 69 

has 100-percent abundance, mass 219 ion is 40 ± 20 percent, 

and mass 502 is 3 ± 2-percent relative abundance. Check 

mass assignments to ensure accuracy to ± 0.15 atomic mass 

unit. Adjust peak widths measured at half height for ions 69, 

219, and 502 so that peak widths range from about 0.5 to 0.65 

atomic mass unit. Adjustment of tune settings normally will 

require recalibrating the method compounds.

10. Instrument calibration

10.1 Inject 2 μL of each calibration solution into the GC/MS 

and acquire data by using the previously described GC/MS 

conditions. Enter the calibration concentration levels, compound names, quantitation ion, confirmation ions, and approximate retention times listed in table 2 into the data-processing 

method. Use the GC/MS data-processing software to calculate 

the relative retention time (RRT) and relative response factor 

(RRF) for each compound and surrogate in relation to the 

designated internal standard reference compound (table 2) in 

the calibration solution. A calibration curve is calculated by 

the data-processing software for each compound. A linear calibration curve is suggested for most of the method compounds; 

however, other curve-fitting routines (quadratic curves and 

power curves) might be used for polar compounds. The same 

calibration curve type that demonstrates the “best fit” to the 

data for each compound must always be used in the method. 

Typical equations used to calculate calibration curves for this 

method are similar to other NWQL methods (Sandstrom and 

others, 2001).

10.2 Prior to the analysis of each sample set and every 10 

samples thereafter during a series of analyses, analyze and 

evaluate a calibration solution (or solutions) that contain all of 

the method compounds to ensure that GC/MS performance is 

in compliance with established performance criteria (see section 9.2). The calculated concentration of method compounds 

in the continuing calibration verification solutions (CCVs), 

using the initial calibration curve, must be within ± 25 percent 

of the expected concentration before analysis of environmental 

samples.

10.3 The multicomponent compounds (4-NPs, NPEO1, 

and NPEO2 mixtures each composed of 10 to 20 discernible 

isomers) are calibrated by manually integrating the area of 

their respective quantitation ion peaks that are present in the 

expected retention time window range (table 2). This approach 

was used for determining the concentration of compounds 

when preparing stock standard solutions (section 6.1, fig. 2) 
Table 2. Wastewater method compound retention time, quantitation ion, confirmation ions, surrogate compounds, and internal 

standard reference compound.

[Compounds are listed in order of retention time. min, minutes; m/z, mass-to-charge ratio; IS, internal standard; --, not used]

Compound name Retention

time (min)

Quantitation

ion (m/z)

Confirmation

ion (m/z)

Confirmation

ion (m/z)

Internal standard 

reference

Tetrachloroethylene 7.021 164 166 131 IS1

Bromoform 10.233 173 171 175 IS1

Isopropylbenzene (cumene) 11.507 105 120 -- IS1

Phenol 13.651 94 66 65 IS1

1,4-Dichlorobenzene 15.212 146 148 111 IS1

d-Limonene 15.819 93 136 121 IS1

Acetophenone 17.234 105 120 77 IS1

p-Cresol 17.460 107 108 77 IS1

Isophorone 19.298 82 138 -- IS2

Camphor 20.135 90 105 152 IS2

Isoborneol 20.582 95 136 140 IS2

Menthol 20.921 95 123 138 IS2

Naphthalene 21.123 128 127 102 IS2

Methyl salicylate 21.269 120 152 92 IS2

Dichlorvos 22.374 109 85 220 IS2

Isoquinoline 22.834 129 102 -- IS2

Indole 23.418 117 89 -- IS2

2-Methylnaphthalene 23.568 142 141 115 IS3

3,4-Dichlorophenyl isocyanate 23.639 187 189 124 IS3

1-Methylnaphthalene 23.869 142 141 115 IS3

3-Methyl-1H-indole (skatol) 25.120 130 131 -- IS3

2,6-Dimethylnaphthalene 25.519 156 141 -- IS3

3-tert-Butyl-4-hydroxyanisole (BHA) 26.606 180 165 137 IS3

5-Methyl-1H-benzotriazole 27.918 103 104 77 IS3

N,N-Diethyl-meta-toluamide (DEET) 27.983 119 190 91 IS3

Diethyl phthalate 28.064 149 177 -- IS3

4-tert-Octylphenol 28.320 135 206 107 IS3

Benzophenone 28.806 182 105 77 IS3

Tributyl phosphate 28.830 99 155 211 IS3

Triethyl citrate (ethyl citrate) 28.914 157 115 203 IS4

Analytical Method 11
12  Determination of Wastewater Compounds in Whole Water

Table 2. Wastewater method compound retention time, quantitation ion, confirmation ions, surrogate compounds, and internal 

standard reference compound.—Continued

[Compounds are listed in order of retention time. min, minutes; m/z, mass-to-charge ratio; IS, internal standard; --, not used]

Compound name Retention

time (min)

Quantitation

ion (m/z)

Confirmation

ion (m/z)

Confirmation

ion (m/z)

Internal standard reference

Cotinine 29.761 98 176 147 IS4

4-Nonylphenol (total) 29.7–30.6 135 220 107 IS4

Prometon 30.099 210 225 168 IS4

Atrazine 30.218 200 215 202 IS4

Tri(2-chloroethyl) phosphate 30.311 249 251 205 IS4

Pentachlorophenol 30.394 266 264 268 IS4

4-n-Octylphenol 30.448 107 206 -- IS4

Diazinon 30.673 304 179 199 IS4

Phenanthrene 30.903 178 176 89 IS4

Octylphenol, monoethoxy- (OPEO1) 30.903 135 107 179 IS4

Anthracene 31.044 178 176 89 IS4

Caffeine 31.444 194 109 82 IS4

Acetyl-hexamethyl-tetrahydro-naphthalene 

(AHTN)

31.468 243 258 213 IS4

Carbazole 31.524 167 139 166 IS4

Hexahydrohexamethyl 

cyclopentabenzopyran (HHCB)

31.538 243 258 197 IS4

4-Cumylphenol 31.576 197 212 -- IS4

Carbaryl 32.120 144 115 116 IS4

Metalaxyl 32.135 206 220 249 IS4

Bromacil 32.587 205 207 -- IS4

Metolachlor 32.850 162 138 240 IS4

Chlorpyrifos 32.878 314 316 197 IS4

Anthraquinone 33.095 208 180 152 IS4

Nonylphenol, monoethoxy- (total, NPEO1) 33.1–33.9 179 193 207 IS4

Fluoranthene 34.134 202 101 203 IS4

Triclosan 34.378 288 290 218 IS4

Pyrene 34.731 202 101 203 IS5

Bisphenol A 34.994 213 228 119 IS5

Octylphenol, diethoxy-(OPEO2) 35.168 223 135 294 IS5

Nonylphenol, diethoxy- (total, NPEO2) 35.7–36.5 237 223 279 IS5

Tri(dichloroisopropyl) phosphate 36.400 379 383 381 IS5

Tri(2-butoxyethyl) phosphate 37.054 299 199 125 IS5

Triphenyl phosphate 37.176 326 325 215 IS5

Diethylhexyl phthalate 38.282 149 167 279 IS5
Table 2. Wastewater method compound retention time, quantitation ion, confirmation ions, surrogate compounds, and internal 

standard reference compound.—Continued

[Compounds are listed in order of retention time. min, minutes; m/z, mass-to-charge ratio; IS, internal standard; --, not used]

Compound name Retention

time (min)

