SPECIAL REPORT 95-16

Preservation of Water Samples

Containing Nitroaromatics and

Nitramines

Thomas F. Jenkins, Philip G. Thorne, Erika F. McCormick

and Karen F. Myers May 1995
Abstract

This study was conducted to develop a method for stabilizing water samples

to be analyzed for nitroaromatic and nitramine explosives using SW846

Method 8330. Several options were tested using river water fortified with

15 nitroaromatic, nitramine, and aminonitroaromatic analytes. Acidification to pH 2 using sodium bisulfate was selected based on its ability to

retard microbiological and chemical transformations, its ease of use under

field conditions, and its usability with both the direct and preconcentration

procedures in Method 8330. Holding-time studies were performed over a

64-day storage period using fortified river water and groundwaters with

and without chemical stabilization. Nonacidified samples showed rapid

loss of tetryl, TNB, and TNT and slower loss of the dinitroaromatics. These

losses were accompanied by increasing concentrations of transformation

products. Losses of these nitroaromatics were completely eliminated by

acidification to pH 2. Nitramines were stable over the entire period whether

samples were acidified or not. A small loss of the aminodinitroaromatics

was observed for both acidified and unacidified samples. The rate of loss

for acidified samples was initially greater than for nonacidified samples.

Sample acidification caused no adverse effects on SW846 Method 8330,

although samples to be preconcentrated using salting-out solvent extraction should be neutralized prior to extraction to prevent additional loss of

aminodinitroaromatics.The use of sample acidification was tested in a production laboratory scenario using field-contaminated water samples. The

loss of aminodinitrotoluenes due to acidification was again observed for

some samples but not for others. A small interference near the retention

time of TNT was observed and traced to the disposable syringes used for

sample filtration prior to HPLC determination. Acidification completely eliminated

losses of TNT and TNB over the 28-day study. As observed for fortified

samples, HMX and RDX were stable whether samples were acidified or not.

For conversion of SI units to non-SI units of measurement consult ASTM

Standard E380-93, Standard Practice for Use of the International System

of Units, published by the American Society for Testing and Materials,

1916 Race St., Philadelphia, Pa. 19103.

This report is printed on paper that contains a minimum of 50% recycled

material.
Special Report 95-16

Preservation of Water Samples

Containing Nitroaromatics and

Nitramines

Thomas F. Jenkins, Philip G. Thorne, Erika F. McCormick

and Karen F. Myers May 1995

Prepared for

U.S. ARMY ENVIRONMENTAL CENTER

SFIM-AEC-ET-CR-95009

Approved for public release; distribution is unlimited.

US Army Corps


of Engineers

Cold Regions Research &


Engineering Laboratory
PREFACE

This report was prepared by Dr. Thomas F. Jenkins, Research Chemist, and Philip

G. Thorne, Physical Sciences Technician, Geological Sciences Division, Research and

Engineering Directorate, U.S. Army Cold Regions Research and Engineering Laboratory (CRREL), and Erika F. McCormick, Chemist, and Karen F. Myers, Biologist, Environmental Chemistry Branch, Environmental Engineering Division, Environmental

Laboratory, U.S. Army Engineer Waterways Experiment Station (WES), Vicksburg, Mississippi.

Funding was provided by the U.S. Army Environmental Center, Aberdeen Proving

Ground, Maryland, Martin H. Stutz, Project Monitor.

The authors gratefully acknowledge Dr. C.L. Grant, Professor Emeritus, University

of New Hampshire, and Alan D. Hewitt, CRREL, for their technical review of this manuscript. The authors acknowledge useful discussions with Dr. Edouard S.P. Bouvier,

Waters Corporation; Dr. Ronald J. Spanggord, SRI International; and Daniel C. Leggett,

CRREL, regarding the behavior of the amino-containing analytes when samples were

acidified. The authors also wish to acknowledge Donald E. Parker and B. Lynn Escalon

of the AScI Corporation, Vicksburg, Mississippi, for their technical assistance, and Linda

K. Stevenson of WES for sample management.

This publication reflects the personal views of the authors and does not suggest or

reflect the policy, practices, programs, or doctrine of the U.S. Army or the Government

of the United States. The contents of this report are not to be used for advertising or

promotional purposes. Citation of brand names does not constitute an official endorsement or approval of the use of such commercial products.

ii
CONTENTS

Page

Preface ........................................................................................................................ ii

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

Objective .................................................................................................................... 2

Evaluation of alternatives ....................................................................................... 2

Experimental materials............................................................................................ 3

Chemicals ........................................................................................................... 3

Solid-phase extraction materials..................................................................... 3

Water samples .................................................................................................... 3

Analyte spiking ................................................................................................. 3

Analysis...................................................................................................................... 3

Experimental design and results ........................................................................... 4

Selection of initial test matrix .......................................................................... 4

Preliminary evaluation of sodium bisulfate and percentages of

acetonitrile necessary to stabilize samples ............................................ 5

Further evaluation of successful methods of preservation ........................ 8

Evaluation of selected preservatives on EPA Method 8330

determination ............................................................................................. 9

Holding-time study using pH 2 stabilization for fortified

river water ................................................................................................... 11

Holding-time study using pH 2 stabilization for fortified

groundwaters.............................................................................................. 16

Evaluation of acidification using field-contaminated groundwater ........ 17

Problems with acidification for the amino compounds.............................. 21

Conclusions ............................................................................................................... 22

Literature cited .......................................................................................................... 22

Abstract ...................................................................................................................... 31

ILLUSTRATIONS

Figure

1. Stability of TNB in samples acidified to pH2, pH 3.5, and left

unacidified .................................................................................................. 5

2. Concentration of 3,5-DNA for samples acidified to pH 2, pH 3.5, and

left unacidified............................................................................................ 6

3. Stability of TNB as a function of the acetonitrile concentration for

unacidified samples ................................................................................... 6

4. Stability of TNB as a function of the acetonitrile concentration for

samples also acidified to pH 3.5 .............................................................. 7

5. Stability of TNT in samples acidified to pH2, pH 3.5, and left

unacidified .................................................................................................. 7

6. Production of 2ADNT in samples acidified to pH 2, pH 3.5, or left

unacidified .................................................................................................. 8

7. Stability of tetryl in samples preserved at pH 2, pH 3.5, with 2.5%

acetonitrile, and with 7.5% acetonitrile .................................................. 9

iii
Figure Page

8. Stability of HMX and RDX in fortified Connecticut River water as a

function of storage time for samples acidified to pH 2 or left

unacidified .................................................................................................. 13

9. Stability of TNB, TNT, and DNB in fortified Connecticut River

water as a function of storage time for samples acidified to pH 2

or left unacidified ....................................................................................... 13

10. RP-HPLC chromatograms for Connecticut River water samples

fortified with DNB, tetryl, 4ADNT, and 3NT ........................................ 14

11. RP-HPLC chromatograms for Connecticut River water samples

fortified with HMX, RDX, TNB, TNT, 2,4-DNT, and 2NT .................. 14

12. Stability of 2ADNT, 4ADNT, and 3,5-DNA in fortified Connecticut

River water as a function of storage time for samples acidified to

pH 2 or left unacidified ............................................................................. 15

13. Stability of 2NT, 3NT, 4NT, and NB in fortified Connecticut River

water as a function of storage time for samples acidified to pH 2

or left unacidified ....................................................................................... 15

14. Losses of TNB in acidified and unacidified groundwater samples

as a function of storage time .................................................................... 16

15. Losses of TNT in unacidified groundwaters and Connecticut River

water samples as a function of storage time ......................................... 17

16. Losses of 4ADNT in acidified groundwaters and Connecticut River

water samples as a function of storage time ......................................... 17

17. Stability of HMX and RDX in groundwater samples from the Naval

Surface Warfare Center ............................................................................. 18

18. Stability of 4ADNT in three groundwater samples from the Naval

Surface Warfare Center ............................................................................. 19

19. Instability of 4ADNT in three other groundwater samples from

the Naval Surface Warfare Center ........................................................... 19

20. Behavior of TNT in several groundwater samples from the Naval

Surface Warfare Center ............................................................................. 20

21. Behavior of TNB in several groundwater samples from the Naval

Surface Warfare Center ............................................................................. 20

22. Chromatograms for the acidified groundwater samples from the

Naval Surface Warfare Center ................................................................. 21

TABLES

Table

1. Retention times for various separations used at CRREL ........................ 4

2. Retention times for various separations used at WES ............................ 4

3. Recovery of target analytes from fortified Milli-Q water ....................... 10

4. Concentrations of various analytes fortified into Connecticut River

water for replicate study ........................................................................... 10

5. Results of direct analysis of preserved and unpreserved Connecticut

River water using the direct analysis procedure of SW846

Method 8330................................................................................................ 11

iv
Table Page

6. Results of holding-time study using fortified Connecticut River

water ............................................................................................................ 12

7. Recoveries of analytes from polyethylene cap liners .............................. 15

8. Results of holding-time study using three fortified groundwaters ...... 16

v
INTRODUCTION

There is an unavoidable delay between sample

collection at hazardous waste sites and analysis at

off-site environmental testing laboratories.

Analytes must be sufficiently stable in the matrix

under investigation using prescribed storage conditions or they must be stabilized, usually by the

addition of chemical preservatives. Matrix-specific

analytical methods typically include a section describing storage conditions and acceptable holding times between sample collection and analysis.

The most commonly used analytical method in

the United States for nitroaromatics and nitramines

in water is SW846 Method 8330 (EPA 1992). The

preextraction holding time prescribed in this

method is seven days, with samples maintained

under refrigeration and no preservative specified.

However, two recent holding-time studies have

indicated that the nitroaromatics frequently associated with munitions wastes can be subject to

significant losses while being held prior to analysis. Maskarinec et al. (1991) found rapid loss of

2,4-dinitrotoluene (2,4-DNT) in both surface water and groundwater even when stored at 4°C in

the dark; they recommended a preextraction holding time of only four days for water samples to be

analyzed for 2,4-DNT. Grant et al. (1993) found

very rapid losses of 1,3,5-trinitrobenzene (TNB)

and 2,4,6-trinitrotoluene (TNT) for surface water

maintained at 2°C in the dark, but loss of 2,4-DNT

was very small. Losses of 55% for TNB and 30%

for TNT were observed after only seven days’ storage, and the losses reached 77% for TNB and 45%

for TNT after 14 days. The major loss mechanism

appears to be microbiological transformation resulting in the reduction of one nitro group on the

ring to amino (Won et al. 1974, McCormick et al.

1976). For TNB and TNT this results in a series of

transformation products, with the most stable being 3,5-dinitroaniline (3,5-DNA) (from TNB) and

the 2- and 4-amino-dinitrotoluenes (2ADNT and

4ADNT) (from TNT). Elsewhere, Goerlitz (1992)

reported TNT losses as high as 75% after 20 days

of storage for unpreserved groundwater samples

from Hawthorne Army Ammunition Plant. Miller

et al. (1983) reported that 2,4-DNT, TNB, and several other nitroaromatics fortified into groundwater from Sunflower Army Ammunition Plant demonstrated significant instability when stored at 4°C

in the dark without preservation. Given the differences in results for different waters, it appears

that variations in physical, chemical, and microbiological properties of specific waters can produce a wide range of effects, i.e., degradation

mechanisms and rates are somewhat specific to a

given water. Thus, preservation techniques appropriate for nitroaromatics in water are needed and

should be used on a routine basis.

