Methods of Analysis by the U.S. Geological Survey

National Water Quality Laboratory—Determination of 

Wastewater Compounds by Polystyrene-Divinylbenzene 

Solid-Phase Extraction and Capillary-Column Gas 

Chromatography/Mass Spectrometry

Water-Resources Investigations Report 01-4186 

 

U.S. Department of the Interior 

U.S. Geological Survey
Methods of Analysis by the U.S. Geological Survey 

National Water Quality Laboratory—Determination of 

Wastewater Compounds by Polystyrene-Divinylbenzene 

Solid-Phase Extraction and Capillary-Column Gas 

Chromatography/Mass Spectrometry

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

Larry B. Barber, and Mark R. Burkhardt 

_______________________________________________________

U.S. GEOLOGICAL SURVEY

Water-Resources Investigations Report 01-4186 

Denver, Colorado 

2002
U.S. Department of the Interior

Gale A. Norton, Secretary

U.S. Geological Survey

Charles G. Groat, Director

The use of trade, product, or firm names in this report is for descriptive 

purposes only and does not imply endorsement by the U.S. Government. 

For additional information write to: Copies of this report can be purchased from: 

Chief, National Water Quality Laboratory U.S. Geological Survey

U.S. Geological Survey Branch of Information Services

Box 25046, Mail Stop 407 Box 25286 

Federal Center Federal Center

Denver, CO 80225-0046 Denver, CO 80225-0286 
CONTENTS 

Abstract 1 

Introduction 1 

Analytical method 3 

1. Scope and application 3 

2. Summary of method 7 

3. Interferences 8 

4. Apparatus and instrumentation 8 

5. Reagents and consumable materials 9 

6. Standards 10 

7. Procedure 12 

8. Safety precautions and waste disposal 16 

9. Gas chromatograph/mass spectrometer performance 17 

9.1 Gas chromatograph performance evaluation 17 

9.2 Mass spectrometer performance evaluation 17 

10. Calibration 18 

11. Quality assurance and quality control 18 

12. Calculation of results 23 

12.1 Qualitative identification 23 

12.2 Quantitation 23 

12.3 Reporting results 24 

Method performance 25 

Sample preservation and holding times 33 

Summary 33 

References cited 34 

FIGURES 

1. Graph showing manual integration of the extracted ion profile for the 

quantitation ion (mass-to-charge ratio 135) of para-nonylphenol from 

 the 2-nanogram-per-microliter calibration solution for the wastewater method 10 

2. Manifold assembly cap on 20-liter carboy for collecting extracted sample

water from the wastewater method 14 

3. Solid-phase extraction sample assembly for the wastewater method 15 

4. Solid-phase cartridge elution apparatus for the wastewater method 15 

TABLES 

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

parameter/method codes, and possible compound uses 4 

2. Wastewater method compound retention time, quantitation ion, 

confirmation ions, surrogate compounds, and internal standard 

reference compound 19 

3. Wastewater method compounds detected in unfortified reagent-water, 

ground-water, and surface-water samples 26

Contents iii
4. 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 27 

5. Wastewater method initial detection limits calculated from the recovery

variability data reported in table 4 using the eight replicate reagent-water 

samples with compound concentrations that range from 0.5 to 80 micrograms 

per liter 31 

iv Contents 
CONVERSION FACTORS 

Multiply By To obtain

Length

centimeter (cm) 3.94 x 10-1 inch 

micrometer (μm) 3.94 x 10-5 inch 

millimeter (mm) 3.94 x 10-2 inch 

meter (m) 3.94 x 10-1 inch 

Mass 

gram (g) 3.53 x 10-2 ounce, avoirdupois 

kilogram (kg) 3.53 x 10-1 ounce, avoirdupois 

microgram (μg) 3.53 x 10-8 ounce, avoirdupois 

milligram (mg) 3.53 x 10-5 ounce, avoirdupois 

milliliter per minute (mL/min) 3.38 x 10-2 ounce per minute 

nanogram (ng) 3.53 x 10-11 ounce, avoirdupois 

Volume

liter (L) 2.64 x 10-1 gallon 

microliter (μL) 2.64 x 10-7 gallon 

milliliter (mL) 2.64 x 10-4 gallon 

Degree Celsius (oC) may be converted to degree Fahrenheit (oF) by using the following

equation: 

oF = 9/5 (oC) + 32. 

ACRONYMS AND ABBREVIATIONS

in. inch 

min minute

mg/kg milligram per kilogram

mg/mL milligram per milliliter

μg/L microgram per liter

ng/μL nanogram per microliter

AHTM acetyl-hexamethyl-tetrahydronaphthalene 

AP alkylphenol 

APEC alkylphenol ethoxycarboxylate 

APEO alkylphenol polyethoxylate 

CAS Chemical Abstracts Service 

CCV continuing calibration verification solution 

DCM dichloromethane

CLLE continuous liquid-liquid extraction 

EE diethyl ether 

ETFE ethylenetetrafluoroethylene 

GC gas chromatograph 

GCC glass bottle, amber

GC/MS gas chromatography/mass spectrometry

Contents v
HHCB hexahydrohexamethyl-cyclopentabenzopyran 

K-D Kuderna-Danish 

LT–MDL long-term method detection level 

MDL method detection limit 

MRL minimum reporting level 

MS mass spectrometry

m/z mass-to-charge ratio 

N-Evap nitrogen evaporative concentrator 

NP nonylphenol 

NPEO nonylphenol ethoxylate 

NWQL National Water Quality Laboratory

N/A not applicable 

OPEO octylphenol ethoxylate 

PAH polycyclicaromatic hydrocarbon 

PCB polychlorinated biphenyl 

PSDVB polystyrene-divinylbenzene 

QA/QC quality assurance and quality control 

RSD relative standard deviation 

SPE solid-phase extraction 

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey

GLOSSARY

Continuing calibration verification – The continuing calibration verification (CCV) is a 

calibration standard that 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. 

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. 

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 variability introduced by multiple 

instruments, multiple analysts, and multiple calibrations from 6 to 12 months (Childress and others, 

1999). 

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, 1997). 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 variability and establish more realistic minimum reporting levels.

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

that may be reliably reported by using a given analytical method (Timme, 1995). 

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. 

Surrogate – A compound not expected to be found in any environmental sample, which is 

added to every sample in a known amount prior to sample processing. The surrogate is used to 

monitor method performance for each sample. 

Contents vii
Methods of Analysis by the U.S. Geological Survey 

National Water Quality Laboratory—Determination of Wastewater 

Compounds by Polystryrene-Divinylbenzene Solid-Phase Extraction 

and Capillary-Column Gas Chromatography/Mass Spectrometry

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

Mark R. Burkhardt

Abstract

A method for the determination of 67 

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 in wastewater 

on aquatic organisms. This method also may

be useful for evaluating the impact of 

combined sanitary and storm-sewer overflow 

on the water quality of urban streams. The

method focuses on the determination of 

compounds that are an indicator of 

wastewater or that have been chosen on the 

basis of their endocrine-disrupting potential or 

toxicity. These compounds include the 

alkylphenol ethoxylate nonionic surfactants 

and their degradates, food additives, 

fragrances, antioxidants, flame retardants, 

plasticizers, industrial solvents, disinfectants, 

fecal sterols, polycyclicaromatic

hydrocarbons, and high-use domestic 

pesticides. 

Water samples are filtered to remove

suspended particulate matter and then are

extracted by vacuum through disposable 

solid-phase cartridges that contain 

polystyrene-divinylbenzene resin. Cartridges 

are dried with nitrogen gas, and then sorbed 

compounds are eluted with dichloromethanediethyl ether (4:1) and determined by

capillary-column gas chromatography/mass 

spectrometry. Recoveries in reagent-water 

samples fortified at 4 micrograms per liter

averaged 74 percent ± 7 percent relative 

standard deviation for all method compounds. 

Initial method detection limits for singlecomponent compounds (excluding hormones 

and sterols) averaged 0.15 microgram per 

liter. Samples are preserved by filtration, the 

addition of 60 grams NaCl, and storage at 4 

degrees Celsius. The laboratory has 

established a sample-holding time (prior to 

sample extraction) of 14 days from the date of 

sample collection until a statistically accepted 

method can be used to determine the 

effectiveness of these sample-preservation 

procedures. 

INTRODUCTION 

Industrial and domestic waste must be 

managed effectively to meet the challenges of 

increasing population, stringent regulatory

requirements, and aging wastewater-treatment

facilities in the United States. Specific

analytical methods are available to monitor 

chemical compounds in wastewater to meet 

these challenges. Many compounds are 

regulated by the U.S. Environmental 

Protection Agency (USEPA), and appropriate 

analytical methods generally are available 

(U.S. Environmental Protection Agency, 

1995) to monitor them in industrial wastes or 

in discharge from wastewater-treatment

facilities. However, because of the complexity

of the sample matrix, specific analytical

methods are required to determine polar and 

nonpolar organic compounds that might

INTRODUCTION 1
affect water quality. Other compounds 

known to be toxic to aquatic life currently

are unregulated even though some, such as 

nonylphenol ethoxylates (NPEOs), are on 

the USEPA Toxic Substance Control Act 

Priority Testing List (U.S. Environmental 

Protection Agency, 1996). To meet some of 

the challenges of assessing the effect of 

wastewater discharge on water quality, the

U.S. Geological Survey (USGS) National 

Water Quality Laboratory (NWQL) and 

National Research Program have developed 

an analytical method that uses representative 

compounds from various chemical classes to 

monitor unregulated and regulated 

contaminants. 

Traditional methods for determining

organic compounds in natural-water samples 

generally are optimized for one or two 

classes of compounds and use liquid-liquid 

extraction with an organic solvent followed 

by analysis with gas chromatography (GC) 

and nitrogen-phosphorus, electron-capture, 

or mass spectrometry (MS) detection. 

Analytical methods that use solid-phase 

extraction (SPE) as an alternative to liquidliquid extraction have been implemented at 

the NWQL for the determination of

pesticides in water (Lindley and others, 

1996; Werner and others, 1996; Furlong and 

others, 2001; Sandstrom and others, 1992 

and 2001; Zaugg and others, 1995). These 

SPE methods are attractive because they are 

rapid, efficient, use much less solvent than 

liquid-liquid extraction, and, consequently, 

are more affordable and produce less toxic 

waste. 

This report describes a method for 

determining a broad range of wastewater 

compounds in filtered natural-water 

samples. It is rapid and efficient compared 

to older USGS methods (Wershaw and 

others, 1987) and was developed potentially

to replace or augment separatory-funnel or 

continuous liquid-liquid extraction (CLLE) 

sample preparation techniques in use at the 

NWQL. The method supplements other 

methods of the USGS for the determination 

of organic substances in water that have 

been described previously by Wershaw and 

others (1987) and by Fishman (1993). A 

prototype wastewater method that uses 

CLLE instead of SPE for sample preparation 

was implemented at the NWQL in October

1998 on a custom basis (Brown and others, 

1999). A comparison of results for over 30 

environmental samples between the CLLE

and the SPE methods demonstrated, as 

expected, that concentrations for 

hydrophobic (low-water solubility) 

compounds were much less (up to 400 

percent) when determined by SPE because 

of sample filtration, whereas results for 

hydrophilic compounds were identical. 

Application of this SPE method will provide 

data for dissolved wastewater compounds to 

supplement available whole-water CLLE

data used to identify urban sanitary-sewer 

and storm-sewer problems (Wilkison and 

others, 2000), as well as emerging

contaminants in concentrated animal feedlot 

runoff for the USGS’s Toxic Substances 

Hydrology Program. This new SPE method 

has been used at the NWQL as a conditional 

method since January 1, 2001. It officially

was approved and implemented at the

NWQL on June 27, 2001. 

This report provides a detailed 

description of all aspects of the method, 

including the equipment, reagents, sampling

protocol, instrument calibration, and SPE 

procedure required for sample analysis. 

Method performance (bias and variability) 

and estimated method detection limits

1

 for 

67 compounds are presented. 

 

1

Words in boldface are defined in Glossary.

