AD-A269 721 72 REL69 

Experimental Assessment of

* Analytical Holding Times for

Nitroaromatic and Nitramine

Explosives in Soil

Clarence L. Grant, Thomas F. Jenkins and Susan M. Golden June 1993

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A study was conducted to experimentally evaluate the maximum acceptable

preextraction analytical holding times (MHTs) for three nitroaromatic compounds

and two nitramines in soil. Three spiked soils and a field-contaminated soil were

utilized in the study. Analytes investigated were HMX, RDX, TNB, TNT and 2,4-

DNT, all at the low gg/g level. Subsamples of each soil were extracted with

acetonitrile in an ultrasonic bath after being held for periods of Q, 3, 7, 14, 28

and 56 days at either room temperature (220C), under refrigeration (20C) or

frozen (-15°C). Extracts were analyzed by RP-HPLC. The two nitramines, HMX

and RDX, were stable over the entire period for all soils under all storage

temperatures. For the three nitroaromatics (TNB, TNT and 2,4-DNT) the results

were very different, in that all three analytes rapidly degraded in spiked soils at

room temperature, more slowly degraded under refrigerator temperature and

remained quite stable when frozen. Of the three, TNB degraded most rapidly,

followed by TNT and 2,4-DNT, The deqradation at room temperature and in the

refrigerator was much faster for one soil than for the others. Even when frozen

there was a small loss of 2,4-DNT in the soil showing the most rapid

degradation. For the field-contaminated soil, the nitroaromatics were much

more stable, even at room temperature, although some degradation occurred.

Because of the large stability difference between fortified and field- contaminated

soils, the efficacy of using fortified soils to estimate MHTs is discussed. The

recommended MHT for soils containing only nitramines is eight weeks under

refrigeration. When nitroaromatics are present, refrigeration is inadequate and

soils should be frozen to preserve analyte integrity. When frozen, an MHT of eight

weeks is recommended.

For conversion of SI metric units to U.S./British customary units of measurement

consult ASTM Standard E380-89a, 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 93-11 -

US Army Corps

of Engineers

Cold Regions Research &

Engineering Laboratory

Experimental Assessment of

Analytical Holding Times for

Nitroaromatic and Nitramine

Explosives in Soil

Clarence L. Grant, Thomas F. Jenkins and Susan M. Golden June 1993

Prepared for

U.S. ARMY ENVIRONMENTAL CENTER

CETHA-TS-CR-93045

Approved for public rpf-,-- et4rlbtbtgon Is unlimited.
PREFACE

This report was prepared by Dr. Clarence L. Grant, Professor Emeritus, University of New

Hampshire, and Dr. Thomas F. Jenkins, Research Chemist, Geological Sciences Branch,

Research Division, U.S. Army Cold Regions Research and Engineering Laboratory, and

Susan M. Golden, Chemist, Science and Technology Corporation. Funding was provided by

the U.S. Army Environmental Center (formerly the U.S. Army Toxic and Hazardous

Materials Agency), Aberdeen Proving Ground, Maryland, Martin H. Stutz, Project Monitor.

The authors gratefully acknowledge Marianne E. Walsh and Daniel C. Leggett, CRREL,

for their technical review of this manuscript. The authors also acknowledge Patricia

Schumacher, Lawrence Perry, and Ginger Boitnott for assistance in preparation of analytical

standards, providing test soils and measurement of soil respiration, respectively. Karen

Myers, U.S. Army Engineer Waterways Experiment Station, is acknowledged for supplying

the field-contaminated test soil used in this work. Brandee Roemholt is acknowledged for

her assistance in the laboratory.

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 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.