A protocol for preservation of 12 munitionsrelated chemicals in water was developed by Miller

et al. (1983) at the Midwest Research Institute.

Several parameters were evaluated relative to their

effect on analyte stability with time, including pH,

temperature, light, containers, and the presence

of sediment, salt, and acetonitrile (used as an antibacterial agent). Several alternative preservation

techniques were tested using munitions-fortified

water. The resulting data did not demonstrate large

differences among treatments, nevertheless, the

following preservation technique was selected:

water samples are preserved by adding acetonitrile

to the sample to achieve a 10% solution, the pH is

adjusted to 3.5 with glacial acetic acid, suspended

particles are removed by centrifugation, and

samples are stored at 4°C in amber glass bottles.

Preservation of Water Samples Containing

Nitroaromatics and Nitramines

THOMAS F. JENKINS, PHILIP G. THORNE,

ERIKA F. MCCORMICK AND KAREN F. MYERS
Tests using munitions-fortified tap water, monitoring well water, and sediment-deionized water

were used to demonstrate the effectiveness of this

procedure. The procedure, however, does not appear to be very practical on a large scale, particularly for routine use in the field.

Goerlitz and Franks (1989) tested chloroform

and mercuric chloride as preservatives and found

that addition of mercuric chloride at 60 mg/L was

successful in significantly reducing the rate of alteration of TNT in groundwater samples from

Hawthorne AAP. They attribute the alterations to

aerobic microbiological transformation resulting

in the generation of the 2- and 4-aminodinitrotoluene transformation products.

Maskarinec et al. (1986) investigated the use of

solid-phase extraction, not only as a method of

preconcentration of nitroorganics from water, but

also as a means for storage/preservation prior to

analysis. In their study, three groundwaters were

fortified with a set of nitroaromatics, nitramines,

and nitrate esters, extracted with Porapak R (a

styrene-divinylbenzene copolymer with polyvinylpyrrolidone) and held for periods of up to 32

weeks prior to analysis. The authors report that

the resin-adsorbed analytes were stable for at least

16 weeks. The analytical precision of the results

for TNT in particular was quite poor, however,

and drawing firm conclusions from these data

seems risky. Nevertheless, the use of solid-phase

extraction for this purpose was an innovative idea

worthy of serious consideration, since preconcentration prior to determination is often necessary anyway to obtain the desired detection limits

for these analytes in water.

Recently Maskarinec et al. (1990) reported on

the use of acidification to pH 2 with sodium bisulfate as a method of preservation for volatile organics in water. This method proved very effective at extending the acceptable analytical holding times for aromatic hydrocarbons and ketones,

both of which are subject to microbiological degradation. Acidification with sodium bisulfate appears to be a method that could be used conveniently in the field during sample collection.

OBJECTIVE

The objective of the studies described here was

to evaluate several different approaches to preservation with regard to their effectiveness in maintaining the integrity of nitroaromatics in water

samples without inducing instability in nitramines,

often present in the same samples. In addition to

preserving these analytes successfully for at least

seven days after collection, acceptable techniques

must be simple and inexpensive to implement,

must not introduce chemicals that increase the

cost of disposal, and must be compatible with

SW846 Method 8330 (EPA 1992). Specifically,

Method 8330 allows two analytical sequences depending on the detection limits required. If detection limits in the range of 10–20 μg/L are adequate,

a direct injection, reversed-phase high-performance liquid chromatographic (RP-HPLC)

method is used. If lower detection limits are required, water samples must first be preconcentrated prior to analysis by RP-HPLC. The method

currently specified in Method 8330 is salting-out

solvent extraction with acetonitrile, but the use of

either cartridge or membrane solid-phase extraction (SPE) may be allowed as an alternative in the

future. It was our hope to develop a stabilization

method appropriate for samples analyzed by any

of these procedures.

EVALUATION OF

ALTERNATIVES

A number of potential options for preservation

were considered, including the use of mercuric

chloride, sodium azide, chloroform, sodium thiosulfate, acetonitrile, acetonitrile with acidification

to pH 3.5 using acetic acid, and acidification to pH

2 using sodium bisulfate. The stabilization procedures that we decided to test were acidification to

pH 2 using sodium bisulfate (Maskarinec et al.

1990) and the addition of acetonitrile combined

with or without acidification to pH 3.5 with acetic

acid (Miller et al. 1983). The use of acidification

with sodium bisulfate was selected because it

would only require addition of a preweighed

amount of a solid material to each water sample

and could be easily implemented in the field. It

has also been tested for its effectiveness with other

analytes such as volatile organics in water and

was found to be effective at preventing biodegradation of aromatic hydrocarbons (Maskarinec et

al. 1990). Acidification with sodium bisulfate

should not result in disposal difficulties for residual samples since acidification is a common

laboratory practice for sample pretreatment and

is also often used for cleaning of containers and

other glassware. Water samples at pH 2 may cause

some difficulties in either the direct method of

analysis or the two alternative preconcentration

2
schemes, but neutralization prior to analysis can

be performed if necessary.

The second stabilization method selected for

evaluation was addition of acetonitrile as a biocide by itself or in combination with pH adjustment to 3.5 with acetic acid. Miller et al. (1983)

tested and recommended a variation of this

method. Addition of acetonitrile is already a part

of the salting-out solvent extraction method and

hence disposal problems should not be increased.

This method would be less easily implemented in

the field, however, and the minimum concentration of acetonitrile required for effective preservation is not known.

EXPERIMENTAL MATERIALS

Chemicals

All standards and test solutions were prepared

from Standard Analytical Reference Materials

(SARMs) obtained from the U.S. Army Environmental Center, Aberdeen Proving Ground, Maryland. Methanol used in preparation of analytical

standards and HPLC eluent was HPLC grade from

Baker. HPLC eluent was prepared by combining

equal volumes of methanol and water and vacuum

filtering through a 0.45-μm nylon membrane to

degas and remove particulate matter. Acetonitrile

used in preparation of stock standards and as a

chemical preservative was HPLC-grade from

Baker. Glacial acetic acid, used to acidify water

samples, was reagent-grade from Baker. Sodium

bisulfate (NaHSO4), used as a preservative, was

obtained from Aldrich. Humic acid, sodium salt,

was technical-grade from Aldrich.

Solid-phase extraction materials

Prepacked cartridges of Porapak RDX (Sep-Pak,

6 cc, 500 mg) were obtained from Waters Corporation; they were precleaned by eluting with 15 mL

of acetonitrile followed by 30 mL of reagent-grade

water. Empore SDB extraction membranes (47 mm)

were obtained from 3M Corporation; they were

precleaned in the same manner as the Porapak

cartridges. Experimental Empore SDB-LS (extra

clean) membranes were used for samples from

the Naval Surface Warfare Center; they were

cleaned as described above.

Water samples

Blank water samples were either reagent-grade

water from a Milli-Q Type I water system, surface

water from the Connecticut River in West Lebanon, New Hampshire, or groundwater samples

from Vershire (PT), Hartland (TR), or Weathersfield

(MW), Vermont. Groundwater samples were obtained in duplicate from the Naval Surface Warfare Center in Crane, Indiana. One sample of each

duplicate was collected in a bottle containing sodium bisulfate such that the solution concentration was 1.5 g/L. A second sample of each duplicate was collected without acidification.

Analyte spiking

All analyte spiking solutions were prepared in

water. SARMs for each analyte were placed in individual brown glass jugs, reagent-grade water

was added, and the contents were stirred continuously at room temperature for a week. The solutions were then filtered through 0.45-μm nylon

membranes into clean, brown glass jugs. No solvents other than water were used in the preparation of these solutions. The concentration of analyte

in each aqueous spike solution was determined

against standards prepared in methanol or

acetonitrile and diluted 1:1 with reagent-grade

water prior to analysis (EPA 1992).

ANALYSIS

All water samples were analyzed by RP-HPLC

at either the U.S. Army Cold Regions Research

and Engineering Laboratory (CRREL) or the U.S.

Army Engineer Waterways Experiment Station

(WES). At CRREL, analysis was conducted on a

modular system composed of a Spectra-Physics

Model SP8810 isocratic HPLC pump, a SpectraPhysics SP8490 UV variable-wavelength detector

set at 254 nm (cell path 1 cm), a Spectra-Physics

SP8875 autosampler equipped with a Rheodyne

Model 7010 Sample Loop Injector (100 μL), a

Hewlett Packard 3376 digital integrator, and a

Linear strip-chart recorder. At WES, primary

analysis was conducted using a Waters Model

600 system controller, Model 610 fluid unit,

Model 717 plus Auto Injector set for a 50-μL injection, a 486 UV Variable Wavelength Detector

set at 245 nm, and a Maxima Chromatography

Work Station. Confirmation was conducted on

a Waters LC Module 1 with a 486 UV Variable

Wavelength Detector (245 nm), a 717 plus Auto

Injector (50 μL), and a Maxima 820 Chromatography Work Station.

Depending on the specific test conducted, water samples were either analyzed using the direct

method specified in SW846 Method 8330 (EPA

3
Table 2. Retention times for various separations used at WES.

Retention time (min)

Analyte LC-18* LC-CN*

HMX 0.3 13.0

RDX 0.1 8.3

TNB 0.7 5.2

DNB 0.3 5.2

3,5-DNA 9.2 6.6

NB 9.8 4.6

TNT 1.2 6.3

4ADNT 2.4 6.9

2ADNT 2.4 7.4

2,6-DNT 3.3 5.8

2,4-DNT 3.6 6.1

*Separations were conducted at 1.2

mL/min with an eluent of 1:1 methanol/water.

Table 1. Retention times for various separations used

at CRREL.

Retention time (min)

Analyte LC-18* LC-CN*

HMX 2.4 1.4

RDX 3.5 7.3

TNB 4.6 4.3

DNB 5.6 4.4

3,5-DNA 6.1 5.6

tetryl 6.2 9.2

NB 6.6 4.0

TNT 7.4 5.2

4ADNT 8.0 6.0

2ADNT 8.4 6.4

2,6-DNT 8.8 4.9

2,4-DNT 8.9 5.2

2NT 0.6 4.5

4NT 1.8 4.7

3NT 2.4 4.8

tetryl breakdown product 4.6 8.1

2-amino-4-nitrotoluene 5.6 4.2

3-nitroaniline 4.2 3.8

2,4-diamino-6-nitrotoluene 4.0 6.3

2,6-diamino-4-nitrotoluene 2.1 4.8

4-amino-2-nitrotoluene 8.1 4.3

*Separations were conducted at 1.5 mL/min with an eluent

of 1:1 methanol/water.