2 Determination of Wastewater Compounds
The scope of the study includes 

determination of method performance in 

reagent-water and filtered natural-water

samples from ground water obtained in a 

domestic well near Evergreen, Colorado, 

and a surface-water sample obtained from

the Platte River near Confluence Park in

Denver, Colorado. 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, 1997). 

ANALYTICAL METHOD

Organic Compounds and Parameter 

Codes: Pesticides and degradates, 

filtered water, gas chromatography/ 

mass spectrometry, O-1433-01 (see 

table 1)

1. Scope and Application 

This manual method is suitable for the 

determination of microgram-per-liter

concentrations of compounds in filtered 

wastewater and natural-water samples. The

method is applicable to compounds that are 

efficiently partitioned from the water phase

onto the polystyrene-divinylbenzene 

(PSDVB) organic phase, and are sufficiently

volatile and thermally stable for gas 

chromatography. Samples are filtered to 

remove suspended particulate matter, so this 

method is suitable only for dissolved-phase 

compounds. 

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 environmental implications of 

nonionic surfactants is unknown, thus, 

making it important to determine as many

compounds and their degradates as possible 

in this chemical class (Barber and others, 

2000). The alkylphenol polyethoxylates 

(APEOs) are nonionic surfactants, and those 

of particular interest consist of the 

nonylphenol polyethoxylates (NPEOs) and 

the octylphenol polyethoxylates (OPEOs). 

The APEO surfactants generally contain up 

to about 20 ethoxy-units. These compounds 

eventually degrade in the environment to

form alkylphenol diethoxylates (APEO2), 

which are represented in the method by

OPEO2 and NPEO2 (total), and monoethoxylates, which are represented in the 

method by OPEO1 (see table 1). All of these 

compounds are known for their estrogenic 

activity in aquatic organisms (Jobling and 

Sumpter, 1993). The alkylphenol monoand diethoxylates degrade further to form

alkylphenols (APs) and alkylphenol 

ethoxycarboxylates (APEC). The APs are 

represented in the method (see table 1) by

para-nonylphenol, 4-cumylphenol, 4-noctylphenol, and 4-tert-octylphenol, and are 

less estrogenic than their ethoxylated 

counterparts. Unfortunately, the APECs are 

too polar to be analyzed by this method. A 

comprehensive analysis of this family of

compounds (APEOs and their degradates) is 

needed to understand their fate and transport 

in the environment. The hormonal 

disrupting effect of this compound class on 

aquatic life has been documented, and is 

exemplified by a study with rainbow trout: 

male fish exposed to concentrations of APs 

greater than about 20 μg/L produced 

vitellogenin, a yolk protein normally found 

only in females (Jobling and Sumpter,

1993). 

ANALYTICAL METHOD 3
Table 1. Wastewater method compound names, endocrine-disrupting potential, 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; WW, wastewater; Manuf, manufacturing; %, percent; >, greater 

than; CP, combustion product; NA, not available; UV, ultraviolet; --, no data; PAH, polycyclicaromatic hydrocarbon] 

Compound name EDP1 CAS 

number

Parameter/

method 

codes2

Possible compound uses or sources3

1,4-Dichlorobenzene

4 S 106-46-7 34572A Moth repellant, fumigant, deodorant

17beta-Estradiol K 50-28-2 62053A Estrogen replacement therapy, estrogen

metabolite 

1-Methylnaphthalene -- 90-12-0 62054A 2-5% of gasoline, diesel fuel, or crude oil

2,6-Dimethylnaphthalene

4 -- 581-42-0 62055A Present in diesel/kerosene (trace in gasoline) 

2-Methylnaphthalene -- 91-57-6 62056A 2-5% of gasoline, diesel fuel, or crude oil

3beta-Coprostanol -- 360-68-9 62057A Carnivore fecal indicator 

3-Methyl-1H-indole (skatol) -- 83-34-1 62058A Fragrance, stench in feces and coal tar 

3-tert-Butyl-4-hydroxyanisole (BHA) K 25013-16-5 62059A Antioxidant, general preservative 

4-Cumylphenol K 599-64-4 62060A Nonionic detergent metabolite 

4-n-Octylphenol K 1806-26-4 62061A Nonionic detergent metabolite 

4-tert-Octylphenol K 140-66-9 62062A Nonionic detergent metabolite 

5-Methyl-1H-benzotriazole -- 136-85-6 62063A Antioxidant in antifreeze and deicers

Acetophenone -- 98-86-2 62064A Fragrance in detergent and tobacco, flavor in

beverages 

Acetyl-hexamethyl-tetrahydronaphthalene (AHTN) -- 21145-77-7 62065A Musk fragrance (widespread usage) persistent in

ground water

Anthracene

4 -- 120-12-7 34221A Wood preservative, component of tar, diesel, or 

crude oil, CP

Anthraquinone

4 -- 84-65-1 62066A Manuf dye/textiles, seed treatment, bird repellant

Benzo[a]pyrene

4 K 50-32-8 34248A Regulated PAH, used in cancer research, CP

Benzophenone S 119-61-9 62067A Fixative for perfumes and soaps

beta-Sitosterol -- 83-46-5 62068A Plant sterol

beta-Stigmastanol -- 19466-47-8 62086A Plant sterol

Bisphenol A K 80-05-7 62069A Manuf polycarbonate resins, antioxidant, FR

Bromacil4 -- 314-40-9 04029E H (GUP), >80% noncrop usage on grass/brush

Bromoform4 -- 75-25-2 34288A WW ozination byproduct, military/explosives

Caffeine

4 -- 58-08-2 50305B Beverages, diuretic, very mobile/biodegradable

Camphor -- 76-22-2 62070A Flavor, odorant, ointments

Carbaryl

4

 K 63-25-2 82680F I, crop and garden uses, low persistence 

Carbazole -- 86-74-8 62071A I, Manuf dyes, explosives, and lubricants

Chlorpyrifos

4 K 2921-88-2 38933F I, domestic pest and termite control (domestic

use restricted as of 2001) 

Cholesterol -- 57-88-5 62072A Often a fecal indicator, also a plant sterol 

Cotinine -- 486-56-6 62005A Primary nicotine metabolite 

Diazinon4 K 333-41-5 39572F I, > 40% nonagricultural usage, ants, flies

Dichlorvos

4

S 62-73-7 38775B I, pet collars, flies, also a degradate of naled or 

trichlofon

d-Limonene -- 5989-27-5 62073A F, antimicrobial, antiviral, fragrance in aerosols

Equilenin -- 517-09-9 62074A Hormone replacement therapy drug

Estrone -- 53-16-7 62484A Biogenic hormone

Ethynyl estradiol K 57-63-6 62052A Oral contraceptive 

4 Determination of Wastewater Compounds
Table 1. Wastewater method compound names, endocrine-disrupting potential, parameter/method codes, and 

possible compound uses—Continued 

Compound name EDP1 CAS 

number

Parameter/

method 

codes2

Possible compound uses or sources3

Fluoranthene4 -- 206-44-0 34377A Component of coal tar and asphalt (only traces

in gasoline or diesel fuel), CP

Hexahydrohexamethylcyclopentabenzopyran (HHCB)-- 1222-05-5 62075A Musk fragrance (widespread usage) persistent

in ground water

Indole -- 120-72-9 62076A Pesticide inert ingredient, fragrance in coffee 

Isoborneol -- 124-76-5 62077A Fragrance in perfumery, in disinfectants

Isophorone

4 -- 78-59-1 34409A Solvent for lacquer, plastic, oil, silicon, resin

Isopropylbenzene (cumene) -- 98-82-8 62078A Manuf phenol/acetone, fuels and paint thinner 

Isoquinoline

4 -- 119-65-3 62079A Flavors and fragrances

Menthol -- 89-78-1 62080A Cigarettes, cough drops, liniment, mouthwash 

Metalaxyl

4 -- 57837-19-1 50359B H, F (GUP), mildew, blight, pathogens,

golf/turf

Methyl salicylate -- 119-36-8 62081A Liniment, food, beverage, UV-absorbing lotion

Metolachlor

4 -- 51218-45-2 39415F H (GUP), indicator of agricultural drainage

N,N-diethyl-meta-toluamide (Deet) -- 134-62-3 62082A I, urban uses, mosquito repellent

Naphthalene

4 -- 91-20-3 34443A Fumigant, moth repellent, major component 

(about 10%) of gasoline 

Nonylphenol, diethoxy- (total, NPEO2) K NA 62083A Nonionic detergent metabolite 

Octylphenol, diethoxy- (OPEO2) K NA 61705A Nonionic detergent metabolite 

Octylphenol, monoethoxy- (OPEO1) K NA 61706A Nonionic detergent metabolite 

para-Cresol

4 S 106-44-5 62084A Wood preservative

para-Nonylphenol (total) K 84852-15-3 62085A Nonionic detergent metabolite 

Pentachlorophenol4 S 87-86-5 34459A H, F, wood preservative, termite control

Phenanthrene

4 -- 85-01-8 34462A Manuf explosives, component of tar, diesel

fuel, or crude oil, CP

Phenol4 -- 108-95-2 34466A Disinfectant, manuf several products, leachate 

Prometon4 -- 1610-18-0 04037F H (noncrop only), applied prior to blacktop 

Pyrene

4 -- 129-00-0 34470A Component of coal tar and asphalt (only traces

in gasoline or diesel fuel), CP

Tetrachloroethylene

4 -- 127-18-4 34476A Solvent, degreaser, veterinary anthelmintic

Tri(2-chloroethyl) phosphate S 115-96-8 62087A Plasticizer, flame retardant

Tri(dichloroisopropyl) phosphate S 13674-87-8 62088A Flame retardant

Tributyl phosphate -- 126-73-8 62089A Antifoaming agent, flame retardant

Triclosan S 3380-34-5 62090A Disinfectant, antimicrobial (concern for 

acquired microbial resistance) 

Triethyl citrate (ethyl citrate) -- 77-93-0 62091A Cosmetics, pharmaceuticals

Triphenyl phosphate -- 115-86-6 62092A Plasticizer, resin, wax, finish, roofing paper, FR

Tri(2-butoxyethyl) phosphate -- 78-51-3 62093A Flame retardant

1

World Wildlife Fund Canada (1999). 2

Parameter codes define sample constituent variables linked to compound analytical results stored in the National Water 

Information System data base. 3

ChemFinder Webserver (2001); National Toxicology Program (2001); National Institute of Standards and Technology

(2001); Spectrum Laboratories, Inc. (2001); HealthCentral.com (2001); EXtension TOXicology NETwork (2001). 4

Compound determined by at least one other method at the National Water Quality Laboratory.

ANALYTICAL METHOD 5
The APEOs also are degraded during

sewage treatment (Geiger and others, 1984). 

Therefore, sewage-treatment plant effluents 

can be major point sources for the 

hydrophobic APs and APECs. The more polar 

APECs cannot be determined by this method 

because they thermally degrade on the GC, 

which is unfortunate, because they have a 

higher potential for leaching into ground 

water than do APs. The volatility and 

availability of reference standard APEOs limit 

the method to the determination of OPEO1, 

OPEO2, and NPEO2, even though APEOs 

typically contain up to about 20 ethoxy-units. 

Alkylphenols exhibit long-term 

persistence and bioaccumulation potential in

the environment. Research on the distribution 

of alkylphenols in rivers and estuaries of 

England (Blackburn and Waldock, 1995) 

suggests that the majority of APs entering

rivers will sorb onto particulate matter and 

ultimately accumulate in sediments. Water 

solubility and sediment/water partition data

for these compounds indicate that if the total 

concentration of APs in the particulate plus 

dissolved phases exceeds about 20 μg/L, then

the majority of the APs are present in the

particulate phase, and, at concentrations less 

than 20 μg/L, the higher concentration 

remains in solution (Blackburn and Waldock, 

1995). Various investigators have reported 

concentrations of APs ranging from 10 to 100 

mg/kg in sediment samples near sewagetreatment plants. Hale and others (2000) 

detected concentrations of NPs at more than 

50 mg/kg in some sediment samples near a

treatment plant that had ceased operation 20 

years earlier. Complementary methods are 

needed at the NWQL to determine APs and 

other hydrophobic compounds associated 

with suspended particles and sediments 

because this filtered-water method is 

applicable to the determination of compounds 

in the dissolved phase. 