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CONTENTS

Page

P reface ................................................................................................................................. ii

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

E xperim ental ...................................................................................................................... 3

C h em icals ........................................................................................................................ 3

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

So ils .................................................................................................................................. 3

Soil wetting and analyte spiking ................................................................................. 4

Soil respiration .............................................................................................................. . 4

Soil holding time test parameters ............................................................................... 4

Soil extraction ................................................................................................................. 5

R P-H PLC analysis ........................................................................................................ 5

D ata analysis ................................................................................................................. . 6

Results and discussion ..................................................................................................... 6

Initial analyte concentrations ....................................................................................... 6

Behavior of analytes in fortified soil as a function of holding time ....................... 8

Holding time behavior of analytes in a field-contaminated soil .............. 15

Additional comments ........................................................................................................ 16

Literature cited ................................................................................................................... 17

ILLUSTRATIONS

Figure

1. Illustration of ASTM method for estimating maximum holding time .............. 2

2. Illustration of ESE method for estimating maximum holding time .................. 2

3. Chromatograms for soil extracts, day zero ........................................................... 7

4. Chromatograms for extracts of Crane soil after seven days of soil storage

at different temperatures ................................................................................. 8

5. Chromatograms for extracts of Windsor soil after three days of soil storage

at different temperatures ................................................................................. 9

6. Chromatograms for extracts of Windsor soil after seven days of soil storage

at different temperatures ................................................................................ 10

7. Chromatograms for extracts of Windsor soil after 14 days of soil storage

at different temperatures ................................................................................ 10

8. Chromatograms for extracts of Windsor soil after 28 days of soil storage

at different temperatures ................................................................................ 12

9. Refrigerator storage effects on TNB for three fortified soils ............... 12

10. Time variation of recoveries of TNT from spiked Charlton soil and Kewanee

sediment samples ............................................................................................ 13

11. Refrigerator storage effects on TNT for three fortified soils ............... 14

12. Refrigerator storage effects on TNB and 3,5-DNA field-contaminated soil ...... 16

TABLES

Table

1. Physical and chemical properties of test soils ...................................................... 4

z. Concentration of combined analyte spiking solution and initial analyte

concentrations in test sr'Js .............................................. ......................... 4

3. Soil respiration measurements, 0-14 days at room temperature ....................... 5

iii
4. Experimental factors for soil hoiding time study ............................. u................... 5

5. Retention times of test analytes and transformation products for two

reversed-phase columns .................................... 5

6. Initial concentration of nitroaromatics and nitramines in fortified and

field-contaminated soils estimated by RP-HPLC ................... 6

7. Concentrations of analytes and transformation products as a function of

holding time and storage condition, Windsor sandy loam ............ 11

8. Concentrations of analytes and transformation products as a function of

holding time and storage condition, Charlton silty loam ............. 13

9. Concentrations of analytes and transformation products as a function of

holding time and storage condition, Ft. Edwards clay ............................... 14

10. Concentrations of analytes and transformation products as a function of

holding time and storage condition, Crane-Rockeye soil ............. 15

iv
Experimental Assessment of Analytical Holding Times for

Nitroaromatic and Nitramine Explosives in Soil

CLARENCE L. GRANT, THOMAS F. JENKINS AND SUSAN M. GOLDEN

INTRODUCTION studied, a total of 2 mL of acetonutrile was added to

each 2-g portion of soil. The spiked soils were then

Several years ago, CRREL developed a labora- vortex mixed for 30 seconds and stored at room

tory method for the determination of nitroaromatic temperature (+20°C), refrigerator temperature

and nitramine explosives in soil (Jenkins et al. (+4*C) or freezer temperature (-20°C) for eight

1989). This method was collaboratively tested time periods ranging up to 365 days. Quadrupli-

(Bauer et al. 1990) and subsequently accepted by cate subsamples for each combination of soil type

the American Society for Testing and Materials and storage temperature were analyzed at each

(ASTM 1991), theAssociation of Official Analytical time period and the resulting concentrations of

Chemists (AOAC 1990) and the Environmental each analyte plotted as a function of holding time.

Protection Agency (EPA 1992) as a standard labo- While the effect of this large amount of acetonitrile

ratory method for this determination, on the soil biota is unknown, storage of soils under

Onecriterionthatwasnotexperimentallyevalu- acetonitrile does not mimic the manner in which

ated during this method development process was normal soil samples are stored prior to analysis for

an acceptable preextraction sample holding time. nitroaromatics and nitramines. In fact, acetonitrile

Lacking available experimental data, the EPA is the extraction solvent of choice for analysis of

method established a preextraction holding time of soils for these analytes (Jenkins et al. 1989). In

seven days for soil in SW846 Method 8330 (EPA summary, while the Maskarinec et al. (1991) study

1992). This holding time was chosen to be consis- seems to be carefully done and the statistical treattent with those for other organics in a soil matrix ment of the data is extensive, we feel it suffers a

and for contractual compliance. flaw because of the use of acetonitrile for fortificaSubsequently, the U.S. Army Environmental tion and the resulting MIT estimates may not be

Center (USAEC) (formerly the Toxic and Hazard- appropriate for customary soil sample storage proous Materials Agency), the U.S. EPA and the U.S. cedures.