Quantitation

ion (m/z)

Confirmation

ion (m/z)

Confirmation

ion (m/z)

Internal standard reference

2,2',4,4'-Tetrabromodiphenyl ether 38.375 328 326 324 IS5

Benzo[a]pyrene 41.431 252 250 126 IS6

3beta-Coprostanol 42.927 373 355 388 IS6

Cholesterol 43.209 386 301 275 IS6

beta-Sitosterol 45.038 414 396 381 IS6

beta-Stigmastanol 45.193 416 401 233 IS6

Surrogate Compounds

Decafluorobiphenyl 18.786 334 265 -- IS2

Caffeine-d9 31.444 197 110 -- IS4

Fluoranthene-d10 34.087 212 106 -- IS4

Bisphenol A-d11 34.947 216 234 -- IS4

Internal Standards

1,4-Dichlorobenzene-d4

 (IS1) 15.132 150 152 -- --

Naphthalene-d8 (IS2) 21.048 136 -- -- --

Acenaphthene-d10 (IS3) 26.700 164 162 160 --

Phenanthrene-d10 (IS4) 30.842 188 -- -- --

Chrysene-d12 (IS5) 38.010 240 -- -- --

Perylene-d12 (IS6) 41.558 264 132 -- --

Analytical Method 13
14 Determination of Wastewater Compounds in Whole Water

and also has been used in other studies (Blackburn and Waldock, 1995).

10.4 A third-party check (TPC) solution of the method 

compounds obtained from an independent source from the 

calibration mixture must be analyzed to verify the calibration 

solution concentrations (see section 6.7). The compound concentrations calculated in the TPC must be within ± 30 percent 

of the expected concentration. If the TPC sample concentrations do not fall within the ± 30-percent window, the source 

of the error must be determined and corrected before environmental samples are analyzed. 

11. Quality Assurance and Quality Control

The NWQL has prepared a laboratory quality management document (Maloney, 2005) and quality-assurance and 

quality-control (QA/QC) guidance document (M.R. Burkhardt 

and T.J. Maloney, U.S. Geological Survey, written commun., 

1998) for processing and analyzing samples for laboratory 

analytical services. These documents are followed for this 

method to establish and verify data quality. The samplematrix, sample-preparation, and sample-analysis steps are 

evaluated to determine data quality for each sample individually, and for all samples as part of a sample-preparation set and 

a sample-analysis set.

Problematic sample matrices will affect the performance of the method during sample preparation and analysis. 

Extremely complex sample matrices, such as raw sewage, are 

discouraged because they require additional equipment and 

instrument cleaning as outlined in section 7.10. 

Before sample holding-time expiration, each environmental 

sample is prepared for analysis as part of a sample-preparation set 

that contains as many as 18 environmental samples, a laboratory 

reagent spike, and blank (control) to monitor method performance. It is unlikely, however, that a unique sample matrix will 

perform similarly to the recoveries of compounds and surrogates 

in the set spike and blank. Control limits for laboratory spikes in 

reagent water are set at the mean percent recovery ± 3 standard 

deviations.

Surrogate compounds (chemically representative of 

most method compounds) are added to each sample prior to 

preparation. Surrogate standard recoveries are used to measure 

gross sample-processing problems and matrix effects. Control 

limits for surrogates are set at the mean percent recovery ± 3 

standard deviations as compiled from reagent water laboratory 

set spike and blank sample data. The recoveries of caffeine-d9

, 

fluoranthene-d10, and bisphenol A-d11 may be used to monitor sample preparation and potential matrix effects for their 

respective nonisotopically labeled analogs, as well as other 

chemically similar (by functionality, reactivity, or volatility) 

compounds. Surrogate recoveries generally need to be used to 

evaluate specific sample-preparation steps and are of limited 

use for assessing method compound recoveries. Concentrations reported by the NWQL for compounds and surrogates 

in environmental samples are never corrected for spike or 

surrogate recoveries. However, spike and surrogate recoveries 

provide data on method performance that is useful for interpretation of environmental sample data.

Set blanks provide information regarding possible contamination introduced to the sample at the laboratory. Possible 

contamination from field and sample handling is not monitored unless the appropriate field blanks are submitted to the 

laboratory for analysis. Some compounds in this method are 

common in personal-care products and might be detected in 

laboratory blanks. If compounds are detected in more than 10 

percent of the historical laboratory blanks, they are treated as 

though they are always potentially present in sample background. 

As a part of a field quality-control program, the NWQL 

suggests that customers include some laboratory matrix spike 

samples in their projects. The NWQL has prepared a spiking solution that is available at One-Stop Shopping (catalog 

number N1430). Laboratory matrix spike samples also can be 

requested if a duplicate sample is submitted (U.S. Geological 

Survey, 2005).

NOTE: These matrix spike samples can be requested by 

shipping a duplicate sample and separate analytical services 

request (ASR) form with “NWQL SPIKE” indicated in the 

field “Comment to NWQL.” An amount equivalent to 2 μg/L 

(for most compounds) is spiked at the laboratory. 

Sample extracts are analyzed in an instrument batch or 

sequence (table 3) to provide additional information for quality assurance and facilitate corrective actions that might be 

required if performance criteria are not met.

 The analytical sequence includes a laboratory set spike 

and set blank sample. Continuing calibration verification solutions are analyzed at designated intervals (every sample preparation set) to check that the instrument is in compliance with 

initial calibration criteria. Compounds that are quantitatively 

reported (not permanently flagged as an estimated concentration in data reports) must have a calculated concentration within 

± 25 percent of the expected CCV concentration. The Quality 

Management System document (Maloney, 2005) requires three 

CCV check solutions to be analyzed every 24 hours. Finally, 

low-concentration standards equivalent to 1 μg/L (or less) are 

analyzed in each sequence before and after the environmental 

samples to ensure that instrument sensitivity is maintained 

throughout the sample sequence. If the instrument does not meet 

acceptance criteria, then follow suggested procedures of cleaning and maintenance (see Section 9.1, GC performance evaluation) to bring the instrument into compliance.

Each sample also has a PAH procedural internal standard 

solution added to correct for slight differences in extract volume and to adjust for slight variations in instrumental performance. The procedural internal standards are added prior to 

instrument analysis and are used to monitor instrument conditions, such as extract injection-volume variations, unexpected 

GC compound retention time shifts, or instrument abnormalities caused by power interruptions or component malfunctions.
It is difficult to troubleshoot partial QA/QC problems 

caused by a combination of a complex sample matrix, samplepreparation errors, or a marginally acceptable analysis. Certain 

process failures require sample preparation to be repeated if 

a duplicate sample has been provided to the NWQL. Other 

failures might be identified as “matrix-induced” and be 

impossible to correct, thus requiring associated data qualifiers 

for reporting results. In rare cases, certain failures, such as 

surrogate recoveries that are not within control limits, might 

indicate that sample results should not be reported, or be qualified. If this is the case, the data can be transmitted three ways: 

estimated (“E” remark coded), a raised reporting level, or the 

data are reported as deleted – ruined (D – R).

12. Calculation of Results

Before quantitative results are reported, each compound 

first must meet qualitative criteria.