Detection of caffeine can be an 

important indicator of wastewater 

contamination in surface-water samples, 

however, caffeine is not persistent in the 

environment because of rapid degradation by

bacteria. Caffeine usually will not be detected

in ground-water samples even though its 

detection might be expected on the basis of 

surface-water concentrations (Seiler and 

others, 1999). 

Coprostanol has been a traditional 

indicator of sewage contamination, because it 

is produced almost exclusively in the 

digestive tract of higher mammals (humans, 

pigs, and cats) and often correlates with the

presence of other sewage-derived pollutants, 

such as pathogens, toxic metals, organic 

compounds, and hormones. Coprostanol is 

persistent in the environment under anaerobic 

conditions and is degraded by aerobic bacteria 

(Shigenaka and Price, 1988). Although the 

method is not particularly sensitive for fecal 

sterols, mainly because compound 

derivatization is not used, their presence with 

polychlorinated-biphenyls (PCBs), 

polycyclicaromatic hydrocarbons (PAHs), 

nonylphenols (NPs) and other hydrophobic, 

environmentally “resistant” compounds might 

be useful to determine sources of 

contaminants associated with suspended 

sediment. The need to detect sterols in the 

suspended-sediment phase also indicates the 

need to implement a complementary

suspended-phase method at the NWQL. 

The detection of beta-stigmastanol and 

beta-sitosterol in the same sample generally

indicates a substantial contribution from plant 

sterols. The detection of beta-coprostanol 

generally is associated with higher mammals. 

Cholesterol, on the other hand, can originate 

from plant and animal sources. 

Other important compounds determined 

by this method are organochlorine flame 

retardants, tri(2-chloroethyl) phosphate (Fyrol 

6 Determination of Wastewater Compounds
CEF TM) and tri(dichloroisopropyl) phosphate 

(Fyrol FR 2 TM). These compounds have been 

detected frequently during custom 

implementation of this method at the NWQL, 

even though they have been replaced in most 

industrial applications by the polybrominated, 

flame retardants because they are less toxic if

they ignite. The polybrominated

diphenylethers and other polybrominated 

flame-retarding chemicals have the potential

to become a widespread, persistent 

environmental problem similar to the PCBs

(de Boer and others, 1998) because they have 

been used extensively in many common 

products. Moreover, these compounds are 

difficult to analyze because of their extremely

low water solubilities and high molecular

weights. The NWQL currently (2002) is 

evaluating several specific brominated fireretardant compounds to include in this 

method because of their environmental 

significance. Generally, water treatment is not 

sufficient to remove halogenated flame 

retardants, and the FyrolsTM have been 

detected at 0.5 to 3 μg/L in samples before

and after wastewater treatment (van Stee and 

others, 1999). 

Triclosan is another chlorinated 

compound that is widely used as an 

antimicrobial agent in household products, 

such as hand soaps, shampoos, antiperspirant 

products, and toothpaste. In contrast to the 

halogenated flame retardant compounds, van 

Stee and others (1999) reported that about 80 

percent of triclosan is removed during water 

treatment. There is recent evidence (McMurry

and others, 1998) that bacteria might become 

resistant to triclosan, which is cause for

concern, especially considering its widespread 

use. Triclosan also has been detected in 

greater than 25 percent of surface-water 

samples analyzed by using the CLLE

wastewater method at the NWQL. 

Several fragrance compounds have been 

included in the wastewater method because of 

their widespread use in numerous domestic 

products and detergents. Use of the synthetic 

polycyclic musks, acetyl-hexamethyltetrahydro-naphthalene (AHTN) and 

hexahydrohexamethyl-cyclopentabenzopyran

(HHCB), has surpassed that of the once used 

(prior to 1978) and highly controversial nitromusks (Fromme and others, 1999). There also 

is recent evidence (Franke and others, 1999) 

concerning bioaccumulation and potential

toxicity of the synthetic polycyclic musk 

compounds in aquatic life. Treatment of 

wastewater in a survey of 25 sewagetreatment plants near the Ruhr River in 

Germany indicated that primary water 

treatment only reduces the concentration of 

polycyclic musk compounds by about 30 

percent, whereas secondary water treatment 

removes about 85 percent (Simonich and 

others, 2000). AHTN and HHCB have been 

detected in wastewater samples of the United 

States (Standley and others, 2000), and 

because of their persistence and widespread 

use, are expected to be more reliable than 

caffeine for correlating and evaluating

potential wastewater problems. The synthetic 

musks tentatively were identified in about 5 

percent of wastewater samples (both groundwater and surface-water) at the NWQL, but

until standard compounds were obtained, 

positive identification was impossible. These

compounds should prove to be invaluable for 

hydrologic studies because of their mobility, 

widespread use, and recalcitrant properties. 

2. Summary of Method 

Water samples are filtered in the field 

by using glass-fiber filters (0.7-μm nominal 

pore diameter) to remove suspended 

particulate matter (Sandstrom, 1995) before 

they are sent to the NWQL. Filtered 1-L

water samples are extracted with disposable, 

polypropylene SPE cartridges that contain 

polystyrene-divinylbenzene phase. The SPE 

ANALYTICAL METHOD 7
cartridges are dried thoroughly by using a 

flow rate of 2 L/min of pressurized nitrogen, 

which takes about 45 minutes. 

After the SPE cartridges have dried, the

sample bottles are rinsed thoroughly with a

mixture of 15 mL dichloromethane (DCM)

and diethyl ether (EE), at 4:1. The DCM-EE 

rinsate also is used to elute sorbed compounds 

from the corresponding SPE cartridges. Next, 

the extract is evaporated by using a gentle

stream of nitrogen to a final volume of 0.4 

mL and then is transferred to an autosampler

vial that contains a 400-L glass insert. 

Finally, the concentrated extracts are determined by capillary-column gas chromatography/mass spectrometry (GC/MS).

3. Interferences 

Organic compounds that have gaschromatographic retention times and 

characteristic ions with a mass identical to 

those of the compounds of interest might 

interfere, and because of the complex nature 

of wastewater, there are often unknown 

compounds that interfere. 

Phthalates and preservatives (BHT and 

related compounds) in the cartridge material 

often contribute to low-concentration 

contamination. Samples, collection 

equipment, or SPE cartridges that are handled 

improperly also might become contaminated 

with soaps, caffeine, and fragrances. 

Precautions are necessary to avoid 

contamination during sample collection (see

section 7.1, Field Sampling) because some 

method compounds are contained in 

commonly used products. Sample-collection 

protocols and cleaning procedures for field 

equipment (Radke and others, 1998a and 

1998b) need to be followed to reduce 

interference. 

4. Apparatus and Instrumentation 

4.1 Cleaning and elution module — For 

cleaning and preparation of SPE cartridges, 

Supelco, Inc., Visiprep Solid-Phase 

Extraction Vacuum Manifold or equivalent. 

4.2 O-rings — Viton, 3.25 cm (1 9/32 

in.) outside diameter by 2.62 cm (1 1/32 in.) 

inside diameter. 

4.3 Teflon tubing — 0.317 cm (1⁄8 in.) 

outside diameter by about 1 m (39.4 in.) 

length, for vacuum pumping samples through 

SPE cartridges. 

4.4 Extraction caps — Teflon, 32/133 

threads to fit sample bottles and tapered 

opening to fit the Luer end of SPE cartridges. 

4.5 Luer stopcock — Teflon flowcontrol valves (on-off valves). 

4.6 Carboy — NalgeneTM, thick-walled, 

capable of maintaining 200 kPa (29 in.) 

mercury vacuum, 20-L volume, VWR 

Scientific Inc., catalog number 36494-092 or 

equivalent. 

4.7 Tefzel-ethylenetetrafluoroethylene 

(Tefzel-ETFE) female Luer connector — 1/4-

28 thread, Tefzel-ETFE union with 1/4-28 

thread; Upchurch Scientific or equivalent. 

4.8 Bottle-top solvent dispensers — 

Adjustable from 2 to 5 mL and 5 to 25 mL; 

Brinkman Dispensette, Van Waters & Rogers 

(VWR) Scientific or equivalent. 

4.9 25-mL graduated Kuderna-Danish 

receivers (concentrator tubes) — Kontes part 

number 570081-2526 or equivalent. 

4.10 Solvent reservoirs — Polypropylene, 

30-mL column. 

4.11 Adapters — Teflon, connects SPE 

cartridge barrel to male Luer fitting.

8 Determination of Wastewater Compounds
4.12 Vacuum pump — Any vacuum 

pump with sufficient capacity to maintain a

vacuum of 200 kPa (29 in.) of mercury.

4.13 Analytical balances — Balance for 

samples accurately weighs 1,400 ±1 g.

Balance for standard preparation accurately

weighs 10 ±0.01 mg. 

4.14 Nitrogen evaporative concentrator

— Organomation N-Evap or equivalent. 

4.15 Micropipets — 50-, 100-, and 200-

μL fixed-volume and variable-volume 

micropipets with disposable glass bores; 

VWR Scientific or equivalent. 

4.16 Glass syringes — 10-, 50-, 100-, 

250-, and 500-μL volume. 

4.17 Fused-silica capillary column — 

Any column that provides adequate 

resolution, capacity, accuracy, and precision. 

A 30-m by 0.25-mm inside diameter fusedsilica capillary column coated with a 0.50-μm 

bonded film of 5-percent polyphenylmethylsilicone; Hewlett-Packard HP-trace 

analysis column or equivalent. 

4.18 GC/MS bench-top system — 

Agilent Technologies, Model 5973 or 

equivalent. 

4.18.1 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 9 

mL/min; injection volume, 2 μL, splitless 

injection. 

4.18.2 Recommended MS 

conditions. Source, 200°C; 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 Helium carrier gas (99.999 

percent). 

5.2 Sodium sulfate drying cartridges, 

Luer-Lok, 3-mL volume, 2.5 g Na2SO4 ; 

International Sorbent Technology, Ltd., 

catalog number 802-0250-M or equivalent. 

5.3 Nitrogen gas, for evaporation, 

ultrapure. 

5.4 Sodium chloride, reagent grade, 

baked 8 hours at 450°C, VWR or equivalent. 

5.5 Acetic acid, glacial, reagent grade, 

Sigma Scientific Inc. or equivalent. 

5.6 Sodium acetate, anhydrous, reagent 

grade, Sigma Scientific Inc. or equivalent. 

5.7 SPE cartridges, 6-mL barrel, packed 

with 500 mg of PSDVB; Oasis polystyrenedivinylbenzene packing material, Waters Inc., 

catalog number 186000115 or equivalent. 

5.8 Glass-fiber filters, 0.7-μm nominal 

pore diameter (GF/F grade), baked at 450°C 

for 2 hours; Whatman, Inc. or equivalent. 

5.9 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. 

5.10 Solvents, dichloromethane (DCM) 

and diethyl ether (EE); B&J Brand pesticide

grade or equivalent. 

5.11 Solution 2000 water, prepared by

Solution 2000 purification system or 

equivalent. 

ANALYTICAL METHOD 9
5.12 Acetic acid/sodium acetate buffer, 

pH 4.3 (dilute 30 g acetic acid and 15 g

sodium acetate in 1 L reagent water). 

5.13 Potassium phosphate buffer, pH 

7.0 (dilute 30 g dipotassium hydrogen 

phosphate and 20 g potassium dihydrogen 

phosphate in 1 L reagent water). 

5.14 Dichloromethane: diethyl ether 

mixture, 80:20 volume per volume. 

6. Standards 

6.1 Stock standard solutions at 10,000 

ng/μL —Obtain method compounds and 

surrogate compounds at greater than 99-

percent purity if available 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 (para-NP, OPEO1, and OPEO2) 

are only available in technical mixtures. For 

the technical grade nonylphenol (NP) mixture 

(ortho-NP and para-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, Calibration). 