Navy jointly funded Oak Ridge National Labora- We also have concerns with the data treatment.

tory to conduct an experimental study to recom- MHTs were estimated using two definitions: a

mend appropriate maximum preextraction hold- modified version of an ASTM procedure (1986)

ing times (MHTs) for soils contaminated with and one reported by Prentice et al. (1986). In ASTM,

nitroaromatic and nitramine explosives (Maskari- MHT is defined as the "maximum period of time

nec et al. 1991). In this study replicate 2-g aliquots during which a properly preserved sample can be

of three different soils were placed in 40-mL glass stored before such degradation of the constituent

vials and, three days before fortification, the soils of interest occurs or change in sample matrix ocwere wetted with 0.5 mL of reagent grade water. curs that the systematic error exceeds the 99%

This was done to allow bacterial activity to come to confidence interval (not to exceed 15%) of the test

a steady state prior to fortification with the explo- about the mean concentration found at zero time."

sives. On the day the study began, each subsample The zero time mean concentration and standard

was spiked with a 0.5-mL aliquot of each indi- deviation are estimated from an appropriate numvidual explosive stock solution. These stock solu- ber of samples (usually 10) analyzed immediately

tions were in an acetonitrile matrix (Maskarinec et after collection. If an analyte concentration is less

al. 1991) and since four different analytes were than one order of magnitude higher than the crite-
1.0 I I I 1 I

•..--• Mean coacentration for day zero

S• __ MHT (21 days)

0.8 -- • v/ ".

SLower two-sided 99 % confidence "Best fit" line for change •

S0.6 m limit on day zero concentration in concentration with time

c

Oo 0.4 --

¢p

c 0.2 --

m

] J ]

20 40 60

Days of Storage

Figure 1. Illustration of ASTM method for estimating maximum holding time.

rion of detection, a bulk sample is fortified and the time mean. Fighre I is an illustration of this definizero time mean and standard deviation are rede- tion using a hypothetical example. Note that the

termined. Concentrations are then measured after number of replicates used in the confidence intervarious time intervals using a number of replicate• val calculation is the number used for each time

calculated from the percent relative standard de- interval measurement rather than the 10 replicates

viation (RSD) of the zero time results. The average used to estimate the zero time mean.

concentration found at each analysis point is plot- According to Maskarinec et al. (1991), their

ted vs. time on linear graph paper and the "best "working definition differed slightly from the exgraphical fit" to the data points is drawn. A MHT is act ASTMdefinition .... " We believe it differs greatly.

the point where the "best fit" line intersects the Their data are fitted via least squares to linear zerotwo-sided 99% confidence interval about the zero order or first-order kinetic models or, in some

1.0 f. Intercept I I I I ]

S0.8 • " • ,.... ---. --.. Least squares fit

S_ 10%intercept MHT (19 days) "" • ..•

,- change Lower one-sided

9Q % confidence intewal 0.6 --

I:::

0o 0.4 •

S0.2 •

I I ..... J I J

20 40 60

Days of Storage

Figure 2. Illustration of ESE method for estimating maximum holding time.

2
cases, to other models such as a cubic spline. The EXPERIMENTAL

zero time concentration is estimated as the intercept of the fitted model rather than the zero time Chemicals

mean, and the 99% confidence interval on the inter- All standards and test solutions were prepared

cept is calculated using the standard deviation of from Standard Analytical Reference Materials

the intercept. We are not passing judgment on the (SARMs) obtained from the USAEC. Aqueous stanappropriateness of the Maskarinec et al. (1991) dards and test solutions were prepared in reagent

procedure, but it should be clearly understood that grade water obtained from a Milli-Q Type I Reit differs markedly from the ASTM method. In agent Grade Water System (Millipore Corp.).