12.1 Qualitative identification

The retention time of the quantitation ion for the compound of interest should be within 0.1 minute of the expected 

relative retention time in the absence of any obvious matrix 

effects. Furthermore, the profiles of the qualification and 

quantification ion peaks must maximize within two scans of 

each other (in the absence of any obvious interference). Visually compare the sample compound spectra to the reference 

standard spectra and confirm that they match. The GC/MS 

operator must visually compare reference spectra, integrated 

ion ratios of the quantitation ion to two confirmation ions, and 

the elution of ion profiles to determine if the spectral match 

between standards and samples is reasonable. Peak area ratios 

of the quantification and qualifier ions must agree within 

± 20 percent between standards and samples for concentrations greater than the minimum reporting level (MRL) in the 

absence of any obvious coeluting interference.

NOTE: Occasionally, ion(s) can appear to be missing or ion 

abundance ratios can appear to be distorted in the spectrum 

of a compound in a sample when compared to the reference 

spectrum, especially at concentrations near the MDL if there is 

interfering spectral contamination. A distorted sample spectrum often results from automatic data-processing routines 

that subtract the average of the two spectra before and after the 

spectrum at the apex of the peak. In this situation, a compound 

can be positively identified if the ion profiles maximize within 

two scans of each other (after accounting for any obvious 

interferences to ion profiles, if necessary). 

12.2 Quantitation

12.2.1 Determination of single-component compounds. After 

a compound has passed qualitative criteria, the concentration 

is calculated according to the calibration curve used to establish the best fit with the calibration points. Curve-fitting routines provided by the instrument manufacturer and described 

in a similar NWQL method report (Sandstrom and others, 

2001) are used to obtain a calibration curve for each compound. If the calculated concentration of a compound exceeds 

Table 3. Typical analytical sequence suggested for use with this method.

[CCV, continuing calibration verification; ng/μL, nanogram per microliter] 

Injection vial

sequence Sample description

1 Calibration level 7 (10 ng/μL)1

2 Calibration level 6 (5 ng/μL)1

3 Calibration level 5 (2 ng/μL)1

4 Calibration level 4 (1 ng/μL)1

5 Calibration level 3 (0.5 ng/μL)1

6 Calibration level 2 (0.2 ng/μL)1

7 Calibration level 1 (0.1 ng/μL)1

8 Instrument detection level standard (1 ng/μL, calibration level 4)

9 Preparation set one method spike 

10 Preparation set one method blank 

11–20 Set of 10 or less environmental samples for set one

21 CCV (calibration level 3, 4, or 5; for set one and set two)

22 Preparation set two method spike 

23 Preparation set two method blank 

24–33 Set of 10 or less environmental samples for set two 

34 CCV (calibration level 3, 4, or 5; for set two)

35 Instrument detection level standard (1 ng/μL, calibration level 4)

1

 The sequence order of calibration solutions is not important.

Analytical Method 15
16  Determination of Wastewater Compounds in Whole Water

the highest concentration point of the calibration curve, add 

higher concentration calibration solutions to the curve or dilute 

the extract to bring the compound response within the range of 

the calibration curve. Concentration results must be reported 

as estimated with the “E” remark code if compound response 

is less than the lowest point on the calibration curve or the 

interim reporting level (IRL). If curve-fitting routines (quadratic curves and power curves) are used for calibration, verify 

that the sample compound response is not outside the working 

range of the calibration curve (or in a region of unexpected 

deviations in the calibration curve), or recalculate the concentration by using another type of calibration curve.

12.2.2 Determination of multicomponent compounds. The 

4-NPs, NPEO1, and NPEO2 technical mixtures are each 

composed of 10 to 20 discernible isomers. Manually integrate 

the isomeric peak areas of their respective quantitation ions 

present in their expected retention time window range (table 

2), similar to the calibration process described earlier (Section 

10, Instrument Calibration) for these compounds. The spectra 

of each isomeric GC peak of a multicomponent compound are 

checked for interference. If interferences cause the ratios of 

the confirmation ions to the quantification ion to be unreasonable, then integrate that portion of the ion chromatogram that 

is caused by the contamination or interference and subtract the 

interference from the total.

NOTE: This procedure seldom is necessary because the quantitation and confirmation ions for 4-NP, NPEO1, and NPEO2 

normally are free from coeluting interference.

12.3 Reporting results

Data are reported according to the NWQL laboratory 

quality management document (Maloney, 2005; Childress and 

others, 1999) and a quality-assurance and quality-control (QA/

QC) guidance document (M.R. Burkhardt and T.J. Maloney, 

U.S. Geological Survey, written commun., 1998). Alphanumeric 

data-qualifier codes are used to report information about the 

presence and concentration of a compound when concentrations 

are less certain because of matrix effects, interferences, and 

other unexpected circumstances.

The wastewater method is considered to be “informationrich” (Childress and others, 1999) because compound identifications are determined by mass spectrometry; consequently, 

results are not censored at the IRL. Compound concentrations, 

therefore, are reported as follows.

If the concentration is equal to or greater than the IRL, 

the concentration is reported to three significant figures. If the 

concentration is less than either the IRL or the lowest calibration standard (usually 0.2-μg/L), results are reported by using 

the “E” code to indicate that it has been estimated. Other 

instances where it is appropriate to use the “E” code have been 

documented (Maloney, 2005; Childress and others, 1999). 

They include matrix interferences, compounds that have 

been permanently assigned an “E” remark code, and those 

compounds that do not meet quality-assurance criteria, such 

as being out of calibration by more than ± 25 percent. If the 

calculated result is greater than the highest concentration standard in the calibration curve, then the sample is diluted into 

the range of the calibration curve and reanalyzed, or higher 

calibration standards are added to the curve to bracket the 

sample result. If dilution is not possible, concentration results 

can be reported with an “E” remark code.

Reporting compound results as estimated, because their 

concentrations are less than the IRL, does not decrease confidence in qualitative identification. However, concentrations 

reported near or less than the MDL need to be interpreted cautiously. If compounds are barely discernible in mass spectra 

and responses are near or less than the MDL, then the potential 

for reporting false detections (false positives) or mistakenly 

reporting compounds as not present (false negatives) increases. 

When there is doubt about qualitative identification, reporting 

conservative results (less than IRL) is appropriate. 

Compounds present in over 10 percent of the historical 

laboratory blanks are referred to as chronic blank compounds 

and are treated as if present in all samples. Consequently, no 

data (not even estimated data with an “E” remark code) will be 

reported less than the 95th percentile of the historical laboratory blank concentration levels for chronic blank compounds. 

If the 95th percentile is greater than the IRL derived from the 

calculated MDL, the IRL is raised to the 95th percentile. 

NOTE: Laboratory blanks are evaluated annually for contamination.

Results and Discussion of Method 

Validation

Reagent-water (Solution 2000 water) samples, surfacewater samples collected from the Platte River in Confluence 

Park, Denver, Colo., and ground-water samples collected 

from a domestic well near Evergreen, Colo., were used to test 

method performance. One set of 1-L subsamples was fortified 

at a lower concentration (0.5 – 10 μg/L) of each compound 

and another set was fortified at a concentration 8 times higher 

(4.0 – 80 μg/L) of each compound. In addition, the three sample matrices were extracted and analyzed unfortified (table 4) 

to determine the natural presence of any method compounds. 

 The presence of 10 compounds in the unspiked reagentwater sample (table 4) reemphasizes the ubiquitous nature of 

many of the method compounds, as well as the importance of 

avoiding contamination throughout sample collection, preparation, and analysis. Therefore, if contamination is detected in 

the set blank, or if the compound is chronic, the concentration 

of the compound in an environmental sample is not reported 

less than the set blank concentration or 95th percentile concentration (for chronic compounds), or whichever concentration is 

greater.
Table 4. Wastewater method compounds detected in unfortified reagent-water, ground-water, 

and surface-water samples.

