United States
Environmental Protection
Agency
Office of
Research and
Development
Office of Solid
Waste and
Emergency
Response
EPA/600/S-99/002
May 19, 1999
Federal Facilities Forum Issue
Field Sampling And Selecting On-site Analytical
Methods For Explosives In Water
A. B. Crockett1, H. D. Craig2, and T. F. Jenkins3
The Federal Facilities Forum is a group of
U.S. Environmental Protection Agency (EPA)
scientists and engineers who represent EPA
regional offices and are committed to the
identification and resolution of issues affecting
the characterization and remediation of federal
facility Superfund, Resource Conservation and
Recovery Act, and Base Realignment and
Closure sites. Current forum members are
identified at the end of this paper. The forum
members identified a need to provide remedial
project managers and other federal, state, and
private personnel working on hazardous waste
sites with a technical issue paper that identifies
screening procedures for characterizing
groundwater and surface water contaminated
with explosive and propellant compounds. Some
Forum members provided technical guidance
and direction in the development of this issue
paper, and other members provided comments.
This paper was prepared by A. B. Crockett,
H. D. Craig, and T. F. Jenkins. Support for this
project was provided by the EPA National
Exposure Research Laboratory, Environmental
Sciences Division with the assistance of the
Superfund Project's Technology Support Center
for Monitoring and Site Characterization. For
further information, contact Ken Brown,
Technology Support Center Director, at (702)
798-2270, Alan B. Crockett at (208) 526-1574,
or Harry D. Craig at (503) 326-3689.
It is imperative that any persons working
on sites believed to be contaminated with
explosive residues thoroughly familiarize
themselves with the physical and toxic
properties of the materials potentially
present and take all measures as may be
prudent and/or prescribed by law to protect
life, health, and property. This publication is
not intended to include discussions of the safety
issues associated with sites contaminated with
explosive residues. Examples of safety issues to
be considered include but are not limited to
geophysical detection methods, explosion
(detonation) hazards, toxicity of secondary
explosives, and personal protective equipment.
Information pertaining to toxicity concerns can
be found in Roberts and Hartley (1992) and
Yinon (1990). Specifically, this paper is not
intended to serve as a guide for sampling and
analysis of unexploded ordnance (UXO), bulk
high explosives, or secondary explosives in soil
where concentrations exceed 100,000 mg/kg
(10%). These conditions present a
potential detonation hazard; therefore,
explosive safety procedures and safety
precautions should be identified before
initiating site characterization activities in
such environments. It also does not serve as
a guide to installation of groundwater wells in
areas in which such hazards exist.
-*
echnology
upport
reject
1 Idaho National Engineering and Environmental Laboratory, Lockheed Martin Idaho Technologies Company
2 U.S. Environmental Protection Agency, Region 10
3 U.S. Army Corps of Engineers, Cold Regions Research and Engineering Laboratory
Technology Support Center for
Monitoring and Site Characterization
National Exposure Research Laboratory
Environmental Sciences Division
Las Vegas, NV 89193-3478
Technology Innovation Office
Office of Solid Waste and Emergency Response
U.S. EPA, Washington, B.C.
Walter W. Kovalick, Jr., Ph.D., Director
Printed on Recycled Paper
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Purpose and Scope
This issue paper provides guidance to remedial
project managers on field sampling and selecting
on-site analytical methods for detecting and
quantifying secondary explosive compounds in
water (see Table 1). A similar issue paper was
previously prepared on explosives in soils
(Crockett et al. 1996), and updated as all.S. Army
Corps of Engineers Cold Regions Research and
Engineering Laboratory (CRREL) report
(Crockett et al. 1998). The paper also includes a
brief discussion of the reference analytical method
for the determination of 14 explosives and
co-contaminants in water, soil, and sediments,
EPA Method 8330 (EPA 1998).
Table 1. Analytical Methods for Commonly Occurring Explosives, Propellants, and
Impurities/Degradation Products.
Acronym
Compound Name
Nitroaromatics
TNT
TNB
DNB
2,4-DNT
2,6-DNT
Tetryl
2AmDNT
4AmDNT
NT
NB
Nitramines
RDX
HMX
NQ
2,4,6-trinitrotoluene
1,3,5-trinitrobenzene
1,3-dinitrobenzene
2,4-dinitrotoluene
2,6-dinitrotoluene
Methyl-2,4,6-trinitrophenylnitramine
2-amino-4,6-dinitrotoluene
4-amino-2,6-dinitrotoluene
Nitrotoluene (three isomers)
Nitrobenzene
Hexahydro-1 ,3,5-trinitro-1 ,3,5-triazine
Octahydro-1 ,3,5,7-tetranitro-1 ,3,5,7-tetrazocine
Nitroguanidine
Field Laboratory
Method Method
Cs
Cp, Ip, CFIp, FOBp
Cs, Is, CFIs
Cs
Cs
Cs, Is
Cs
Is
Cs
Cp, Ip, CFIp, FOBp
Cp
Cs
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Q
Nitrate Esters
NC Nitrocellulose
NG Nitroglycerin
PETN Pentaerythritol tetranitrate
Ammonium Picrate/Picric Acid
AP/PA Ammonium 2,4,6-trinitrophenoxide/2,4,6-trinitrophenol
Cs
Cs
Cs
Cs
Cp, Is
*L
G,*P
*p
A = Ammonium Picrate/Picric Acid (Thorne and Jenkins 1995a)
C = Colorimetric field method(s)
CFI = Continuous flow immunosensor
FOB = Fiber-optic biosensor
Q = Nitroguanidine (Walsh 1989)
I = Immunoassay field method(s)
L = Nitrocellulose (Walsh unpublished CRREL method)
N = EPA SW-846, Nitroaromatics and Nitramines by HPLC, Method 8330 (EPA 1998)
G = EPA SW-846, Nitroglycerin by HPLC, Method 8332 (EPA 1998)
P = PETN and NG (Walsh unpublished CRREL method)
p = Primary target analyte
s = Secondary target analyte
* The performance of a number of field methods has not been assessed using "approved" laboratory methods. It is
recommended that verification of the performance of any analytical method be an integral part of a sampling/analysis
projects quality assurance program.
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This issue paper is divided into the following
major sections: (1) purpose and scope, (2) back-
ground, (3) objectives of water sampling and
monitoring water, (4) an overview of sampling
and analysis for explosives in water, (5) proce-
dures for statistically comparing on-site and
reference analytical methods, (6) a summary of
on-site analytical methods for explosives in water,
and (7) a summary of the EPA reference method
for explosive compounds in water, Method 8330.
While some sections may be used independently,
joint use of the field sampling and on-site
analytical methods sections is recommended to
develop a sampling and analytical approach that
achieves project objectives.
Many of the explosives listed in Table 1 are not
specific target compounds of on-site methods, yet
they may be detected by one or more on-site
methods because of their similar chemical struc-
ture. The explosive and propellant compounds
targeted by high-performance liquid chromato-
graphy (HPLC) methods such as EPA Method
8330 also are listed in the table.
Background
Evaluating sites potentially contaminated with
explosives is necessary to carry out EPA,
U.S. Department of Defense, and U.S. Department
of Energy policies for site characterization and
remediation under the Superfund, Resource
Conservation and Recovery Act, Installation
Restoration, Base Realignment and Closure, and
Formerly Used Defense Site environmental
programs. Facilities that may be contaminated
with explosives include, for example, active and
former manufacturing plants, ordnance works,
Army ammunition plants, Naval ordnance plants,
Army depots, Naval ammunition depots, and
Army and Naval proving grounds, burning
grounds, artillery impact ranges, explosive
ordnance disposal sites, bombing ranges, firing
ranges, and ordnance test and evaluation facilities.
Historical disposal practices from manufac-
turing, spills, ordnance demilitarization, lagoon
disposal of explosives-contaminated wastewater,
and open burn and/or open detonation (OB/OD)
of explosive sludge, waste explosives, excess
propellants, and unexploded ordnance often
resulted in soil and groundwater contamination.
Common munitions fillers and their associated
secondary explosives (indicated in parentheses
[see Table 1 for definitions of acronyms used in
the following paragraphs]) include Amatol
(ammonium nitrate/TNT), Baratol (barium
nitrate/TNT), Cyclonite or Hexogen (RDX),
Cyclotols (RDX/TNT), Composition A-3 (RDX),
Composition B (TNT/RDX), Composition C-4
(RDX), Explosive D or Yellow D (AP/PA),
Octogen (HMX), Octols (HMX/TNT), Pentolite
(PETN/TNT), Picratol (AP/TNT), tritonal (TNT),
tetrytols (tetryl/TNT), and Torpex (RDX/TNT).
Propellant compounds include DNTs and
single-base (NC), double-base (NC/NG), and
triple-base (NC/NG/NQ) smokeless powders. In
addition, NC is frequently spiked with other
compounds (e.g., TNT, DNT, and DNB) to
increase its explosive properties. Explosive D or
Yellow D is used primarily in Naval munitions
such as mines, depth charges, and medium to large
caliber projectiles. Tetryl is used primarily as a
booster charge, and PETN is used in detonation
cord.
Although on-site waste disposal of munitions-
related compounds was discontinued 20 to 50
years ago, a number of munitions facilities have
high levels of soil and groundwater contamin-
ation. Under ambient environmental conditions,
explosives are highly persistent in groundwater
and soil, exhibiting a resistance to naturally
occurring volatilization, biodegradation, and hy-
drolysis. Talmage et al. (1999) reviewed the
environmental fate of several explosive com-
pounds as discussed below. Data indicate that
explosives in weathered, contaminated soils
exhibit slower degradation and desorption kinetics
than explosive residues in spiked soil samples
(Grant et al. 1995). Desorption of explosives
from soil depends on environmental factors
including soil chemistry, contaminant concen-
tration, and the number of pore volumes leached
through the soil (Pennington et al. 1995; EPA
1995).
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Biological degradation products of TNT in
water, soil, or sediments include 2AmDNT;
4AmDNT; and 2,6-diamino-4-nitrotoluene; and
2,4-diamino-6-nitrotoluene. Photolysis of TNT in
water results in formation of 1,3,5-trinitrobenzene
(TNB) and several other compounds. The com-
pound TNB biologically degrades into 3,5-
dinitroanaline, which has been recommended as
an additional analyte for EPA Method 8330
(Grant et al. 1993). In surface waters, TNT is
degraded by photolysis and has a half-life of 0.5
to many hours. The biological half-life of TNT is
much longer, ranging from several weeks to
6 months. Spanggord et al. (1980) reviewed
studies on the sorption of TNT by soils and
sediments and reported the soil-water partitioning
coefficients (Kp) to range from 5.5 to 19.3 ([(ig
chemical in soil/g of soil]/[(ig chemical in water/g
of water]). Recent data show that irreversible
binding may be a significant long-term fate of
TNT that has sometimes not been considered in
older studies (Brannon and Myers 1997; Comfort
et al. 1995). Studies of compost residues (Thome
and Leggett 1997) and C-14 labeled TNT spiked
into soil (Comfort et al. 1995; Hundal et al. 1997)
show that, overtime, solvent-extractable TNT and
metabolic products decrease, but not all of the
original TNT can be accounted for. As the
solvent-extractable TNT decreases, the concen-
tration of hydrolyzable TNT degradation products
increases. Acid hydrolysis is able to break some
chemical bonds between TNT degradation
products and humus or soils. However, overtime,
those bonds seem to become even stronger and
cannot be chemically broken to recover TNT
degradation products (Hundal et al. 1997).
Although the water solubility of RDX is only
low to moderate, the compound is moderately to
highly mobile in the environment. When released
to the environment, RDX can be expected to leach
to and persist in groundwater (Talmage et al.
1999). In surface water, RDX is degraded by
photolysis to formaldehyde, nitrate and nitrite
ions, and nitroso compounds, for which the half-
lives range from hours to many days, depending
on the environmental conditions. As shown by
measured soil-water Kp ranging from 0.80 to 4.15
for sandy loam, clay loam, and organic clay, RDX
does not strongly partition to sediments. In soils,
RDX is quite persistent and is biodegraded very
slowly aerobically, and about an order of magni-
tude faster anaerobically (Brannon and Myers
1997). The limited biodegradation of RDX in
water has been accompanied with the identifi-
cation of hexahydro-l-nitroso-3,5-dinitro-l,3,5-
triazine (MNX), hexahydro-l,3-dinitroso-5-nitro-
1,3,5-triazine (DNX), and hexahydro-1,3,5-
trinitroso-l,3,5-triazine (TNX), which are RDX
intermediates formed by sequential reductions of
the nitro groups to nitroso groups (McCormick et
al. 1981; Kitts et al. 1994; Sikora et al. 1997).
These mono-, di-, and trinitroso intermediates of
RDX are environmentally undesirable. Additional
products formed were hydrazine, 1,1-dimethyl -
hydrazine, 1,2-dimethylhydrazine, formaldehyde,
and methanol.
The compound HMX has a low to moderate
affinity for soil and suspended material, which
accounts for the ready migration of HMX to
groundwater. However, the low solubility of
HMX limits migration of HMX to groundwater.
The primary mechanism of removal of HMX from
surface water is through photolysis. The photol-
ysis half-life of HMX is from 2 to 17 days and
adsorption to suspended material and biosorption
is not significant. While aerobic and anaerobic
degradation of HMX to 1,1-dimethyl-hydrazine
has been demonstrated in enriched media,
biodegradation is not expected to contribute
significantly to the loss of HMX under ambient
conditions. While HMX contamination is not
detected as commonly as TNT or RDX, military-
grade RDX contains approximately 10% HMX as
a manufacturing impurity (Army 1984).
Tetryl is primarily degraded by hydrolysis in
groundwater in which it is sometimes detected and
by photolysis in surface water in which it is
seldom detected. Photolysis is about an order of
magnitude faster than hydrolysis, and the latter
rate has been estimated at about 300 days at 20 °C
with a pH of 6.8. The solubility of tetryl in water
is 75 ppm at 20 °C, which may impede leaching to
groundwater. The primary hydrolysis product,
picric acid, has a solubility of 11,000 mg/L and
may leach to groundwater.
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The frequency of occurrence of specific explo-
sives in groundwater was assessed by Walsh et al.