some cases, the intercept differs substantially from Methanol used in the preparation of HPLC eluent

the day zero mean and the standard deviation of and acetonitrile used for soil extraction were HPLC

the intercept differs greatly from the standard de- grade from Ailtech and Baker, respectively. Eluent

viation of the day zero results. was prepared by combining equal volumes of

The second method (ESE method) of estimating methanol and water and vacuum filtering through

MHTs (after Prentice et al. 1986) is defined as the a nylon membrane (0.45 pm) to degas and remove

time when a one-sided 90% confidence interval on particulate matter.

the concentration predicted by the least squares

model selected to represent the concentration vs. Analyte spiking solutions

time data falls below a 10% change in the intercept All analyte spiking solutions were prepared in

of the model. This definition is illustrated in Figure water. SARMs for 2,4,6-trinitrotoluene (TNT), 2,4-

2, again using a hypothetical example. When a dinitrotoluene (2,4-DNT), 1,3,5-trinitrobenzene

linear model is used, the slope is tested to see if it (TNB), 1,3,5-hexahydro-1,3,5-trinitrotriazine

differs significantly from zero. If it doesn't, the (RDX) and 1,3,5,7-octahydro-1,3,5,7-tetranitroMHT is the longest time tested. Based on statistical tetrazocine (HMX) were placed in individual brown

considerations, Maskarinec et al. (1991) found that glass jugs, reagent grade water was added, and the

there was not much consistency in the pattern of contents were stirred at room temperature for a

models chosen. Often three different models were week. The solutions were then filtered through

chosen for the same soil when tested at different 0.45-pm nylon membranes into clean, brown glass

storage temperatures but a given temperature pat- jugs. No solvents, other than water, were used in

terndid nothold for other soils or even for the same the preparation of these solutions.

soil at a different analyte concentration. For high The concentration of analyte in each aqueous

concentrations of explosives in soils the Prentice et spike solution was determined against standards

al. method gave MHT estimates that were always prepared in methanol or acetonitrile diluted 1:1

longer (up to 2.5 times longer) than corresponding with reagent grade water prior to analysis (Jenkins

estimates using the modified ASTM definition. et al. 1986, EPA 1992). A multianalyte spiking soluHowever, for low explosives concentrations the tion was prepared by combining appropriate volmodified ASTM definition sometimes gave longer umes of these individual analyte solutions and

estimates by as much as a factor of 6, although the filtering through a 0.2-pm nylon membrane. The

trend was not consistent. combined analyte spike solution was stored in a

Because of these very large inconsistencies, brown glass bottle in the refrigerator until used.

Maskarinec et al. (1991) had to interpret their results very conservatively. Briefly, they recom- Soils

mended storage of RDX, HMX, and 2,4-DNT con- Blank test soils were obtained locally from Vertaminated soils at 4'C (refrigerator) with a MHT of mont (Windsor), New Hampshire (Charlton) and

six weeks and TNT contaminated soils at -20'C New York (Ft. Edwards). These soils were air dried,

(freezer) also with a MHT of six weeks. groundwithamortarandpestleandpassedthrough

In the following study, we re-examine the issue a 30-mesh sieve (590 m). Some physical and chemiof MHTs for explosives-contaminated soils with cal properties of these soils are presented in Table

emphasis on 1) avoidance of organic solvent addi- 1. Replicate 5.0 ± 0.1-g subsamples of each blank

tion during soil fortification, 2) alternative ap- soil were placed in individual 20-mL glass scintilproaches to data analysis/interpretation, and 3) lation vials.

comparison of stability of fortified soils to a field- A field-contaminated soil was obtained from

contaminated soil. the Rockeye site at the Naval Surface Warfare

3
Table 1. Physical and chemical properties of test soils, exposure" and those to be stored frozen, the

Soil spiked soils were immediately placed in the

Ft. Edwards Windsor sandy Charlton silty appropriate storage temperature in the dark.