EXPERIMENTAL DESIGN

AND RESULTS

Selection of initial test matrix

In a previous study (Grant et al. 1993), the

preextraction holding times for nitroaromatics and

nitramines in water were evaluated using three

sample matrices: reagent-grade water, groundwater from Enfield, New Hampshire, and surface

water from the Connecticut River in West Lebanon, New Hampshire. Of these, the most rapid

rate of analyte transformation occurred in the Connecticut River water. After only seven days of refrigerator storage of Connecticut River water fortified with TNB and TNT, only 45% of the initially

fortified TNB and 70% of the initially fortified TNT

remained. The expected microbiological transformation products, 3,5-DNA from TNB and 2ADNT

and 4ADNT from TNT (Won et al. 1974, McCormick et al. 1976), appeared as the concentrations

of TNB and TNT decreased with time. Thus, fortified Connecticut River water appears to be a good

choice as a test matrix for evaluation of alternative

preservation techniques.

To ensure that Connecticut River water in the

vicinity of West Lebanon, New Hampshire, could

be relied upon to be degradative for nitroaromatics,

a short holding-time study was conducted in a

1992) or were preconcentrated using either salting-out solvent extraction (SOE), cartridge solidphase extraction (SPE-C), or membrane solidphase extraction (SPE-M) (Jenkins et al. 1992).

Primary analysis was conducted on a 25-cm ×

4.6-mm (5-μm) LC-18 column (Supelco) eluted

with 1:1 methanol/water (v/v) at 1.5 mL/min at

CRREL or 1.2 mL/min at WES (EPA 1992). At

CRREL, samples were introduced by overfilling a

100-μL sampling loop. At WES, 50-μL samples

were introduced using a 200-μL loop. Retention

times of the analytes of interest are shown in Tables

1 and 2 for analyses conducted at CRREL and

WES, respectively. Concentration estimates were

obtained for most analytes from peak heights from

the digital integrator. In some instances, particularly for samples preconcentrated using Porapak

RDX SPE cartridges, better quantitative results

were obtained using peak areas. At CRREL, the

identities of transformation products were confirmed by analysis of some of the samples on LCCN, using a 25-cm × 4.6-mm (5-μm) LC-CN column from Supelco eluted with 1:1 methanol/water (v/v) at 1.5 mL/min (EPA 1992). At WES, confirmation of analyte identities as well as quantitative results for 2ADNT and 4ADNT were obtained

on an LC-CN column (Supelco) eluted with 1:1

methanol/water at 1.2 mL/min.

4
manner similar to that described by Grant et al.

(1993). Since TNB was the least stable analyte in

the earlier study, it was selected as the test analyte

and was fortified at 50 μg/L. Results indicated

that after three days’ storage at room temperature, the concentration of TNB was reduced by

80% and a buildup of 3,5-DNA had occurred. Thus,

as observed previously, fortified Connecticut River

water should be an excellent test matrix for the

assessment of various stabilization techniques.

Preliminary evaluation of sodium

bisulfate and percentages of acetonitrile

necessary to stabilize samples

Connecticut River water was fortified with

RDX, TNB, and TNT at 42, 26, and 41 μg/L, respectively. One subsample of the fortified water

was stored under refrigeration without addition

of any chemical preservative, and this sample

served as the control sample for judging the effectiveness of the various chemical stabilization procedures examined. The water was analyzed at day

0, 4, 8, 20, and 34. A second subsample was acidified to pH 3.5 with acetic acid and stored over the

same period under refrigeration. Acetonitrile

(ACN) was added to 14 subsamples in the appropriate amounts to achieve acetonitrile concentrations in duplicate at 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, and

10.0% (v/v). One subsample at each acetonitrile

concentration was acidified to pH 3.5 with acetic

acid, and the pH of the remaining subsample at

each ACN concentration was left unmodified.

These 14 samples were stored and analyzed as

described above. A final subsample of the fortified Connecticut River water was acidified to pH

2.0 with sodium bisulfate and held under refrigeration for 30 days. This sample was analyzed in

the same manner as described above after 0, 4, 8,

16, and 30 days of storage.

Analytical results for the control sample agreed

with those presented in Grant et al. (1993) for

nitroaromatic and nitramine fortified Connecticut

River water. The rate of loss of TNB was found to

be very rapid (Fig. 1), the rate of loss of TNT was

somewhat slower, and there was no evidence for

loss of RDX. After only four days, the concentration of TNB in the unacidified control sample was

reduced from 26.0 μg/L to 8.3 μg/L. The concentration of 3,5-DNA, the associated, relatively stable

transformation product, increased as the TNB was

lost, with a maximum concentration of 6.1 μg/L

at 20 days (Fig. 2). The rate of loss of TNB was

reduced by acidification to pH 3.5 with acetic acid

(Fig. 1), and 3,5-DNA was not detected in this

sample until day 20. By day 34, however, the TNB

concentration had been reduced to 2.7 μg/L, which

is about a 90% reduction in concentration relative

to day 0, and the 3,5-DNA concentration was about

10 μg/L. This 3,5-DNA concentration was higher

than that determined for the unpreserved sample

30

20

10

0 20 40

Storage Time (days)

TNB Concentration (μg/L)

pH 2.0

3.5

Unacidified

Figure 1. Stability of TNB in samples acidified to pH2, pH 3.5, and left

unacidified.

5
6

4

2

0 20 40

Storage Time (days)

3,5-DNA Concentration (μg/L)

pH 3.5

Unacidified

Control

8

10

pH 2.0

Figure 2. Concentration of 3,5-DNA for samples acidified to pH 2, pH 3.5,

and left unacidified.

vation depended on the ACN concentration used.

For concentrations at or below 5.0%, increasing

losses of TNB were found as a function of storage

time (Fig. 3). For all of these samples, the loss of

TNB was accompanied by an increase in the concentration of 3,5-DNA. For ACN concentrations

below 5.0%, the rate of loss of TNB was inversely

related to ACN concentration. The fastest rate of

loss was observed for the control (no addition of

ACN). No loss of TNB, or observable 3,5-DNA,

were found for samples containing concentrations

at day 20 and probably indicates that acidification

to pH 3.5 is having a greater effect on the destructive mechanism for 3,5-DNA than on the mechanism of production. Acidification to pH 2 with

sodium bisulfate, however, was very effective in

preserving the TNB concentration over the entire

30-day period (Fig. 1), and no detectable concentration of 3,5-DNA was observed. The small increase in TNB concentration with time is attributed to day-to-day calibration error.

The effects of the addition of ACN for preser30

20

10

0 20 40

Storage Time (days)

TNB Concentration (μg/L)

7.5% ACN

5.0%

1.0%

0.1%

Control (0%)

Figure 3. Stability of TNB as a function of the acetonitrile concentration

for unacidified samples.

6
of ACN of 7.5% (Fig. 3) or 10% (not shown), which

appeared to be as effective as acidification to pH 2

for TNB stabilization in this matrix.

The concentration at which ACN was effective

in retarding TNB loss was reduced when the

samples were also acidified to pH 3.5 with acetic

acid (Fig. 4). For this combination, no observable

TNB loss or increase in 3,5-DNA concentration

was found for samples containing ACN concentrations of 2.5% or higher. For samples containing

1.0%, 0.5%, and 0.1% ACN, TNB concentrations at

34 days were 7.6 μg/L, 6.3 μg/L, and <d, respectively. Acidification did reduce the rate of loss of

TNB relative to samples containing the same percent ACN but without acidification (Fig. 3 and 4).

The effects of the various chemical treatments

were very similar for TNT, except the rates of

change were reduced in all cases. For example,

after four days, the TNT concentration in the control sample was reduced from 41.3 μg/L to 26.5

μg/L for a reduction of about 36% (Fig. 5). For

TNB over the same period, the reduction was about

30

20

10

0 20 40

Storage Time (days)

TNB Concentration (μg/L)

Unacidified

Control (%)

2.5% ACN

1.0%

0.5%

Acidified

Control (%)

Figure 4. Stability of TNB as a function of the acetonitrile concentration

for samples also acidified to pH 3.5.

30

20

10

0 20 40

Storage Time (days)

TNT Concentration (μg/L)

pH 2.0

3.5

Unacidified

40

50

Figure 5. Stability of TNT in samples acidified to pH2, pH 3.5, and left

unacidified.

7
a manner similar to that discussed

above, except the analytes tested

were tetryl, 2ADNT, 4ADNT, and

3,5-DNA, an analyte recommended

for inclusion in Method 8330 (Walsh

et al. 1993). Tetryl was chosen because it had been demonstrated to

be unstable with regard to both reduction and hydrolysis when held

in a soil matrix for very short periods (Jenkins 1994). The three amino

compounds were selected because

of the potential for protenation at

the low pH used in two of the preservation techniques, which could

affect their long-term stability.

The results for tetryl were similar

to that reported earlier for TNB. The concentration of tetryl rapidly declined in the unpreserved

control such that after seven days only about 50%

remained (Fig. 7). The loss of tetryl was accompanied by the production of a transformation product that eluted about 1.6 min prior to tetryl (4.6

min vs. 6.2 min for tetryl). This transformation

product was noted in the tetryl soil holding-time

study discussed above, but the compound was

not identified. Acidification to pH 2 and acidification to pH 3.5 with an acetonitrile concentration

of 2.5% were very successful in stabilizing tetryl

over the entire 31-day study. Stabilization using

an acetonitrile concentration of 7.5% without acidification appeared to be a slightly less effective preservative, although no transformation products

were observed even after 31 days of storage. The

small differences in concentration for the three

preservatives shown in Figure 7 could be a result

of poor quantitation due to the development of a

noisy baseline as the samples aged. This appears

to be due to long-term storage of samples containing acetonitrile.

The results for 3,5-DNA were quite different

from the results with tetryl. For the unpreserved

control, the concentration only declined from 57

μg/L to 41 μg/L after 31 days. Concentrations of

3,5-DNA in the samples stabilized using the three

different preservatives did not appear to be substantially different from one another. For the

2ADNT and 4ADNT, no apparent losses of these

two compounds were observed with the unpreserved control sample held for 31 days. There appeared to be a slightly lower recovery of both compounds for the sample preserved at pH 2 using

sodium bisulfate. This result was obtained using

the direct-injection RP-HPLC method without neu12

8

4

0 20 40

Storage Time (days)

2-ADNT Concentration (μg/L)

pH 3.5

Unacidified

Control

16

pH 2.0

Figure 6. Production of 2ADNT in samples acidified to pH 2, pH 3.5, or

left unacidified.

68%. Acidification to pH 3.5 with acetic acid resulted in increased stability of TNT relative to TNB

with the concentration at day 34 of 32.6 μg/L (Fig.

5). No loss of TNT was observed for the sample

acidified to pH 2 with sodium bisulfate over the

entire 30-day storage period. As with TNB, the

loss of TNT was accompanied by the production

of the monoamino transformation products (Fig.

6). The effects of storage with various levels of

ACN, with and without acidification to pH 3.5,

were similar to that described above for TNB.

Overall, the three storage conditions that were

successful in preserving TNB were also successful

in preserving TNT. As discussed above, RDX was

stable in the control sample and was unaffected

by any of the chemical preservatives tested.

Thus, of the various stabilization techniques

investigated in this initial study, three appeared to

be quite successful:

(1) acidification to pH 2 with sodium bisulfate,

(2) acidification to pH 3.5 with addition of ACN

to a concentration of 2.5% or greater, and

(3) addition of ACN without acidification to

achieve a concentration of 7.5% or greater.

In all cases examined, TNB, TNT, and RDX were

stable for at least 30 days when samples were preserved using these three techniques. Without

preservation, TNB and TNT were unstable in these

matrices.