(1993), who compiled analytical data on water
samples collected from 32 military installations.
Of the 812 samples analyzed by EPA Method
8330 (EPA 1998), a total of 114 samples (14%)
contained detectable levels of explosives. The
frequency of occurrence and the maximum
concentrations detected are shown in Table 2.
The most commonly occurring compound in
contaminated samples, RDX, was detected in 61 %
of the contaminated samples. The compound
TNT was detected in 56% of the contaminated
samples. Overall, RDX or TNT or both were
detected in 94% of the samples containing
explosive residues. Thus, by analyzing for RDX
and TNT at the facilities sampled, 94% of the
contaminated samples could have been identified.
This demonstrates the feasibility of screening for
one or two compounds or classes of compounds to
identify the extent of groundwater and surface
water contamination at munitions sites assuming
that the method detection limits are adequate. At
locations in which RDX-contaminated wastewater
has been disposed of in lagoons or in which spills
have occurred, there is a significant likelihood
that the groundwater has been contaminated with
explosives.
The U.S. Army conducted a study from 1984 to
1985 to evaluate the impact of selected open burn
and/or open detonation facilities on groundwater
quality under various site-specific conditions
(AEHA 1986). A total of 109 wells were sampled
at 17 individual facilities. The facilities were
selected to represent a reasonably large cross-
section of OB/OD sites with fairly diverse
environmental settings. Samples were analyzed
for TNT, 2,4-DNT, 2,6-DNT, RDX, HMX, and
tetryl. The results (see Table 3) show that
explosives were detected in groundwater at 9 of
19 (47%) sites. The compound most frequently
detected was TNT, but RDX was detected at
considerably higher levels. Detected in just two
wells, HMX was detected at high concentration,
and tetryl was never detected. The study
examined factors potentially contributing to
groundwater contamination including soil
permeability, depth to groundwater, temperature,
the level of surface soil contamination, the size
and age of the facility, and annual precipitation
and evaporation rates. The conclusions were that
(1) in the eastern half of the country, the
"predominant factor precluding significant
contamination is low soil permeability" and (2) in
the West, the major factor that precludes
groundwater contamination is "apparently the
significant excess of evaporation over
precipitation". With the exception of the level of
surface soil contamination, the other factors
showed little or no association with resultant
groundwater quality. Recent studies by AEHA
(1994) and Jenkins et al. (1997) also have
identified explosives groundwater contamination
at OB/OD and target impact areas resulting from
active firing range activities.
Other recent studies have shown that explosives
in surface water may migrate considerable
distances from moderate to highly contaminated
disposal areas (LANL 1996; Murphy and Wade
1998). Elevated levels of explosives in surface
waters and sediments have been detected from 1.0
to 1.5 mi downstream from source areas.
Moderately to highly contaminated soils often
leach explosives into groundwater, and
contaminated groundwater may re-emerge into
surface water, particularly for nitramines such as
RDX and HMX. Contaminated sediments also
may serve as a source of recontamination to
surface water because of the low affinity of most
explosives to sediments.
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Table 2. Occurrence of Analytes Detected in Groundwater Contaminated
with Explosives.
Compound
Samples with
Analyte Present
(%)
Maximum and
(Median Levels)
(M9/L)
Nitroaromatics
TNT
1,3,5-TNB
2-AmDNT
2,4-DNT
4-AmDNT
DNB
Tetryl
2,6-DNT
56
28
23
21
15
13
13
9
981
46
218
6.7
217
8.7
12
29
(3.5)
(1.5)
(11)
(1.2)
(4.6)
(0.78)
(0.92)
(0.10)
Nitramines
RDX
HMX
TNT and/or RDX
61
14
94
1400
673
(3.0)
(76)
Derived from Walsh et al. (1993).
Table 3. Occurrence and Concentration of Explosive Residues in Groundwater
at Open Burning Open Detonation Sites.
Type
Explosive
TNT
RDX
2,4-DNT
2,6-DNT
HMX
Tetryl
Facilities
(%)
41
35
35
18
12
0
Wells
(%)
12
10
6
4
2
0
Maximum Geometric Mean
(ug/L) (ug/L)
306
1195
1788
651
583
NA
32
168
14
13
365
NA
Derived from AEHA (1986)
17 facilities, 109 wells total
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Objectives of Sampling and Monitoring
Water
Data Quality Objectives
The EPA data quality objective (DQO) process
is designed to facilitate the planning of
environmental data collection activities by
specifying the intended use of the data (i.e., the
decision that is to be made), the decision criteria
(i.e., the action level), and the tolerable error rates
(EPA 1994a; ASTM D 5792, "Standard Practice
for Generation of Environmental Data Related to
Waste Management Activities: Development of
Data Quality Objectives"). Integrated use of on-
site and laboratory methods for explosives in
water facilitates achieving objectives such as
determining the horizontal and vertical extent of
contamination, obtaining data to conduct a risk
assessment, identifying candidate waste for
treatability studies and pumping tests, identifying
the amount of groundwater or surface water to be
remediated, monitoring and optimizing treatment
systems, and determining whether remedial
actions have met cleanup criteria.
Objectives in Sampling Water
The frequency of occurrence and the coefficient
of variation of a contaminant determine the
number of samples required to adequately char-
acterize exposure pathways, and both are essential
in designing sampling plans. Low frequencies of
occurrence and high coefficients of variation, such
as with explosives, require more samples to char-
acterize the exposure pathways of interest. Sam-
pling variability typically contributes much more
to total error than analytical variability. Under
these conditions, the major effort should be to re-
duce sampling variability by taking more samples
using less expensive methods (EPA 1992).
Environmental data such as the rates of occur-
rence, average concentrations, and coefficients of
variation are typically highly variable for
contaminants associated with explosive sites.
Solidatambienttemperatures, explosives dissolve
slowly and sparingly in aqueous solutions and
have low vapor pressures. These chemical
properties limit the modes of mobility compared
to other contaminants such as fuels or solvents.
The differences between explosives and most
other organic contaminants are a function of
contaminant fate and transport properties, occur-
rence in different media, interactions with other
chemicals, and use and disposal practices. Areas
of high concentrations that serve as sources for
contamination of groundwater remain at or near
the surface where deposited, unless the soils
themselves are moved (Jenkins et al. 1996a).
The EPA guidance for data usability in risk
assessment (EPA 1992) indicates that on-site
methods can produce legally defensible data if
appropriate method quality control is available
and if documentation is adequate. Field analyses
can be used to decrease cost and analytical time as
long as supplemental data are available from an
analytical method capable of quantifying multiple
explosive analytes (e.g., Method 8330) (EPA
1992). Significant quality assurance oversight of
field analysis is recommended to ensure data
usability. The accuracy (i.e., the correctness of
the concentration value and a combination of both
systematic error [bias] and random error [preci-
sion]) of on-site measurements may not be as high
as in fixed laboratories, but the quicker turn-
around and the possibility of analyzing a larger
number of samples more than compensates for
this potential lack in accuracy. Remedial project
managers, in consultation with chemists and
quality assurance personnel, should set accuracy
levels for each method and proficiency standards
for the on-site analyst.
Drinking Water Health Advisories and Water
Quality Criteria for Explosives
In 1985, the EPA and the Department of the
Army established a Memorandum of Under-
standing (MOU) to develop EPA Drinking Water
Health Advisories for Army environmental
contaminants (Roberts and Hartley 1992). The
(MOU) memo resulted in a review of the
toxicological database for selected munitions
chemicals and the development of recommended
exposure limits for specific durations (1 day, 10
days, longer term [7 years], and lifetime [70
-------
years]) (Roberts and Hartley 1992; Roberts et al.
1993; EPA 1996b). Both cancer and noncancer
toxicity endpoints were considered in the assess-
ment. The EPA Drinking Water Health Advi-
sories values for lifetime exposure to selected
explosives or 1E-04 lifetime excess cancer risk
levels for EPA Group B (probable human) carcin-
ogens are presented in Table 4.
The EPA has not established water quality
criteria for munitions compounds, but a series of
unpublished reports by Oak Ridge National
Laboratory have been compiled (Talmage et al.
1999) in which acute and chronic water quality
criteria were calculated for TNT in accordance
with EPA guidelines (Stephan et al. 1985). How-
ever, the available data on other explosive
compounds were not sufficient to meet these
guidelines so Tier II or secondary values were
calculated in accordance with EPA guidance for
the Great Lakes System (EPA 1993c). These
water quality criteria are summarized in Table 4
along with sediment quality criteria (Talmage et
al. 1999) normalized to organic carbon (milligram
explosive/kilogram organic carbon).
Table 4. Water Quality Criteria for Munitions-Related Chemicals
Drinking Water
Water and Sediment Quality Criteria/Screening
Benchmarks3
Compound
TNT
RDX
HMX
1,3-DNB
1,3,5-TNB
2,4-DNT
2,6-DNT
NC
NG
NQ
Health Advisories
(ug/L)
2d
2d
400d
1d
5e
5e
Nontoxic
5d
700d
Acute"
(ug/L)
570
700
1880
110
30
Chronicb
(ug/L)
90
190
330
20
10
Sediment0
(mg/kgoc)
9.2
1.3
0.47
0.67
0.24
a Talmage et al. (1999)
b Calculated in accordance with EPA Tier I (TNT) or Tier II guidelines (other chemicals) (EPA 1993c)
c Milligrams chemical/kg organic carbon in the sediment; calculated in accordance with EPA
guidelines (Stephan et al. 1985)
d Lifetime exposure (EPA 1996b)
e Lifetime excess carcinogenic risk of 1E-04 (EPA 1996b).
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Advantages of On-Site Analytical Methods
Monitoring Remediation Measures
On-site methods may be useful for analysis of
water treatment processes for explosives, such as
granular activated carbon (GAC) or chemical and
ultraviolet (UV) oxidation treatment systems
(EPA 1993a, 1996a; AEC 1997). However, on-
site methods should be evaluated against labor-
atory methods on a site and matrix-specific basis
because of the possibility of matrix interferences.
Treatability studies may be used to evaluate the
potential of different treatment technologies to
remove and degrade target and intermediate ex-
plosive compounds and to evaluate whether clean-
up levels can be achieved for site remediation.
Treatability study waste for explosives-contam-
inated waters should be of higher than average
concentration to evaluate removal rates for target
and intermediate compounds. The potential
effects from compounds related to treatment pro-
cesses, such as TNB from chemical and UV oxi-
dation systems (AEC 1997) and MNX, DNX, and
TNX from biological and phytoremediation
systems (Sikora et al. 1997), also should be
evaluated.
On-site analytical methods are a valuable, cost-
effective tool to assess the nature and extent of
contamination (EPA 1997b). Because costs per
sample are lower, more samples can be analyzed.
In addition, the availability of near-real-time
results permits redesign of the sampling scheme
while in the field. On-site analysis also facilitates
more effective use of off-site laboratories using
more robust analytical methods. Even if on-site
methods are only used to determine the presence
or absence of contamination and the contaminated
samples are sent off-site for laboratory analysis,
total analytical costs can be reduced considerably,
provided that the on-site methods have low
enough detection limits to meet site DQOs. Be-
cause on-site methods provide near-real-time
feedback, they can be used to focus additional
sampling on areas of known contamination, thus
possibly saving additional mobilization and
sampling efforts.
During site remediation, such as Superfund
remedial actions, data may be needed on a
near-real-time basis to assess the progress of
pump-and-treat remedial actions (EPA 1994b;
Craig et al. 1996). These treatment systems are
often estimated to operate for a period of 10 to 30
years. On-site methods can be used during
remediation to monitor individual extraction wells
and combined influent explosives concentrations,
as well as to evaluate GAC breakthrough and
determine when to replace the GAC bed. Final
attainment of groundwater or surface water clean-
up levels should be determined by an approved
method, such as EPA Method 8330 (EPA 1998).
Figure 1 shows the time series extraction well
concentrations of TNT and RDX for a GAC treat-
ment system for a 110-acre groundwater plume at
the U.S. Naval Submarine Base in Bangor,
Washington. The influent for a typical single-
stage fixed bed GAC system enters the top of the
carbon column, and the explosives are adsorbed as
the waste stream flows through the column. The
treated liquid stream (effluent) exits the bottom of
the column. Once the effluent no longer meets the
treatment criterion, the spent carbon is reacti-
vated, regenerated, or replaced. As the GAC
system continues to operate, the mass-transfer
zone moves down the column. Figure 2 shows the
adsorption pattern and the corresponding effluent
breakthrough curve. The breakthrough curve is a
plot of the ratio of effluent concentration (Ce) to
influent concentration (C0) as a function of the
water volume treated per unit time. When a pre-
determined concentration appears in the effluent
(CB), breakthrough has occurred. At this point,
the effluent quality no longer meets treatment
objectives. When the carbon becomes so satur-
ated with explosives that they can no longer be
adsorbed, the carbon is said to be spent (Ce = C0).
Alternate design arrangements may allow indi-
vidual adsorbers in multi-adsorber systems to be
operated beyond the breakpoint as far as complete
exhaustion. This condition of operation is defined
as the operating limit (Ce = CL).
-------
On-site colorimetric methods for system moni-
toring and determination of breakthrough curves
are being used at a Superfund remedial action for
an explosives washout lagoon groundwater GAC
pump-and-treat system (ACOE 1998). Figure 3
shows the RDX breakthrough curve for between
bed samples in a two-bed GAC system in series
for a 350-acre groundwater plume at the Umatilla
Chemical Depot in Hermiston, Oregon. Influent
concentrations into the system are 97 (ig/L of
TNT, 29 ng/L of TNB, 710 ng/L RDX, and 63
(ig/L of HMX. Final effluent concentrations also
are monitored using the on-site colorimetric
methods. The only compound detected in the
between bed samples and final effluent samples
was RDX. The GAC system exhibits preferential
adsorption for explosives compounds (TNT >
TNB > HMX > RDX) in the same waste stream.
Other explosives compounds are progressively
displaced in favor of TNT adsorption. The pres-
ence of multiple explosives will reduce the carbon
bed life in relation to single compound isotherms,
particularly for the breakthrough of RDX
(Vlahakis 1974; Lee and Stenstrom 1996).