[μg/L, micrograms per liter; --, not detected; N, number of samples]

Median concentration (μg/L)

 Compound name Reagent

(N=7)

Ground

(N=3)

Surface

(N=3)

 1,4-Dichlorobenzene -- -- --

 1-Methylnaphthalene -- -- --

 2,2',4,4'-Tetradibromodiphenyl ether -- -- --

 2,6-Dimethylnaphthalene -- -- --

 2-Methylnaphthalene 0.01 -- 0.02

 3beta-Coprostanol -- -- .26

 3-Methyl-1H-indole (skatol) -- -- --

 3-tert-Butyl-4-hydroxyanisole (BHA) -- -- --

 3,4-Dichlorophenyl isocyanate -- -- .08

 4-Cumylphenol -- -- --

 4-n-Octylphenol -- -- --

 4-Nonylphenol (total) -- -- 1.20

 4-tert-Octylphenol .10 -- .16

 5-Methyl-1H-benzotriazole -- -- 1.30

 Acetophenone .04 -- --

 Acetyl-hexamethyl-tetrahydro-naphthalene 

(AHTN) -- -- .21

 Anthracene -- -- --

 Anthraquinone -- 0.02 --

 Atrazine -- -- --

 Benzo[a]pyrene -- -- --

 Benzophenone -- .09 .17

 beta-Sitosterol -- -- --

 beta-Stigmastanol -- -- --

 Bisphenol A -- -- .21

 Bromacil -- -- --

 Bromoform -- -- --

 Caffeine -- .03 .20

 Camphor .04 -- .04

 Carbaryl -- -- --

 Carbazole -- -- --

 Chlorpyrifos -- -- --

 Cholesterol .50 -- .96

 Cotinine -- -- .41

 Diazinon -- -- --

Results and Discussion of Method Validation 17
18  Determination of Wastewater Compounds in Whole Water

Table 4. Wastewater method compounds detected in unfortified reagent-water, ground-water, 

and surface-water samples—Continued.

[μg/L, micrograms per liter; --, not detected; N, number of samples]

Median concentration (μg/L)

 Compound name Reagent

(N=7)

Ground

(N=3)

Surface

(N=3)

 Dichlorvos -- -- --

 Diethylhexyl phthalate 0.15 -- 0.33

 Diethyl phthalate .08 0.13 .11

 d-Limonene -- -- --

 Fluoranthene -- .03 .04

 Hexahydrohexamethyl cyclopentabenzopyran (HHCB) -- -- .89

 Indole -- -- --

 Isoborneol -- -- --

 Isophorone -- -- --

 Isopropylbenzene (cumene) -- -- --

 Isoquinoline -- -- --

 Menthol -- -- --

 Metalaxyl -- -- --

 Methyl salicylate -- -- --

 Metolachlor -- -- --

 N,N-Diethyl-meta-toluamide (DEET) -- -- .24

 Naphthalene .01 -- .03

 Nonylphenol, diethoxy- (total, NPEO2) -- -- 2.80

 Nonylphenol, monoethoxy- (total, NPEO1) -- -- 1.60

 Octylphenol, diethoxy- (OPEO2) -- -- .19

 Octylphenol, monoethoxy- (OPEO1) -- -- .88

 p-Cresol -- -- --

 Pentachlorophenol -- -- .40

 Phenanthrene .02 .04 .03

 Phenol .05 -- .07

 Prometon -- -- --

 Pyrene -- .02 .04

 Tetrachloroethylene -- .30 .01

 Tri(2-chloroethyl) phosphate -- -- .21

 Tri(dichloroisopropyl) phosphate -- -- .23

 Tributyl phosphate -- -- --

 Triclosan -- -- .15

 Triethyl citrate (ethyl citrate) -- -- .13

 Triphenyl phosphate -- -- .10

 Tri(2-butoxyethyl) phosphate -- .10 .40
Mean bias and variability data from analyses of the fortified matrices (background corrected as necessary) are listed in 

table 5.

Recoveries in reagent-water samples fortified at 0.5 μg/L 

averaged 72 percent ± 8 percent relative standard deviation. 

The concentration of 21 compounds is always reported as 

estimated for one of three reasons: unacceptably low-biased 

recovery (less than 60 percent) or highly variable method performance (greater than 25 percent RSD), unstable instrument 

response, or reference standards prepared from technical mixtures. Furthermore, nine additional compounds (see footnote 

4, table 5) demonstrated the potential for variable performance 

during some portion of this initial method validation. The 

concentration of these compounds also will be estimated if the 

set spike recovery or expected set CCV concentrations are not 

within control limits (see section 11). The initial MDLs were 

calculated for compounds in reagent water by using the corresponding spike concentration as listed in table 5.

The MDL is defined as the minimum concentration of a 

substance that can be measured and reported with 99-percent 

confidence that the compound concentration is greater than 

zero (U.S. Environmental Protection Agency, 1997b). Initial 

MDLs were determined according to the procedure outlined 

by the U.S. Environmental Protection Agency (1997b). 

The MDL was calculated according to the equation

MDL = S ∙ t(n–1, 1–α= 0.99), (1)

where S = standard deviation of 

 replicate analyses, in 

 microgram per liter, at the

 lowest spike concentration;

n = number of replicate analyses;

and

t(n–1, 1–α = 0.99) = Student’s t-value for the 99-

percent confidence level 

with n–1 degrees of freedom.

According to the USEPA procedure, at least seven 

replicate reagent-water samples are fortified with compounds 

at concentrations of two to five times the calculated MDL. 

This concentration range was used to calculate initial MDLs 

(table 6). Initial MDLs for various method compounds were 

calculated by using concentrations higher than the desired 

spiking level so that the compound would be detected in each 

of the replicate reagent-water samples. Initial MDLs that 

were calculated by using fortified concentrations higher than 

the approved spiking amount have been defined as estimated 

MDLs, and have been footnoted as such in table 6. Initial 

MDLs that were calculated from this procedure ranged from 

0.02 to 1.35 μg/L and averaged 0.18 μg/L.

 The IRLs have been set higher (two to ten times for 

most compounds) than the calculated initial MDLs (table 6). 

This precaution reduces the risk of reporting that a compound 

is undetected (less than the IRL), when it is actually in the 

sample near the MDL concentration (Childress and others, 

1999). All qualitatively identified compounds detected less 

than the IRL are reported as estimated, regardless of the established IRL, because this wastewater method is classified as an 

“information-rich” method, as are other mass spectrometric 

methods (Childress and others, 1999). Furthermore, there was 

no need to raise any of the initial IRLs because of chronic 

blank contamination (see table 4).

The IRLs will be updated annually as necessary by using 

data acquired from the NWQL long-term method detection level (LT–MDL) program (Childress and others, 1999). 

The LT–MDL is calculated with 6 to 12 months of data, and 

includes results from multiple instruments, analysts, and calibration curves. Laboratory blank data also will be evaluated 

annually.

A boxplot of the CLLE extraction results for 140 environmental samples collected throughout the United States, 

with the majority from urban sampling sites, is shown in figure 

3. The data are reported using a log scale to accommodate 

the large concentration ranges for each compound. The data 

include estimated concentrations reported less than the IRL. At 

least one method compound was found in every environmental 

sample analyzed. The most frequently detected compounds 

were caffeine and DEET, which were detected in 67 percent 

(94 out of 140) of the environmental samples. 