Further evaluation of successful

methods of preservation

To further evaluate these options, the stability

of several other SW846 8330 target analytes were

evaluated over a 31-day period. The test was conducted using fortified Connecticut River water in

8
phase extraction (SPE) using Porapak RDX, and membrane SPE using Empore SDB membranes, according to the methods described in

detail in Jenkins et al. (1994). The

resulting extracts were analyzed using RP-HPLC as specified in Method

8330, and percent recoveries were

calculated vs. initial spiked concentrations (Table 3). As you will note,

the results for the membrane SPE

method for the solution preserved

with 7.5% ACN indicated that recoveries of the analytes ranged from

only 3% for HMX and RDX to 45%

for 2,4-DNT. Since these recoveries

are unacceptably low, no additional

experiments were conducted using

this preservative.

Results for the SOE procedure,

when samples were acidified to pH

2, indicated that low recovery was

obtained for the two amino-containing analytes,

3,5-DNA (35%) and 2ADNT (54%). These results

are consistent with some protenation of these compounds to form the corresponding ammonium

cations, which would not be expected to partition

favorably into the salted-out acetonitrile. A further test of the SOE procedure was conducted

when the initially pH-2-preserved solution was

neutralized with aqueous KOH to pH 6.6 before

extraction. The recovery of 3,5-DNA and 2ADNT

improves to 100 and 97%, respectively, after neutralization (Table 3). No problems were encountered with the pH-2-preserved solution with either the cartridge or membrane SPE methods,

where recovery of the amino-containing compounds appears to be unaffected by the low pH.

Recovery of HMX using the SDB membrane

method, however, is low for both preserved solutions and the unpreserved solution, as was observed in our earlier study (Jenkins et al. 1992,

1994). An apparent high recovery of RDX was

found for the cartridge SPE method for all three

solutions. This again had been observed previously; part of the problem appears to be due to a

narrowing of the peak width for RDX for cartridge

SPE extracts compared with the unextracted standards used for establishing response factors. All

three extraction methods appear to give good recovery for the portion of solution preserved with

2.5% ACN at pH 3.5.

These results indicate that acidification using

sodium bisulfate to pH 2 or addition of ACN to

120

80

40

0 20 40

Storage Time (days)

Tetryl Concentration (μg/L)

Unpreserved

Control 

7.5% ACN

pH 2.0

pH 3.5, 2.5% ACN

Figure 7. Stability of tetryl in samples preserved at pH 2, pH 3.5, with

2.5% acetonitrile, and with 7.5% acetonitrile.

tralization. Low recovery could be due to some

protenation of the amines at pH 2, although pKa

values for 2ADNT and 4ADNT have been reported

as 0.59 and 1.23, respectively (Glover et al. 1977),

indicating that most of both compounds should

exist in the nonionized amino form at pH 2. Overall, the three stabilization techniques appear to be

quite adequate in preserving these four analytes.

Evaluation of

selected preservatives on

EPA Method 8330 determination

Effects on preconcentration methods

The next phase of this study was to evaluate

the effects these three potential stabilization techniques have on the most commonly used analytical method for nitroaromatics and nitramines,

SW846 Method 8330. The following experiment

was conducted to determine the effects of these

preservatives on the extraction/preconcentration

procedures used for low-level determination.

A solution was prepared containing HMX,

RDX, TNB, DNB, 3,5-DNA, TNT, 2ADNT, and 2,4-

DNT in reagent-grade water at concentrations

ranging from 10.7 to 12.2 μg/L. The solution was

divided into four portions; three were preserved

as described above, and the fourth was left

unpreserved to serve as a control for comparison

of analytical results. Aliquots of each portion were

immediately preconcentrated using salting-out

solvent extraction with acetonitrile, cartridge solid9
achieve a 2.5% solution along with acidification

to pH 3.5 do not cause major analytical problems for Method 8330. Neutralization will be required if SOE is used for preconcentration of pH2-preserved samples, but this is not difficult to

achieve and the pH need only be raised to 3.5 to

enable complete recovery of the amino compounds.

Effects on the direct method

To assess the effects of acidification to pH 2

with sodium bisulfate on the direct analysis procedure of Method 8330, a sample of Connecticut

River water was divided into four portions and

fortified with the 15 target analytes in the four

groups indicated in Table 4. Each of these four

solutions was then divided into two aliquots; one

aliquot was acidified to pH 2 with sodium bisulfate and the other was left unacidified. Six replicate portions of each solution were then processed

as described in Method 8330 except that samples

were diluted 1:1 with methanol rather than ace–

tonitrile and analyzed by RP-HPLC (EPA 1992).

The decision to use methanol rather than aceton–

itrile was made to matrix-match the sample and

eluent to improve the quality of the chromatograms near the regions where HMX and RDX elute.

The mean value and standard deviation determined for each analyte in the preserved and

unpreserved solutions are presented in Table 5.

An F-test was conducted to compare the variances

for acidified and unacidified portions for each

analyte to assess whether acidification affected the

analytical precision obtained for the acidified solution relative to the unacidified control. For all

analytes, except TNT and 2ADNT, the variances

obtained were not significantly different at the

95% confidence level. Thus, for these analytes, no

detrimental effect on analytical precision was

Table 3. Recovery of target analytes from fortified Milli-Q water preserved with either

sodium bisulfate (pH 2), 2.5% acetonitrile at pH 3.5, or 7.5% acetonitrile without pH adjustment using SOE, SPE-C, and SPE-M.

Analyte (% recovery)

Treatment/preservation HMX RDX TNB DNB 3,5-DNA TNT 2ADNT 2,4-DNT

SOE*

control 100 112 102 102 106 107 104 100

pH 2 90 93 93 104 35 93 54 101

pH 2(neutralized) 95 103 96 96 100 95 97 93

2.5% ACN 5 103 96 95 98 96 102 98

SPE-C*

control 06 132 107 114 112 106 110 104

pH 2 05 138 104 110 112 92 106 92

2.5% ACN 03 126 101 111 126 109 120 110

SPE-M*

control 1 102 98 101 106 91 98 89

pH 2 2 106 103 107 112 96 104 94

2.5% ACN 9 76 103 108 114 96 105 96

7.5% ACN 3 3 11 14 19 40 40 45

* SOE – salting-out solvent extraction

SPE-C – cartridge solid-phase extraction (Porapak RDX)

SPE-M – membrane solid-phase extraction (Empore-SDVB)

Table 4. Concentrations of various analytes fortified into Connecticut River water for replicate

study.

Fortified Connecticut River

concentration (μg/L)

Group Analyte 

X ± S

1 DNB 39.9 ± 0.52

tetryl 41.2 ± 0.58

4ADNT 57.2 ± 0.42

3NT 59.9 ± 1.95

2 HMX 64.6 ± 1.33

RDX 100.5 ± 0.94

TNB 52.7 ± 0.85

TNT 91.4 ± 0.37

2,4-DNT 69.7 ± 0.38

2NT 74.4 ± 0.69

3 3,5-DNA 59.5 ± 0.32

2ADNT 79.5 ± 1.03

4NT 178.4 ± 0.96

4 NB 43.6 ± 0.48

2,6-DNT 99.2 ± 1.34

* 

X = mean; S = standard deviation

10
found due to acidification. For 2ADNT, the F ratio

was only 5.68, compared with a critical value of

5.05, and this marginally significant value may be

a result of the unusually good precision obtained

for this experiment. For TNT, the cause of the significant F ratio was traced to a small and variable

coeluting peak in the chromatogram obtained for

the acidified Connecticut River water. This peak

was not found in the unacidified water, nor was it

found in the methanol or the sodium bisulfate.

Subsequently this interference was found to be

due to the use of a disposable syringe containing a

rubber-tipped plunger for sample filtration prior

to RP-HPLC analysis. This interference is only produced with acidified samples, and the degree of

interference increases with time as samples are

held prior to analysis. Since various samples

are analyzed at different times after filtration, the size of this interference varies from

analysis to analysis. We did not fully appreciate or understand the significance of this

problem until after nearly all of the experimental work in this report was completed,

including the evaluation using explosivescontaminated groundwater samples. This

interference can be eliminated by the use of

disposable plastic syringes that do not have

a rubber-tipped plunger. This interference

will be discussed again in a later section on

the results of acidification on field-contaminated groundwaters from the Naval Surface Warfare Center.

Since variances, except for TNT and

2ADNT, were not significantly different, further analysis of these results was conducted

by pooling the variances obtained for the

acidified and unacidified samples for a given

analyte and performing a t test to assess

whether the mean values obtained for the

acidified and unacidified solutions were statistically different. Since the variances were

significantly different for TNT and 2ADNT,

variances were not pooled and t values were

calculated using the approach presented in

Miller and Miller (1984). Except for 2ADNT

and 3,5-DNA, means of acidified and

unacidified samples were found to be “not

significantly different” at the 95% confidence

level. Thus, for these 13 analytes, there is no

indication of a measurable effect on the accuracy of the direct analysis method under

Method 8330 due to acidification of samples

to pH 2. The significant differences in means

for the 2ADNT and 3,5-DNA were not surprising in light of the problems discussed above

for the amino compounds and others to be presented later. Nevertheless, these differences are

too small to be of practical significance.

Holding-time study using

pH 2 stabilization for

fortified river water

Based on the results above, acidification to pH

2 with sodium bisulfate was selected as the option

with the greatest potential for conveniently stabilizing waters containing nitroaromatics and

nitramines. To evaluate this procedure more thoroughly, a replicated holding-time study was conducted using all 15 target analytes (14 current

SW846 Method 8330 target analytes plus 3,5-DNA).

Table 5. Results of direct analysis of preserved (pH 2) and

unpreserved Connecticut River water (six replicates each) using the direct analysis procedure of SW846 Method 8330.

Preserved

Analyte (pH 2) 

X S F* t*

HMX No 64.6 0.33 2.55 0.82

Yes 66.2 0.83

RDX No 00.5 0.94 1.89 1.20

Yes 02.8 1.30

TNB No 52.7 0.63 2.77 0.55

Yes 53.4 0.97

DNB No 39.9 0.52 4.98 0.03

Yes 39.9 0.23

3,5-DNA No 59.5 0.32 4.49 3.04

Yes 58.2 0.15

tetryl No 41.2 0.58 2.28 0.61

Yes 40.4 0.87

NB No 43.6 0.48 1.74 0.40

Yes 43.2 0.63

TNT No 91.4 0.37 31.8 1.61†

Yes 92.9 2.05

4ADNT No 57.2 0.42 4.14 0.21

Yes 57.4 0.86

2ADNT No 79.5 0.02 5.68 3.43††

Yes 77.8 0.43

2,6-DNT No 99.2 0.34 2.40 0.65

Yes 97.2 2.08

2,4-DNT No 69.7 0.38 2.39 0.29

Yes 69.9 0.59

2NT No 74.4 0.69 2.12 0.21

Yes 74.1 1.01

4NT No 78.4 0.97 3.17 0.80

Yes 76.5 1.73

3NT No 59.9 0.95 3.37 0.06

Yes 60.1 1.06

 

X = mean; S = standard deviation

* Critical values for F and t at the 95% confidence level are 5.05 and

2.23, respectively.