The contribution of the paranonylphenols (total) in the NP technical 

mixture is determined by manually integrating

the sum of the peaks within the expected 

retention time window (fig. 1) for the 

quantitation ion (m/z 135, see table 2, Section 

10). Also, note how the qualification ion 

profiles (m/z 220 and 107) must coincide in a 

similar pattern with ion 135. The ortho-NPs

elute prior to the para-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 paraNP isomers (total), which is 16 times the

concentration of the stock standard solutions 

of the single-component compounds in the 

method. To prepare this stock standard 

solution, calculate the necessary amount of 

the technical mixture needed (about 180 

mg/mL). 

Figure 1. Manual integration of the extracted ion 

profile for the quantitation ion (mass-to-charge ratio 

135) of para-nonylphenol from the 2 nanogram-permicroliter calibration solution for the wastewater

method.

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 

10 Determination of Wastewater Compounds
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. 

During development of the custom 

wastewater method, NPEO1 and NPEO2 only

were available in a technical mixture. A 

source of NPEO2 standard has been identified 

and evaluated for purity for inclusion in this 

method. Standards of the NPEO1 compounds 

also recently have become available and 

currently (2002) are being evaluated for 

composition and purity. It is anticipated that 

they will be included in the method in 2002. 

6.2 Intermediate method compound 

standard solution at 100 ng/μL — Prepare a 

mixed stock standard solution at 100 ng/μL

that contains each method compound stock 

standard solution at 100 ng/μL (not 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 spike 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, 

Calibration) in a 50-mL volumetric flask and 

dilute with methanol. Add 100 μL of the 20- 

ng/μL standard solution to a 1-L sample to 

obtain a surrogate spike solution of 2.0 μg/L. 

A surrogate concentration of 5.0 ng/μL is 

expected in a 0.40-mL 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 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 Compound spike solution at 

20 ng/μL — Dilute 2.0 mL of the 

intermediate method compound standard 

solution at 100 ng/μL in a 10-mL volumetric 

flask with methanol. 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 0.40-mL 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, 

Calibration) are obtained from Supelco 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 PAH procedural 

internal standard solution at 100 ng/μL in a 

0.40-mL 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 singlecomponent compounds in the calibration 

mixture that respond poorly by GC/MS 

(cholesterol, 3beta-coprostanol, 17betaestradiol, beta-stigmastanol, beta-stitosterol, 

ethynyl estradiol, OPEO1, and 5-methyl-1Hbenzotriazole) is 4 times that of the other

single-component compounds. The 

concentration of the multicomponent 

compounds in the calibration mixture, NP 

(total) and NPEO2 (total), is 20 and 16 times, 

respectively, that of the single-component 

compounds in the calibration mixture. The 

concentration of the PAH procedural internal 

standard compounds in the calibration 

ANALYTICAL METHOD 11
mixtures 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. 

7. Procedure 

7.1 Field sampling — Collect samples 

and filter them through 0.7-μm nominal poresize diameter glass-fiber filters (Sandstrom, 

1995) into 1-L amber glass pesticide bottles 

(NWQL type GCC) before shipping at 4oC to 

the NWQL, preferably by express mail. 

Field-sampling procedures should follow 

those typically used to collect samples for

trace organic compound analyses (Hardy and 

others, 1989; Ward and Harr, 1990; Radke 

and others, 1998a, 1998b). Some of the 

compounds that are determined by this 

method are found in commonly used 

products, such as coffee, tea, cola, soap, and 

insecticide repellant. 

CAUTION: 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. The probability of

sample contamination with compounds 

determined by this method is higher than for 

other NWQL methods. For this reason, field 

blanks should be analyzed routinely by using

EM Science, Inc. Omni Solv® water to 

monitor for potential sample contamination. 

7.2 Initial sample preparation — 

Samples received at the NWQL must be 

visually examined to determine if they have

been filtered in the field, and if not, they must 

be filtered (Sandstrom, 1995). If the sample 

bottle has less than about 30 mL of headspace 

after filtration, then discard a minimum 

amount of water to provide enough headspace 

for later addition of 60 g NaCl. Weigh the 

sample and bottle and record the gross sample

weight (±1 g). After sample filtration, and the 

gross weight has been recorded, add 60 10 g

NaCl (baked) to make the sample about 1 

molar NaCl. Store filtered 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. Addition of NaCl also might 

augment sample filtration to preserve samples 

from biodegradation. 

7.3 SPE cartridge cleaning — 

Assemble the Na2SO4 drying cartridges, 

stopcocks, and SPE cartridges vertically in 

that order. Attach them to the Luer-Lok 

fittings on the vacuum/elution manifold by

twisting them clockwise to ensure that the

fittings are closed. Add 5 mL (the SPE barrel 

volume) of the elution solvent (DCM-EE, 4:1) 

to rinse the SPE and Na2SO4 drying

cartridges. Allow the solvent to drain by

gravity until the phase is completely saturated 

before applying vacuum. Then open the LuerLok fittings on the vacuum manifold by

turning them counterclockwise to allow the 

solvent to be removed from the cartridges by

vacuum. Rinse the cartridges with an 

additional 5 mL of DCM-EE (4:1) and allow 

at least 10 minutes for the vacuum to remove 

any residual solvent. Attach the

polypropylene 25-mL empty sample 

reservoirs to the Luer-Lok fittings on the

vacuum/elution manifold and clean them by

rinsing with DCM-EE (4:1).

NOTE: Unlike most other SPE phases, it is 

permissible for PSDVB to dry prior to sample

extraction, thus making it possible to prepare 

SPE cartridges up to a day in advance of 

sample extraction. 

12 Determination of Wastewater Compounds 
7.4 Sample extraction — Before the 

14-day holding time expires, obtain the

samples from the refrigerator and acidify with

3 mL of the acetic acid/sodium acetate buffer 

(pH 4.3). Prepare a laboratory reagent water 

set blank and set spike sample with each set

of samples. Add 100 μL of the surrogate 

spike solution (20 ng/μL; see section 6.3) to 

each sample, set spike, and set blank by using

a stepper syringe to make the 100-μL

additions. Fortify the spike sample with 

100 μL of the compound spike solution (20 

ng/μL; see section 6.5) by using a micropipet 

dispenser and 100-μL disposable glass bore. 

(This step results in a concentration of 2.0 

μg/L for the fortified spike compounds and 

surrogate compounds in a 1-L sample.) 

NOTE: Allow the spike and surrogate

solutions to come to room temperature, and 

then shake well before adding them to 

samples. 

Immediately prior to using the SPE 

cartridges, visually inspect by rotating them to 

ensure that there is no substantial voidvolume between the polyethylene frits and the 

PSDVB phase. 

CAUTION: Loosely packed PSDVB phase 

can cause uneven flow or channeling during

the SPE process and result in reduced 

compound recoveries. Therefore, it is 

essential to ensure that the PSDVB phase is 

packed firmly before using SPE cartridges. 

Attach the SPE cartridges to the vacuum 

lines from the waste carboy manifold 

assembly (fig. 2) by using an adapter (not 

shown). 

CAUTION: Ensure that the 20-L carboys 

have enough headspace (empty volume) 

remaining to accommodate the total water 

volume from all samples before a new sample

set is extracted, otherwise it might be difficult 

to exchange carboys during the extraction. 

Attach Teflon bottle caps to sample

bottles. Turn on the vacuum pump, and attach

the male Luer end of the SPE cartridge to the

bottle cap before inverting the sample bottles 

and placing them in the rack (fig. 3). 

Ensure that there are no leaks or sources 

of bubbles in the system. Small bubbles 

might form as the sample is pumped through 

the tubing and will slow the extraction 

process. Large air bubbles can be a problem 

if they cause uneven flow (channeling)

through the cartridge packing. One advantage 

of operating under vacuum, as opposed to 

positive pressure, is that system leaks usually

do not result in sample loss. The desired 

extraction flow-rate range (between 25 to 50 

mL/min) is obtained by loosening or

tightening the sample bottle caps, which 

might need to be done once or twice during

sample extraction. 

NOTE: Reasonable extraction times range

from 20 to 40 minutes for 1-L samples and 

correspond to flow rates between 25 and 50 

mL/min. No adverse effects on compound 

recoveries have been observed when flow 

rates are maintained in this operating range.

Pump the entire sample through the SPE 

cartridge. Unscrew the sample bottle from the

Teflon bottle cap. Place the Teflon bottle cap 

back on the rack. Add 20 mL of phosphate 

buffer (pH 7) to the cap and draw it by

vacuum through the cartridge; this buffer is 

added to remove excess salt from the 

cartridge prior to the drying step and adjusts 

the pH of the cartridge to neutral prior to the

elution step. Salt deposits formed while 

drying the SPE cartridge might lead to 

channeling and variable recovery during the 

subsequent elution step. Adjusting the pH of 

the cartridge to neutral will improve the

elution of basic compounds that otherwise 

remain partially bound to the sorbent. Turn 

off the vacuum pump after all of the buffer 

solution has passed through the cartridge. 

ANALYTICAL METHOD 13
Figure 2. Manifold assembly cap on a 20-liter carboy for collecting extracted sample water 

from the wastewater method. 

NOTE: Equipment cleanup is minimized 

because there is no possibility for sample 

cross-contamination except from the Teflon 

bottle caps, an advantage of using this 

procedure. 

7.5 Cartridge drying — Attach the 

male Luer end of the SPE cartridge to the

female Luer-Lok fitting on the gas-pressure

module of the SPE vacuum manifold. Dry the 

cartridges by using a positive pressure of

nitrogen (2 L/min through each cartridge for 

about 45 minutes) to remove all interstitial 

water. 

NOTE: The color of the PSDVB phase 

becomes lighter as it dries, and the wet/dry

boundary layer is noticeable if carefully

observed. It is important to ensure that the 

cartridge is completely dry prior to elution, or 

compound recoveries might be unacceptable. 

Remove cartridges from nitrogen gas as

soon as possible after determining that they

are dry. Excessive gas-flow time results in 

compound losses caused by volatilization. 

NOTE: Recoveries of compounds more 

volatile than naphthalene have been observed 

to decrease by about 10 to 20 percent if 

cartridges are allowed to remain under 

nitrogen gas flow beyond 20 minutes past dry.

14 Determination of Wastewater Compounds 
Figure 3. Solid-phase extraction sample assembly

for the wastewater method. 

7.6 Compound elution — The sample

bottle is rinsed with DCM-EE (4:1) to remove

hydrophobic compounds that adhere to the 

glass surface. The solvent rinse from the 

bottle then is used to elute the dry SPE 

cartridge. 

Slide the Viton O-ring over the mouth 

and just past the threads of the sample bottle. 

Screw on the Teflon bottle cap until it comes 

in contact with the Viton O-ring to form an 

air-tight seal. In a hood, add 15 mL of DCMEE (4:1) to the sample bottles through the

hole in the top of each bottle cap. Attach a 

stopcock and Na2SO4 drying cartridge to each 

bottle cap by twisting slightly clockwise to 

ensure that the Luer-Lok connections are 

secure (fig. 4). Set stopcocks in the closed 

position before rinsing sample bottles. Rinse

each bottle thoroughly by rotating the bottle, 

thus ensuring that the solvent contacts all

inside surfaces. 

Figure 4. Solid-phase cartridge elution apparatus

for the wastewater method.

NOTE: Thoroughly rinsing sample bottles is 

important because as much as 30 to 40 percent

of some hydrophobic compounds (particularly

PAHs, sterols, and organochlorine compounds) 

might adhere to glass walls. 

ANALYTICAL METHOD 15
Attach 25-mL solvent reservoirs to dried 

SPE cartridges with a Teflon adapter and 

place in 25-mL K-D receivers. Invert the 

sample bottles and place them in the elution 

rack with the male Luer end of the drying

cartridge positioned above the appropriately

labeled receiver (fig. 4). 