Summary and Conclusions

The U.S. Geological Survey (USGS) National Water 

Quality Laboratory has developed an analytical method 

(O-4433-06) for the determination of 69 compounds typically 

found in domestic and industrial wastewater. The method 

provides an efficient means for detecting emerging toxic and 

estrogenic compounds that otherwise might not be reported 

because they are unregulated or not included in other USGS or 

U.S. Environmental Protection Agency methods. Whole-water 

samples are extracted using continuous liquid–liquid extraction with dichloromethane, and compounds are determined 

by capillary-column gas chromatography/mass spectrometry. 

The method focuses on the determination of compounds that 

are an indicator of wastewater, exhibit endocrine-disrupting 

potential or toxicity, or have widespread use. Determination 

of the alkylphenol ethoxylate nonionic surfactant compounds 

is particularly important because they are persistent indicators 

of wastewater. Other method compounds are representative 

of fragrances, food additives, antioxidants, flame retardants, 

plasticizers, industrial solvents, disinfectants, fecal sterols, 

polycyclic aromatic hydrocarbons, and high-use domestic 

pesticides. 

Average recovery of all method compounds for shortterm single-operator results in reagent-water samples fortified 

at 0.5 microgram per liter was 72 ± 8 percent relative standard 

deviation and initial method detection limits averaged 0.18 

microgram per liter. The compound recoveries and interim 

reporting levels for this whole-water continuous liquid–liquid 

Summary and Conclusions 19
20 Determination of Wastewater Compounds in Whole Water

Table 5. Wastewater method mean bias and variability of spike-recovery data for eight replicates with compounds spiked at two 

concentrations that range from 0.5 to 80 micrograms per liter in reagent-water (including calculated method detection limits), groundwater, and surface-water samples.

[μg/L, micrograms per liter; %, percent; CCV, continuing calibration verification solution; RSD, relative standard deviation; MDL, method detection limit; --, 

not applicable]

 Compound name

Spike

amount

(μg/L)

Mean % recovery % RSD_ Initial 

MDL

 Reagent Ground Surface Reagent Ground Surface (μg/L)

 2,2',4,4'-Tetrabromodiphenyl 

ether4

 4.0

 .5

48.82

68.86

 64.86

 62.29

 56.43

 48.57

 8.81

 9.76

 6.72

 6.27

 9.38

14.21 0.11

 3beta-Coprostanol4

 16.0 55.94 67.73 60.73* 8.93 26.15 34.90

 2.0 62.50 88.93 109.91* 10.58 20.49 19.25 .38

 3-Methyl-1H-indole (skatol)4 4.0 67.91 76.04 70.36 38.70 25.61 43.33

 .5 81.71 97.14 86.86 5.55 6.16 11.19 .07

 4-Cumylphenol 4.0 83.19 87.46 92.25 8.56 8.48 9.75

 .5 74.57 84.00 92.57 10.92 6.59 11.41 .13

 4-n-Octylphenol 4.0 83.22 93.43 91.61 8.14 8.77 9.91

 .5 78.00 99.71 103.71 9.48 6.17 9.73 .11

 4-tert-Octylphenol 4.0 82.31* 94.68 81.62* 3.03 9.87 7.52

 .5 3.71* 101.71 80.82* 8.85 5.95 9.41 .11

 5-Methyl-1H-benzotriazle4 16.0 42.96 109.32 139.82* 28.20 26.67 13.39

 2.0 53.50 83.07 108.00* 10.48 9.26 5.59 .35

 Acetophenone 4.0 88.38* 88.54 102.64 8.38 10.24 11.24

 .5 77.28* 97.71 129.71 5.83 6.08 10.44 .07

 Acetyl-hexamethyl-tetrahydronaphthalene (AHTN)  4.0

 .5

81.41

73.43

 89.11

 86.57

 85.68

 109.14

 8.36

 10.03

 7.47

 6.22

 9.75

12.15 .11

 Anthracene 4.0 78.72 86.32 82.82 8.45 8.80 9.50

 .5 70.00 82.29 85.43 7.38 6.65 11.92 .08

 Anthraquinone 4.0 66.31 66.41* 75.18 8.86 9.92 8.21

 .5 66.00 64.00* 88.57 7.82 6.98 8.52 .08

 Atrazine4 4.0

 .5

71.22 

78.00

 81.71

 85.43

 67.68

 82.29

 39.90

 6.94

 16.13

 7.49

34.68

10.63 .08

 Benzo[a]pyrene 4.0 86.72 90.96 82.61 13.40 8.66 9.53

 .5 74.57 87.43 77.43 5.30 5.86 15.19 .06

 Benzophenone 4.0 86.38 80.66* 100.67* 7.86 10.38 7.67

 .5 76.00 78.48* 86.18* 8.32 6.55 8.74 .10

 beta-Stigmastanol4

 16.0 45.30 62.73 49.54 36.6 28.92 33.32

 2.0 67.00 85.93 95.64 5.24 11.56 19.60 .22

 Bisphenol A4 4.0 68.13 82.96 76.58* 3.98 13.46 11.24 

 .5 46.29 101.14 63.14* 2.24 7.81 10.81 .22

 Bromacil 4.0 78.45 85.38 89.04 10.10 14.30 8.46

 .5 70.57 93.79 114.39 7.76 7.41 10.10 .10
Table 5. Wastewater method mean bias and variability of spike-recovery data for eight replicates with compounds spiked at two 

concentrations that range from 0.5 to 80 micrograms per liter in reagent-water (including calculated method detection limits), groundwater, and surface-water samples—Continued.

[μg/L, micrograms per liter; %, percent; CCV, continuing calibration verification solution; RSD, relative standard deviation; MDL, method detection limit; --, 

not applicable

 Compound name

 

Spike

amount

(μg/L)

Mean % recovery % RSD Initial 

MDL

 Reagent Ground Surface Reagent Ground Surface (μg/L)