Typical loading rates achieve 1 to 4% total
explosives loading onto the lead GAC bed before
breakthrough occurs.
Overview of Sampling and Analysis
for Explosives in Water
Explosive Hazards During Well Installation
The explosives safety procedures necessary for
geophysical detection, handling, and disposal of
UXO and geotechnical operations such as well
installation in areas that potentially contain high
levels of explosives in soil are beyond the scope
of this document. These conditions present a
potential detonation hazard; therefore, explosive
safety procedures and safety precautions should
be identified before initiating site characterization
activities in these environments (EPA 1993a). A
qualified explosives safety expert should be con-
sulted in preparing field sampling procedures for
operations under these conditions (ACOE 1996a,
1996b).
Water Sampling
Except for the significant hazards of installing
wells and working in areas that may contain UXO,
bulk high explosives, or highly contaminated
surface soils such as explosives washout lagoons,
procedures for sampling groundwater and surface
water for explosive residues are similar to sam-
pling for other semi volatile organic compounds.
The EPA guidance on groundwater sampling can
be found in Subsurface Characterization and
Monitoring Techniques, Volume 1: Solids and
Ground Water (EPA 1993b, Chapter 5). The
American Society for Testing and Materials
(ASTM) provides guidance for sampling surface
and groundwater: "Guide for Sampling
Groundwater Monitoring Wells," Standard D
4448; "Guide for Planning and Implementing a
Groundwater Monitoring Program," Standard D
5851; "Practice for Sampling Wastes from Pipes
and Other Discharge Points," Standard D 5013;
and "Practice for Sampling with a Dipper or Pond
Sampler," Standard D 5358. Other standard
procedures can be located on the ASTM World
Wide Web site, http://www.astm.org.
Well Screens and Casing Materials
Parker and Ranney (1993) and Parker et al.
(1989) demonstrated that none of the explosives
evaluated sorb to well casings. There were no
significant differences among polytetra-
fluoroethylene, rigid polyvinyl chloride, and
stainless steel used as well casing materials for
RDX, TNT, HMX, TNB, DNB, NB, 2AmDNT,
DNT, or NTs.
Containers, Holding Times, and Preservation
Methods
The EPA guidance (EPA 1998) on sampling
containers for semivolatile organic compounds
specifies 1-gal, two 0.5-gal, or four 1-L amber
glass containers with Teflon-lined lids. These
containers and volumes were designed for
laboratory procedures such as Method 8330 for
which significant sample concentration may be
required. Similar bottles, of adequate volume for
the method, should be satisfactory for on-site
analytical methods.
10
-------
TNT Concentration Changes Over Time
T T T
Wells Located <350 Feet Downgjadfent of Former Lagoon
^••^.4EOI^-^-4I.4E0lf^-^-4E4Ea^-a-4E0lf»^-^4Ea^--^-COI
Hate
RDX Concentration Changes Over Time
Near-Field Extraction Wdls
Date
Wells Located 350-700 Feet Down gradient of Farmer Lagoon
MO-
I
s
t»
C !
CJ"
Date
14X
Far-Field Extraction Wdls
Date
Figure 1. Extraction Well Concentrations of TNT and RDX for a GAC Treatment System for a 110-acre Groundwater Plume at the U.S.
Naval Submarine Base in Bangor, Washington.
-------
c,
I
~
Cr
T
Ce 100
g 50
(
Granular Activated Charcoal System
:- - -Jf~
: ^^X
> 5 1D152U253D35404& 5O 55 «0 G& 7O 75 £0 85 90 9
Days
&
Figure 3. RDX Breakthrough Curve
The EPA-specified holding time for nitroaro-
matic compounds in water is 7 days until extrac-
tion, and extracts must be analyzed within the
following 40 days (EPA 1998). The specified
sample preservation procedure is cooling to 4» C.
This criterion was based on professional judgment
rather than experimental data. While recent,
scientifically based data have been generated on
improved preservation procedures for explosives
in water (see below), the ramifications of using
such procedures for legally defensible data should
be considered during the DQO process. Deviation
from EPA procedures may require the user to
justify such changes and might result in the data
being deemed unfit for the intended use.
Because of the short holding times between
sample collection and analysis using on-site
12
-------
analytical methods, sample preservation is typically
not a concern. However, if samples will be held
before analysis, sample preservation may need to
be considered. For split samples sent to an off-site
analytical laboratory, sample holding times and
improved preservation methods become an
important consideration.
Two recent studies, by Maskarinec et al. (1991)
and Grant et al. (1993), have shown that nitramines
are stable in water, without any form of
preservation for 30 and 50 days. However, both
studies also demonstrated that nitroaromatics can
undergo significant degradation within days.
Maskarinec recommended a maximum holding time
of 4 days for DNT in groundwater while Grant
found DNT relatively stable and recommended a
holding time of 30 days for surface water. Grant's
work on TNB and TNT showed losses of 55% and
35%, respectively, in 7 days for spiked surface
water samples stored under refrigeration. Jenkins
et al. (1995a) showed that tetryl also can degrade
rapidly in surface waters with 73% being lost in
7 days despite refrigeration. Degradation was
much faster in surface water than it was in
groundwater or reagent water.
Because nitroaromatics can degrade rapidly in
water samples, Jenkins et al. (1995a) evaluated
possible sample preservation procedures. Sample
acidification to a pH of 2 with sodium bisulfate was
demonstrated to retard microbiological and
chemical transformations, is relatively easy to
conduct in the field, and does not interfere with
solid phase extraction preconcentration procedures
in Method 3535A (EPA 1998). Acidification of
spiked surface water samples eliminated losses of
TNT and TNB for 64 days and at least 28 days for
tetryl. Nitramines (RDX and HMX) were stable in
spiked, refrigerated surface water, with or without
preservation, for at least 64 days. Small losses of
aminodinitro-toluenes were observed for both
acidified and unacidified samples, and the loss rate
was initially higher for acidified samples. Acidified
samples must be neutralized if the samples will be
concentrated using the salting-out procedure in
Method 8330.
Procedures For Statistically Comparing
On-site And Reference Analytical
Methods
When on-site methods are used, their perfor-
mance needs to be evaluated, which is commonly
done by analyzing the splits of some water samples
by both the on-site method and a reference method
(commonly Method 8330). The performance of the
on-site method is then statistically compared to the
reference method using a variety of criteria,
depending upon the objective and the
characteristics of the data. In most cases,
measures of precision and bias are determined.
Precision refers to the agreement among a set of
replicate measurements and is commonly reported
as the relative standard deviation (standard
deviation divided by the mean and expressed as a
percent), the coefficient of variation (standard
deviation divided by the mean), or the relative
percent difference (sample value minus the
reference method value divided by the mean and
expressed as a percent). Bias refers to systematic
deviation from the true value.
The following discussion of statistical methods
applies to comparisons of analytical results based
on paired sample data (e.g., duplicate or split water
samples are analyzed by both an on-site method
and a reference method or water sample extracts
are analyzed by a reference and on-site method).
Precision and Bias Tests for Measurements of
Relatively Homogenous Material—When
multiple splits of well-homogenized samples are
analyzed using different analytical methods,
statistical procedures summarized in Grubbs (1973),
Blackwood and Bradley (1991), and Christensen
and Blackwood (1993) can be used to compare the
precision and bias of the methods. Grubbs
described a statistical approach appropriate for
comparing the precision of two methods that takes
into account the high correlation between the
measurements from each method. An advantage
of Grubbs' approach is that it provides unbiased
estimates of the precision of each method by
partitioning the variance of the measurement results
13
-------
into its component parts (e.g., variance caused by
subsampling and by the analytical method).
Blackwood and Bradley (1991) extended Grubbs'
approach to a simultaneous test for equal precision
and bias of two methods. Similar tests are provided
in Christensen and Blackwood (1993) for
evaluating more than two methods.
For comparisons involving bias alone, t-tests or
analysis of variance may be performed. For com-
paring two methods, paired t-tests are appropriate
for assessing relative bias in normally distributed
data (otherwise data are transformed to achieve
normality or nonparametric tests are used). A
paired t-test can be used to test whether the con-
centration as determined by an on-site method is
significantly different from Method 8330 or any
other reference method. For comparing multiple
methods, a randomized complete block analysis of
variance can be used in which the methods are the
treatments and each set of split samples consti-
tutes a block.
These tests are best applied when the concen-
trations of explosives are all of approximately the
same magnitude. As the variability in the sample
concentration increases, the capability of these tests
for detecting differences in precision or bias
decreases. The variability in the true quantities in
the samples is of concern, and high variability in
sample results caused by poor precision rather than
variability in the true concentration is handled well
by these methods.
Precision and Bias Tests for Measurements
over Large Value Ranges—When the
concentrations of explosives cover a large range
of values, regression methods for assessing
precision and accuracy become appropriate.
Regression analysis is useful because it allows
characterization of nonconstant precision and bias
effects and predicts intervals for new
measurements (e.g., the results of an on-site
method can be used to predict the concentration if
the samples were analyzed by a reference method).
In a regression analysis, the less precise on-site
method is generally treated as the dependent
variable and the more precise reference analytical
method (e.g., Method 8330) as the independent
variable. A linear relationship and a slope that
differs from a value of 1.0 indicates a constant
relative bias in the on-site method (i.e., the two
methods differ by a fixed percentage). Similarly,
an intercept value significantly different from zero
indicates a constant absolute bias (i.e., the two
methods differ by a fixed absolute quantity). Of
course, both fixed and relative bias components
may be present.
When uncertainty is associated with the concen-
tration of an explosive as measured by the refer-
ence method, standard least squares regression
analysis can produce misleading results. Standard
least squares regression incorporates the assump-
tion that the independent variable values are known
exactly as in standard reference material. When
the reference method results contain appre-ciable
error compared to the on-site method, regression
and variability estimates are biased. Furthermore,
the interpretation of R-squared and uncertainty
intervals are affected, which is known as an errors-
in-variables problem.
Because of the errors-in-variables problem, the
slope coefficient in the regression of the on-site
data on the reference data generally will be biased
low. Hence, a standard regression test to deter-
mine whether the slope is significantly different
from 1 can result in rejection of the null hypothesis
even when there is no difference in the true bias of
the two methods. A similar argument applies to
tests of the intercept value being equal to zero.
To perform a proper errors-in-variables regres-
sion requires consideration of the measurement
errors in both the dependent and independent
variables. The appropriate methods for per-
forming the regression (including some guidance
about how large the error in the reference
analytical method can be before a problem is
encountered) are outlined in Mandel (1984). These
methods require estimating the ratio of the random
error variance for the on-site method to that of the
reference analytical method. With split sample
data, suitable estimates of the variance ratio
generally can be obtained by using variance
14
-------
estimates from Grubbs' test or the related tests
mentioned above.
If the variance ratio is not constant over the
range under study, more complicated models than
those analyzed in Mandel (1984) must be employed.
Alternatively, transformations of the data could
stabilize the variance ratio. Note that it is the
variance ratio, not the individual variances, that
must remain constant. For example, the ratio of
variances for two methods with nonconstant
absolute variances but constant relative variances
will still have a constant variance ratio.
It should be noted that performing regressions on
data sets in which samples with concentrations
below the detection limit (for one or both methods)
have been eliminated also may result in biased
regression estimates, regardless of the regression
analysis method that is used.
Comparison to Regulatory Thresholds and
Action Limits—When the purpose of sampling is
to make a decision based on the comparison of
results to a specific value such as an action level
for cleanup, on-site and reference analytical
method results may be compared simply on the
basis of the degree of agreement between the two
methods. The appropriate statistical tests are based
on the binomial distribution and include tests of
equality of proportions and chi-square tests
comparing the sensitivity and specificity (i.e., false
positive and false negative rates) of the on-site
method relative to the reference analytical method.
Note that any measure of consistency between the
two methods is affected by how close the true
values in the samples are to the action level. The
closer the true values are to the action level, the
less the two methods will agree, even if they are of
equal accuracy. For example, if the action level is
2 • g/L and most samples have levels of above 100
• g/L, the agreement between the on-site method
and reference should be very good. If, however,
the concentration in most samples is 0.5 to 10 • g/L,
the two methods will be much more likely to
disagree. This must be kept in mind when
interpreting results, especially when comparing
across different studies for which samples may
have been collected at considerably different
analyte levels.
Summary of On-site Analytical Methods
for Explosives in Water
There is significant interest in field methods for
rapidly and economically determining the presence
and concentration of secondary explosives in
groundwater and the influent and effluent to
groundwater remediation facilities. Such
procedures allow much greater flexibility in
mapping the extent of contamination, designing
pumping strategies based on near-real-time data,
accruing more detailed characterization for a fixed
cost, and guiding continuous remedial actions.
Ideally, on-site analytical methods would provide
high-quality data on a near-real-time basis at low
cost and of sufficient quality to meet all intended
uses including risk assessments and final remedial
action objectives without the need for more
rigorous procedures. While the currently available
on-site methods are not ideal (i.e., they are not
capable of providing compound-specific
concentrations of multiple compounds simul-
taneously as might be desired in risk assessment),
they have proved to be very valuable during the
characterization and remediation monitoring of
some sites. Currently available field methods that
have been evaluated against standard analytical
methods and demonstrated in the field include
colorimetric, immunoassay, and biosensor methods
(see Table 5). Each method has relative
advantages and disadvantages, so that no method is
optimal for all applications. To assist in the
selection of one or more screening methods for
various users needs, Table 6 (modified and ex-
panded from EPA 1997a) provides information on
on-site test methods for detecting explosives in
water. The selection criteria are discussed in the
following sections.
The three types of on-site methods, colorimetric,
immunoassay, and biosensor, are fundamen-tally
quite different. The CRREL colorimetric methods
were developed by Jenkins for TNT (Jenkins 1990)
15
-------
and RDX (Walsh and Jenkins 1991) in soils. Later
Jenkins et al. (1994a, 1995b) developed a solid-
phase extraction method for TNT and RDX in
water in which the extraction disks could be
extracted with acetone and analyzed by the soil
analytical procedures. The same methods are now
used in the Strategic Diagnostics, Inc., EnSys
procedure for extraction of TNT and RDX + HMX
from groundwater, and the EnSys TNT and RDX
soil test kits are used to complete the analysis (see
below). (Note that the EnSys procedure refers to
the RDX + HMX kit as simply the RDX kit while
the draft EPA Method 8510 refers to it as RDX +
HMX. The latter designation is used throughout
this document.) The commercial versions of the
methods are the most commonly used. Therefore,
the tables and the text refer only to the EnSys
procedures for TNT and RDX + HMX but the
CRREL methods can provide equivalent results.