† Critical value for t is 2.78 (d.f. = 5), thus the t value for TNT is not

significant at the 95% confidence level.

†† Critical value for t is 2.37 (d.f. = 7), thus the t value for 2ADNT is

significant at the 95% confidence level.

11
Connecticut River water was selected as the principal test matrix based on the results discussed

above. The analytes were divided into four groups

as described above (Table 4). The river water was

fortified at concentrations ranging from 50 to 150

μg/L and divided into two portions. The first portion was divided into 11 subsamples and stored in

22-mL glass vials. These samples served as control samples to assess analyte losses without preservation. The second portion was acidified to pH

2.0 by addition of sodium bisulfate (1.5 g/L) and

was also divided into 11 subsamples. On day 0

(about 4–16 hours after preparation), six replicates

each of the control and preserved samples were

analyzed to establish initial concentrations for later

comparison. The remaining samples of control and

preserved water were held under refrigeration in

the dark for up to 64 days. One replicate sample of

the control and acidified water was analyzed in

triplicate on days 3, 7, 14, 28, and 64 (Table 6).

The behavior of the 15 analytes in this study

varied significantly, falling into four different

groups. In the first group, analyte concentrations

appeared to be stable with or without acidification over the entire 64-day period. This group included HMX and RDX (Fig. 8), and their behavior

is consistent with the results of our earlier study

(Grant et al. 1993) and that of Maskarinec et al.

(1991).

For the second group, the concentrations in the

acidified samples remained stable for at least 28

days (and for all but tetryl for 64 days), but concentrations in the unacidified samples declined at

variable rates over the entire period. This group

includes tetryl, TNB, TNT, 1,3-dinitrobenzene

(DNB), 2,6-DNT, and 2,4-DNT in decreasing order

of the rate of concentration decline (Table 6, Fig.

9). The difference in stability of these analytes for

acidified and unacidified samples is clearly shown

in the chromatograms for fortification groups 1

Table 6. Results of holding-time study using fortified Connecticut River water.

Concentration ( 

X ± S)

Preserved (μg/L)

Analyte (pH 2.0) Day 0 Day 3 Day 7 Day 14 Day 28 Day 64

HMX No 64.6 ±1.33 66.1 ±0.58 67.1 ±0.79 65.8 ±1.92 66.3 ±0.86 63.1 ±0.28

Yes 66.2 ±0.83 64.3 ±2.85 62.2 ±0.94 64.2 ±0.86 63.3 ±0.34 59.5 ±0.48

RDX No 100.5 ±0.9 97.1 ±1.0 97.0 ±0.6 98.9 ±2.9 100.5 ±0.7 97.0 ±0.38

Yes 102.8 ±1.3 100.9 ±1.9 97.3 ±0.97 100.7 ±0.17 99.7 ±1.86 97.6 ±0.63

tetryl No 41.2 ±0.58 10.3 ±0.44 11.0 ±0.33 <d <d <d

Yes 40.4 ±0.87 41.8 ±0.76 42.4 ±0.37 41.7 ±0.90 41.2 ±0.58 32.5 ±5.10

TNB No 52.7 ±1.63 29.8 ±0.48 17.7 ±0.08 7.5 ±0.59 0.7 ±0.0 <d

Yes 53.4 ±0.97 52.0 ±1.06 51.3 ±0.22 54.1 ±0.63 51.8 ±0.23 45.9 ±2.72

TNT No 91.4 ±0.37 75.4 ±1.24 65.9 ±0.36 50.8 ±3.42 32.7 ±0.31 13.4 ±0.45

Yes 92.9 ±2.04 91.0 ±1.75 89.3 ±2.52 95.4 ±0.70 90.4 ±1.18 89.6 ±0.12

DNB No 39.9 ±0.52 37.2 ±0.32 33.3 ±0.11 28.5 ±0.17 23.8 ±0.38 11.3 ±0.25

Yes 39.9 ±0.58 39.8 ±0.29 39.0 ±0.76 39.1 ±0.20 39.0 ±0.18 38.5 ±0.37

2,6-DNT No 99.2 ±1.34 96.2 ±1.38 90.7 ±0.60 84.4 ±1.38 84.9 ±0.82 73.5 ±0.88

Yes 97.2 ±2.14 95.9 ±0.53 91.9 ±0.31 94.8 ±0.43 94.6 ±0.46 94.3 ±1.02

2,4-DNT No 69.7 ±0.38 68.8 ±0.15 68.3 ±0.04 66.1 ±4.73 63.7 ±0.27 58.8 ±0.38

Yes 69.9 ±0.59 68.6 ±0.51 67.2 ±0.48 71.5 ±1.07 67.3 ±0.44 66.1 ±0.05

4ADNT No 57.2 ±0.42 57.5 ±0.09 55.3 ±0.23 54.0 ±2.39 54.0 ±0.80 50.4 ±0.30

Yes 57.4 ±1.50 46.4 ±1.16 44.3 ±0.38 43.1 ±2.16 43.8 ±0.25 41.4 ±0.13

3,5-DNA No 59.5 ±0.32 54.4 ±0.37 48.4 ±0.16 45.9 ±0.05 40.0 ±0.44 30.9 ±0.31

Yes 58.2 ±0.15 53.3 ±2.29 52.1 ±0.49 52.7 ±0.84 51.8 ±0.21 51.3 ±0.11

2ADNT No 79.5 ±1.03 77.0 ±0.68 73.1 ±0.89 73.4 ±0.49 69.0 ±0.53 62.9 ±0.30

Yes 77.8 ±0.43 73.4 ±2.38 71.1 ±0.38 73.0 ±0.72 71.5 ±0.41 70.8 ±0.36

2NT No 74.4 ±0.69 71.2 ±1.18 65.6 ±0.79 64.1 ±5.47 61.1 ±0.57 53.7 ±1.06

Yes 74.1 ±1.01 68.4 ±0.55 63.5 ±0.98 68.7 ±0.76 60.9 ±1.13 49.5 ±4.35

3NT No 59.9 ±1.95 54.4 ±0.44 51.3 ±0.24 49.5 ±0.41 46.4 ±1.10 40.9 ±0.61

Yes 60.1 ±1.07 53.8 ±0.07 50.5 ±0.64 48.6 ±1.32 45.6 ±1.06 40.4 ±1.35

4NT No 178.4 ±1.0 163.2 ±2.5 153.3 ±0.9 150.2 ±0.3 136.3 ±4.6 129.7 ±0.9

Yes 176.5 ±1.7 161.6 ±4.8 150.4 ±0.78 149.0 ±0.50 138.6 ±0.69 126.8 ±2.61

NB No 43.6 ±0.48 42.4 ±0.62 40.6 ±0.25 40.8 ±0.45 40.3 ±0.35 37.7 ±0.53

Yes 43.2 ±1.46 41.2 ±0.58 40.2 ±0.86 40.6 ±0.27 39.6 ±0.90 37.9 ±0.64

* 

X = mean; S = standard deviation

12
tion declined from about 91 μg/L to 13.4

μg/L over 64 days. Concentrations of tetryl,

TNB, TNT, and DNB in the unacidified

samples dropped by 73%, 66%, 28%, and

17%, respectively, during the currently accepted holding time of seven days. Transformation products from TNB, TNT, tetryl,

DNB, and 2,6-DNT were observed in

increasing concentrations as the concentrations of the fortified analytes declined. For

TNB and TNT these transformation products were positively identified as the monoamino reduction products (McCormick et

al. 1977). In the case of tetryl, however, a

major transformation product was identified as picrate ion, and lesser amounts of a

transformation product thought to be due

to reduction of a nitro on the ring to amino

was also observed (Fig. 10). For DNB and

2,6-DNT, the observed retention times of

the transformation products were consistent with those expected for the monoamino

transformation products, but we did not

have the authentic compounds available to

verify this assignment.

The third group of analytes included

4ADNT, 2ADNT, and 3,5-DNA. For solutions where these analytes were fortified,

there appeared to be a rapid step-drop in

concentration for acidified samples within

the first several days, which differed substantially from their behavior in the

unacidified samples (Fig. 12). For acidified

samples, losses after seven days of storage

were 9%, 10%, and 23% for 2ADNT, 3,5-

DNA, and 4ADNT, respectively. After 64

days, losses were 9%, 12%, and 28% for the

same three compounds, indicating that no

significant additional loss had taken place.

Unacidified controls were somewhat more

stable initially and, after seven days of storage, losses were 8%, 19%, and 3% for

2ADNT, 3,5-DNA, and 4ADNT, respectively, but these losses increased to 21%,

48%, and 12% for these compounds by day

64. Losses in the unacidified samples are

presumably due to microbial transformation. We will discuss the losses in acidified

samples in more detail later.

The fourth group of analytes includes the three

isomers of nitrotoluene and nitrobenzene (2NT,

3NT, 4NT, NB). Concentrations for a given analyte

declined slowly in acidified and unacidified solutions at identical rates over the entire 56-day peFigure 8. Stability of HMX and RDX in fortified Connecticut

River water as a function of storage time for samples acidified to

pH 2 or left unacidified.

Control

Acidified

60

40

20

0 20 40 60

Storage Time (days)

 Concentration (μg/L)

80

100

TNT

TNB

DNB

TNT

DNB

TNB

Figure 9. Stability of TNB, TNT, and DNB in fortified Connecticut River water as a function of storage time for samples

acidified to pH 2 or left unacidified.

and 2 as a function of storage time (Fig. 10 and 11).

In the worst case, tetryl declined when not acidified from about 41 μg/L to “not detected” in less

than 14 days. For TNB, the concentration in the

unacidified sample declined from about 53 μg/L

to 0.7 μg/L in 28 days, and for TNT the concentra60

40

20

0 20 40 60

Storage Time (days)

 Concentration (μg/L)

80

100

Control

Acidified

RDX

HMX

13
Retention Time (min)

CONTROL

Day 0

DNB

4ADNT

3NT

Tetryl

DNB

ACIDIFIED

4ADNT

3NT

Tetryl

DNB

4ADNT

3NT

Tetryl

DNB

4ADNT

3NT

Tetryl

0 4 8 12 DNB 4ADNT 3NT Tetryl16

Day 7

DNB

4ADNT

3NT

Tetryl

Degradation Products

Tetryl

Day 28

DNB

4ADNT

3NT

Tetryl

Degradation Products

Tetryl

0 4 8 12 16

Day 64

DNB

4ADNT

3NT

Tetryl

Tetryl

Degradation Products

riod (Fig. 13). A mechanism that could explain

this loss is partitioning into the polyethylene cap

liner. This behavior has been observed elsewhere

for Teflon in contact with hydrophobic organics,

including these three nitrotoluenes (Parker et al.

1990, Leggett and Parker 1994).

To test the hypothesis that losses of the

mononitrotoluenes was due to partitioning into

the polyethylene cap liners, caps taken from vials

stored with and without acidification for 28 days

were rinsed thoroughly with reagent-grade water

Figure 10. RP-HPLC chromatograms for Connecticut

River water samples fortified with DNB, tetryl,

4ADNT, and 3NT.