 Caffeine 4.0 73.00 77.17* 80.55* 13.80 13.61 8.89

 .5 74.86 84.60* 72.00* 5.31 7.48 8.98 0.06

 Camphor 4.0 79.58* 80.39 82.42* 8.26 12.41 9.16

 .5 71.49* 82.00 78.32* 5.84 6.90 11.58 .09

 Carbazole 4.0 78.16 89.43 88.25 14.60 9.89 12.16

 .5 96.00 93.94 104.86 7.68 5.40 10.32 .12

 Chlorpyrifos 4.0 83.28 91.18 85.00 8.42 6.02 10.53

 .5 78.57 87.14 78.29 9.61 5.44 15.36 .12

 Cholesterol4

 16.0 55.58* 69.08 63.52* 20.30 29.93 33.74

 2.0 60.14* 88.43 85.80* 7.77 17.13 15.38 .30

 Diazinon 4.0 77.28 83.07 89.82 10.00 6.74 10.56

 .5 77.43 88.00 96.29 9.63 10.42 11.59 .11

 Diethylhexyl phthalate (DEHP) 4.0

 .5

 81.06*

 90.20*

159.57

225.33

 82.08*

 49.45*

12.00

40.78

76.26

 7.73

18.76

18.21 .85

 Diethyl phthalate (DEP) 4.0 86.31* 90.82 97.05* 7.89 10.86 9.00

 .5 76.08* 104.29 91.15* 8.03 6.50 11.93 .10

 Fluoranthene 4.0 80.78 89.34* 82.86* 8.19 8.75 9.17

 .5 79.43 84.32* 80.44* 6.35 5.22 11.03 .08

 Hexahydrohexamethyl-cyclopentabenzopyran (HHCB)4 4.0

 .5

 80.59

 74.86

 88.57

 88.29

 81.53*

124.57* 9.23 9.74

 8.17

 5.62

 9.25

12.15 .11

 Indole 4.0 69.75 70.11 62.86 38.80 35.39 41.13

 .5 80.86 91.71 67.14 6.36 7.17 19.06 .08

 Isoborneol 4.0 71.69 76.18 79.07 38.40 15.68 8.15

 .5 78.00 85.71 95.14 3.92 7.55 10.14 .05

 Isophorone 4.0 81.97 86.64 85.43 9.09 16.58 8.84

 .5 79.14 87.71 97.71 6.34 6.37 11.18 .08

 Isoquinoline 4.0 75.44 79.96 90.79 14.20 16.72 9.61

 .5 76.00 95.43 115.71 7.75 10.53 11.91 .09

 Menthol 4.0 79.13 79.47 82.50 10.70 14.89 8.15

 .5 76.00 81.71 98.00 4.56 7.80 7.99 .05

 Metalaxyl 4.0 85.38 90.82 100.36 9.03 10.32 9.96

 .5 79.14 98.00 107.14 10.30 7.64 12.09 .13

 Methyl salicylate 4.0 81.53 84.61 93.54 8.06 11.93 9.07

 .5 73.14 91.43 106.57 5.88 6.15 9.91 .07

 Metolachlor 4.0 81.44 86.82 92.71 3.86 8.94 9.92

 .5 76.86 87.71 94.86 9.95 6.64 11.08 .12

 N,N-Diethyl-meta-toluamide (DEET) 4.0

 .5

 84.38

 77.71

109.32

 98.57

 96.01*

 71.31*

 8.91

 9.90

26.67

 7.57

 9.62

 9.18 .12

Summary and Conclusions  21
22  Determination of Wastewater Compounds in Whole Water

Table 5. Wastewater method mean bias and variability of spike-recovery data for eight replicates with compounds spiked at two 

concentrations that range from 0.5 to 80 micrograms per liter in reagent-water (including calculated method detection limits), groundwater, and surface-water samples—Continued.

[μg/L, micrograms per liter; %, percent; CCV, continuing calibration verification solution; RSD, relative standard deviation; MDL, method detection limit; --, 

not applicable]

 Compound name

 

Spike

amount

(μg/L)

Mean % recovery_ % RSD Initial 

 Reagent Ground Surface Reagent Ground Surface MDL

(μg/L)

 p-Cresol 4.0 87.91 88.96 97.54 9.95 13.77 8.82

 .5 81.14 99.71 118.29 6.50 6.28 11.28 0.08

 Phenanthrene 4.0 76.74* 84.39* 83.32* 8.15 10.04 9.06

0.5 68.29* 74.71* 76.43* 6.26 7.19 11.14 .07

 Phenol 4.0 68.41* 72.04 77.30* 2.87 14.59 9.62

 .5 64.57* 86.86 93.86* 6.14 7.26 9.09 .07

 Prometon 4.0 64.03 73.00 79.32 34.10 6.40 17.04

 .5 70.29 81.14 92.86 7.78 6.80 11.59 .08

 Pyrene 4.0 79.59 76.77* 81.04* 8.24 8.43 9.02

 .5 76.29 85.03* 79.38* 6.50 5.00 10.99 .08

 Tri(2-chloroethyl) phosphate 4.0 77.91 85.26 79.07* 8.35 5.81 1.21

 .5 103.10 91.77 77.80* 5.04 4.87 4.09 .08

 Tri(dichloroisopropyl) phosphate 4.0

 .5

 79.86

 74.86

 82.89

 88.86

 85.26*

 64.18*

 5.40

 6.70

12.28

 7.45

 7.85

 9.41 .08

 Tributyl phosphate 4.0 81.81 87.21 97.79 9.02 10.18 9.91

 .5 76.86 96.86 181.14 8.87 6.06 9.56 .11

 Triclosan 4.0 67.63 73.54 74.70* 8.58 9.21 7.74

 .5 75.71 87.71 84.00* 7.99 8.27 10.54 .09

 Triethyl citrate (ethyl citrate) 4.0 71.09 78.18 82.48* 9.37 14.37 7.85

 .5 66.29 82.86 77.80* 6.38 7.49 8.71 .07

 Triphenyl phosphate 4.0 78.94 91.32 82.98* 9.26 7.79 9.36

 .5 75.71 99.14 82.51* 8.13 5.06 10.19 .10

 Tri(2-butoxyethyl) phosphate4 4.0 79.81 93.98* 169.32* 6.37 14.21 6.34

 .5 85.43 84.11* 164.97* 3.99 6.10 4.29 .05

 Compounds with low recovery1

, high variable recovery (RSD)2, or from a technical mixture3

 to be reported with an E remark code (estimated concentration)

 1-Methylnaphthalene1 4.0 60.53 57.50 54.29 7.53 9.80 12.84

 .5 44.86 49.43 68.34 4.35 8.00 5.78 .03

 1,4-Dichlorobenzene1 4.0 47.16 30.25 36.82 7.16 12.77 11.01

0.5 20.85 23.43 26.57 5.13 6.45 16.09 .03

 2,6-Dimethylnaphthalene1 4.0 59.06 56.89 60.57 8.23 8.85 11.30

 .5 37.43 41.71 47.71 7.99 9.76 15.58 .05

 2-Methylnaphthalene1 4.0 54.81* 52.43 56.75* 7.83 9.89 11.72

 .5 40.04* 45.43 48.86* 4.74 7.17 12.33 .03

 3-tert-Butyl-4-hydroxyanisole (BHA)1 4.0

 .5

 51.75

 37.14

 62.14

 31.67

 9.93

 23.00

49.50

28.80

43.80

39.43

10.35

18.10 .16

 3,4-Dichlorophenyl isocyanate1 4.0

 .5

 63.41

 37.43

 69.43

 64.57

 57.78*

 59.28*

11.90

 7.99

10.12

 5.28

10.92

14.24 .06

 beta-Sitosterol1 16.0 21.66 109.79 90.32 40.30 30.95 35.59

 2.0 52.07 51.86 57.29 3.30 7.59 17.94 .11
Table 5. Wastewater method mean bias and variability of spike-recovery data for eight replicates with compounds spiked at two 

concentrations that range from 0.5 to 80 micrograms per liter in reagent-water (including calculated method detection limits), groundwater, and surface-water samples—Continued.