Add 3 mL of EE to the 25-mL solvent 

reservoir to elute the SPE cartridge and ensure 

that any residual water is removed from the

SPE sorbent prior to solvent elution with 

DCM-EE (4:1) from the bottle rinse. This first 

diethyl ether fraction is collected in the K-D 

receiver and is processed later as part of the 

sample extract. After EE has drained from the

SPE cartridge, open the stopcock and allow 

the DCM-EE (4:1) solvent rinse from the

sample bottle to drain into the solvent 

reservoir so that compounds from the SPE 

cartridge can be eluted by gravity into the 

K-D receiver (about 20 minutes). Finally, add 

another 3 mL of EE to the 25-mL solvent 

reservoir to displace any remaining DCM-EE 

(4:1) solvent from the SPE sorbent. This final 

EE fraction also is collected in the K-D 

receiver with the extract. 

NOTE: During the developmental stages of 

this method, it was observed that most 

problems with compound recovery were 

associated with difficulties in the elution step, 

usually related to incomplete removal of

water from the SPE cartridges. 

7.7 Weigh bottles — Record empty

sample bottle weights to ± 1 g.

7.8 Solvent evaporation — 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. When 

the extract volume is between 2.5 and 4 mL, 

add 20 μL of the PAH procedural internal 

standard solution (100 ng/μL) by using a 

1.0-mL stepper syringe. Remove the extracts 

when the final volume is 0.4 mL. Maintain a 

consistent flow rate by leaving the needles 

attached to the N-Evap with the flow of

nitrogen remaining unaltered. Concentrate the

DCM-EE extract at ambient temperature and 

periodically check the extract; at no time

should the extract be allowed to evaporate 

completely. Allow about 40 to 50 minutes for 

evaporation of nearly 15 mL of DCM-EE to 

0.4 L.

7.9 Vial extracts — 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 

extracts to appropriately labeled GC vials that

contain 400-L glass inserts. Store extracts in 

a freezer prior to GC/MS analysis. 

8. Safety Precautions and Waste 

Disposal 

8.1 Method compounds may be 

extracted on a laboratory bench, but all steps 

in the method that require the use of organic 

solvents, such as cartridge cleaning, bottle

rinsing, cartridge elution, and extract 

concentration, must be conducted in a fume 

hood. Eye protection, gloves, and protective 

clothing must be worn in the laboratory area 

and when handling reagents, solvents, or any

corrosive materials, such as acetic acid buffer. 

Typical laboratory disposable, nitrile gloves 

do not provide adequate protection from 

DCM, so avoid contact with DCM. 

8.2 The waste stream produced during

sample preparation is about 1 molar NaCl and 

pH 7. It is collected in thick-walled carboys, 

and must be disposed of according to local

regulations. Solvent used to clean or rinse 

glassware, equipment, and cartridges also 

must be disposed of in the appropriate waste 

containers. 

16 Determination of Wastewater Compounds 
9. Gas Chromatograph/Mass 

Spectrometer Performance 

Check instrument performance 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

9.1.1 Gas chromatograph 

performance normally is indicated by peak 

shape, compound resolution, and variation of 

selected-compound response factors relative to 

response factors obtained by using a new 

capillary column and freshly prepared

calibration solutions. An example of the 

separation and peak shape for the complex

mixture of NP compounds is shown in the 

selected ion chromatogram (fig. 1) 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. 1) or if compounds fail to meet the 

calibration criteria (see section 10, Calibration), 

change the injection port liner or perform 

maintenance on the capillary column to bring

the gas chromatograph into compliance. About 

0.6 m (one column loop) of the capillarycolumn 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. 

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, locate and fix the leaks. Also, 

check the instrument every 24 hours during a 

series of sample analyses to ensure that mass 

spectrometer performance is in accordance 

with the perfluorotributylamine tuning criteria

outlined below. In addition, initially adjust 

the mass spectrometer response (also outlined 

below) to ensure that the established 

minimum reporting level (MRL) for each 

selected compound can be achieved. 

9.2.2 Check the mass spectrometer 

tune daily. 

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. 

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 they range 

from about 0.5 to 0.65 atomic mass unit. 

Adjustment of tune settings normally will 

require recalibrating the method compounds. 

ANALYTICAL METHOD 17
10. Calibration 

10.1 Acquire initial calibration data by

using a new capillary column and the latest 

calibration solutions. Use these data in 

subsequent evaluation of the GC/MS 

performance. 

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, generally needs to be within ±20 

percent of the expected concentration. 

10.3 Inject 2 μL of each calibration 

solution into the GC/MS and acquire data by

using the previously described GC/MS 

conditions. Enter the compound names, mass 

spectral ions, approximate retention times 

(table 2), and calibration concentration levels 

into the data system. The GC/MS dataprocessing software then calculates the 

relative retention time and response factors 

for each compound and surrogate in relation 

to their designated internal standards in the

calibration solution. The data-processing

software also uses linear regression routines 

to calculate and plot calibration curves for 

each compound. Typical equations used to 

calculate calibration curves for this method 

are similar to other NWQL methods 

(Sandstrom, 2001). 

10.4 See table 2 for compound 

quantitation ions and their respective PAH 

internal-standard reference compounds used 

for these calculations. The concentration of 

chrysene-d12 in the procedural internal 

standard solution was changed inadvertently

so that it was not consistent with the

concentration of the other internal standard 

compounds and could no longer be used 

during method development. Phenanthrened10 was used successfully in its place, but it is 

anticipated that chrysene-d12 might be used 

when new standard solutions are prepared. 

10.5 Calibration of multicomponent 

compounds — The para-NPs and NPEO2 

mixtures are each composed of 10 to 20 

discernible isomers. They are calibrated by

manually integrating the area of their

respective quantitation ion peaks that are

present in the expected range of the retention 

time window (table 2). This approach also 

was used for determining the concentration of

compounds in the preparation of stock 

standard solutions (section 6.1, fig. 1) and 

also has been used in other studies (Blackburn 

and Waldock, 1995). 

11. Quality Assurance and Quality

Control 

The NWQL has prepared a qualityassurance and quality-control (QA/QC)

guidance document (Merle Shockey, U.S. 

Geological Survey, written commun., 1998) 

for the Organic Chemistry Program that is 

followed for this wastewater method to ensure 

that QC standards are correctly established 

and consistently met. The sample matrix, 

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. 

First, the sample matrix must be

consistent with the requirements of the 

method (filtered water). Problematic sample 

matrices will affect the performance of the

method during sample preparation and 

analysis. Extremely dirty sample matrices, 

such as raw sewage, are discouraged because 

18 Determination of Wastewater Compounds 
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

para-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

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

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

Cotinine 29.761 98 176 147 IS4

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

Prometon 30.099 210 225 168 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

ANALYTICAL METHOD 19
Table 2. Wastewater method compound retention time, quantitation ion, confirmation ions, surrogate 

compounds, and internal standard reference compound—Continued 

Compound name

Retention

time

(min) 

Quantitation 

ion 

(m/z) 

Confirmation 

ion 

(m/z) 

Confirmation 

ion 

(m/z) 

Internal 

standard

reference 

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-tetrahydronaphthalene (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

Fluoranthene 34.134 202 101 203 IS4

Triclosan 34.378 288 290 218 IS4

Pyrene 34.731 202 101 203 IS4

Bisphenol A 34.994 213 228 119 IS4

Octylphenol, diethoxy- (OPEO2) 35.168 223 135 294 IS4

Nonylphenol, diethoxy- (total,

NPEO2) 35.7–36.5 237 223 279 IS4

Tri(dichloroisopropyl) phosphate 36.400 379 383 381 IS4

Tri(2-butoxyethyl) phosphate 37.054 299 199 125 IS4

Triphenyl phosphate 37.176 326 325 215 IS4

Estrone 39.395 270 185 146 IS4

17beta-Estradiol 39.574 272 213 172 IS4

Ethynyl estradiol 40.120 213 296 160 IS6

Equilenin 40.294 266 223 210 IS6

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

20 Determination of Wastewater Compounds 
Table 2. Wastewater method compound retention time, quantitation ion, confirmation ions, surrogate 

compounds, and internal standard reference compound—Continued 

Compound name

Retention

time

(min) 

Quantitation 

ion 

(m/z) 

Confirmation 

ion 

(m/z) 

Confirmation 

ion 

(m/z) 

Internal 

standard

reference 

Surrogates

Decafluorobiphenyl 18.786 334 265 -- IS2

Caffeine13C3 31.444 197 110 -- IS4

Fluoranthene-d10 34.087 212 106 -- IS4

Bisphenol A-d3 34.947 216 234 -- IS4

Internal Standards

1,4-Dichlorobenzene-d4 (IS1) 15.132 150 152 -- --

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

Acenapthene-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 -- --

they contaminate sample preparation 

equipment and instrumentation, thus 

affecting the results of subsequent samples. 

Unknowingly processing raw sewage or 

unfiltered samples with this method could 

have deleterious effects on subsequent 

sample results. On the other hand, reagent 

water used for set spikes and blanks is not 

representative (lacking dissolved organic 

carbon) of an environmental sample matrix. 

Consequently, recoveries of surrogate and 

method compounds from reagent water 

often are less than or greater than recoveries 

obtained from environmental sample

matrices, which demonstrate some of the

limitations of making comparisons among

different sample matrices. 

Before sample expiration, each 

environmental sample is prepared for 

analysis as part of a sample preparation set 

that contains a laboratory reagent spike and 

blank (control) to monitor general laboratory

conditions and procedures. There is no 

guarantee, however, that each unique sample

matrix will perform similarly to the

recoveries of compounds and surrogates 

obtained from the set spike and blank. 

Consequently, spiking at the laboratory of 

field duplicate samples is encouraged to gain

some indication of how method compounds 

perform in a particular sample matrix. 

Laboratory set spikes are of limited use

because they are prepared in reagent water, 

which usually is not indicative of the sample

matrix. Furthermore, just because surrogate

compounds perform well in a given sample 

matrix does not necessarily mean that

method compounds will perform equally

well. Historical, statistical data for set spikes

may be used to anticipate method compound 

recovery, but they are no substitute for field 

sample spikes to determine specific matrix

effects. Control limits for laboratory spikes

in reagent water are set at the mean percent

recovery ± 3 standard deviations. 

Surrogate compounds (not necessarily

chemically representative of every method 

compound) 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 

ANALYTICAL METHOD 21
spike and blank samples. The recoveries of 

caffeine--13

C3 and fluoranthene-d10 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 should 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. 

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. Even then, only limited information 

can be inferred because each individual

sample is handled separately. Some

compounds in the wastewater method are 

common in personal-care products, and might 

be detected occasionally in laboratory blanks. 

If compounds are detected in more than 10 

percent of the historical laboratory blanks, 

they are treated as though they always are 

potentially present in sample background. If

this is the case, use the 95th percentile of

historical laboratory blank concentrations to 

establish a higher MRL for the specific 

compound than might otherwise be derived 

from the MDL calculation. 

Sample extract(s) are analyzed in an 

instrument batch or sequence 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 set spike 

and blank sample, as well as bracketing

continuing calibration verification solutions 

(CCVs) to check periodically at designated 

intervals (10 environmental samples or less)

that the instrument is in compliance with 

initial calibration criteria. For those

compounds that are quantitatively reported 

(not permanently assigned an estimated 

concentration) by using this method, a 

calculated concentration within ± 20 percent 

of the expected CCV concentration is

acceptable. Finally, a low-concentration 

standard equivalent to 1 μg/L (or less) is

analyzed in each sequence after the 

environmental samples to ensure that

instrument sensitivity is maintained

throughout the sample set. If available, field 

matrix spike samples at concentrations 

between 2 and 5 times the expected MDL also 

can be analyzed to ensure method sensitivity

for different sample matrices. 

If the instrument does not meet 

acceptance criteria, then follow recommended 

procedures of cleaning and maintenance (see 

section 9.1, GC performance evaluation) to 

bring the instrument back into compliance. It

might be possible to reanalyze only that 

portion of the sequence corresponding to the 

instrumental failure between bracketing

CCVs because samples are analyzed in a 

specific sequence. In some cases, identifying

and removing problematic sample extracts 

from sequences might be required to meet 

performance criteria for other sample results. 