Retention Time (min)

CONTROL

Day 0

HMX

RDX

TNB

TNT

2,4-DNT

2NT

ACIDIFIED

HMX

RDX

TNB

TNT

2,4-DNT

2NT

HMX

RDX

TNB

TNT

2,4-DNT

2NT

HMX

RDX

TNB

TNT

2,4-DNT

2NT

Day 28

HMX

RDX

TNB

TNT

2,4-DNT

DNA

2NT

ADNTs

0 4 8 12 16

Day 64

HMX

RDX

TNT

2,4-DNT

2NT

DNA

ADNTs

HMX

0 4 8 12 16 RDX TNBTNT 2,4-DNT 2NTDNA

Day 7

HMX

RDX

TNB

TNT

2,4-DNT

2NT

DNA

ADNTs

Figure 11. RP-HPLC chromatograms for Connecticut

River water samples fortified with HMX, RDX, TNB,

TNT, 2,4-DNT, and 2NT.

14
Control

Acidified

60

40

20

0 20 40 60

Storage Time (days)

 Concentration (μg/L)

80

2ADNT

2ADNT

4ADNT

3,5-DNA

4ADNT

3,5-DNA

Figure 12. Stability of 2ADNT, 4ADNT, and 3,5-DNA in

fortified Connecticut River water as a function of storage

time for samples acidified to pH 2 or left unacidified.

and placed in new vials filled to capacity with

blank reagent-grade water. The vials were inverted to ensure that solution contacted the cap

liner, and they were held under refrigeration for

36 days. At 7, 19, and 36 days, a small portion of

the water was withdrawn and analyzed. Detectable concentrations of the three isomers of nitrotoluene were found, and the concentrations

increased as exposure times increased (Table 7).

Cap liners from the 64-day acidified samples

were rinsed with reagent-grade water and placed

in new vials containing 2 mL of acetonitrile and

inverted to extract sorbed analytes. These samples were stored overnight in the dark at room

temperature. Detectable concentrations of the

three isomers of mononitrotoluenes, NB, and the

two isomers of the dinitrotoluenes were found

(Table 7). The percentage recovery ranged from

21 to 70% of the mass lost from the water for the

various compounds. Thus, sorption by the cap

liners appears to be responsible for the slow

losses of the nitrotoluenes and nitrobenzene observed and accounts for the loss of the dinitrotoluenes from acidified samples as well.

Table 7. Recoveries of analytes from polyethylene cap liners (0.44 cm3).

Mass of analytes recovered when caps (day 28) were

equilibrated with reagent-grade water for various lengths

of time.

Mass (μg) recovered

Day 7 Day 19 Day 36

C/A* C/A C/A

2NT 0.18/0.15 0.18/0.19 0.19/0.21

3NT 0.15/0.11 0.17/0.15 0.21/0.19

4NT 0.31/0.37 0.55/0.57 0.65/0.64

* C/A—Control/Acidified

Mass of analytes recovered when caps (day 64) were extracted for 18 hours with ACN.

Mass of analyte lost Mass of analyte recovered

from solution (μg) from cap liners (μg)

2NT 0.56 0.20

3NT 0.48 0.10

4NT 1.17 0.32

NB 0.14 0.06

2,4-DNT 0.10 0.06

2,6-DNT 0.10 0.07

Figure 13. Stability of 2NT, 3NT, 4NT, and NB in fortified

Connecticut River water as a function of storage time for

samples acidified to pH 2 or left unacidified

200

160

120

80

40

0 20 40 60

Storage Time (days)

Concentration (μg/L)

4NT

2NT

3NT

NB

Control

Acidified

15
Table 8. Results of holding-time study using three fortified ground waters.

Concentration ( 

X ± S)**

Preserved (μg/L)

Analyte/matrix* (pH 2.0) Day 0 Day 3 Day 7 Day 14 Day 28 Day 64

TNB 1 No 53.1 ±1.39 51.4 ±0.30 51.3 ±0.41 35.2 ±0.20 18.4 ±0.22 7.9 ±0.04

Yes 51.8 ±0.69 51.2 ±0.07 56.7 ±1.96 53.9 ±0.30 53.5 ±0.47 51.8 ±0.56

2 No 52.4 ±0.58 52.3 ±0.49 54.7 ±0.18 45.2 ±0.51 33.8 ±0.93 16.5 ±1.20

Yes 52.8 ±1.56 52.0 ±0.10 54.9 ±0.13 54.0 ±0.35 54.6 ±1.42 53.4 ±0.62

3 No 53.6 ±0.62 52.5 ±0.09 53.4 ±0.54 42.2 ±0.85 20.7 ±0.68 4.2 ±0.60

Yes 52.7 ±0.81 52.9 ±0.04 55.6 ±0.15 54.8 ±0.46 56.6 ±0.26 54.7 ±0.16

tetryl 1 No 42.5 ±1.17 39.2 ±0.02 29.0 ±0.28 13.9 ±0.58 7.2 ±0.01 3.0 ±0.13

Yes 41.5 ±0.67 41.6 ±0.53 45.8 ±0.39 43.1 ±0.13 42.7 ±0.41 39.5 ±0.44

2 No 41.4 ±0.48 39.7 ±0.48 38.6 ±0.48 25.8 ±0.27 14.4 ±0.02 3.7 ±0.80

Yes 41.5 ±1.54 41.6 ±0.40 44.4 ±0.53 42.6 ±0.72 42.4 ±0.11 40.4 ±0.42

3 No 170.3 ±0.1 168.5 ±0.0 147.4 ±2.5 83.1 ±0.48 70.2 ±0.01 26.3 ±0.53

Yes 170.8 ±0.6 173.7 ±2.0 179.9 ±3.5 174.7 ±2.2 176.3 ±0.1 158.4 ±0.63

TNT 1 No 89.4 ±2.27 89.1 ±0.24 93.3 ±0.45 80.9 ±0.43 74.6 ±0.89 61.8 ±1.05

Yes 88.3 ±0.71 91.0 ±0.75 96.1 ±2.96 92.6 ±0.98 90.7 ±1.15 91.6 ±0.60

2 No 89.9 ±1.18 91.8 ±1.45 94.0 ±0.40 85.4 ±1.05 74.1 ±2.25 63.4 ±0.37

Yes 89.1 ±2.82 92.1 ±0.96 94.6 ±0.76 92.5 ±0.02 92.3 ±1.73 91.5 ±0.29

3 No 90.5 ±1.51 91.3 ±0.26 94.7 ±0.96 88.1 ±0.84 80.2 ±0.18 62.5 ±0.51

Yes 88.5 ±0.07 93.5 ±1.82 95.0 ±0.15 89.9 ±0.48 94.3 ±0.11 92.7 ±0.27

4-ADNT 1 No 56.0 ±1.67 55.7 ±0.32 57.5 ±0.16 56.9 ±0.01 56.5 ±0.04 56.4 ±0.87

Yes 55.3 ±1.29 45.2 ±0.47 39.6 ±0.22 34.8 ±0.16 35.0 ±0.31 31.1 ±0.10

2 No 55.5 ±0.94 55.7 ±0.23 57.0 ±0.46 56.5 ±0.17 54.6 ±1.13 54.5 ±0.42

Yes 56.2 ±1.69 45.9 ±0.32 41.0 ±0.71 36.1 ±1.56 32.7 ±1.00 29.8 ±0.14

3 No 54.3 ±2.07 51.1 ±2.79 55.8 ±1.80 54.9 ±0.28 53.4 ±0.38 54.5 ±0.25

Yes 54.6 ±0.46 47.8 ±1.85 45.2 ±0.52 41.4 ±0.76 40.9 ±1.02 38.7 ±0.15

 * Matrix 1 = PT, matrix 2 = MW, matrix 3 = TR

** 

X = mean; S = standard deviation

To determine if these losses could be eliminated

using Teflon cap liners, a study was conducted as

follows. Reagent-grade water was fortified with

TNT, NB, the three nitrotoluenes, and the two

DNTs. Samples were stored in an inverted position, under refrigeration, and analyzed after 7, 14,

28, and 64 days. No measurable losses were observed. Thus, the losses for this group of analytes

would have been eliminated if vials using

Teflon cap liners had been used in the holdingtime study.

Holding-time study using

pH 2 stabilization for

fortified groundwaters

Since preservation by acidification to pH 2

was successful for most of these compounds in

Connecticut River water, an additional study was

conducted using three local groundwaters. These

groundwaters were fortified with TNB, TNT,

tetryl, and 4ADNT at the same initial concentrations as used above. The concentration of tetryl

was inadvertently fortified at a higher concentration for one groundwater matrix. We studied

TNB, tetryl, and TNT because they were the least

stable of those tested in the river water matrix.

4ADNT was of interest because of its behavior in

Figure 14. Losses of TNB in acidified and unacidified

groundwater samples as a function of storage time.

60

40

20

0 20 40 60

Storage Time (days)

TNB Concentration (μg/L)

PT Groundwater

MW Groundwater

TR Groundwater

Control

Acidified

acidified samples compared with unacidified. Preserved and unpreserved samples were prepared

and treated as described for the previous study,

except that only duplicates were analyzed for each

storage time. The results of this study are shown

in Table 8.

16
was similar, with the rate of change being greatest for TNB. Both analytes were stable for the

first seven days, but substantial losses were observed by day 14 (Fig. 14). This behavior is somewhat different from that found in the fortified

Connecticut River water, where major losses of

these two analytes were observed by day 3 (Fig.

15). The behavior in both matrices is consistent

with microbiological transformation being the

major loss mechanism for these compounds.

For Connecticut River water, a large population

of aerobic microorganisms is undoubtedly

present initially that requires little or no acclimation time before being capable of transforming these compounds. In the groundwater, the

initial population of aerobic microorganisms is

probably many orders of magnitude lower, and

it appears that several days are required for the

populations to increase sufficiently to result in

significant analyte losses.

The behavior of tetryl in the unacidified

groundwaters is somewhat different from TNB

and TNT. For tetryl, a small loss in all three

groundwaters was observed by day 3, with substantial loss for two of the three groundwaters

by day 7 (Table 8). This behavior is consistent

with earlier research showing that losses of tetryl

can occur by hydrolysis as well as microbiological transformation (Kayser et al. 1984,

Jenkins 1994).

The behavior of 4ADNT in these fortified

groundwaters is similar to that observed in fortified Connecticut River water for acidified

samples, but somewhat different for unacidified

controls. For unacidified samples, no losses of

4ADNT were observed over the 64-day study.

For the acidified samples, substantial losses

were observed for all three groundwaters that

amounted to 29%, 44%, and 47% for the TR, PT,

and MW groundwaters, respectively, after 64

days (Fig. 16). Percent loss of 4ADNT in the TR

groundwater was similar to that found in the

Connecticut River water, and losses in the other

two matrices were greater.