Property clay loam loam The day zero samples and the samples to be

frozen were permitted to stand for two hours

pH 8.4 6.2 6.0 after fortification to allow time for the analytes

TOC (%) 0.5 1.1 1.8 to interact with the soils prior to either extracClay (%) 70 30 20

CEC (meq/ 100 g)** >150 3.5 7.3tionor freezing. The vialscontaining the fieldcontaminated soil were treated and stored in

"Total organic carbon an identical manner as described above ex-

* Cation exchange capacity cept that no fortification was made. An estimate of the initial analyte concentrations in

Center, Crane, Indiana, courtesy of Karen Myers the field-contaminated soil is also presented in

from the U.S. Army Engineer Waterways Experi- Table 2.

ment Station, Vicksburg, Mississippi. This soil contained measurable concentrations of HMX, RDX, Soil respiration

TNT, TNB, two isomeric microbiological transfor- To ensure that the rewetted, fortified and fieldmation products of TNT (McCormick et al. 1976, contaminated soils had regained microbial activWalsh 1990), 2-amino-4,6--dinitrotoluene (2-Am- ity, three vials of each soil were placed in separate

DNT) and 4-amino-2,6-dinitrotoluene (4-Am- 250-mL Erlenmeyer flasks enclosed with a twoDNT) and 3,5-dinitroaniline (3,5-DNA), which is a hole rubber stopper. Air was slowly drawn through

microbiological transformation product of TNB. two aqueous NaOH scrubbers, through an ErlenThis bulk soil was air dried, ground with a mortar meyer containing a given soil and into a CO, collecand pestle and sieved. Subsamples of this soil were tion tube containing standard aqueous NaOH. The

weighed into separate glass scintillation vials in an CO 2 evolved from the soils was cUllected as carbonidentical manner as described above except that ate over a period of two weeks and the carbonate

since less of this soil was available, only 2.00 ± 0.01 level determined by back titration with 0.5 N HCI.

g subsamples were used. The levels of CO2 evolved are shown in Table 3.

Soil wetting and analyte spiking Soil holding time test paramcters

Prior to the onset of the experiment, previously A summary of the test parameters used for the

air-dried test soils were rewetted. Because the tex- soil holding time study is presented in Table 4. For

ture and water holding capacity of the various soils both the fortified and field-contaminated soils, three

differed, the volume of water added to each soil storage conditions were examined, room temperawas varied such that after spike additions were ture (22 ± 2°C), refrigerator storage (2 ± 2

0C) and

made, there was no evidence of free-standing water. For the three initially blank soils,

0.20 mL of reagent grade water was added to Table 2. Concentration of combined analyte spiking solution

the Windsor sandy loam and 1.00 mL was and initial analyte concentrations in test soils.

added to the Ft. Edwards Clay and Charlton Concentration

silty loam. For the field-contaminated soil Spiked Windsor,

from Crane, 0.50 mL of reagent grade water Soil spiking Ft. Edwards and Field contaminated

was added. After water addition, all soils Solution Charlton Cranesoil

were allowed to stand at room temperature Analyle (rag/L) (Pg/g)_ (Pg/g)

in the dark for three days to allow micro- lMX 1.48 0.30 2.60

biological activity to be reestablished RDX 6.68 1.33 0.44

(Maskarinec et al. 1991). TNB 4.92 0.98 0.83

Fortification of the three initially blank TNT 5.06 1.01 2.32

soils was made by carefully adding 1.00 mL 2,4-DNT 4.13 0.83 -

of a combined aqueous spiking solution with 4-Am-DNT - - 1185

2-Am-DNT • -1.18

known concentrations of HMX, RDX, TNB, 3.5-DNA - - 0.67

TNT and 2,4-DNT (Table 2) to each test vial. ---... ..

Except for the soils designated as "day zero Calculated based on measured composition of spiking solution.

4
Table 3. Soil respiration measurements, loss and confound the effect of the holding time

0-14 days at room temperature. t -mperatures. Second, a 5-g portion of soil was

used for the fortified samples instead of the usual

CO2 evolved sample size of 2 g This was necessary because the

Soil (mg evolved as C/g of soil) solubility of HMX and RDX in the aqueous spiking

Windsor sandy loam 0.93 solution is limited (4 mg/L and 42 mg/L, respecChariton silty loam 0.33 tively) as was the moisture-holding capacity of the

Ft. Edwards clay 0.48 test soils. Thus to obtain sufficiently high extract

Crane 0.31 concentrations of these analytes without exceeding

the moisture-holding capacTable 4. Experimental factors for soil holding time study. ity of the soils, larger soil

samples were required.