Researchers at CRREL also developed a
colorimetric analytical procedure for quantifying
ammonium picrate and picric acid in soil and water
(Thorne and Jenkins 1995a, 1995b). In the
procedure, 2 L of water are drawn through an
anion extraction disk under vacuum and the disk is
washed to remove interferences. Picrate ions are
converted to picric acid and are eluted from the
disk, and absorbance measurements are made
before and after conversion to the yellow picrate
ion.
In the EnSys colorimetric method for water, solid-
phase extraction is used to remove and concentrate
analytes from water. A 2-L water sample is
passed through a stack of two membranes to
preconcentrate TNT on the top membrane and
RDX on the bottom membrane. Acetone is used to
elute RDX + HMX from the bottom disk, and a
chemical reaction is induced that causes a color
change indicative of RDX in the solution. The
RDX + HMX concentration is estimated from the
absorbance at 510 nm on a portable
spectrophotometer. The top disk is eluted with
acetone, and a different chemical reaction is
induced causing a color change indicative of TNT.
The TNT concentration is estimated from the
absorbance at 540 nm.
Table 5. Available On-Site Analytical Methods for Explosives in Water.
Analyte(s)
Nitroaromatics
1.
2.
3.
4.
TNT
TNB
DNT
Tetryl
Nitramines
1.
2.
3.
RDX
HMX
NQ
Nitrate Esters
1.
2.
3.
NC
NG
PETN
AP/PA
Type Test
Colorimetric
Colorimetric
Immunoassay
Biosensor
Colorimetric
Immunoassay
Biosensor
Colorimetric
Colorimetric
Colorimetric
Colorimetric
Immunoassay
Biosensor
Colorimetric
Colorimetric
Colorimetric
Colorimetric
Colorimetric
Colorimetric
Colorimetric
Developer/Test Kit
EnSys - TNT
EnSys - TNT
D TECH - TNT
RaPID Assay
Continuous Flow Immunosensor - TNT
Fiber-Optic Biosensor - TNT
CRREL, EnSys - TNT
RaPID Assay
Continuous Flow Immunosensor - TNT
EnSys - TNT
EnSys - TNT
EnSys - RDX + HMX
EnSys - RDX + HMX
D TECH - RDX
Continuous Flow Immunosensor - RDX
Fiber-Optic Biosensor - RDX
EnSys - RDX + HMX
EnSys - RDX + HMX
EnSys - RDX + HMX
EnSys - RDX + HMX
EnSys - RDX + HMX
EnSys - RDX + HMX
CRREL 1
1 U.S. Army Corps of Engineers, Cold Regions Research and Engineering Laboratory
16
-------
Table 6. Comparative Data for Selecting On-Site Analytical Methods for Explosives in Water.3
Method/Kit
CRREL
EnSys
(Commercial
version
CRREL, TNT
and RDX
methods)
DTECH
RaPID Assay
Continuous
Flow
Immunosensor
Fiber-Optic
Biosensor with
Fluidics Unit
Criteria
Method Type,
Analytes, and EPA
Method #
Colorimetric
Ammonium Picrate
/Picric Acid
Colorimetric
TNT
RDX + HMX
Draft Method 8510
Immunoassay - ELISA
TNT
RDX
Immunoassay - ELISA
Magnetic particle/tube kit
TNT
Immunosensor
TNT
RDX
Proposed Method 4655
Immunosensor
TNT
RDX
Proposed Method 4656
Detection Range and
Range Factor
AP/PA: 3.6 to 200 • g/L (56
X)
TNT: 1 to 30 • g/L (30 X)
RDX: 5 to 200 • g/L (40 X)
H MX: 15 to 300- g/L (20 X)
TNT & RDX:
5 to 45 • g/L (9 X)
with DETECHTOR
TNT: 0.07 to 5 • g/L (71 X)
TNT and RDX:
10to1,000-g/L(100X)
TNT: 10 to 150- g/L (15 X)
RDX: 10 to 100- g/L (10 X)
Type of
Results
Quantitative
Quantitative
Semiquantitative
(concentration
range)
Quantitative
Quantitative
Quantitative
Samples per
Batch
AP/PA: Single or
batched
Single
Four (single or
batch)
Batch up to
51 samples
Sequential
Single up to a
batch of four
Water
Sample
Size
2L
2L
1 mL
100|jL
150|jL
1.7mL
for four
fiber
analyses
Sample Preparation
and Extraction
Filtration if the sample
is cloudy, solid-phase
extraction using anion
extraction disk, eluted
with methanol and
sulfuric acid.
Solid-phase extraction
using two membranes
filters, elution of filters
with acetone.
None
Filter (0.2- m) if gross
particulates are
present
To 955 |jL sample,
add 25 |jL ethanol and
20 |jL of SOX buffer
To 1.7 mL of sample,
add 200 |jL buffer and
100 |jL acetone.
Analysis Time/
Production Rate
(one person)
20 minutes to hours to filter, faster
per sample if batched;
recommended only for low turbidity
waters. 20 mins./ sample to
analyze.
20 minutes to a few hours for
filtering, recommended only for low
turbidity waters.
TNT : 35 mins./10 samples
RDX: 50 mins./10 samples
40 minutes for eight samples for
TNT and RDX.
10 to 15 mins. for single sample
70 minutes for 51 samples
3 to 4 minutes per sample, plus 3 to
4 minutes for internal standard, plus
1 minute peak analysis.
Total time < 20 minutes for typical 2-
3 analyses/sample
TNT: 8 minutes per quadruplicate
sample or batch of four.
RDX: 16 minutes per quadruplicate
sample or batch of four.
Double times to run reference
analysis. Typically each sample is
analyzed 2 to 4 times.
' Expanded and modified from EPA 1997a
-------
TableG. (Continued)
Method/Kit
CRREL
EnSys
DTECH
RaPID Assay
Continuous
Flow
Immunosensor
Fiber-Optic
Biosensor with
Fluidics unit
Criteria
Interferences and Cross-reactivities > 1% based on IC50
Relatively free of humic and nitroaromatic interferences.
TNT = TNT + TNB + DNB + DNTs + tetryl;
RDX + HMX = RDX + HMX + PETN + NQ + NC + NG
Humics interfere with TNT and RDX; nitrate and nitrite interfere
with RDX.
TNT interferes with RDX method only when both are present.
Cross-reactivity:
TNT: tetryl = 35%; TNB = 23%; 2AmDNT = 1 1 %; 2,4-DNT = 4%;
AP/PA unknown but -100% at lower limit of detection
RDX: HMX = 3%
Cross-reactivity:
TNB = 65%; 2,4-Dinitroaniline = 6%; tetryl = 5%; 2,4-DNT = 4%;
2AmDNT = 3%;
DNB = 2%
TNT Method: TNB = 600%, tetryl = 38%, 2-AmDNT = 21 %, 2,4-DNT=
20%, NB = 16%, 2-NT = 9%, HMX = 5%, 2,6-DNT = 4%
RDX: 1 ,2-dinitroglycerin = 18%, HMX = 5%, TNB = 4%, 2,4-DNT =
3%, 1,3-DNB = 3%
TNT Method: TNB = 9%. All other tested explosives <4%
RDX Method: no explosive related interferences, (<3%)
no nitrate/nitrite interference
Supplier Recommended QA/QC '
Blank and spiked water samples analyzed daily.
Method and water blanks and a control sample
daily, one duplicate/20 samples. Some positive
field results (1:10) should be confirmed.
Samples testing positive should be confirmed
using standard methods.
Duplicate standard curves; positive control
sample supplied. Positive results requiring action
may need confirmation by another method.
Internal standards used for quantification, blank
matrix sample for background subtraction.
Reference analysis every other sample, run
blank once per set of fiber probes.
Storage Conditions and
Shelf Life of Kit or Reagents
Store at room temperature.
Store at room temperature.
Shelf life:
TNT = 2 to 24 months at 27- C
RDX = 2 to 12 months at 27- C
Store at room temperature or
refrigerate; do not freeze or
exceed 37- C for prolonged
period. Shelf life: 9 months at
room temperature.
Refrigerate reagents 2 to 8- C.
Do not freeze.
Shelf life 3 to 12 months.
Store activated membranes
moist at 4- C and away from
light.
Shelf life ~ 1 month
Fiber probes: Shelf life > 1 year
when stored < 27- C
Skill Level
Medium high
Medium
Low
Med-high,
initial training
recommended
Medium
Medium
00
a Expanded and modified from EPA 1997a
1 Site specific DQOs should always be used to select appropriate QA/QC
-------
Table 6. (Continued)
Method or
Kit
CRREL
EnSys
DTECH
RaPID Assay
Continuous
Flow
Immunosensor
Fiber-Optic
Biosensor
with Fluidics
unit
Criteria
Training Availability
None
Applicable video on
CRREL soil method
available only,
address in text.
Training available.
Applicable video on
CRREL soil and
groundwater methods
are available,
addresses in text.
Training available
Training available
No formalized training
available at this time.
No formalized training
available at this time.
Costs
(not including labor)
$15/sample plus $1,500
for Hach spectrometer.
Vacuum filtration
apparatus needed.
$21/sampleforTNT,
$25/sample for RDX
plus$175/day or
$450/wk, $800/mo for
lab station. Lab station
cost = $1 ,950.
Vacuum filtration
apparatus needed.
$32.50/sample for TNT
or RDX plus $300 for
DTECHTOR (optional).
$13to$20/sample plus
$4,000 for equip.
(purchase), 175/day,
$450/wk or $800 for
first month, $400 each
additional month
(rental).
$50/coupon which lasts
for -20 to 30 samples
plus $21 ,000 for
instrument
(FAST 2000).
$3 to 5/sample plus
$18,000 for instrument
(Analyte 2000 from
Research International)
and $-8,000 for Fluidics
unit.
Comparisons to
Method 8330 References
Thorne and Jenkins 1995a
Craig etal. 1996; EPA
1997a; Jenkins and
Schumacher 1990; Jenkins
eta!1994b
Craig etal. 1996; EPA
1997a;
Teaney and Hudak 1994
Thorne and Myers 1997
Craig etal. 1996; EPA
1997a; Rubioetal. 1996
Craig etal. 1996; EPA
1997a; Bart etal. 1997 a,
1997b;ESTCP1998
Craig etal. 1996; EPA
1997a; Shriver-Lake et al.
1995, 1997; ESTCP 1998
Other
References
Thorne and
Jenkins
1995b
Jenkins et
al. 1995b
USAGE
1999
Calif. EPA
1996a,
1996b
Calif. EPA
1996c
Narang et al
1998,
Whelan et
al. 1993
Shriver-
Lake et al.
1998,
Golden et al.
1997
Developer Information
Tom Jenkins
CRREL
72 Lyme Road
Hanover, NH 03755-1290
(603) 646-4385
Strategic Diagnostics
1 1 1 Pencader Dr.
Newark, DE 19702-3322
(800) 544-8881
www.sdix.com
Strategic Diagnostics
1 1 1 Pencader Dr.
Newark, DE 19702-3322
(800) 544-8881
www.sdix.com
Strategic Diagnostics
1 1 1 Pencader Dr.
Newark, DE 19702-3322
(800) 544-8881
www.sdix.com
Anne Kusterbeck
Naval Research Lab.
4555 Overlook Ave. SW
Washington, D.C. 20375
(202) 404-6042
Lisa Shriver-Lake
Naval Research Lab.
4555 Overlook Ave. SW
Washington, D.C. 20375
(202) 404-6045
Additional Considerations
Large work area (two large desks);
requires the most setup; electricity required;
deionized, methanol, and sulfuric acid
required; must assemble materials;
glassware must be rinsed between
analyses; vacuum filtration apparatus
needed.
Large work area (desk size) power supply
required to charge Hach spectrometer;
possible TNB interference; color indication
of other compounds; requires acetone and
deionized water; cuvettes must be rinsed
between analyses. Nitrate and nitrite
interferences with RDX kit can be corrected
using alumin-a-cartridges. Vacuum filtration
apparatus needed.
Small working area; few setup
requirements; no electricity or refrigeration
required; temperature dependent
development time (effect can be reduced by
changing DTECHTOR setting); significant
amount of packing; relatively narrow range;
no check on test; easy to transport or carry;
kits can be customized. Out-of- range
reruns require use of another kit.
Large work area (desk); requires setup
time, electricity and refrigeration; less
temperature dependent; low detection limit;
all reagents supplied; reagents and kit need
refrigeration. Out-of-range reruns require
dilution and full reanalysis.
Desk size work area. Less packaging
waste, requires electricity and refrigeration.
Instrumentation available from: Research
International, 18706 142nd Ave. NE,
Woodinville, WA 98072, (206) 486-7831
Desk size work area, can be operated
without fuidics unit, requires electricity,
refrigeration recommended. Less packaging
waste. Quantification requires sample
dilution when percent inhibition is >60% for
TNT or > 80% for RDX
' Expanded and modified from EPA 1997a
-------
The steps of the various immunoassay methods
differ considerably. The simplest of the methods
is the D TECH method. In the D TECH kit,
antibodies specific for TNT and closely related
compounds are linked to solid particles. The TNT
molecules in water samples are captured by the
solid particles and collected on the membrane of a
cup assembly. A color-developing solution is
added to the cup assembly, and the presence (or
absence) of TNT is determined by comparing the
solution in the assembly cup to a color card or by
using the simple field test meter. The color is
inversely proportional to the concentration of TNT.