Evaluation of acidification using

field-contaminated groundwater

Since chemical preservation using acidification

to pH 2 with sodium bisulfate looked promising,

this technique was further evaluated with 36

groundwater samples from monitoring wells at

the Naval Surface Warfare Center (NSWC) in

Crane, Indiana. At each well, two water samples

were collected in 125-mL glass bottles. A 0.2-g

60

40

20

0 20 40 60

Storage Time (days)

4ADNT Concentration (μg/L)

MW Groundwater

PT Groundwater

TR Groundwater

Connecticut River Water

Acidification to pH 2 eliminated losses of TNB

and TNT over the entire 64-day study for all three

fortified groundwaters. Acidification also eliminated losses of tetryl for two of the three groundwaters over the 64-day period, but some loss of

tetryl was observed after day 28 for tetryl in matrix no. 3 (Table 8). Substantial losses of tetryl,

TNB, and TNT were observed in all of the unacidified samples, however. The pattern of TNB and

TNT loss in the three unacidified groundwaters

MW Groundwater

PT Groundwater

TR Groundwater

Connecticut River Water

60

40

20

0 20 40 60

Storage Time (days)

TNT Concentration (μg/L)

80

100

Groundwaters

Connecticut River

Water

Figure 15. Losses of TNT in unacidified groundwaters and

Connecticut River water samples as a function of storage time.

Figure 16. Losses of 4ADNT in acidified groundwaters and

Connecticut River water samples as a function of storage time.

17
tion. All of these samples were stored in 40-mL

glass vials with Teflon-lined caps and analyzed

on day 0 (the day they arrived in the laboratory)

and at 7, 14, 21, and 28 days after holding at 4°C in

the dark. Samples were diluted 1:1 with methanol

and filtered prior to analysis. Analytical results

are presented in Appendix A.

Of the nine unfortified groundwater samples

from the NSWC, seven contained detectable concentrations of HMX and RDX, four had detectable

TNT, six had detectable 2ADNT/4ADNT, two had

a very low but detectable TNB concentration, and

one acidified sample had a very low concentration of 2,4-DNT. As observed for the fortified waters discussed earlier, HMX and RDX were stable

in all seven samples over the entire 28-day holding time, whether samples were acidified to pH 2

or not (Fig. 17). The behavior of 2ADNT and

4ADNT was sample-dependent. For three samples,

the concentrations of these compounds in the acidified subsamples were substantially lower than for

the unacidified subsamples (Fig. 18). In three others, some at nearly identical initial concentrations

and measured pH, no loss of 2ADNT or 4ADNT

was found due to acidification (Fig. 19). As observed previously, when loss occurred, the major

portion occurred rapidly over the first few days.

For TNT, acidification to pH 2 proved to be

effective in preserving TNT whether samples initially had TNT present or were fortified with TNT.

For their unacidified counterparts, the results were

mixed: TNT was stable over the 28-day holding

time in two unfortified samples but declined in

two others. The worst case was for well F (Table

A6), where the acidified sample had a mean concentration of about 22 μg/L over the study, but

the unacidified sample showed a consistent decline from 14 μg/L to less than detectable (detection limit estimated at 2 μg/L). The concentration

at 7 days, the currently accepted holding time,

was 9 μg/L, indicating that nearly two-thirds of

the TNT had been lost over this period. The concentration of TNT in the unacidified portion, even

in the day 0 sample, was reduced relative to the

acidified portion, apparently due to loss occurring during the one-day shipping time from the

field to the laboratory. TNT stability in the fortified samples was similarly unpredictable; TNT

concentrations in some remained stable but declined significantly in others (Fig. 20) over the 28-

day holding time.

The behavior of TNB in fortified samples paralleled that of TNT, but the rate of loss appeared to

be faster in samples that showed losses (Fig. 21).

Figure 17. Stability of HMX and RDX in groundwater

samples from the Naval Surface Warfare Center.

portion of sodium bisulfate had been added to

one bottle in each pair so that an acidified and an

unacidified subsample from each well were returned to the laboratory. Samples were shipped

cold by overnight carrier.

Upon receipt in the laboratory the day after

sample collection, the pH of all samples was measured and all 36 unacidified samples were screened

using several commercial enzyme immuno-assay

kits to estimate the TNT concentration (Thorne and

Myers, in press). Based upon the TNT concentration obtained, nine samples were selected to conduct holding-time studies, and 40-mL aliquots of

both the acidified and unacidified portions of these

samples were fortified with additional TNT and

TNB. Fortification was accomplished by addition

of TNT and TNB in aqueous solution prepared

without use of organic solvents. The pH for each

sample and the fortification level for TNT and

TNB is given in Appendix A. A second set of 40-

mL aliquots of each pair of acidified and unacidified samples was also retained without fortifica0 10 20 30

Storage Time (days)

200

160

120

80

40

Acidified

Control

Concentration ( μg/L)

RDX

HMX

Well B

Well 

I

18
Figure 19. Instability of 4ADNT in three other

groundwater samples from the Naval Surface

Warfare Center.

Figure 18. Stability of 4ADNT in three groundwater samples from the Naval Surface Warfare

Center.

4ADNT Concentration (μ g/L)

Well B

80

60

40

20

0 10 20 30

Storage Time (days)

Well F

Well D

Acidified

Control

0 10 20 30

Storage Time (days)

40

30

20

10

4ADNT Concentration (μg/L)

Well E

Well A

Well C

Control

Acidified

19
Figure 21. Behavior of TNB in several groundwater samples from the Naval Surface Warfare

Center.

80

60

40

20

0 10 20 30

Storage Time (days)

Control

Acidified

TNB Concentration ( μg/L)

Well F

Well A

0 10 20 30

Storage Time (days)

40

30

20

10

TNT Concentration (μ g/L)

Control

Acidified

Well F

Well C

Figure 20. Behavior of TNT in several groundwater samples from the Naval Surface Warfare

Center.

20
Well J; Unacidified

TNT Interference

Well J; Acidified to pH 2

0 4 8 12 16

Retention Time (min)

tion with acetonitrile or solid-phase extraction

where the retained compounds were eluted with

acetonitrile, this peak was not observed, which

was also consistent with the hypothesis that it

was due to the rubber-tipped plunger.

Problems with acidification for

the amino compounds

As discussed above, some decrease in concentration was found for the amino-containing compounds in acidified samples in the fortified

Connecticut River water, the three fortified

groundwaters, and some, but not all, of the fieldcontaminated groundwaters from the NSWC. We

initially suspected that this behavior was due to

protenation of a significant portion of the amino

functional groups to form the corresponding ammonium ions (HADNT+) at pH 2 (eq 1). This could

result in low recovery of the parent amine when

conducting direct analysis using RP-HPLC.

HADNT+ ADNT + H+ (1)

Ka = [ADNT] [H+] / [HADNT+] (2)

The pKa values (eq 2) for the ammonium compounds corresponding to 4ADNT and 2ADNT are

The loss of TNB in several samples was accompanied by the appearance of 3,5-DNA, the expected

transformation product. When TNT was either already present, or fortified, at concentrations above

about 150 μg/L, no losses of TNT or TNB were

observed. This may be due to a toxicity effect on

the microorganisms present. Well F (Table A6) had

a detectable concentration of 2,4-DNT, but only in

the acidified portion. Apparently this analyte was

unstable in this sample if not acidified. This result

is consistent with observations of Maskarinec et

al. (1991). These results confirm that the stability

of TNT and TNB in unacidified groundwater

samples is very sample-specific, but that acidification to pH 2 is a very effective stabilization technique for nitroaromatics.

Chromatograms obtained for the acidified

groundwater samples on the LC-18 column revealed the presence of a small but detectable interference with a similar retention time to TNT

(Fig. 22). This interference had been observed earlier with acidified groundwater samples that had

been filtered using a syringe containing a rubbertipped plunger. Analysis of these samples on the

LC-CN confirmation column indicated that this

peak was definitely not TNT. When these samples

were preconcentrated by a factor of 100 prior to

analysis, using either salting-out solvent extracFigure 22. Chromatograms for the acidified groundwater samples from

the Naval Surface Warfare Center.

21
reported to be 1.23 ±0.02 and 0.59 ±0.03, respectively (Glover and Hoffsommer 1977). If we assume an initial concentration of 50 μg/L for

4ADNT, the equilibrium concentrations of 4ADNT

and 4HADNT+ for a [H+] of 0.01M would be 42.7

μg/L and 7.3 μg/L, respectively. Similarly, for an

initial concentration of 50 μg/L of 2ADNT, the

equilibrium concentrations would be 48.1 μg/L

and 1.9 μg/L, respectively. If the protenated species were separated from the unprotenated amino

compounds during RP-HPLC analysis, recoveries

of 85.4% and 97.2% for 4ADNT and 2ADNT, respectively, would result. When solutions of the

amino compounds at about 50 μg/L in reagentgrade water were prepared and analyzed quickly

after acidification, we observed no losses of the

amino compounds relative to unacidified samples.

Since acid–base reactions are kinetically very fast,

the pH of the sample, once injected into the HPLC,

must shift sufficiently that essentially all of the

ADNT reverts to the amino form regardless of initial

sample pH. Thus, the protenation of the 4ADNT is

not directly responsible for the losses we observed.

Figures 16, 18, and 19 indicate that the amount

of 4ADNT lost for various water matrices varies

substantially, but for matrices where loss is found,

the pattern is quite similar. In these cases, loss is

fairly rapid over the first several days and then

the concentrations become fairly stable at a reduced level. Thus, the loss mechanism appears to

have a finite capacity, but the capacity differs from

matrix to matrix. Losses were not found to correlate with the actual pH achieved upon acidification for the well waters from NSWC. However,

problems with sorption of dinitroaniline herbicides from water on surfaces such as glass, stainless steel, Nalgene, and Teflon have been reported

by Strachan and Hess (1982). While there was no

association of this sorption with acidification, these

results demonstrate how sorptive these types of

compounds can be even in the absence of particulate matter. Variations in the colloidal/suspended

matter content for specific water samples may be

responsible for the apparently inconsistent behavior observed. It is possible that acidification to pH

2 activates surfaces on the colloidal/suspended

matter thereby making it more sorptive for these

amino compounds.

CONCLUSIONS

The most important conclusion from this study

is that holding times for water samples containing

nitroaromatics such as TNT, TNB, and tetryl can

be extended to at least 28 days by acidification to

pH 2 using sodium bisulfate. Acidification does

not affect the stability of nitramines that were stable

over this period with or without preservation. Unfortunately, in some samples there can be a loss of

aminodinitroaromatics such as 4ADNT, 2ADNT,

and 3,5-DNA due to acidification to pH 2, and the

lost analytes cannot be recovered by neutralization. The maximum loss due to this process after

28 days was 71% for 4ADNT in well F from NSWC

(Table A6). These amino-containing compounds

can also be lost in unacidified samples, probably

as a result of microbiological transformation, but

at a slower rate than that due to acidification during the first few days. If samples are to be preconcentrated using salting-out solvent extraction, they

must be neutralized prior to extraction, or incomplete recovery of the amino compounds will be

found. If preconcentration is to be accomplished

using solid-phase extraction, neutralization prior

to extraction does not appear to be necessary.

A slow loss of the mononitrotoluenes and nitrobenzene was also found during sample storage

whether samples were acidified or not, and the

loss was found to be due to sorption into the polyethylene cap liners used for sample storage. When

samples were stored in vials with caps containing

Teflon liners, this loss was drastically reduced.

If acidified samples are to be analyzed using

the direct-analysis protocol in SW846 Method 8330,

it is important that filtration be conducted using

disposable syringes that do not have a rubbertipped plunger. If a rubber-tipped plunger is used,

a small but detectable interference will be found

near the retention time of TNT, and the size of this

interference will depend on the time between filtration and analysis.