Each sample also has procedural 

internal standard compounds added to correct 

automatically for any differences (generally

less than ± 10 percent) in extract volume 

analyzed, as well as automatically 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 errors, unexpected GC compound 

retention time shifts, or instrument 

abnormalities caused by power interruptions 

or component malfunctions. 

22 Determination of Wastewater Compounds 
It is difficult to troubleshoot partial 

QA/QC problems caused by a combination of 

a dirty sample matrix, sample preparation 

errors, or a marginally acceptable analysis. 

Certain process failures require sample

preparation to be repeated if a duplicate 

sample has been received. 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 

unacceptable surrogate recoveries, might

indicate that sample results are unsalvageable

and should not be reported. 

12. Calculation of Results 

Before quantitative results are reported, 

each compound first must meet qualitative 

criteria. 

12.1 Qualitative Identification 

12.1.1 The retention time of the 

quantitation ion for the compound of interest 

should be within 0.1 minute (±6 seconds) of 

the expected retention time (as calculated 

from the relative retention time of calibration 

standards and the retention time of the

internal standard in the sample) in the absence

of any obvious matrix effects. Furthermore, 

ensure that the profiles of the qualification 

and quantification ion peaks 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 a reasonable 

match. 

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. Subtracting the spectral

background of a well-defined sample peak 

usually enhances the spectrum, whereas 

subtracting interfering ions with substantial ion 

abundances from a poorly defined sample peak 

can result in a nonsensical spectrum. In this 

situation, the main consideration for positive 

identification of a compound is the requirement 

for the ion profiles (see table 2) to maximize

within two scans of each other (after 

accounting for interfering ion profiles, if 

necessary). If the compound is present, an 

improved spectrum also should be obtained 

after manually subtracting appropriate

background scan(s) that are free from the ions 

of the interfering peak. 

12.2 Quantitation 

12.2.1 Determination of singlecomponent compounds. The concentration of 

a compound is calculated after a compound 

has passed qualitative criteria according to the 

calibration curve used to establish the best fit 

between the calibration points. Curve-fitting

routines provided by the instrument 

manufacturer, and summarized in a similar

NWQL method report (Sandstrom, 2001), are 

used to obtain a calibration curve for each 

compound. If the calculated concentration of 

a compound exceeds the highest 

concentration point of the calibration curve by

20 percent or more, add higher concentration 

calibration standards 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” qualifier code if

compound response is less than the lowest 

point on the calibration curve or the minimum 

reporting level (MRL). If curve-fitting

routines (quadratic curves and power curves) 

are used for calibration, verify that the sample 

compound response is not outside the 

ANALYTICAL METHOD 23
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 para-NPs

and NPEO2 mixtures are each composed of 

10 to 20 discernable 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, Calibration) for 

these compounds. If interferences cause the 

ratios of the qualification ions to the

quantification ion to be unreasonable, then 

integrate that portion of the ion chromatogram

that is caused by the contamination or 

interference (peaks in the sample

chromatogram that are not in the calibration 

standard chromatogram) and subtract the 

interference from the total. 

NOTE: This procedure seldom is necessary

because the quantitation and qualification ions 

for para-NP and NPEO2 compounds 

normally are unique from coeluting

interference.

12.3 Reporting Results 

The wastewater method requires that the 

most accurate information be transmitted to 

project investigators and data interpreters

because it is used to report data for calculating

the presence, fate, and transport of important 

compounds in the environment. Therefore, data 

are reported according to the latest laboratory

quality-assurance information (Merle

Shockey, U.S. Geological Survey, written 

commun., 1998; Childress and others, 1999). 

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 “information-rich” (Childress and others, 

1999) because compound identifications are 

determined by mass spectrometry; consequently, results are not censored at the 

MRL. Compound concentrations, therefore, 

are reported as follows. 

If the concentration is equal to or greater 

than the MRL, the concentration is reported to 

three significant figures. If the concentration 

is less than either the MRL or the lowest 

calibration standard (usually 0.05 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 (Merle 

Shockey, U.S. Geological Survey, written 

commun., 1998; Childress and others, 1999). 

They include such situations as matrix

interferences, method compounds that have 

been permanently assigned an “E” code, and 

those compounds that do not meet qualityassurance criteria, such as being out of 

calibration by more than 20 percent. If the 

result is greater than 120 percent of the 

highest concentration standard in the 

calibration curve, then the sample is diluted 

into the range of the calibration curve and 

reanalyzed. 

NOTE: MRL data are subject to annual 

change in conjunction with the NWQL longterm method detection level (LT–MDL)

program (Childress and others, 1999). 

The attempt to report consistent data

near the MDL is difficult, especially with the

intention to transmit as much information as

possible in complex samples and also avoid 

data censoring. Reporting compound results 

as estimated because their concentrations are 

less than the MRL should not decrease 

24 Determination of Wastewater Compounds 
confidence in qualitative identification. 

However, concentrations reported near or less 

than the MDL must 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. In most of these 

instances, when there is considerable doubt 

about qualitative identification, reporting

conservative results (less than MRL) is 

appropriate. 

METHOD PERFORMANCE

Reagent-water (Solution 2000 water) 

samples and surface-water samples collected 

from the Platte River in Confluence Park, 

Denver, Colo., and ground-water samples 

collected in a domestic well near Evergreen, 

Colo., were used to test method performance. 

The surface-water and ground-water samples 

were filtered into 1-L sample bottles prior to 

extraction according to the method protocol. 

One set of the filtered 1-L subsamples was 

fortified at a lower concentration (0.5 – 8 

μg/L) of each compound and the other set was 

fortified at a higher concentration (4.0 – 80 

μg/L) of each compound. In addition, the 

three sample matrices were extracted and 

analyzed (unfortified) to determine the natural 

presence of any method compounds (table 3). 

The presence of 10 compounds in the reagentwater sample at barely detectable

concentrations reemphasizes the ubiquitous 

nature of about half of the method 

compounds, as well as the importance of 

avoiding contamination throughout sample 

collection, preparation, and analysis. 

Isophorone, for example, was detected in all 

three sample matrices (see table 3) and since 

has been found at trace levels in opened 

bottles of methylene chloride. The subsequent 

isolation of methylene chloride from other 

laboratory solvents has reduced this 

contamination problem. 

Each fortified sample set was extracted 

and analyzed on different days, so comparisons of different matrices and concentrations 

include day-to-day variation. Mean bias and 

variability data from the analyses are listed 

in table 4. 

The concentration of 16 compounds 

always is 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. Initial MDLs were

calculated for compounds in reagent water by

using the corresponding spike concentration 

as indicated in table 4. 

Method detection limits and minimum 

reporting levels — Initial MDLs were

determined according to the procedure 

outlined by the U.S. Environmental Protection 

Agency (1997). 

The MDL was calculated according to

the equation 

 MDL = S x 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 

METHOD PERFORMANCE 25
Table 3. Wastewater method compounds detected in unfortified reagent-water, ground-water, and 

surface-water samples 

[ųg/L, micrograms per liter; --, not detected]

Concentration (μg/L) Compound name

Reagent Ground Surface

1,4-Dichlorobenzene -- -- --

17beta-Estradiol -- -- --

1-Methylnaphthalene -- -- --

2,6-Dimethylnaphthalene -- -- --

2-Methylnaphthalene -- -- --

3beta-Coprostanol -- -- 0.26

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

3-tert-Butyl-4-

hydroxyanisole (BHA) 

-- -- --

4-Cumylphenol -- -- --

4-n-Octylphenol -- -- --

4-tert-Octylphenol -- -- .04 

5-Methyl-1H-benzotriazole -- -- 1.92 

Acetophenone 0.08 -- --

Acetyl-hexamethyltetrahydro-naphthalene

(AHTN) 

.01 -- .48

Anthracene -- -- --

Anthraquinone -- -- --

Benzo[a]pyrene -- -- --

Benzophenone -- -- .06

beta-Sitosterol -- -- --

beta-Stigmastanol -- -- --

Bisphenol A .04 -- --

Bromacil -- -- --

Bromoform -- -- --

Caffeine .01 -- .33

Camphor -- -- .11

Carbaryl -- -- .08

Carbazole -- -- .02

Chlorpyrifos -- -- --

Cholesterol .50 -- .96

Cotinine -- -- .11

Diazinon -- -- .15 

Dichlorvos -- -- --

d-Limonene -- -- --

Equilenin -- -- --

Estrone -- -- --

Concentration (μg/L) Compound name

Reagent Ground Surface

Ethynyl estradiol -- -- --

Fluoranthene -- -- --

Hexahydrohexamethyl

cyclopentabenzopyran 

(HHCB)

-- -- --

Indole -- -- --

Isoborneol -- -- --

Isophorone 0.11 0.40 0.26

Isopropylbenzene (cumene) -- -- --

Isoquinoline -- -- --

Menthol -- -- --

Metalaxyl -- -- --

Methyl salicylate -- -- -- 

Metolachlor -- -- --

N,N-diethyl-meta-toluamide

(Deet) -- -- .12

Naphthalene .03 -- --

Naphthalene .03 -- --

Nonylphenol, diethoxy-

(total, NPEO2) -- -- 6.64

Octylphenol, diethoxy-

(OPEO2) -- -- 1.10

Octylphenol, monoethoxy-

(OPEO1) -- -- --

para-Cresol -- -- --

para-Nonylphenol (total) .31 -- .80 

Pentachlorophenol -- -- --

Phenanthrene -- -- .02

Phenol -- -- --

Prometon -- -- --

Pyrene -- -- --

Tetrachloroethylene -- -- --

Tri(2-chloroethyl) phosphate -- -- .19 

Tri(dichloroisopropyl)

phosphate -- -- .15

Tributyl phosphate -- -- --

Triclosan .11 -- .25

Triethyl citrate (ethyl citrate) -- -- -- 

Triphenyl phosphate .01 -- .07 

Tri(2-butoxyethyl)

phosphate -- -- .71

26 Determination of Wastewater Compounds
Table 4. 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 reagentwater (including calculated method detection limits), ground-water, and surface-water samples

[μg/L, micrograms per liter; %, percent; RSD, relative standard deviation; MDL, method detection limit; 

--, not applicable; <, less than; ND, not determined] 

Mean% recovery % RSD 

Compound name

Spike 

amount 

(μg/L) Reagent Ground Surface Reagent Ground Surface

Initial 

MDL

(μg/L) 

1,4-Dichlorobenzene 4.0

.5 

44.38 

29.94 

48.34 

45.15 

64.62 

49.63 

10.53 

13.77 

11.03 

20.55 

7.46 

6.65 0.28 

17beta-Estradiol 16.0

2.0 

75.03 

35.77 

67.03 

39.73 

79.10 

38.11 

3.93 

52.29 

10.06 

84.25 

3.16 

77.13 

1.64 

1-Methylnaphthalene 4.0

0.5 

69.71 

58.17 

68.09 

69.53 

77.35 

68.34 

2.58 

14.98 

3.47 

5.67 

3.87 

5.78 .13 

2,6-Dimethylnaphthalene 4.0

.5 

69.70 

54.47 

67.82 

65.55 

76.86 

67.50 

2.36 

11.85 

3.69 

9.21 

5.12 

6.29 .10 

2-Methylnaphthalene 4.0

.5 

67.78 

55.44 

66.20

68.34 

77.62 

68.23 

3.06 

15.12 

3.49 

7.17 

4.12 

6.51 .13 

3beta-Coprostanol 16.0

2.0 

76.38 

81.98 

76.72 

84.11 

56.90*

56.95*

8.93 

10.58 

26.15 

20.49 

9.85 

11.80 .52 

3-Methyl-1H-indole 

(skatol) 