The continuous flow immunosensor (CFI) and
the fiber-optic biosensor (FOB) methods were
developed by the Naval Research Laboratory
(NRL). The CFI method (Bart et al. 1997a,
1997b; EPA 1997a) is an antibody-based bio-
sensor capable of detecting low molecular weight
molecules in aqueous solutions. With the CFI
method, TNT or RDX antibodies are immobilized
onto a solid phase (beads, membrane or glass
capillary) saturated with a fluorescent-dye labeled
antigen. The solid phase is placed in a support and
an aqueous buffer solution is pumped through the
support to establish flow. Samples are prepared
in the buffer solution and injected upstream of the
support. When a sample containing TNT or RDX
is introduced, the TNT or RDX binds to the
immobilized antibody, displacing some of the
labeled antigen, which is subsequently detected by
a fluorometer. The concentration is proportional to
the fluorometer signal. A portable version of the
CFI (FAST 2000) has been engineered by
Researchlnternational, Inc. which incorporates the
solid phase, sample injection, pump, fluidics control,
and a fluorometer into a single instru-ment, with
associated software for data acquisition and
analysis using a lap top computer.
The FOB method (Shriver-Lake et al. 1995,
1997; EPA 1997a) is based on a competitive
immunoassay using a fluorescent dye as the
reporter molecule. Fluorescent-dye labeled TNB
is used as the competitor on the surface of an
optical probe. The labeled TNB is exposed to an
antibody-coated optical fiber for 4 minutes,
generating a specific signal that corresponds to the
100% signal. The reference signal is defined as
the signal change associated with the labeled TNB
alone. Inhibition of this signal is observed when
TNT is present in a sample. The percent inhibition
observed is proportional to the TNT concentration
in the sample. The reference signal is determined
both before and after running the sample to
normalize for the gradual decrease in antibody
activity. A portable version of the FOB (Analyte
2000) has been engineered by Research Interna-
tional, Inc. Originally developed only for TNT, the
FOB method has been modified and is now
available for RDX as well.
Method Type, Analytes, and EPA Method
Number
The first column of the criteria listed in Table 6
identifies the type of water screening method, the
analytes it detects, and the EPA draft method
number.
The CRREL colorimetric method for Explosive
D or Yellow D (AP/PA) has been formally docu-
mented (Thorne and Jenkins 1995 a) but it is not
under consideration for incorporation into SW-846
(EPA 1998), nor is it being evaluated by any
method certification organization.
Commercially available colorimetric kits mar-
keted under the EnSys trade name and manu-
factured by Strategic Diagnostics, Inc., are
available for determining nitroaromatics (TNT) and
nitramines (RDX + HMX) in soils. The same
analytical methods can be used to analyze acetone
extracts of filter disks that have extracted
nitroaromatics and nitramines from 2 L of water
via vacuum filtration. The water-extraction step
requires at least a small field laboratory. There-
fore, Strategic Diagnostics has not promoted the
EnSys TNT and RDX + HMX procedures for
analysis of water although the company will
provide procedures upon request. The EnSys
TNT colorimetric method detects nitroaromatics
(i.e., TNT, TNB, DNB, DNTs, and terry 1), and the
RDX + HMX method detects nitramines (RDX,
HMX, and NQ), and nitrate esters (NC, NG, and
PETN). While NC is detected by the actual
analytical method, it is not clear that acetone will
elute NC from the membrane filter disks with the
20
-------
other explosives. The EnSys RDX + HMX
method is draft EPA Method 8510 and is designed
for soil and water. The EPA Method 8515 is the
EnSys TNT method specific to soil (summarized in
soils issue paper [Crockett et al. 1996, 1998]).
However, the water extraction step of the RDX +
HMX method (draft Method 8510) extracts TNT
on the first filter of a two-filter stack, and the first
filter can be extracted with acetone and analyzed
using the TNT soils method (Method 8515). The
EPA currently has no plans to revise Method 8515
to include analysis of water samples.
Strategic Diagnostics, Inc., also manufacturers
commercial enzyme-linked immunosorbent assay
(ELISA) kits, including the D TECH and RaPID
Assay kits to detect TNT in water. D TECH
immunoassay kits also are available for RDX.
Other explosives compounds can sometimes be
detected using immunoassay kits because of their
cross-reactivity (see the Interferences and Cross-
Reactivity section). The California Environmental
Protection Agency has certified the D TECH kits
for TNT (California EPA 1996a) and RDX
(California EPA 1996b) for both water and soil.
The California EPA also has certified the RaPID
Assay kit for TNT (California EPA 1996c) for
water and soil.
The Naval Research Laboratory's two bio-
sensor-based methods have been evaluated
although they are not yet fully commercially
available. The FAST 2000 and Analyte 2000
instruments are commercially available but the
coated membranes and optical fibers are not.
However, the draft EPA methods describe how to
prepare membranes and fibers. Both the CFI and
FOB methods are capable of determining TNT and
RDX. A recent report on both methods (ESTCP
1998) has been submitted to EPA with the intent of
establishing new methods to be incorporated into
SW-846 (EPA 1998) and EPA has assigned draft
method numbers, Method 4655 for the CFI and
4656 for the FOB method. The draft methods are
written for both TNT and RDX in water only but
a soils method may follow.
Detection Range and Range Factor
The lower detection limits of the on-site meth-
ods for water range considerably, from 0.07 ug/L
to 15 ug/L. The detection range of a test kit can
be important and a broad range is generally more
desirable. The importance of the range of the test
kit depends on the range of concentrations
expected in samples, the ability to estimate the
approximate concentration from the sample ex-
tract, the amount of effort required to dilute and
rerun a sample, and the sampling and analytical
objective. Some test kits have a range factor
(upper limit of range /lower limit) of just one order
of magnitude (10X), while other methods span two
orders of magnitude (100X). Because the
concentration of explosives may range widely,
reanalysis of many out-of-range samples may be
necessary if the objective is to determine the
concentration of an explosive in water. The
D TECH immunoassay methods require an
additional complete analysis for each sample
dilution. Other immunoassay methods can run
multiple dilutions in the same analytical run, but the
dilutions must be prepared without knowing
whether they are needed. The EnSys colorimetric
procedure for RDX provides sufficient reagent to
allow running several dilutions at no additional cost.
For the EnSys kits, the analyzed sample can simply
be diluted and reread in the spectrophotometer.
Research results indicate that dilution ratios of as
high as 1 to 10,000 may be necessary to keep
concentrations in the linear range of the tests, and
these dilutions can be conveniently made in one
step using glass microliter syringes (Jenkins
et al 1996b). The procedures that the test methods
use for sample dilution should be considered during
method selection.
The detection range of a kit becomes much less
relevant when the objective is to determine
whether a sample is above or below a single action
limit because the same dilution can be used for all
samples. In some cases, changing the range of a
kit may be desirable to facilitate decision making.
Cleanup levels for explosives in water vary
considerably depending on, for example, the site
conditions, compounds present and their relative
concentration, use of the water, results of risk
assessments, and the selected remedial technology
(EPA 1993a, 1996a; Craig et al. 1996). Typical
remediation goals for water are less than one
microgram/liter for DNTs; low micrograms/liter for
RDX, TNT, TNB, DNB, and NG; and hundreds of
micrograms/liter for HMX and NQ (EPA 1996a).
21
-------
Type of Results
The type of results provided by the various
screening methods are, depending on the concen-
tration range, quantitative or semiquantitative.
The CRREL 2,4-DNT, EnSys, RaPID Assay, the
CFI, and the FOB are quantitative methods that
provide a numerical result. The D TECH kits are
semiquantitative and indicate that the concen-
tration level of an analyte is within one of several
ranges. For example, the D TECH RDX water kit,
without dilution, indicates a concentration within
one of the following ranges: less than 5,5 to 15,15
to 25, 25 to 45, 45 to 60, and greater than 60 ug/L.
Samples per Batch
Several of the available test kits are designed to
run batches of samples, single samples, or both.
However, using a test kit designed for analyzing a
large batch to analyze one or two samples may not
be cost-effective or efficient. For methods requir-
ing filtration to concentrate the analyte, multiple
samples can be simultaneously extracted using a
filtration manifold.
Water Sample Size
The test methods use samples of either 1 mL or
less of water, or 2 L of water.
Sample Preparation and Extraction
Sample preparation and extraction only applies to
the colorimetric methods, which require filtration
and extraction of a 2-L water sample.
Analysis Time
The filtration and extraction step associated with
both the CRREL and EnSys methods requires a
minimum of 20 minutes exclusive of extracting the
filter and may take well over an hour depending on
the amount of particulate matter in the sample. For
this reason, these methods are not recommended
for turbid waters.
Actual sample analysis time for a single sample
ranges from 3 minutes to about 70 minutes
although as many as 10 samples can be
batched and analyzed in the same 70 minutes. For
the NRL methods, eight or more subsamples from
each sample were analyzed and the results were
averaged for the recent method validation study
(ESTCP 1998). However, for routine use, it is
expected that two to four subsamples would
typically be analyzed from each sample. The
effective production rate also depends on the
number of reruns required for samples out of the
detection range.
Interferences and Cross-Reactivity
One of the major differences among the field
methods is interference for colorimetric methods
and cross-reactivity for the immunoassay and bio-
sensor methods. The colorimetric methods for
TNT and RDX are broadly class sensitive—that is,
they are not only able to detect the presence of the
target analyte but also respond to many other
similar compounds (nitroaromatics, and nitra-mines
and nitrate esters, respectively). For colorimetric
methods, interference is defined as the positive
response of the method to secondary target
analytes or co-contaminants similar to the primary
target analyte. The immunoassay and biosensor
methods are relatively specific for the primary
target analytes that they are designed to detect.
For the immunoassay and biosensor meth-ods,
cross-reactivity is defined as the positive response
of the method to secondary target analytes or
co-contaminants similar to the primary target
analyte.
Depending on the sampling objectives, broad
sensitivity or specificity can be an advantage or a
disadvantage. If the objective is to determine
whether explosive compounds are present, then
broad sensitivity is an advantage. Another advan-
tage of the broad response of colorimetric meth-
ods is that they may be used to detect compounds
other than the primary target analyte. For exam-
ple, the colorimetric RDX +HMX method may be
used to determine PETN when RDX + HMX
levels are relatively low or absent. If a secondary
target analyte is present at only low concen-
trations in a sample, the effect on the analytical
result is minimal. If the objective is to determine
the concentration of TNT when relatively high
levels of other nitroaromatics or RDX when
22
-------
elevated levels of other nitramines or nitrate esters
are present, other methods may be more
appropriate.
Extremes of temperature and pH can interfere
with on-site analytical methods. According to the
California Military Environmental Coordination
Committee, physical conditions comprising tem-
peratures outside the range of 4 to 32* C and pH
levels less than 3 or greater than 11 (CMECC
1996) are generally not recommended for both
colorimetric and immunoassay methods. Specific
product literature should be consulted for more
information.
Colorimetric Methods—For TNT methods,
the primary target analyte is TNT and the
secondary target analytes are other nitroaromatics
such as TNB, DNB, 2,4-DNT, 2,6-DNT, and
tetryl. For RDX methods, the primary target
analyte is RDX + HMX, and the secondary target
analytes include PETN, NG, and NQ. If the
primary target analyte is the only compound
present, the colorimetric methods measure the
concentration of that compound. If multiple
analytes are present, the field methods measure
the primary target analyte plus the secondary
target analytes. The response of colorimetric
methods to the secondary target analytes is similar
to the response of the primary target analyte and
remains constant throughout the concentration
range of the methods although the observed colors
may be different.
If multiple analytes are present in water, colori-
metric field results can be roughly compared with
EPA Method 8330 results (EPA 1998). For exam-
ple, if a water sample (as analyzed by Method
8330) contains 100 ug/L each of TNT, TNB, RDX,
and HMX, the EnSys colorimetric methods for
TNT would measure approximately 200 ug/L (100
TNT + 100 TNB), and the RDX test kit would
measure approximately 200 ug/L (100 RDX+ 100
HMX). This example is somewhat simplistic
because each compound has a somewhat
different response factor.
While colorimetric kits are not compound speci-
fic, the color development of the extracts often can
provide an indication of the types of compounds
that may be present. For example, with the TNT
kit, TNT and TNB turn red; DNB turns purple;
2,4-DNT turns blue; and 2,6-DNT turns pink and
tetryl turns orange. For the EnSys RDX + HMX
kit, RDX and HMX turn pink as do nitroglycerine,
PETN, and nitrocellulose. An orange color
indicates a mixture of TNT and nitramines or
nitrate esters.
Immunoassay Methods —For TNT immuno-
assay kits, the primary target analyte is TNT, and
the secondary target analytes are nitroaromatics
TNB, DNTs, AmDNTs, and tetryl. For the RDX
kit, the primary target analyte is RDX, and there is
but little cross-reactivity with HMX (3%). If the
primary target analyte is the only compound
present in water, the immunoassay methods mea-
sure the concentration of that compound.
If multiple analytes are present in water, the
immunoassay kits measure the primary target
analyte plus some percentage of the cross-reactive
secondary target analytes. The response of immu-
noassay kits to the secondary target analytes is not
equivalent to that of the primary target analyte and
does not remain constant throughout the concen-
tration range of the kits. In addition, different
immunoassay kits have different cross-reactivities
to secondary target analytes based on the anti-
bodies used to develop each method. Cross-
reactivities for immunoassay kits are usually
reported at the 50% response level (IC50), typically
the midpoint of the concentration range of the kits.
Table 7 shows the reported cross-reactivities at
IC50 for the immunoassay kits. A complete cross-
reactivity curve for the entire concentration range
should be obtained from the manufacturers for the
immunoassay kits being considered. Where mul-
tiple analytes exist in water samples, immuno-
assay results may not directly compare with EPA
Method 8330 (EPA 1998) results. For example, an
immunoassay kit may have cross-reactivities of
23% for TNB for the TNT test kit and 3% HMX
cross-reactivity for the RDX test kit. The follow-
ing simple example illustrates cross-reactivity;
however, in practice, it is not practical to calculate
contaminant concentrations in this manner because
of synergistic effects and cross-reactivity is
nonlinear. Using the same sample as the
23
-------
colorimetric example above, if a water sample (as
analyzed by Method 8330) contains 100 ug/L each
of TNT, TNB, RDX, and HMX, the TNT field
immunoassay kit would measure approximately
123 ug/L (100 TNT + 23 TNB), and the RDX field
method would measure approximately 103 ug/L
(100 RDX + 3 HMX).