[μg/L, micrograms per liter; %, percent; CCV, continuing calibration verification solution; RSD, relative standard deviation; MDL, method detection limit; --, 

not applicable]

 Compound name

 

Spike

amount

(μg/L)

Mean % recovery % RSD Initial 

MDL

 Reagent Ground Surface Reagent Ground Surface (μg/L)

 4-Nonylphenol (total)3 72.0 81.35 89.80 85.65* 8.58 8.96 9.65

 9.0 88.37 95.08 83.26* 5.03 6.16 11.92 1.23

 Bromoform1 4.0 64.38 67.36 76.96 6.95 10.56 9.20

 .5 45.14 53.71 61.71 4.32 7.27 9.98 .03

 Carbaryl2 4.0 104.50 124.11 134.79 10.30 14.67 8.57

 .5 76.85 143.43 163.71 11.13 5.73 5.44 .13

 Cotinine1 16.0 38.36 39.69 42.22* 40.90 45.73 40.60

 2.0 53.07 64.21 66.57* 8.91 13.21 8.22 .29

 Dichlorvos 4.0 90.94 93.75 103.50 9.47 14.79 7.04

 .5 82.57 106.29 140.57 8.12 6.65 10.08 .11

 d-Limonene1 4.0 35.25 10.36 14.86 39.90 20.76 33.78

 .5 10.86 12.86 13.14 9.85 12.24 19.36 .02

 Isopropylbenzene (cumene) 1 4.0 42.13 14.50 19.79 7.18 17.16 20.04

 .5 9.43 10.57 12.29 10.35 9.23 25.62 .02

 Naphthalene1

 4.0 62.08* 59.61 66.46* 7.27 11.40 8.68

 .5 48.00* 52.00 62.04* 4.17 5.44 10.93 .03

 Nonylphenol, diethoxy- (total, NPEO2) 3 64.0

 8.0

 69.95

 84.38

 83.22

 86.79

 84.87*

 54.10*

18.50

 7.63

 8.46

 6.98

10.35

 4.42 1.23

 Nonylphenol, monoethoxy- 

 (total, NPEO1)3 64.0

 8.0

 72.42

 86.03

 82.48

 82.88

 86.39*

 86.26*

 9.78

 6.33

 8.56

 7.60

 9.01

15.24 1.35

 Octylphenol, diethoxy- (OPEO2)3 3.2

 .4

 63.01

 95.41

 84.13

 64.86

 71.14*

 47.40*

14.40

 5.17

 8.63

 4.66

10.50

 8.99 .05

 Octylphenol, monoethoxy- (OPEO1)3 25.6

 3.2

 69.95

 95.71

 78.65

 99.91

 78.39*

 82.34*

 8.13

10.46

 6.97

 6.47

10.09

10.21 .50

 Pentachlorophenol2 16.0 54.19 58.91 46.16* 14.20 18.61 8.99 

 2.0 65.64 66.21 44.51* 8.18 9.27 19.00 .33

 Tetrachloroethylene1 4.0 30.81 10.43* 13.82* 6.34 14.50 17.02

 .5 2.57 8.86* 5.43* 37.95 22.04 35.04 .24

 Surrogate Compounds

 Caffeine-d6

 2.0 72.26 80.28 82.86 9.67 13.07 9.28 --

 2.0 60.57 63.28 64.07 3.07 3.12 3.59

 Fluoranthene-d10 2.0 81.06 89.86 82.78 6.35 10.09 8.65 --

 2.0 80.12 82.07 75.92 7.59 5.22 10.58

 Bisphenol A-d6

 2.0 68.13 43.50 41.61 33.30 12.45 14.44 --

 2.0 46.29 149.42 144.86 2.24 10.64 22.26

 Decafluorobiphenyl 2.0 26.31 31.79 33.21 5.30 14.10 10.42 --

 2.0 56.50 66.57 62.07 5.60 7.09 13.75 1

 Concentration will be flagged with the “E” remark code because recovery is less than 60 percent or percision is greater than ± 25 percent RSD.

2

 Concentration will be flagged with the “E” remark code because of unstable instrument response.

3

 Concentration will be flagged with the “E” remark code because the reference standard is from a technical mixture.

4

Concentration will be flagged with the “E” remark code if the set spike recovery or set CCV concentration is not within control limits (see section 11).

*

 Percent recovery corrected for background concentration in the unspiked sample.

Summary and Conclusions  23
24 Determination of Wastewater Compounds in Whole Water

Table 6. Wastewater initial method detection limits calculated from the spike-recovery data reported in table 5 using the eight 

replicate reagent-water samples with compound concentrations that range from 0.5 to 80 micrograms per liter.

[μg/L, micrograms per liter; RSD, relative standard deviation; MDL, method detection limit; IRL, interim reporting level; --, not calculated]

Compound name

Spike 

amount

(μg/L) 

Mean

recovery

(μg/L) 

RSD

(μg/L)

Initial

MDL

(μg/L)

Initial

IRL

(μg/L)

 2,2',4,4'-Tetrabromodiphenyl ether 0.5 0.344 0.034 0.11 0.5

 3beta-Coprostanol1 2.0 1.250 .131 .38 2.0

 3-Methyl-1H-indole (skatol) 1 .5 .409 .023 .07 1.0

 4-Cumylphenol .5 .373 .041 .13 1.0

 4-n-Octylphenol .5 .390 .037 .11 1.0

 4-tert-Octylphenol .5 .419 .037 .11 1.0

 5-Methyl-1H-benzotriazle1 2.0 1.070 .112 .35 2.0

 Acetophenone1

 .5 .390 .024 .07 .5

 Acetyl-hexamethyl-tetrahydro-naphthalene

(AHTN) 1 .5 .367 .037 .11 .5

 Anthracene1

 .5 .350 .026 .08 .5

 Anthraquinone1

 .5 .330 .026 .08 .5

 Atrazine1 .5 .390 .027 .08 .5

 Benzo[a]pyrene1

 .5 .373 .020 .06 .5

 Benzophenone .5 .380 .032 .10 .5

 beta-Stigmastanol1

 2.0 1.340 .070 .22 2.0

 Bisphenol A .5 .232 .070 .22 1.0

 Bromacil .5 .353 .028 .10 .5

 Caffeine1

 .5 .375 .020 .06 .5

 Camphor1

 .5 .358 .023 .09 .5

 Carbazole .5 .480 .037 .12 .5

 Chlorpyrifos .5 .393 .038 .12 .5

 Cholesterol1 2.0 1.203 .094 .30 2.0

 Diazinon .5 .387 .037 .11 .5

 Diethylhexyl phthalate (DEHP) 4.0 3.442 .272 .85 2.0

 Diethyl phthalate (DEP) .5 .380 .030 .10 .5

 Fluoranthene1

 .5 .397 .026 .08 .5

 Hexahydrohexamethyl-cyclopentabenzopyran (HHCB) .5 .374 .036 .11 .5

 Indole1

 .5 .404 .026 .08 .5

 Isoborneol1

 .5 .390 .015 .05 .5

 Isophorone1

 .5 .396 .025 .08 .5

 Isoquinoline1

 .5 .380 .029 .09 .5
Table 6. Wastewater method initial detection limits calculated from the spike-recovery data reported in table 5 using the eight 

replicate reagent-water samples with compound concentrations that range from 0.5 to 80 micrograms per liter—Continued.