4.0 

.5 

81.66 

50.42 

67.04 

45.39 

82.42 

33.92 

5.89 

39.49 

10.91 

64.38 

7.27 

66.59 .30 

4-Cumylphenol 4.0

.5 

78.49 

58.85 

79.38 

52.27 

71.98 

50.94 

2.44 

41.38 

7.84 

66.64 

4.05 

50.59 .37 

4-n-Octylphenol 4.0

.5 

79.04 

38.49 

71.78 

39.97 

71.22 

42.55 

2.81 

57.43 

7.83 

75.09 

7.50 

72.90 .33 

4-tert-Octylphenol 4.0

.5 

82.62 

43.06 

85.15 

42.09 

81.62*

42.02*

3.03 

56.19 

8.56 

77.60 

7.52 

76.32 .37 

5-Methyl-1H-benzotriazle 16.0

2.0 

99.03 

107.15 

88.25 

123.20 

108.46*

353.25*

2.40 

22.14 

14.05 

16.87 

11.42 

27.56 

.92 

Acetophenone 4.0

.5 

82.30 

73.40 

79.46 

85.94 

83.05 

94.40 

1.50 

6.14 

3.30 

3.94 

3.25 

6.08 .09 

Acetyl-hexamethyltetrahydro-naphthalene

(AHTN) 

4.0 

.5 

73.02*

77.59*

75.70 

73.76 

66.64*

56.10*

3.77 

6.85 

5.64 

4.63 

5.82 

6.83 .08 

Anthracene 4.0

.5 

72.86 

72.93 

71.53 

61.30 

73.45 

59.71 

3.27 

9.67 

3.07 

14.68 

6.35 

16.98 .11 

Anthraquinone 4.0

.5 

83.75 

94.80 

77.09 

97.60 

81.89 

109.10 

4.10 

7.20 

5.35 

6.37 

6.15 

7.13 .11 

Benzo[a]pyrene 4.0

.5 

75.95 

65.06 

70.76 

68.51 

69.38 

51.64 

3.36 

7.81 

4.18 

24.16 

10.49 

11.86 .08 

Benzophenone 4.0

.5 

87.42 

95.90 

88.66 

90.74 

89.91*

90.70*

3.59 

7.99 

4.18 

5.50 

4.65 

4.84 .12 

beta-Sitosterol 16.0

2.0 

100.70 

92.55 

97.89 

100.54 

70.16 

84.25 

9.33 

11.10 

16.73 

19.24 

7.75 

8.04 .60 

beta-Stigmastanol 16.0

2.0 

97.3 

101.40 

98.44 

96.09 

63.71 

73.35 

10.13 

11.71 

16.31 

19.58 

6.00 

12.65 .72 

Bisphenol A 4.0

.5 

74.25*

28.16*

68.36 

37.51 

65.15 

55.56 

3.98 

55.91 

8.92 

89.92 

5.46 

61.15 

.38 

Bromacil 4.0

.5 

85.02 

119.30 

83.29 

116.11 

85.51 

110.40 

3.72 

5.39 

4.20 

5.66 

2.03 

3.17 .10 

METHOD PERFORMANCE 27
Table 4. 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 reagentwater (including calculated method detection limits), ground-water, and surface-water samples—Continued 

Mean% recovery % RSD 

Compound name

Spike 

amount 

(μg/L) Reagent Ground Surface Reagent Ground Surface

Initial 

MDL

(μg/L) 

Caffeine 4.0

.5 

83.04*

89.90*

87.20 

87.20 

82.55*

82.30*

2.86 

6.72 

2.96 

6.99 

2.41 

6.22 0.09 

Camphor 4.0

.5 

79.00 

80.05 

77.86 

75.05 

77.33*

51.51*

2.02 

6.95 

2.88 

3.50 

1.54 

3.90 .09 

Carbazole 4.0

.5 

81.60 

93.70 

78.91 

93.94 

84.10*

86.00*

4.55 

7.86 

3.70 

5.65 

6.50 

4.63 .11 

Chlorpyrifos 4.0

.5 

75.66 

91.90 

78.18 

84.79 

69.27 

79.60 

5.26 

5.13 

7.01 

7.00 

6.45 

3.71 .08 

Cholesterol 16.0

2.0 

96.12*

27.48*

96.60 

95.80 

54.66*

52.90*

10.23 

13.27 

22.25 

17.13 

9.37 

47.22 .71 

Cotinine 4.0

.5 

49.82 

65.88 

82.34 

89.09 

75.58*

66.38*

2.32 

8.44 

2.86 

4.85 

2.78 

4.62 .33 

Diazinon 4.0

.5 

78.16 

92.70 

82.25 

89.14 

71.40*

73.90*

3.32 

4.48 

6.88 

5.95 

4.62 

4.31 .07 

Ethynyl estradiol 16.0

2.0 

67.10 

33.09 

66.66 

32.01 

50.75 

35.59 

5.71 

55.05 

26.24 

81.25 

5.42 

74.75 

2.08 

Fluoranthene 4.0

.5 

74.69 

82.39 

77.49 

39.01 

70.29 

71.24 

2.84 

6.35 

3.82 

7.42 

6.11 

3.18 .08 

Hexahydrohexamethylcyclo-pentabenzopyran

(HHCB)

4.0 

.5 

74.80 

76.06 

78.15 

76.95 

65.80 

67.30 

3.97 

7.81 

6.38 

5.22 

6.48 

3.93 .12 

Indole 4.0

.5 

79.44 

73.84 

78.07 

68.53 

85.74 

55.05 

2.43 

12.14 

3.81 

17.63 

4.62 

16.87 .14 

Isoborneol 4.0

.5 

80.93 

88.20 

79.05 

80.86 

81.14 

73.02 

2.35 

8.07 

2.92 

4.36 

1.52 

5.65 .11 

Isophorone 4.0

.5 

86.48*

85.80*

77.35*

43.31*

87.53*

58.60*

14.45 

11.64 

3.52 

28.46 

25.43 

6.30 .19 

Isoquinoline 4.0

.5 

85.41 

84.36 

85.33 

80.98 

83.78 

92.30 

2.60 

8.21 

4.58 

7.83 

4.58 

6.73 .11 

Menthol 4.0

.5 

81.46 

86.70 

79.47 

79.94 

81.15 

68.83 

2.46 

5.75 

2.88 

2.40 

2.02 

6.84 .08 

Metalaxyl 4.0

.5 

80.56 

48.00 

85.51 

90.86 

76.73 

83.84 

3.19 

4.90 

7.02 

5.35 

1.13 

4.25 .08 

Methyl salicylate 4.0

.5 

82.98 

87.50 

77.04 

84.95 

82.16 

84.00 

2.37 

5.94 

2.51 

3.70 

1.86 

4.38 .08 

Metolachlor 4.0

.5 

77.44 

91.40 

82.48 

88.57 

71.1 

79.66 

3.86 

5.53 

6.91 

6.32 

4.09 

4.09 .08 

N,N-diethyl-metatoluamide (Deet) 4.0 

.5 

85.15 

101.90 

96.38 

100.34 

90.45*

87.00*

3.88 

9.25 

4.63 

6.22 

6.92 

4.11 .14 

Naphthalene 4.0

.5 

60.07*

56.14*

66.20 

66.41 

76.72 

66.22 

5.02 

17.56 

3.98 

7.66 

3.24 

4.07 .15 

para-Cresol 4.0

.5 

85.49 

35.70 

71.58 

36.57 

85.44 

41.07 

4.83 

51.73 

7.25 

75.13 

4.14 

71.38 .27 

Phenanthrene 4.0

.5 

77.95 

79.76 

75.47 

73.62 

74.22 

72.17*

1.78 

6.36 

2.58 

3.46 

5.00 

3.01 .08 

Phenol 4.0

.5 

86.25 

92.50 

98.16 

254.65 

89.05*

95.88 

2.87 

7.84 

10.60 

65.64 

3.20 

43.27 .11 

Prometon 4.0

.5 

83.26 

101.00 

84.75 

96.57 

77.67 

91.00 

2.65 

7.54 

6.40 

7.90 

2.25 

5.39 .12 

28 Determination of Wastewater Compounds 
Table 4. 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 reagentwater (including calculated method detection limits), ground-water, and surface-water samples—Continued 

Mean% recovery % RSD 

Compound name

Spike 

amount 

(μg/L) Reagent Ground Surface Reagent Ground Surface

Initial 

MDL

(μg/L) 

Pyrene 4.0

.5 

73.84 

81.18 

77.27 

77.86 

69.26 

71.02 

3.03 

5.81 

3.88 

9.22 

6.21 

3.21 0.08 

Tri(2-chloroethyl) 

phosphate 

4.0 

.5 

84.25 

103.10 

85.26 

91.77 

79.07*

77.80*

3.34 

5.04 

5.81 

4.87 

1.21 

4.09 .08 

Tri(dichloroisopropyl)

phosphate 

4.0 

.5 

79.86 

96.40 

84.62 

96.00 

71.68*

72.30*

5.40 

5.29 

7.52 

10.80 

4.95 

4.41 .08 

Tributyl phosphate 4.0 

.5 

87.53 

112.40 

98.24 

17.37 

87.70 

122.70 

3.88 

5.97 

5.66 

7.42 

7.00 

4.54 .10 

Triclosan 4.0

.5 

84.50*

69.21*

76.93 

78.50 

80.42*

85.63*

5.00 

31.68 

6.73 

61.58 

5.18 

100.65 

.48 

Triethyl citrate 

(ethyl citrate) 

4.0 

.5 

84.02 

98.20 

83.78 

93.37 

78.80 

96.30 

2.12 

5.59 

5.62 

5.63 

2.05 

3.74 .09 

Triphenyl phosphate 4.0 

.5 

74.94*

90.00*

81.98 

93.37 

69.40*

80.30*

6.07 

4.48 

6.31 

11.74 

5.92 

4.25 .06 

Tri(2-butoxyethyl) 

phosphate 

4.0 

.5 

79.47 

103.40 

82.11 

115.43 

78.49*

90.40*

6.37 

12.52 

7.66 

9.44 

5.20 

6.32 .20 

Compounds with low recovery1

, high variable recovery (RSD)1, unstable instrument response2, or from a 

technical mixture3 to be reported with an "E" code (estimated concentration) 

1,4-Dichlorobenzene

1 4.0

.5 

44.38 

29.94 

48.34 

45.15 

64.62 

49.63 

10.53 

13.77 

11.03 

20.55 

7.46 

6.65 .28

17beta-Estradiol1 16.0

2.0 

75.03 

35.77 

67.03 

39.75 

79.10 

38.11 

3.93 

52.29 

10.06 

84.25 

3.16 

77.13 

1.64 

3-tert-Butyl-4-

hydroxyanisole (BHA)

1

4.0 

.5 

51.42 

17.64 

24.88 

19.42 

22.05 

16.82 

46.23 

13.44 

107.68 

51.47 

106.99 

27.53 

3.07 

Bromoform1

 4.0 

.5 

54.58 

43.03 

62.75 

51.14 

71.80 

57.23 

5.39 

27.56 

5.39 

19.74 

4.03 

6.53 .18 

Carbaryl

2 4.0

.5 

101.24 

161.10 

86.00 

132.80 

100.06*

155.20*

4.79 

13.19 

12.06 

18.04 

3.53 

5.01 .44 

Dichlorvos

1 4.0

.5 

6.7 

14.24 

23.17 

9.30 

31.31 

48.55 

44.73 

44.38 

43.07 

27.02 

23.11 

37.25 .10 

d-Limonene

1 4.0

.05 

29.58 

18.51 

34.11 

18.46 

50.70 

23.73 

16.99 

19.06 

17.30 

34.73 

14.35 

26.80 .05 

Equilenin1 4.0

.5 

58.02 

1.79 

27.58 

16.95 

39.73 

21.43 

19.99 

49.15 

81.45 

55.79 

28.61 

110.32 

1.80 

Estrone

1 4.0

.5 

84.33 

21.22 

28.34 

30.67 

129.58 

52.53 

18.91 

46.82 

39.31 

74.20 

45.61 

68.84 1.12 

Isopropylbenzene 

(cumene)1

4.0 

.5 

33.24 

20.64 

39.07 

27.98 

53.2 

34.88 

15.16 

12.42 

13.57 

24.49 

13.14 

13.46 .04 

Nonylphenol, diethoxy- 

(total, NPEO2) 3

64.0 

8.0 

78.30 

83.69 

79.84 

89.55 

61.15*

98.94*

6.34 

12.59 

6.40 

35.26 

7.70 

5.30 2.52 

Octylphenol, diethoxy- 

(OPEO2)3

.8 

.1 

122.59 

110.67 

118.58 

159.20 

61.42*

<0.0*

2.24 

17.33 

14.04 

2.39 

7.63 

ND 

.04 

Octylphenol, 

monoethoxy-(OPEO1)