Biosensor Methods—For the CFI method,
relative to TNT at 100%, the cross-reactivities of
other explosive compounds are TNB 600%;tetryl
38%; 2-AmDNT 21%; 2,4-DNT 20%; NB 16%;
2-NT 9%; HMX 5%; 2,6-DNT 4%; 4-AmDNT
1%; and RDX 1%. The RDX method is much
more compound specific with 18% cross-reac-
tivity for 1,2-dinitroglycerin, 5% for HMX, 4% for
TNB, and about 3% for DNB, 3-NT, and 2-NT.
For the FOB TNT method, TNB is 9% cross-
reactive, and for the RDX method, all of the 17
explosive-related compounds tested are less than
3% cross-reactive.
Matrix Interferences—Colorimetric, immun-
oassay, and biosensor methods may be subject to
positive or negative matrix interferences from
organic and inorganic substances in water. For
colorimetric methods, through careful visual anal-
ysis noted by a positive red or pink color change in
the sample before colorimetric analysis, these
interferences can be evaluated. Inorganic nitrate
and nitrite in water samples interfere with
colorimetric methods unless special procedures are
used to remove these compounds during analysis.
It is important to note that high levels of humic
organics can impart a yellowish coloration to the
acetone extracts. An increase in the intensity of
the yellow color upon reaction with the reagent is
not a positive response for the TNT test, and the
development of a reddish hue to the solution is
necessary before a detection is claimed. Analysis
of a field matrix blank may be useful in identifying
such interference.
Many of the immunoassay methods use a
reverse-coloration process, and nontarget analyte
organic matrix interference results in less color
development. Therefore, on-site method results
are biased high compared to laboratory results.
Both the CFI and FOB NRL methods have been
used at several different sites, but interferents
other than other explosives have not been
determined.
Supplier Recommended
Quality Assurance/Quality Control
The manufacturers or developers recommended
quality assurance/quality control (QA/QC)
procedures vary considerably with the on-site
method. Some test methods do not specify
QA/QC procedures and leave to the investigator
the determination of the numbers of blanks,
duplicates, replicates, and standards that are run.
During field application of these methods, it is
common to send at least 10 to 20% of the positive
samples to an off-site laboratory for analysis by
EPA Method 8330. A smaller fraction of the
nondetect samples also may be verified. In some
cases, field methods are used to identify samples
containing explosive residues, and all such samples
are sent to an off-site laboratory for analysis. In
any case, the QC samples recommended by the
method developer should be used. However, it is
up to the user to determine how much and what
types of QA/QC are needed to achieve the DQOs.
While it is essential to ensure that field methods
perform as intended, requiring laboratory type QC
may be inappropriate for on-site analytical
methods. Because site characterization efforts
may be cost constrained, excess QC samples
reduce the number of field samples that can be
analyzed. Good sample handling procedures and
correlation of the field methods with the laboratory
HPLC method over the concentration range of
interest should be the primary performance
criteria. Documentation of procedures and results
must be emphasized.
During the initial evaluation of on-site and
off-site analytical methods, it may be desirable to
analyze a variety of QC samples to determine
sources of error. The methods can then be modi-
fied to minimize error as efficiently as practical to
include, for example, the collection and analysis of
duplicates, replicates, splits of samples, and splits
of extracts.
24
-------
Table 7. On-Site Analytical Methods for Explosives in Water, Percent Interference, or Cross-Reactivity.
Is)
Test Method
Nitroaromatics
TNT
TNB
DNB
2,4-DNT
2,6-DNT
2AmDNT
4AmDNT
Tetryl
Nitramines
RDX
HMX
Other
PETN
TNT
EnSys a
DTEChT
RaPID Assay3
Flow Immunosensor
Fiber Optic Biosensor a
100
100
100
100
100
100
23
65
600
9
100
_b
2
-
<3
100
4
4
20
<3
100
-
<1
4
<3
NC
11
3
21
<3
NC
<1
1
1
<3
100
35
5
38
<4
NC
<1
<1
1
<1
NC
<1
<1
5
<4
-
-
-
-
-
RDX
EnSys a
DTECH3
Flow Immunosensor
Fiber Optic Biosensor a
NC
<1
2
<3
NC
<1
4
<3
NC
<1
3
<3
NC
<1
3
<3
NC
<1
1
<3
NC
<1
1
<3
NC
<1
2
<3
NC
<1
1
<3
100
100
100
100
100
3
5
<3
100
<1
-
-
a Interference for colorimetric methods, cross-reactivity for immunoassay methods at 50% response
b No data
NC No Color Development
(IC50)
-------
Storage Conditions and Shelf Life of Kit or
Reagents
Storage conditions and the shelf life of irnmuno-
assay kits are more critical than with colorimetric
methods. The reagents for some immunoassay
kits should be refrigerated but not frozen or ex-
posed to high temperatures. Their shelf life can
vary from 3 months to more than 1 year. Color-
imetric reagents can be stored at room temper-
ature. The EnSys colorimetric kits have shelf lives
of at least 2 months and up to 1 or 2 years.
Before ordering test kits, it is important to know
when they will be used to ensure that they will be
used before the expiration date.
For immunoassay kits, D TECH may be stored
at room temperature while the RaPID Assay
reagents should be refrigerated. Neither kit should
be subjected to freezing.
The CFI membranes need to be stored moist at
4» C, and away from light. They have a shelf life
of about 1 month. It is recommended that the
FOB be operated out of direct sunlight and recom-
mended but not required, that stock solutions be
refrigerated. Stock solutions can be freeze dried
for storage up to a year and rehydrated and held
for up to a month unrefrigerated. The antibody-
coated fibers may be preserved and stored for
more than a year at room temperature if freeze
dried or be placed in a buffer solution at 4* C.
These procedures may become simplified if the
CFI and FOB methods become commercialized.
Skill Level
The skill level necessary or required to run these
tests varies from low to high (Table 6), requiring a
few hours to a day of training. The manufacturer
of the commercial kits generally provides on-site
training. A free training videotape on the CRREL
version of the TNT and RDX procedures (which
also is useful for the EnSys colorimetric kits) is
available by submitting a written request to:
Commander U.S. Army Environmental Center
Attn: SFIM-AEC-ETT/Martin H. Stutz
Aberdeen Proving Ground
MD 21010
E-mail: mstutz@,aec2.apgea.army.mil.
A training video on the USAGE Standard
Operating Procedures for Analysis of TNT and
RDX (USAGE 1999) will be available from:
Kira Lynch
Seattle District Corps
PO Box 3755 (EN-TB-ET)
Seattle WA 98124-2255
E-mail: Kira.P.Lynch(g),usace.army.mil.
Training videos are also available for some kits.
Cost
As shown in Table 6, routine sample costs vary
by method. The cost per sample is affected by
the costs of consumable items, analytical
instruments, and reusable apparatuses required to
run the method. In figuring the cost per sample, it
is important to estimate the costs of possible
reruns for out-of-range analyses. With the EnSys
colorimetric kits, or the CRREL AP/PA method,
the color-developed extracts may be simply diluted
and reread with the spectrophotometer. It should
also be noted that the CRREL TNT and RDX +
HMX methods should become more economical
than the EnSys kits as the number of samples
increases. With the other methods, the original
water sample must be diluted and reanalyzed,
which for immunoassay methods requires the use
of an additional kit. Colorimetric methods typically
have sufficient extra acetone for dilution to rerun
samples with no increase in material cost. It
should be noted that the per-sample costs shown
in Table 6 are only for supplies plus equipment.
Labor, data management, data review and data
reporting are not included.
In contrast to the previous methods which have
relatively low initial costs and higher per sample
costs, the two NRL biosensor-based methods,
have high initial capital costs and low per sample
costs. Eventually there is a break-even point at
which, with high numbers of samples, the NRL
methods become more economical than colori-
metric and immunoassay methods.
Comparisons to Laboratory Method 8330
The objectives of the study or investigation, the
site-specific contaminants of concern, the concen-
tration ranges encountered or expected, and their
relative concentration ratios affect the selection of
26
-------
a particular on-site method. The accuracy of an
on-site method is another selection criterion but
care must be used in interpreting accuracy results
from comparisons between reference analytical
methods and on-site methods.
Colorimetric methods actually measure classes
of compounds (i.e., nitroaromatics or nitramines)
and immunoassay methods are more compound
specific. Therefore, the reported accuracy of a
method may depend on the mix of explosives
compounds present in the water sample and the
reference method data used for the comparison
(i.e., data on specific compounds or total
nitroaromatics or nitramines).
The precision and bias of the screening methods
are most appropriately assessed by comparison to
established laboratory methods such as EPA
Method 8330. Methods of comparison that have
been used include relative percent difference
(RPD), linear regression, correlation, and percent
of false positive and false negative results. If
precision and bias are of critical importance, it is
recommended that the reports referenced in this
section be consulted directly. Statistical results
can be misleading when outliers or extreme values
are present. For example, in one linear regression
comparing field and laboratory methods, the slope
reported was 11 (i.e., measured against an ideal
slope of 1) while the correlation coefficient was
0.99 (i.e., measured against an ideal of 1.0) all
because of one very high concentration value
being included with the remaining low values. It
also should be remembered that the contribution of
analytical error may be small compared to total
error. Field error is usually the major contributor
to total error. In comparing results, it should be
noted that all CFI and FOB statistical results are
usually based on the means of several analyses
per sample whereas the regression lines and RPD
for the other on-site methods are based on single
measurements.
Several studies have been conducted comparing
the performance of two or more on-site methods
with Method 8330. Thorne and Myers (1997)
evaluated several immunoassay methods including
the D TECH TNT and RDXkits, and the RaPID
Assay TNT kit. Craig et al. (1996) and EPA
(1997) evaluated (1) the EnSys TNT and RDX +
HMX colorimetric kits, (2) D TECH TNT and
RDX immunoassay kits, (3) the RaPID Assay
TNT immunoassay kit, (4) the CFI methods for
TNT and RDX, and (5) the FOB for TNT. The
results presented include estimates of method bias
as determined by calculated RPDs and linear
regression analysis. Another study was conducted
by NRL to obtain more comprehensive data on the
FOB and CFI methods (ESTCP 1998). The Army
Corps of Engineers compared the EnSys TNT and
RDX + HMX method with Method 8330 during
monitoring at the Umatilla Groundwater site
(ACOE 1998). The results from each of these
studies are summarized below and in Tables 8
and 9.
The Thorne and Myers (1997) study inves-
tigated TNT and RDX levels in 44 groundwater
wells from three sites: the Umatilla Army Depot
in Hermiston, Oregon; the Naval Submarine Base
in Bangor, Washington; and the Naval Surface
Warfare Center in Crane, Indiana. The capability
of immunoassay kits was evaluated to determine
whether groundwater samples exceeded the EPA
lifetime health advisory of 2 ug/L (Roberts and
Hartley 1992; Roberts et al. 1993) and whether
the RPDs (the difference between the field and
reference method concentration divided by the
mean value and expressed as a percent) were
within ± 50% of Method 8330 results. The results
"were disappointing" and "none of the test kits
performed as well as advertised," Thorne and
Myers (1997) reported. "The quantitative assays
were neither accurate nor precise enough to re-
place Method 8330 although they could be used
adequately as screening tools". The D TECH
RDX test "failed badly" by producing 24% false
negative and 18% false positive results relative to
the drinking water advisory limit of 2 ug/L. The D
TECH TNT kit produced 30% false positive and
no false negatives. The detection limit of both D
TECH kits are above the 2 ug/L drinking water
advisory limits. The RaPID Assay method for
TNT demonstrated no false positive or false
negative results. Thorne and Myers also looked at
the percent of sample results within ± 50% of the
Method 8330 results. For D TECH TNT and
RDX kits, 32% and 58% of the results,
27
-------
respectively, were within acceptable limits. For
the RaPID Assay method, 85% of the results
were acceptable. Finally they conducted
regression analyses comparing the RaPID Assay
TNT kit performance with Method 8330 results on
groundwater samples. The ideal regression line
would be Y = mX + b where the slope, m, would
equal 1 and the intercept, b, would equal 0. The
dependent variable is Y (on-site method estimate),
and the independent variable is X (Method 8330
result). A correlation coefficient (r) is typically
calculated that shows the degree of association
between the on-site method and Method 8330 and
can range between -1 and +1. For a perfect posi-
tive correlation r = 1. The Thorne and Myers
RaPID Assay results were Y = 1.48X + 0.0 with
a correlation coefficient of r = 0.93, which is
highly significant (99% probability level).
Results from the EPA study (Craig et al. 1996;
EPA 1997a) are summarized in Tables 8 and 9 for
the groundwater samples from the Umatilla
Chemical Depot in Hermiston, Oregon, and the
Naval Submarine Base in Bangor, Washington.
Groundwater at Umatilla has high nitrates and low
turbidity while groundwater and leachate at
Bangor has relatively high organic carbon and
higher turbidity. Tables 8 and 9 includes the slope
of regression lines for TNT and RDX data
respectively, the correlation coefficient (r), the
mean and median of the absolute RPD values
(indication of precision), and mean of the RPDs
(indication of bias). The mean RPD closest to 0
shows the greatest average agreement with the
reference laboratory method. The study concluded
that no on-site analytical method out performed
the other methods in all comparisons. For the
TNT methods, the EnSys and CFI had the highest
accuracy followed by the FOB, RaPID Assay,
and D TECH methods. All TNT method results
were biased high based on the net RPDs at both
sites and were generally biased slightly low for
RDX. For RDX, the EnSys and CFI methods
showed the highest accuracy followed by D
TECH. In general, the RDX on-site analytical
methods performed better than the TNT method.
The performance may have resulted from the
higher levels of RDX, which necessitated sample
dilution and, thereby, also reduced matrix
interferences.
The EnSys TNT kit accuracies were similar for
both sites and for the RDX kit, results were
slightly more accurate at the Bangor site. Using
an RPD acceptance criterion of ± 50% of the
Method 8330 result, 89% of the EnSys TNT
results were acceptable and 78% of the RDX
results were acceptable. Overall accuracy of the
TNT and RDX EnSys colorimetric methods were
acceptable.
The D-TECH methods were more accurate at
Umatilla than Bangor because of lower
interference from organics and particulate matter.