[μg/L, micrograms per liter; RSD, relative standard deviation; MDL, method detection limit; IRL, interim reporting level; --, not calculated]

Compound name

Spike 

amount

(μg/L) 

Mean

recovery

(μg/L) 

RSD

(μg/L)

Initial

MDL

(μg/L)

Initial

IRL

(μg/L)

 Menthol1

 0.5 0.380 0.017 0.05 0.5

 Metalaxyl .5 .396 .041 .13 .5

 Methyl salicylate1 .5 .366 .021 .07 .5

 Metolachlor .5 .385 .038 .12 .5

 N,N-Diethyl-meta-toluamide (DEET) .5 .389 .038 .12 .5

 p-Cresol1

 .5 .406 .026 .08 1.0

 Phenanthrene1

 .5 .341 .021 .07 .5

 Phenol1

 .5 .323 .023 .07 .5

 Prometon1

 .5 .351 .027 .08 .5

 Pyrene1

 .5 .381 .025 .08 .5

 Tri(2-chloroethyl) phosphate1 .5 .515 .026 .08 .5

 Tri(dichloroisopropyl) phosphate1 .5 .363 .016 .05 .5

 Tributyl phosphate .5 .384 .034 .11 .5

 Triclosan1

 .5 .378 .030 .09 1.0

 Triethyl citrate (ethyl citrate) 1 .5 .331 .021 .07 .5

 Triphenyl phosphate .5 .427 .017 .10 .5

 Tri(2-butoxyethyl) phosphate1 .5 .517 .067 .05 .5

 Compounds with low recovery, high variable recovery (RSD), or from a

 technical mixture to be reported with an E remark code (estimated concentration)

 1-Methylnaphthalene1 .5 .224 .010 .03 .5

 1,4-Dichlorobenzene1 .5 .224 .010 .03 .5

 2,6-Dimethylnaphthalene1 .5 .187 .015 .05 .5

 2-Methylnaphthalene1 .5 .206 .010 .03 .5

 3-tert-Butyl-4-hydroxyanisole 

(BHA)

 .5 .186 .053 .16 5.0

 3,4-Dichlorophenyl isocyanate1 .5 .187 .014 .06 1.0

 4-Nonylphenol (total) 1 9.0 7.953 .396 1.23 5.0

 beta-Sitosterol1

 2.0 1.041 .034 .11 2.0

 Bromoform1

 .5 .226 .010 .03 .5

 Carbaryl .5 .384 .043 .13 .5

 Cotinine1

 2.0 1.061 .095 .29 1.0

 Dichlorvos .5 .413 .034 .11 1.0

 d-Limonene1 .5 .054 .005 .02 .5

 Isopropylbenzene (cumene) 1 .5 .047 .005 .02 .5

 Naphthalene1

 .5 .240 .010 .03 .5

 Nonylphenol, diethoxy- 

 (total, NPEO2) 1 8.0 6.750 .412 1.23 5.0

 Nonylphenol, monoethoxy- 

 (total, NPEO1) 1 8.0 6.882 .435 1.35 2.0

Summary and Conclusions  25
26 Determination of Wastewater Compounds in Whole Water

HHCB(42)

AHTN(51)

Table 6. Wastewater method initial detection limits calculated from the spike-recovery data reported 

in table 5 using the eight replicate reagent-water samples with compound concentrations that range 

from 0.5 to 80 micrograms per liter—Continued.

[μg/L, micrograms per liter; RSD, relative standard deviation; MDL, method detection limit; IRL, interim reporting level; 

--, not calculated]

Compound name

Spike 

amount

(μg/L) 

Mean

recovery

(μg/L) 

RSD

(μg/L)

Initial

MDL

(μg/L)

Initial

IRL

(μg/L)

 Octylphenol, diethoxy- (OPEO2) 1 0.4 0.382 0.014 0.05 1.0

 Octylphenol, monoethoxy- (OPEO1) 1 3.2 3.062 .159 .50 1.0

 Pentachlorophenol1 2.0 1.313 .107 .33 2.0

 Tetrachloroethylene1 4.0 1.220 .078 .24 .5

1

 The compound was fortified at a concentration that was higher than five times the calculated MDL. Therefore, the MDL 

has been defined as an estimated MDL.

Figure 3. Results for analysis of 140 

environmental whole-water samples. 

The compounds are listed in reverse 

alphabetical order. The concentration 

axis is in log scale to accommodate 

the large concentration ranges for the 

compounds of interest. The number 

of samples in which the compound 

was detected is listed after each 

compound name in parenthesis.
extraction method compare well with the National Water 

Quality Laboratory’s filtered-water solid-phase extraction 

waste-water method. 

The assessment of fate and transport for these emerging 

contaminants also is possible with the development of a complementary sediment waste indicator method at the NWQL. 

The high frequency of compounds that have been detected 

for the previous 8 years (1998–2005) has demonstrated the 

capability of this method for identifying anthropogenic contaminants over a wide range of sample matrices. Many method 

compounds are recognized endocrine-system disrupters 

(alkylphenols, alkylphenol polyethoxylates, and bisphenol A, 

for example), whereas others, such as caffeine, musk fragrances, 

and fecal sterols, are excellent indicators of wastewater. 

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Glossary

C

Continuing calibration verification 

(CCV) A standard solution that contains 

method compounds and is used to determine the bias of the present calibration 

curve for the method compounds. The CCV 

is an instrumental standard only and is not 

processed through preparative steps of the 

method.

I

Interim reporting level (IRL) A temporary 

reporting level used for new or custom 

methods when long-term method detectionlevel data are unavailable and a laboratory 

reporting level has not yet been established 

(U.S. Geological Survey, 2003).

Internal standard (IS A compound not 

expected to be found in any environmental 

sample that is added to every sample extract 

in a known amount. The internal standard 

is used to measure the relative gas chromatographic/mass spectrometric (GC/MS) 

responses of other compounds and surrogates in each sample.

L

Long-term method detection level (LT–

MDL) The minimum concentration of a 

substance that can be identified, measured, 

and reported with 99-percent confidence 

that the compound concentration is greater 

than zero. The LT–MDL is calculated from 

replicate analyses of samples fortified with 

all the method compounds, and includes 

precision introduced by multiple instruments, multiple analysts, and multiple 

calibrations from 6 to 12 months (Childress 

and others, 1999).

M

Method detection limit (MDL) The minimum concentration of a substance that can 

be measured and reported with 99-percent 

confidence that the compound concentration is greater than zero (U.S. Environmental Protection Agency, 1997b). The MDL 

is calculated from at least seven replicate 

analyses of samples fortified with all the 

method compounds. The MDL is used to 

establish initial minimum reporting levels, 

until the long-term method detection level 

can be calculated to include day-to-day 

precision.

Minimum reporting level (MRL) The lowest measured concentration of a compound 

that may be reliably reported by using a 

given analytical method (Timme, 1995).

P

Procedural internal standard 

quantitation A quantitation method 

where the internal standard is added during 

sample processing prior to transferring the 

sample extract to a vial. The addition of the 

procedural internal standard during sample 

processing compensates for quantitation 

losses in those processing steps after the 

internal standard is added.

S

Surrogate A compound not expected to be 

found in any environmental sample that is 

added to every sample in a known amount 

prior to sample processing. The surrogate 

is used to monitor method performance for 

each sample.

Glossary  29
30 Determination of Wastewater Compounds in Whole Water

Manuscript approved for publication July 10, 2006

Prepared by the U.S. Geological Survey Publishing Service Center, Denver, Colorado

USGS Publishing Staff

Edited by Jon W. Raese

Report approved by Keith L. Lucey

Cover designed by Suzanne C. Roberts

Report prepared by Margo L. Johnson

Technical Reviews

Vince Matri, U.S. Environmental Protection Agency

Gregory K. Brown, USGS, Branch of Regional Research

Mark R. Burkhardt, USGS, National Water Quality Laboratory

James A. Lewis, USGS, National Water Quality Laboratory

Peter F. Rogerson, USGS, Office of Water Quality

Donald H. Wilkison, USGS, Missouri Water Science Center

For more information concerning the research in this report, contact:

National Water Quality Laboratory, Chief

U.S. Geological Survey

P.O. Box 25046, MS 407

Denver Federal Center

Denver, Colorado 80225-0046

http://nwql.cr.usgs.gov
Zaugg and others—DETERMINATION OF WASTEWATER COMPOUNDS IN WHOLE WATER—Techniques and Methods 5–B4

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