3

16.0 

2.0 

75.31 

45.18 

73.42 

44.41 

68.06 

65.93 

4.86 

42.73 

9.52 

76.05 

7.16 

52.89 

.29 

METHOD PERFORMANCE 29
Table 4. 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 reagentwater (including calculated method detection limits), ground-water, and surface-water samples—Continued 

Mean% recovery % RSD 

Compound name

Spike 

amount 

(μg/L) Reagent Ground Surface Reagent Ground Surface

Initial 

MDL

(μg/L) 

para-Nonylphenol (total)

3

 80.0 

10.0 

73.62*

36.78 

79.56 

36.95 

64.94*

38.40*

5.45 

53.73 

8.44 

88.83 

5.90 

73.91 6.40 

Pentachlorophenol2 4.0

.5 

80.49 

111.35 

76.50 

92.43 

90.66 

62.07 

6.25 

28.06 

9.11 

45.63 

3.14 

43.74 

.39 

Tetrachloroethylene

1 4.0

.5 

25.36 

18.49 

33.05 

36.46 

41.65 

28.29 

16.34 

10.39 

12.91 

22.51 

13.48 

15.05 .03 

Surrogate compounds

Caffeine13C3 2.0

.5 

91.33 

80.10 

90.00 

84.91 

86.82 

82.56 

2.81 

3.75 

2.79 

4.38 

2.58 

6.15 

-- 

Fluoranthene-d10 2.0

.5 

73.17 

71.60 

79.53 

71.43 

68.98 

83.45 

2.66 

2.59 

3.18 

4.76 

2.47 

4.23 

-- 

Bisphenol A-d3 2.0

.5 

40.42 

28.12 

41.27 

65.75 

67.55 

73.13 

3.66 

61.67 

6.33 

100.80 

3.91 

88.18 

-- 

Decafluorobiphenyl 2.0

.5 

35.10 

78.13 

70.90 

60.43 

55.18 

53.64 

4.89 

21.36 

6.06 

24.38 

9.01 

11.36 

-- 

1

Concentration is estimated because recovery is less than 60 percent or variability is greater than 25 percent RSD. 2

Concentration is estimated because of unstable instrument response. 3

Concentration is estimated because the reference standard is from a technical mixture. 

*Percent recovery corrected for background concentration in the unspiked sample. 

was used to calculate initial MDLs for most 

of the compounds. However, initial MDLs

for some method compounds were 

calculated by using concentrations higher 

than the desired spike 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 spike amount have been defined as 

estimated MDLs, and have been footnoted

as such in table 5. Initial MDLs that were

calculated from this procedure for singlecomponent compounds (excluding

hormones and sterols) ranged from 0.03 to 

0.48 μg/L and averaged 0.15 μg/L. Initial 

MDLs for the sterols, hormones, and 

multicomponent para-NP compounds are 

greater than 0.15 μg/L. 

The initial minimum reporting levels 

(MRLs) have been set higher (two to five 

times for most compounds) than the 

calculated initial MDLs (table 5). This 

precaution reduces the risk of reporting that

a compound is undetected (less than the 

MRL), when it is actually in the sample near

the MDL concentration (Childress and 

others, 1999). All qualitatively identified 

compounds detected less than the MRL are 

reported as estimated, regardless of the

established MRL, because the wastewater 

method is classified as an “information-rich” 

method, as are other mass spctrometric 

methods (Childress and others, 1999). 

Calculation of the MDL over a long

time (6 to 12 months), including results from 

multiple instruments, analysts, and 

calibration curves, is referred to as a longterm method detection level (LT–MDL or 

operational MDL) (Childress and others, 

1999). The spiking solution at a concentration of 1 μg/L will be used routinely

throughout the year to calculate LT–MDLs 

for the wastewater method. The initial 

MDLs and initial MRLs will be updated 

30 Determination of Wastewater Compounds 
Table 5. Wastewater method initial detection limits calculated from the recovery

variability data reported in table 4 using the eight replicate reagent-water samples 

with compound concentrations that range from 0.5 to 80 micrograms per liter

[All concentrations in micrograms per liter; RSD, relative standard deviation; 

MDL, method detection limit; MRL, minimum reporting level] 

Compound name Spike 

amount

Mean

recovery

RSD Initial 

MDL

Initial 

MRL

1-Methylnaphthalene 0.5 0.291 0.044 0.13 0.5

2,6-Dimethylnaphthalene .5 .272 .032 .10 .5

2-Methylnaphthalene .5 .277 .042 .13 .5

3beta-Coprostanol 2.0 1.640 .173 .52 2.0

3-Methyl-1H-indole (skatol) .5 .252 .100 .30 1.0 

4-Cumylphenol .5 .294 .122 .37 1.0

4-n-Octylphenol .5 .192 .110 .33 1.0

4-tert-Octylphenol .5 .413 .123 .37 1.0

5-Methyl-1H-benzotriazle

1 16.0 15.848 .307 .92 2.0

Acetophenone

1 .5 .367 .030 .09 .5

Acetyl-hexamethyl-tetrahydronaphthalene (AHTN) 1

.5 .388 .027 .08 .5

Anthracene .5 .365 .035 .11 .5

Anthraquinone .5 .474 .036 .11 .5

Benzo[a]pyrene

1 .5 .325 .025 .08 .5

Benzophenone .5 .479 .039 .12 .5

beta-Sitosterol 2.0 1.851 .199 .60 2.0

beta-Stigmastanol 2.0 2.028 .241 .72 2.0

Bisphenol A1 4.0 2.970 .127 .38 1.0

Bromacil .5 .596 .032 .10 .5

Caffeine

1 .5 .450 .031 .09 .5

Camphor

1 .5 .400 .028 .09 .5

Carbazole .5 .469 .037 .11 .5

Chlorpyrifos

1 .5 .460 .025 .08 .5

Cholesterol 2.0 .550 .237 .71 2.0

Cotinine .5 .329 .111 .33 1.0

Diazinon1 .5 .464 .022 .07 .5

Ethynyl estradiol

1 16.0 1.736 .693 2.08 5.0

Fluoranthene1 .5 .412 .250 .08 .5

Hexahydrohexamethyl-cyclopentabenzopyran (HHCB).5 .380 .041 .12 .5

Indole .5 .369 .045 .14 .5

Isoborneol .5 .441 .036 .11 .5

Isophorone .5 .215 .063 .19 .5

Isoquinoline .5 .422 .035 .11 .5

Menthol

1 .5 .434 .025 .08 .5

Metalaxyl

1 .5 .240 .025 .08 .5

Methyl salicylate1 .5 .438 .025 .08 .5

METHOD PERFORMANCE 31
Table 5. Wastewater method initial detection limits calculated from the recovery variability 

data reported in table 4 using the eight replicate reagent-water samples with compound 

concentrations that range from 0.5 to 80 micrograms per liter—Continued 

Compound name Spike 

amount 

Mean

recovery

RSD Initial 

MDL

Initial 

MRL

Metolachlor

1 0.5 0.457 0.025 0.08 0.5

N,N-diethyl-meta-toluamide (Deet) .5 .510 .048 .14 .5

Naphthalene .5 .281 .049 .15 .5

para-Cresol .5 .179 .092 .27 1.0

Phenanthrene

1 .5 .399 .026 .08 .5

Phenol .5 .463 .036 .11 .5

Prometon .5 .505 .039 .12 .5

Pyrene

1 .5 .406 .025 .08 .5

Tri(2-chloroethyl) phosphate

1 .5 .516 .030 .08 .5

 Tri(dichloroisopropyl) phosphate

1 .5 .482 .027 .08 .5

Tributyl phosphat .5 .562 .033 .10 .5 

Triclosan1 4.0 3.380 .159 .48 1.0

Triethyl citrate (ethyl citrate) 1 .5 .491 .028 .09 .5

Triphenyl phosphate

1 .5 .450 .020 .06 .5

Tri(2-butoxyethyl) phosphate .5 .517 .067 .20 .5 

1,4-Dichlorobenzene .5 .150 .093 .28 .5

Compounds with low recovery, high variable recovery (RSD), unstable instrument 

response, or from a technical mixture to be reported with an "E" code (estimated 

concentration) 

17beta-Estradiol1 16.0 12.000 .547 1.64 5.0

3-tert-Butyl-4-hydroxyanisole (BHA) 4.0 2.057 1.020 3.06 5.0

Bromoform .5 .273 .059 .18 .5

Carbaryl .5 .806 .147 .44 .5

Dichlorvos .5 .071 .032 .10 1.0

d-Limonene

1 .5 .093 .017 .05 .5

Equilenin 4.0 2.321 .598 1.80 5.0

Estrone .5 .106 .373 1.12 5.0

Isopropylbenzene (cumene)1 .5 .103 .013 .04 .5

Nonylphenol, diethoxy-(total, NPEO2) 8.0 6.695 .840 2.52 5.0

Octylphenol, diethoxy-(OPEO2)

1 .8 .981 .014 .04 1.0

Octylphenol, monoethoxy-(OPEO1)

1 16.0 12.050 .096 .29 1.0

para-Nonylphenol (total) 10.0 3.678 2.133 6.40 5.0 

Pentachlorophenol1 4.0 3.220 .128 .39 2.0

Tetrachloroethylene

1 .5 .092 .010 .03 .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.

32 Determination of Wastewater Compounds 
annually by using data acquired from the 

NWQL (Childress and others, 1999). 

SAMPLE PRESERVATION AND

HOLDING TIMES 

Whole-water samples are preserved 

from biodegradation (Ogawa and others, 

1981) in the field by filtering them through a 

0.7-μm nominal pore diameter, glass-fiber 

filter. Removing suspended sediment also is 

required for processing samples by SPE. 

When the sample is received at the laboratory,

60 g of NaCl is added prior to refrigeration at

4oC, further augmenting sample preservation. 

It might be possible to add NaCl to samples in 

the field, thus improving the timeliness of

sample preservation, as well as extending

time requirements for shipping samples to the

laboratory. After these considerations for 

sample preservation have been evaluated 

more thoroughly, a holding-time study is 

planned to calculate a statistically acceptable 

holding time. Until then, to ensure that

samples are processed in a timely manner, the

holding time for the wastewater method has 

been set at 14 days from the date of sample

collection. 

SUMMARY 

The U.S. Geological Survey (USGS)

National Water Quality Laboratory and 

National Research Program have developed 

an analytical method for the determination of 

67 compounds typically found in domestic 

and industrial wastewater. The wastewater 

method provides an efficient means of 

detecting important 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. 

Water samples are filtered and the compounds 

are isolated by solid-phase extraction and 

determined by capillary-column gas 

chromatography/mass spectrometry. The

method focuses on the determination of 

compounds that are an indicator of wastewater 

and compounds that have been chosen on the 

basis of their endocrine-disrupting potential or 

toxicity. Analysis 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, polycyclicaromatic

hydrocarbons, and high-use domestic 

pesticides. 

Average recovery of all method 

compounds for short-term single-operator 

results in reagent-water samples fortified at 4 

micrograms per liter was 74 ± 7 percent 

relative standard deviation. Initial method 

detection limits for single-component 

compounds (excluding hormones and sterols) 

averaged 0.15 microgram per liter. 

The high frequency of compounds that 

were detected at environmentally significant 

concentrations for 3 years with the continuous 

liquid-liquid extraction wastewater method 

demonstrates the capability of identifying

anthropogenic contaminants over a wide range 

of sample matrices. Many 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. 

Even though the method is not particularly

sensitive for the underivatized fecal sterols or 

hormones, it has proven to be effective for 

identifying their presence in wastewater. 

SUMMARY 33
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