The majority of the TNT RPD values were
positive and linear regression slopes were greater
than 1.0, thereby indicating a high bias for the on-
site methods, possibly resulting from TNB
interference or cross-reactivity. Using an RPD
acceptance criterion of ± 50% of the Method 8330
result, 70% of the D TECH results were
acceptable while 56% of the RDX results were
acceptable.
The CFI TNT method performed about the
same at both sites while the FOB method
performed better at the Umatilla site. For RDX,
the CFI performed well at both sites and was
similar in accuracy to the EnSys method.
A recent report (ESTCP 1998) documented the
performance of the NRL CFI and FOB methods
at the Umatilla Army Depot, the Bangor Naval
Submarine Base, and the Crane, Naval Surface
Weapons Center. Both methods were modified
significantly between the Craig et al.(1995) study
and the ESTCP (1998) study. For the CFI, the
instrument was changed and the small columns
were replaced by membranes. The FOB TNT
antibody was changed as well as the fiber
geometry. The statistics provided in Table 8 and
discussed below were calculated after the
analytical results below 10 ug/L, or listed as below
the detection limit, were replaced with 5 ug/L
(one-half of the method detection limit).
However, data were not included if both the NRL
and Method 8330 data would have been replaced
with 5 ug/L. This approach was taken to permit
more data to be included in the analyses yet avoid
producing extreme RPDs near the detection limit.
Because a few very high or low values relative to
28
-------
most of the data can have a misleading impact on
linear regression data, some regression equations
were recalculated with high or low samples
deleted based on the Method 8330 concentration.
The recalculated results are discussed in the text
below.
The CFI data for TNT showed highly significant
correlations with Method 8330 data at the Umatilla
and Bangor sites; however, the mean and median
RPDs were high (Table 8). This apparent
contradiction results from several high
concentration samples. If the three samples at
Umatilla with Method 8330 results greater than
200 ug/L are deleted (367, 846, and 1160 ug/L),
the regression equation changes to Y = 0.61X + 9
and the correlation coefficient drops to 0.51, which
is not significant (95% probability level). For the
Bangor site, two of the seven sample results
showed concentrations greater than 200 ug/L and
most of the remaining Method 8330 values were
below 5 ug/L. Only four samples from the Crane
facility were analyzed by the CFI method;
therefore, no regression line was calculated. The
mean and median RPDs were about 100.
The FOB results for TNT showed regression
slopes of 0.41 and 0.35 for the Umatilla and
Bangor sites, respectively. The corresponding
correlation coefficients were 0.52 and 0.70, which
were significant and not significant, respectively.
For all of the TNT samples analyzed by the FOB
method, the TNT levels were below 200 ug/L. No
Crane samples were analyzed for TNT using the
FOB method.
For RDX, the CFI method showed highly
significant and significant regressions with Method
8330 data collected at Umatilla and Bangor,
respectively (ESTCP 1998). Net RPDs
demonstrated a low bias at both sites (-63 and
-42), and the means and medians of the absolute
RPDs ranged from 68 to 78. At Crane, the CFI
method showed a highly significant regression,
mean and median absolute RPDs of 64 and 42
with a net RPD of -6. If the one especially high
or low 8330 value is deleted from the Bangor and
Crane data sets, the resulting regression lines are
still significant, as is the regression for Umatilla
when the three samples with 8330 values greater
than 200 ug/L are deleted.
The FOB results on RDX at Umatilla and
Bangor showed highly significant regressions,
mean and median RPDs generally below 50, and
low net RPDs. All of the Umatilla 8330 results
were below 200 ug/L, and if two high samples
(356 and 562 ug/L) at Bangor are deleted, the
regression is still significant although the slope of
the regression line changes to 2.5.
The Army Corps of Engineers collected
numerous groundwater samples at Umatilla that
were analyzed for multiple compounds during an
effort to document the conditions in groundwater
wells and the effectiveness of the granular
activated carbon treatment system (ACOE 1998).
After eliminating nonrepresentative data, EnSys
and Method 8330 data were available for 40 RDX
and 36 TNT samples (Table 8). These regression
results for these data show Y = 0.69X + 132 with
r = 0.90 for RDX and Y = 1.3X -15 with r = 0.97
for the TNT data. The averages of the absolute
value of the RPDs were 31 for RDX and 44 for
TNT. For these data sets, the net RPDs (average
when sign is considered) were -6.1 for RDX and
22 for TNT and are relatively unbiased compared
with other study results presented in Table 9.
Ideally, the net RPD should balance out to zero
indicating no bias.
Several projects comparing Method 8330 results
with on-site analytical methods are underway so
additional published data will become available
from EPA and the Corps of Engineers. Also, see
the section on emerging technologies about a
planned demonstration for the summer of 1999 on
current and emerging on-site methods for
explosives in water and soil.
29
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Table 8. Comparison of On-Site Analytical Methods for TNT to EPA Method 8330
Method
EnSys1
DTECH1
RaPID Assay1
Flow Immunosensor1
Fiber-Optic Biosensor1
Flow Immunosensor2'4
Fiber-Optic Biosensor2'4
EnSys3
Method
Flow Immunosensor2'4
TNT
Regression
Umatilla
Y = 1.4X+191
Y = 2.0X+73
Regression
Bangor
Y=1.1X+51
Y=11X-558
Y=1.0X+140
Y = 1.2 + 46
Y=1.7X-267
Y = 0.71X-18
Y = 0.41X + 24
Y=1.3X-15
Y=1.2X + 242
Y = 0.71X + 285
Y=1.6X-7
Y = 0.35X+10
Regression Crane
NA
Correlation
Coefficient (r)
Umatilla/Bangor
0.98**/1.0**
0.88**/1.0**
0.99**combined
0.70*70.84*
0.91**/0.76*
0.92**/0.98**
0.52*70.70
0.97**
Correlation
Coefficient (r)
NA
Mean RPD
(absolute value)
Umatilla/Bangor
66/58
64/143
78 combined
47/52
33/107
114/100
85/55
44
Mean RPD
(absolute value)
103
Median RPD
(absolute value)
Umatilla/Bangor
45/63
48/152
87 combined
47/38
25/116
147/89
74/52
30
Median RPD
(absolute value)
103
Net RPD
Umatilla/Bangor
66/58
58/143
78 combined
32/51
30/100
-41/87
67/40
22
Net RPD
36
Number of
Samples
Umatilla/
Bangor
15/9
15/7
7
combined
11/7
12/8
14/7
16/6
36
Number of
Samples
4
OJ
o
1 EPA1997a
2 ESTCP1998
3 ACOE1998
4
Statistics based on means of usually eight or more analyses of each sample.
Statistically significant at the 95% probability level.
Statistically significant at the 99% probability level.
-------
Table 9. Comparison of On-Site Analytical Methods for RDX to EPA Method 8330
Method
EnSys1
DTECH1
Flow Immunosensor1
Flow Immunosensor2'4
Fiber-Optic Biosensor2'4
EnSys3
Method
Flow Immunosensor2'3
Fiber-Optic Biosensor2'3
RDX
Regression
Umatilla
Y = 0.81X+ 135
Y=1.3X-269
Y = 0.92X+203
Y = 0.72X-30
Y = 0.53X+13
Y = 0.69X+ 132
Regression
Bangor
Y = 0.96X + 6
Y = 1.7X+172
Y = 0.72X+1.1
Y = 0.67X - 3
Y = 0.61X+38
Regression Crane
Y = 0.75X + 40
Y = 0.44X-5
Correlation
Coefficient (r)
Umatilla/Bangor
0.86**/0.92**
0.96**/0.61*
0.72**/0.92**
0.91**/0.69*
0.64**/0.82**
0.90**
Correlation
Coefficient (r)
0.77**
0.94**
Mean RPD
(absolute value)
Umatilla/Bangor
33/21
53/67
26/30
78/76
37/56
31
Mean RPD
(absolute value)
64
100
Median RPD
(absolute value)
Umatilla/Bangor
27/21
32/56
19/23
78/68
33/40
32
Median RPD
(absolute value)
42
104
Net RPD
Umatilla/Bangor
-11/-7.7
-36/61
-11/-30
-6S/-42
10/14
-6.1
Net RPD
-6
-100
Number of
Samples
Umatilla/Bangor
23/12
23/12
20/12
20/11
20/10
40
Number of
Samples
13
11
1 EPA1997a
2 ESTCP1998
3
ACOE1998
4 Statistics based on means of usually eight or more analyses of each sample.
* Statistically significant at the 95% probability level.
** Statistically significant at the 99% probability level.
-------
Additional Considerations
Other important factors in the selection of an
on-site method include, for example, the size and
type of working area required, the temperature of
the working area, the need for electricity and
refrigeration, the amount of waste produced, the
need to transport solvents, and the degree of
portability. Immunoassay methods are more
sensitive than colorimetric methods to freezing and
elevated temperatures, and the ambient
temperature affects the speed at which color
development takes place on some immunoassay
methods. Most tests are best run protected from
the weather, for example, in a van, field trailer, or
nearby building.
Emerging Methods
A GC-nitrogen phosphorus detector method
currently under development at CRREL appears
to offer the ability to provide on-site analysis for
the common suite of nitroaromatics and nitra-
mines in water (Hewitt and Jenkins in press). In
this method, the analytes of interest are precon-
centratedby passing 1 L of water through Empore
SDB-RPS extraction membranes and eluting the
retained compounds with 5 mL of acetone. An
aliquot of the acetone extract is then determined
on a field portable gas chromatograph equipped
with a nitrogen-phosphorus detector. Method
detection limits were demonstrated to be below 1
ppb for TNT, RDX, 4-amino-DNT, 2-amino-DNT,
and 2,4-DNT.
Two German companies are now manufacturing
immunoassay kits for explosives in water but no
literature was found comparing on-site results to
standard methods. The TNT kit by Coring System
uses a 96 well microplate to which TNT conjugate
is bound. Samples, or TNT standards and a TNT
specific antibody (rabbit) are pipetted into wells
and the plate is incubated for an hour. After
rinsing, a rabbit specific antibody is added, the
plate is incubated for an hour, rinsed, and substrate
is added to the wells. After 20 minutes of
incubation, blue color development is stopped and
the resulting yellow color is read at 450 nm using
a photometer. The TNT concentration is inversely
proportional to the color. The method detection
limit is reported to be 0.5 ug/L for water. The
method is cross reactive with 1,3-DNT (650%),
and 2,4-DNT (60%). For more information send
e-mail to: info(g),coring.de.
Coring currently has no plans to market their kit in
the U.S. No information was provided by Bio-
Genes on their immunoassay procedure.
In December, 1998, EPA issued a Notice of an
Intention to Conduct a Demonstration and Per-
formance Verification Study of Explosives Field
Analytical Devices as part of the Environmental
Testing and Verification program. The demon-
stration is planned for the summer of 1999 and will
include analysis of both water and soils. For
further information on the demonstration, contact:
Eric Koglin
National Exposure Research Laboratory
Environmental Sciences Division
P.O. Box 93478, Las Vegas, NV 89193-3478
Phone: 702-798-2432
E-mail: koglin.eric(g),epa. gov
Summary of the EPA Reference
Methods for Explosive Compounds
in Water
Properties of Secondary Explosives
The two secondary explosives used to the
greatest extent by the U.S. military over the past
70 years are TNT and RDX. With their manu-
facturing impurities and environmental trans-
formation products, the two compounds account
for a large part of the explosives contamination at
active and former U.S. military installations.
While all explosive compounds can all be classi-
fied as semivolatile organic chemicals, their
physical and chemical properties require different
analytical approaches than normally is used for
other semivolatiles.
Table 10 presents some of the important
physical and chemical properties for TNT and
32
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RDX, and some of their commonly encountered
manufacturing impurities and environmental
transformation products. The unique properties
that differentiate these chemicals from other
semivolatiles such as polychlorinated biphenyls
(PCBs) and polynuclear aromatic hydrocarbons
(PNAs) are their thermal lability and polarity.
Many of these compounds thermally degrade or
explode at temperatures below 300* C. Thus,
methods based on gas chromatography (GC) have
not gained wide acceptance. However, methods
developed by Hable et al. (1991) and Walsh and
Ranney (1998a, 1998b) have shown that the gas-
chromatography electron-capture detector (GC-
ECD) method can be used successfully for nitro-
aromatics and nitramines in water. In addition, log
KOW values range from 0.06 to 2.01 compared with
values of 4 to 5 for PCBs and PNAs, indicating
that these compounds are quite polar and that the
nonpolar solvents used for other semi-volatile
organics are not the best choice for extraction of
nitroaromatics and nitramines from water. For
most routine analyses, environmental water
samples are extracted with either salting-out
solvent extraction with acetonitrile or using solid-
phase extraction with a styrene-divinyl-benzene-
based solid phase (Method 3535A [EPA 1998]).
The sample extracts are analyzed using reversed-
phase high-performance liquid chromatography
(RP-HPLC), often using Method 8330 (EPA
1998) or a recently adopted GC-ECD method,
Method 8095 (EPA 1998).
Water Extraction
High concentration water samples have
generally been analyzed by diluting an aliquot of
the water 1:1 with methanol, then filtering the
sample through a 0.45 to 0.50-um filter, and
analyzing a 100-uL aliquot of the filtrate by
RP-HPLC-UV. Quantitation limits for this direct
water method (Method 8330) range from 5.7 ug/L
for 2,4-DNT to 14 ug/L for RDX.
Often detection limits that can be obtained using
the direct water method are not sufficient for
project-specific DQOs. In these cases, the target
analytes must be extracted from the water and
preconcentrated before either RP-HPLC-UV
(Method 8330) or GC-ECD (Method 8095) deter-
mination. Extraction is accomplished using either
salting-out solvent extraction (Leggett et al. 1990)
followed by nonevaporative preconcentration
(Jenkins and Miyares 1991), or by solid-phase
extraction, EPA Method 3535A (Jenkins et al.
1995b, 1995c; EPA 1998).
A direct comparison of salting-out solvent
extraction and solid-phase extraction with RP-
HPLC-UV was conducted by Jenkins et al.