Factors No. of levels Levels RP-HPLC analysis

Fortified soils All soil extracts were anaAnalytes 5 HMX,RDX,TNBTNT,2,4-DNT lyzed by reversed-phase high

Soils 3 Ft. Edwards, Charlton, Windsor performance liquid chromaStorage temp. ('C) 3 -15', 2-, 22- tography (RP-HPLC). AnalyStorage time (days) 6 0,3,7,14,28,56 sis was conducted on a moduReplicates 3 a,b,c lar system composed of a

Field-contaminated soils Spe _:tra- Physics Model

Analytes 7 HMX,RDX,TNB,TNT,2-A, i-DNT,4-Am-DNT,3,5-DNA SPUeO ternary HPLC pump,

Soils 1 Crane a Spectra-Physics Spectra 100

Storage temp. ('C) 3 -15', 2-, 22- UV variable wavelength deStorage time (days) 6 0,3,7,14,28,56 tector set at 254 nm (cell path

Replicates 3 a,b,c 1 cm), a Dynatech Model LC

241 auto sampler equipped

freezer storage (-15 ± 20C). Portions stored under with a Rheodyne Model 7125 sample loop injector,

these conditions were extracted after 0, 3, 7, 14, 28 a Hewlett-Packard 3393A digital integrator and a

and 56 days of storage and the analyte concentra- Linear strip chart recorder.

tions determined. Because of expected variability All extracts were analyzed on a 25-cm x 4.6-mm

among subsamples, triplicate portions were ana- (5-pm) LC-18 column (Supelco) eluted with 1:1

lyzed foreachstoragetemperatureforeachstorage methanol/water (v/v) at 1.5 mL/min (Jenkins et

time. al. 1989). Samples were introduced by overfilling a

100-gL sampling loop. Retention times of the

Soil extraction analytes of interest are shown in Table 5. ConfirFor soil extraction, the vials containing the soil mation of identities of analytes and transformation

were warmed to room temperature and 9.00 mL of products were obtained on a 25-cm x 4.6-mm (5-

acetonitrile added. The vials were vortex mixed for

1 minute and placed in a sonic bath for 18 hours. Table 5. Retention times of test analytes and

The temperature of the bath was maintained at less transformation products for two reversed-phase

than 25°C with cooling water. The vials were then columns.

removed from the bath and allowed to stand undis- Retetion htne (6pii)

turbed for 30 minutes. A 10.00-mL aliquot of aque- Co0,.pound LC-18 LC-CN

ous CaC12 (5 g/L) was then added and the soil HMX MX2.6 9.1

particles were allowed to flocculate for 30 minutes RDX 3.8 6.1

before a 5-mL aliquot of the supematant was fil- TNB 4.9 4.0

tered through a 0.5 gm Millex SR filter. 4-amino-2-nitrotoluene 5.1 3.7

This extraction procedure was based on the 2-amino-4-nitrotoluene 5.5 3.8

method developed by Jenkins et al. (1989) (SW846 1,3-dinitrobenzene 6.0 3.9

3,5-dinitro,niline 6.8 5.0

Method 8330) with two differences. First the soils TNT 7.8 4.9

were not air dried prior to extraction, because it 4-amino-2,6-dinitrotoluene 8.7 5.3

was judged that the time required to dry the soil in 2-amino-4,6-dinitrotoluene 9.0 5.6

thevials at room temperaturecould result in analyte 2,4-DNT 9.4 4.7

5
min) LC-CN column (Supelco) under the same op- worked well for the fortified soils where standard

erational conditions (Table 5). deviations were small and the results should be

very comparable to the standard ASTM procedure.

Data analysis For the field-contaminated soil, however, more

The mean and standard deviation for each of replicates would have improved the results. The

416 sets of triplicate measurements were calcu- major weakness of this approach is the larger than

lated. Suspect individual measurements were desirable uncertainty in the day zero mean due to

flagged on the basis of extreme values of the % RSD the small number of replicat2s.