Because acidification can affect the stability of

the amino transformation products in solution,

we do not recommend acidification to pH 2 for

R&D projects studying the fate of explosives under various treatment protocols. In these cases,

samples can be stabilized if necessary by addition

of acetonitrile to achieve 2.5% and acidification to

pH 3.5 using acetic acid.

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22
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Elwell and M.H. Stutz (1993). Evaluation of analytical requirements associated with sites potentially contaminated with residues of high explosives. USA Cold Regions Research and Engineering Laboratory, Special Report 93-5.

Won, W.D., R.J. Heckly, D.G. Glover and J.C.

Hoffsommer (1974) Metabolic disposition of 2,4,6-

trinitrotoluene. Applied Microbiology, 27: 513–516.

23
APPENDIX A: HOLDING-TIME STUDY RESULTS

Table A1. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane, Indiana, well A, samples 48622 and 48635.

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 7.1 0 0 134 365 12 5

Yes pH = 2.2 0 0 135 359 11 5

No 30.0 24.5 133 375 26 24

Yes 30.0 24.5 135 373 27 24

Day 7

No 0 0 144 378 5 4

Yes 0 0 126 370 11 3

No 138 376 22 19 14 4

Yes 128 370 25 25 11 6

Day 14

No 0 0 135 372 14 4

Yes 0 0 135 385 4 13 3

No 137 373 17 1 21 14 3

Yes 132 367 26 26 10 4

Day 21

No 0 0 133 372 1 11 3

Yes 0 0 134 366 3 8 5

No 130 370 12 4 19 11 5

Yes 135 368 26 29 9 4

Day 28

No 0 0 133 377 2 11 2

Yes 0 0 133 373 5 9 5

No 136 380 8 4 17 11 6

Yes 130 371 26 27 8 4

Table A2. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane, Indiana, well B, samples 48725 and 48726.

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 6.0 0 0 165 58 9 7

Yes pH = 2.5 0 0 158 55 6 5

No 75.0 36.7 166 51 70 35 11 7

Yes 75.0 36.7 155 50 69 40 4 3

Day 7

No 0 0 156 57 10 7

Yes 0 0 150 56

No 152 60 57 4 32 7 6

Yes 149 56 69 36

Day 14

No 0 0 160 58 11 6

Yes 0 0 168 58 5

No 159 55 53 5 34 11 5

Yes 163 55 72 39

Day 21

No 0 0 157 56 11 9

Yes 0 0 160 55 6

No 156 55 47 4 31 10 8

Yes 158 56 70 34

Day 28

No 0 0 155 64 11 11

Yes 0 0 159 61

No 156 62 44 7 30 11 10

Yes 177 70 78 40

25
Table A3. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane, Indiana, well C, samples 48727 and 48728.

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 6.40 0 0 173 76 2 17 59 54

Yes pH = 2.47 0 0 173 66 2 25 61 52

No 75.0 61.2 168 69 71 3 77 58 50

Yes 75.0 61.2 170 66 69 1 81 57 51

Day 7

No 0 0 164 72 4 14 59 53

Yes 0 0 170 71 3 21 58 53

No 170 85 67 4 75 58 56

Yes 166 69 68 3 79 57 51

Day 14

No 0 0 167 70 3 13 59 56

Yes 0 0 175 71 3 20 61 54

No 164 75 63 4 75 58 53

Yes 174 71 70 2 81 60 53

Day 21

No 0 0 172 69 4 10 60 53

Yes 0 0 163 65 2 21 56 52

No 169 70 62 4 73 63 54

Yes 176 68 72 2 81 59 53

Day 28

No 0 0 168 82 2 3 11 62 56

Yes 0 0 175 74 3 25 58 52

No 165 83 57 3 71 59 56

Yes 177 76 73 2 84 59 52

Table A4. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane, Indiana, well D, samples 48731 and 48732

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 6.3 0 0 252 157 5 7 110 47 65

Yes pH = 2.5 0 0 247 155 4 6 115 35 54

No 75.0 49.0 243 148 74 8 563 45 61

Yes 75.0 49.0 245 144 73 3 565 36 51

Day 7

No 0 0 249 150 4 7 102 44 63

Yes 0 0 247 156 3 4 109 20 36

No 244 152 72 6 557 44 62

Yes 240 144 72 3 557 16 35

Day 14

No 0 0 258 161 5 7 109 47 64

Yes 0 0 255 158 3 2 115 18 33

No 252 155 75 9 573 50 65

Yes 252 157 76 3 581 19 30

Day 21

No 0 0 268 154 4 7 109 49 66

Yes 0 0 251 153 5 4 118 16 31

No 263 151 74 8 575 41 67

Yes 259 155 78 4 596 22 32

Day 28

No 0 0 255 161 4 7 109 48 65

Yes 0 0 283 175 5 2 123 18 33

No 255 161 74 9 568 46 66

Yes 252 154 74 3 576 16 30

26
Table A5. Results of holding-time study for groundwater samples from the Naval Surface Warfare

Center, Crane, Indiana, well E, samples 48743 and 48744.

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 4.49 0 0 112 608 8 180 10 8

Yes pH = 2.22 0 0 101 555 3 170 9 8

No 75.0 24.5 107 601 80 405 10 6

Yes 75.0 24.5 99 550 73 399 11 8

Day 7

No 0 0 103 598 5 179 11 7

Yes 0 0 99 552 3 167 11 8

No 105 595 72 401 10 9

Yes 98 551 72 394 9 9

Day 14

No 0 0 112 624 5 183 10 8

Yes 0 0 105 587 3 175 11 9

No 111 612 73 411 9 7

Yes 102 575 74 409 11 7

Day 21

No 0 0 116 618 6 3 183 14 9

Yes 0 0 106 590 5 2 184 12 8

No 112 610 76 3 418 14 9

Yes 103 566 75 3 411 13 9

Day 28

No 0 0 114 627 4 3 178 14 10

Yes 0 0 108 590 3 2 178 11 8

No 114 619 73 2 416 14 12

Yes 107 586 76 1 414 12 7

Table A6. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane,

Indiana, well F, samples 48745 and 48746.

Conc. fortified (μg/L) Determined concentration(μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT 2,4-DNT

Day 0

No pH = 5.78 0 0 325 102 7 14 51 40

Yes pH = 2.30 0 0 316 102 2 24 17 21 4

No 75.0 122.3 373 100 69 5 128 45 39

Yes 75.0 122.3 314 97 70 1 135 17 20 3

Day 7

No 0 0 318 102 4 9 49 40

Yes 0 0 306 103 7 18 13 18 4

No 316 101 50 7 122 46 40

Yes 305 100 68 3 129 11 17 5

Day 14

No 0 0 328 104 5 5 47 39

Yes 0 0 317 106 2 22 13 15 6

No 321 104 42 10 114 49 39

Yes 323 109 72 4 140 11 17 4

Day 21

No 0 0 336 100 4 3 52 42

Yes 0 0 333 108 4 19 15 21 4

No 325 102 37 11 115 54 45

Yes 336 114 74 4 142 17 21 4

Day 28

No 0 0 331 108 5 53 9

Yes 0 0 334 114 1 22 14 21 4

No 333 110 32 13 114 55 43

Yes 322 111 73 3 145 15 21 4

27
Table A7. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane, Indiana, well G, samples 48741 and 48742.

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 3.69 0 0

Yes pH = 2.30 0 0

No 75.0 36.7 67 33

Yes 75.0 36.7 70 40

Day 7

No 0 0

Yes 0 0

No 66 33

Yes 70 38

Day 14

No 0 0

Yes 0 0

No 66 36

Yes 72 40

Day 21

No 0 0

Yes 0 0

No 68 35

Yes 74 41

Day 28

No 0 0

Yes 0 0

No 65 35

Yes 73 39

Table A8. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane, Indiana, well H, samples 48719 and 48720.

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 4.50 0 0

Yes pH = 2.27 0 0

No 75.0 36.7 68 32

Yes 75.0 36.7 68 39

Day 7

No 0 0

Yes 0 0

No 71 36

Yes 69 33

Day 14

No 0 0

Yes 0 0

No 68 32

Yes 71 36

Day 21

No 0 0

Yes 0 0

No 63 30

Yes 69 35

Day 28

No 0 0

Yes 0 0

No 64 34

Yes 71 38

28
Table A9. Results of holding-time study for groundwater samples from the Naval Surface Warfare Center, Crane, Indiana, well I, samples 48616 and 48629.

Conc. fortified(μg/L) Determined concentration (μg/L)

Acidified? TNB TNT HMX RDX TNB 3,5-DNA TNT 4ADNT 2ADNT

Day 0

No pH = 7.10 0 0

Yes pH = 2.18 0 0

No 30.0 24.5 65 82 28 23 4

Yes 30.0 24.5 68 79 28 24

Day 7

No 0 0

Yes 0 0

No 74 88 20 20

Yes 69 84 25 23 3

Day 14

No 0 0

Yes 0 0

No 67 83 14 3 19 3

Yes 71 83 26 24 4 3

Day 21

No 0 0

Yes 0 0

No 67 83 6 3 14 3 2

Yes 69 82 27 24 2 3

Day 28

No 0 0

Yes 0 0

No 68 85 3 5 15 5 2

Yes 75 93 29 27 2 2

29
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May 1995

Preservation of Water Samples Containing Nitroaromatics and Nitramines

Thomas F. Jenkins, Philip G. Thorne, Erika F. McCormick and Karen F. Myers

U.S. Army Cold Regions Research and Engineering Laboratory

72 Lyme Road Special Report 95-16

Hanover, New Hampshire 03755-1290

U.S. Army Environmental Center

Aberdeen Proving Ground, Maryland 21010-5401 SFIM-AEC-ET-CR-95009

Approved for public release; distribution is unlimited.

Available from NTIS, Springfield, Virginia 22161

38

Acidification Explosives RDX Water

Analysis Preservation TNT

UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED UL

This study was conducted to develop a method for stabilizing water samples to be analyzed for nitroaromatic and

nitramine explosives using SW846 Method 8330. Several options were tested using river water fortified with 15

nitroaromatic, nitramine, and aminonitroaromatic analytes. Acidification to pH 2 using sodium bisulfate was

selected based on its ability to retard microbiological and chemical transformations, its ease of use under field

conditions, and its usability with both the direct and preconcentration procedures in Method 8330. Holding-time

studies were performed over a 64-day storage period using fortified river water and groundwaters with and

without chemical stabilization. Nonacidified samples showed rapid loss of tetryl, TNB, and TNT and slower loss

of the dinitroaromatics. These losses were accompanied by increasing concentrations of transformation products.

Losses of these nitroaromatics were completely eliminated by acidification to pH 2. Nitramines were stable over

the entire period whether samples were acidified or not. A small loss of the aminodinitroaromatics was observed

for both acidified and unacidified samples. The rate of loss for acidified samples was initially greater than for

nonacidified samples. Sample acidification caused no adverse effects on SW846 Method 8330, although samples

to be preconcentrated using salting-out solvent extraction should be neutralized prior to extraction to prevent

additional loss of aminodinitroaromatics.