(1994a, 1995b, 1995c) using groundwater samples
from the Crane, Indiana, Naval Surface Warfare
Center. The results indicate that excellent
extraction efficiency was achieved using both
procedures (recoveries were generally greater
than 90%). Quantitation limits using these
approaches were similar and ranged from less
than 0.1 ug/L for some target analytes to 0.84
ug/L for RDX. The authors cautioned, however,
that carefully cleaned solid phases must be used or
interferences will be released from the solid
phases by some water matrices (Jenkins et al.
1994a, 1995b, 1995c). A small residual peak that
interfered with RDX was found even with highly
cleaned solid-phase materials. The GC-ECD
method, which was recently given preliminary
approval by the EPA (Method 8095, [EPA 1998]),
specifies that solid-phase extraction should be
used when samples are to be analyzed by
GC-ECD. The salting-out solvent extraction
method was not evaluated for use with GC-ECD.
Method detection limits (MDLs) for the GC-ECD
method range from 0.04 ug/L to 0.4 ug/L for the
various target analytes when a 500-mL sample is
used and preconcentrated into 5 mL of
acetonitrile.
Reversed-Phase High-Performance Liquid
Chromatography Determination
Generally, detection of the analyte within the
proper retention time window on two columns with
different retention orders is required for
confirmation of the presence of these explosives.
Method 8330 specifies primary analysis on an
LC-18 (octadecylsilane) column with confirma-
tion on a cyanopropylsilane (LC-CN) column
(Jenkins et al. 1989).
33
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Table 10. Physical and Chemical Properties of Predominant Nitroaromatics and Nitramines.
Compound
TNT
TNB
2,4-DNT
Tetryl
RDX
HMX
Molecular
Weight
227
213
182
287
222
296
Melting Point
(-C)
80.1 to 81. 6
122.5
69.5 to 70.5
129.5
204.1
286
Boiling Point
(-C)
240 (explodes)
315
300
(decomposes)
(decomposes)
(decomposes)
(decomposes)
Water Solubility
(mg/L at 20- )
130
385
270
80
42
5 at 25-
Vapor Pressure
(to rr at 20-)
4.4E-06
2.2E-04
1.1E-04
5.7E-09
4.1E-09
3.3E-14
iogKow
1.86
1.18
2.01
1.65
0.86
0.061
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Walsh, Chalk, and Merritt (1973) were the first to
report on the use of RP-HPLC for the analysis of
nitroaromatics in munitions waste. Most sub-
sequent HPLC methods for these compounds rely
on UV detection because of its sensitivity and
ruggedness. Initially, determination was specified at
254 nm because of the availability of fixed
wavelength detectors at this wavelength based on
the mercury vapor lamps and a significant absor-
bance of all target analytes. Current instruments
are generally equipped with either variable
wavelength detectors or diode array detectors, and
wavelengths of maximum absorption can be
selected to optimize detection. However, 254 nm is
still often used because it is specified in Method
8330 and because of the low incidence of
interference at this wavelength.
Gas-Chromatography Electron-Capture
Detector Determination
The earliest use of gas chromatography to deter-
mine nitroaromatics dates from the early 1960s
(Parsons et al. 1961). The early methods used the
relatively insensitive flame ionization detector, and it
was not until the early 1970s that the selectivity and
sensitivity of the BCD for nitroaromatics was
realized (Murrmann et al. 1971). The first GC-ECD
method for nitroaromatics and nitramines in water
was developed by Hoffsommer and Rosen (1972).
The introduction of fused silica columns in the early
1980s reduced the problems with thermal
degradation of these thermally labile compounds and
permitted routine determination of nitramines by
GC. Routine analytical methods were developed for
nitroaromatics by Belkin et al. (1985) and for
nitroaromatic and nitramines by Hable et al. (1991)
at the Army Environmental Hygiene Agency.
Hable's method used solvent extraction to extract
and preconcentrate the target analytes and
GC-ECD for determination. Unfortunately, two
separate extractions were needed, one for
nitroaromatics using toluene and a second for
nitramines using isoamylacetate. More recently,
Walsh and Ranney (1998b) combined solid-phase
extraction with GC-ECD, and the results were used
to establish EPA Method 8095 (EPA 1988). One
advantage of Method 8095 compared with Method
8330 is the ability to quantify nitroglycerine and
PETN in the same determination as the
nitroaromatics and nitramines.
Method 8095 specifies that detection of peaks
from the BCD within the proper retention time
window on two columns with different polarity is
required for confirmation of the presence of the
target analytes. Method 8095 specifies DB1 as the
primary analytical column (although DB5 provides
better resolution for target analytes) and either
RTX200 or RTX225 as the confirmation column
(Walsh and Ranney 1998a, 1998b).
It is important that the injector and oven
temperatures, column lengths, and linear velocities
specified in Method 8095 are used for GC-ECD
analysis. Otherwise, poor recovery, particularly for
HMX, will result. The injection port liner must be
thoroughly deactivated and changed frequently, or
performance will be degraded for HMX, RDX, and
the aminodinitrotoluenes. Particular attention also
must be given to thorough drying of the solid phase
used for solid-phase extraction before elution with
acetonitrile.
A comparison of the performance of GC-ECD
with RP-HPLC-UV for these target analytes in
water is presented by Walsh and Ranney (1998a,
1998b). Analysis of extracts by both RP-HPLC-
UV and GC-ECD results in excellent analytical
confirmation, particularly when target analytes are
present at very low concentrations.
Method Specifications and Validation
Based on the research described above, EPA
Method 8330 (EPA 1998) and Method 8095 (EPA
1998) specify the following:
A. Salting-out the Solvent Extraction
1. Place 251.3 g of sodium chloride in a 1-L
(round) volumetric flask. Add a 770-mL
aliquot of the water sample, and stir the flask
with a stirring bar until the salt is dissolved.
2. While stirring the solution, add 164 mL of
acetonitrile to the flask. Stir for at least 15
minutes (30 minutes is safer) to dissolve as
much acetonitrile as possible. Turn off the
35
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stirring bar, and allow the phases to separate
for at least 10 minutes.
3. Remove the upper acetonitrile layer (about 8
mL) with a Pasteur pipette and transfer it to
a 100-mL (round) volumetric flask. Add 10
mL of fresh acetonitrile to the water sample
in the 1-L flask and stir for an additional 15
minutes, followed by 10 minutes to allow the
phases to separate. Remove the upper
acetonitrile layer and combine with the initial
acetonitrile extract in the 100-mL flask.
4. Add 84 mL of salt water (325 g of NaCl per
1,000 mL of reagent-grade water) to the 100-
mL flask, and stir for 15 minutes, followed by
10 minutes for phase separation. Carefully
transfer the top acetonitrile layer to a 10-mL
graduated cylinder using a Pasteur pipette.
Add an additional 1.0 mL of acetonitrile to the
100-mL flask and stir for 15 minutes followed
by 10 minutes for phase separation. Combine
the second extract with the first in the 10-mL
graduated cylinder. Record the volume of
extract and then dilute it 1:1 with
reagent grade water. This extract is analyzed
using RP-HPLC.
B. Cartridge Solid-Phase Extraction
1. Obtain prepacked solid-phase extraction
cartridges (Porapak RDX or Sep-Pak, 6 cc,
500 mg, or equivalent). Clean the cartridges
by placing them on a solid-phase extraction
manifold and passing 15 mL of acetonitrile
through each using gravity flow. Then flush
the acetonitrile from the cartridges using 30
mL of reagent-grade water. Ensure that the
cartridges are never allowed to dry after the
initial cleaning.
2. Place a connector on the top of each car-
tridge and fit the connector with a length of
one-eighth-in.-diameter Teflon tubing. Place
the other end of the tubing in a 1-L beaker
containing 500 mL of sample. Turn on the
vacuum and set the flow rate through each
cartridge at about 10 mL per minute. Adjust
the flow rate if it declines significantly
because of partial plugging from the
suspended material. After extracting the
sample, remove the top plug containing the
fitted tubing from each cartridge and pass 10
mL of reagent-grade water through the
cartridge using gravity flow unless the car-
tridges are sufficiently plugged to require a
vacuum. Use a 5-mL aliquot of acetonitrile to
elute the retained analytes from the car-
tridges under gravity flow. Measure the
volume of the recovered acetonitrile, and
either use directly for GC-ECD determin-
ation (Method 8095) or dilute 1:1 with
reagent-grade water for RP-HPLC-UV
determination.
C. Membrane Solid-Phase Extraction
1. Preclean styrene-divinylbenzene membranes
(47 mm, Empore or equivalent) by centering
on a 47-mm vacuum filter apparatus and add
several milliliters of acetonitrile to swell the
membrane before clamping the reservoir in
place. Add a 15-mL aliquot of acetonitrile to
soak into the membrane for 3 minutes. Then
turn on the vacuum and pull most (but not all)
of the solvent through the membrane.
2. Add a 30-mL aliquot of reagent-grade water
and resume the vacuum. Just before the last
of the water is pulled through the membrane,
remove the vacuum, fill the reservoir with a
500-mL sample, and resume the vacuum.
The sample extraction will take from 5
minutes to an hour depending on the amount
of suspended matter present. Once the water
is eluted, draw air through the membrane for
1 minute to remove excess water. Place a
40-mL vial below the outlet of the membrane,
and add a 5-mL aliquot of acetonitrile on top
of the membrane. Allow the acetonitrile to
soak into the membrane for 3 minutes. Then
apply the vacuum to pull the acetonitrile
through the membranes into the vials.
Remove each resulting extract with a Pasteur
pipette, and measure the volume in a 10-mL
graduated cylinder. Measure the volume of
the recovered acetonitrile, and either use
directly for GC-ECD determination (Method
8095) or diluted 1:1 with reagent-grade water
for RP-HPLC-UV determination.
36
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Summary
A number of defense-related sites are
contaminated with elevated levels of secondary
explosives in groundwater and surface water.
Levels of contamination range from barely
detectable (approximately 1 ug/L) to more than
10,000 ug/L. On-site analytical methods are
essential to more economical and improved
characterization and remediation. What they lack
in accuracy and multi-compound specificity, they
more than make up for in the increased number of
samples that can be analyzed and the utility of
near-real-time data for making decisions on-site.
While verification using a standard laboratory
analytical method such as EPA Method 8330 or
8095 should be part of any quality assurance
program, reducing the number of samples
analyzed by more expensive methodology can
result in reduced costs and more efficient use of
limited resources while still achieving the DQOs.
Two basic types of on-site analytical methods
are in use for explosives in water: colorimetric
and immunoassay. Colorimetric methods genera-
ly detect broad classes of compounds such as
nitroaromatics or nitramines while immun assay
methods are more compound specific. Prototype
biosensor methods for TNT and RDX have been
field tested and are emerging methods for explo-
sives analysis in water (Rogers and Gerlach
1996). Because TNT or RDX is usually present
in explosive-contaminated groundwater or surface
water, the use of field procedures designed to
detect these or similar compounds can be very
effective.
Selection of an on-site analytical method involves
evaluation of many factors including the specific
objectives of the study and DQOs, compounds of
interest, explosives present at the site, the number
of samples to be run, the sample analysis rate,
interferences or cross-reactivity of the method, the
skill required, the analytical cost per sample, and
the need for and availability of support facilities or
services. Other factors that should be considered
are the precision and accuracy of the on-site
analytical method and the required detection limits.
It should be remembered that analytical error may
be small compared to field error and that the
precision and bias of a method is dependent on the
site-specific conditions (compounds present and
relative concentration) as well as the skill of the
analyst.
Federal Facility Forum Members
Region 1
U.S. EPA
JFK Federal Building
Boston, MA 02203
Megan Cassidy, Co-Chair
(617) 573-5785
Region 2
U.S. EPA
290 Broadway
New York, NY 10007-1866
Bill Roach
(212) 637-4335
Region 3
U.S. EPA
341 Chestnut Bldg.
Philadelphia, PA 19107
Steve Hirsh, Co-Chair
(215) 566-3052
Paul Leonard
(215) 566-3350
Region 4
U.S. EPA
345 Courtland Street, N.E.
Atlanta, GA 30303-3415
Carl Froede
(404) 562-8550
Jim Barksdale
(404) 562-8537
Olga Perry
(404) 562-8534
37
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Region 5
U.S. EPA
77 W. Jackson Boulevard
Chicago, IL 60604
Craig Thomas
(312) 886-5907
Carol Witt Smith
(312) 886-6146
Region 6
U.S. EPA
1445 Ross Avenue
Dallas, TX 75202-2733
Nancy Morlock
(214) 665-6650
Chris Villarreal
(214) 665-6758
Ruby Williams
(214) 665-6733
Region 7
U.S. EPA
726 Minnesota Avenue
Kansas City, KS 66101
Scott Marquess
(913)551-7131
Region 8
U.S. EPA
999 18th Street
Denver, CO 80202-2413
Floyd Nichols
(303) 312-6983
Jim Kiefer
(303) 312-6907
Judith McCulley
(303) 312-6667
Region 9
U.S. EPA
75 Hawthorne Street
San Francisco, CA 94105-3901
Glenn Kistner
(415) 744-2210
Sheryl Lauth
(415) 744-2387
Region 10
U.S. EPA
1200 Sixth Avenue
Seattle, WA 98101
Harry Craig
(503) 326-3689
Kathy Stryker
(206)553-1171
Headquarters
U.S. EPA/OSWER
401 M Street, SW
Washington, DC 20460
Allison Abernathy
(202) 260-5646
Doug Bell
(202) 260-8716
Lance Elson
(202) 564-2577
Remi Langum
(202) 260-2457
Diane Lynne
(202) 564-2587
38
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Notice
The U.S. Environmental Protection Agency, publication. Mention of trade names or cornmer-
through its Office of Research and Development, cial products does not constitute endorsement or
funded and prepared this Issue Paper. It has been recommendation by the EPA for use.
peer reviewed by the EPA and approved for
Acknowledgment
Work partly performed under the auspices of wishes to thank the U.S. Army Environmental
the U.S. Department of Energy, Office Contract Center and Cold Regions Research and Engineer-
No. DE-AC07-94ID13223, through Interagency ing Laboratory for assisting in the preparation of
Agreement No. DW89937192-01-2 with the U.S. this document.
Environmental Protection Agency. The EPA
39
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