(> 50%) and inconsistencies in the overall pattern Using the day zero values as true values, perfor that compound. Eachsuspect value was checked cent recoveries were calculated for each time pefor possible computation or transcription errors. riod. Where substantial degradation was absent an

Twelve individual extreme values (four for HMX, estimate of the overall recovery was obtained by

three for RDX, three for TNT and two for 2,4-DNT) averaging across the five periods.

with no assignable cause were arbitrarily excluded

because they produced large distortions of both

means and standard deviations. In no case was RESULTS AND DISCUSSION

more than one datum excluded from a triplicate

set. These exclusions amounted to less than 1% of Initial analyte concentrations

the values. It is instructive to compare the day zero extractA modified version of the ASTM procedure was able analyte concentrations in the three fortified

used to estimate MIHTs where appropriate. Due to soils (Table 6) with the expected concentrations

time constraints and the small amount of field- calculated from the multianalyte spiking solution

contaminated soil available, triplicate measure- (Table 2). Both HMX and RDX gave slightly higher

ments were used throughout. To gain degrees of extractable concentration estimates than expected

freedom and to fairly represent precision for the in Windsor and Charlton soils, while the RDX

entire experiment, pooled standard deviations were value for Ft. Edwards was the expected one. The

calculated for the six sets of triplicates foreach soil / HMX concentration in Ft. Edwards soil could not

storage condition where rapid degradation was be reliably estimated because of a large peak elutabsent. This produced more degrees of freedom for ing very early that tails badly and causes serious

the standard deviation than the nine that wuld quantitation problems for both HMX and RDX. We

have been obtained if we had been able to run ten feel this peak results from a large number of colloireplicates on day zero as suggested by ASTM. dal particles in the extract of this high clay content

Wherea 99%confidence interval exceeded ±15% of soil. All three soils showed very similar 2,4-DNT

the day zero mean, the limits were set at ±15% as concentrations, which were in good agreement

specified in the ASTM procedure. This procedure with the expected value. The precision of these

Table 6. Initial concentration of nitroaromatics and nitramines in fortified and field-contaminated

soils estimated by RP-HPLC. The fortified soils were extracted two hours after spiking solution

was added.

Mean soil concentration and relative standard deviation

Windsor Charlton Ft. Edwards Crane

X RSD X RSD X RSD X RSD

Compound ((Pg/g) (%) (Ug/g) (%) (lg/g) (%) (pg/g) (%)

HMX 0.37 0.7 0.39 2.6 a a 2.60 37.4

RDX 1.50 0.5 1.62 2.4 1.33 6.4 0.44 12.4

TNB 0.91 0.4 0.82 1.8 0.57 26.1 0.83 26.9

TNT 0.97 0.5 0.98 2.1 0.60 15.0 232 15.1

4-Am-DNT - - - - - 1.85 8.3

2-Am-DNT - - .- 1.18 8.7

2,4-DNT 0.85 0.3 0.86 2.4 0.88 1.5 - -

3,5-DNA - .- - -. - .- 0.67 12.3

a - Interference from colloidal particles from high clay content soil.

6
Windsor Sandy Loam ChaOwon SIy Loam Ft. ,warts Ciay

X

a I.-

b.-- #

N N

,Z

L0 2 4 6 a 10 12 0 2 4 6 a 10 12 0 2 4 6 a 10 12 Retent'ionw T(merin)

Figure 3. Chromiatogramis for soil extracts, day zero.

determinations was excellent; only RDX in Ft. s- _nding to 4-Am-DNT and 2-Am-DNT are also

Edwards soil had an RSD greater than 2.6%. present in day zero extracts from Ft. Edwards clay

For TNB and TNIT in Windsor and Charlton (Fig. 3) and the peak for 3,5-DNA is larF-r than

soils, the extractable concentration estimates were observed for the Windsor and Charlton extracts.

only moderately lower than the expected values Since 3,5-DNA is a microbiological transformation

and the RSDs were again very low. The Ft" Edwards product of TNB, and 4-Am-DNT and 2-Am-DNT

soil, however, gave low recoveries of both TNB and are similarly derived from TNT, the most plausible

TNT and the RSDs were much higher than for the explanation for their presence in the day zero exother two soils. Two hypotheses that would ex- tracts is as a consequence of TNB and TNT degraplain low recoveries from Ft. Edwards soil are 1) dation during the two hours between spiking and

TNB and TNT were bound in non-extractable forms extraction.

during the two hours between spiking and extrac- The concentrations of analytes in the Crane soil

tion, and 2) TNB and TNT were partially degraded differ slightly from the fortified soils; HMX is a

during that brief period. Experimental evidenre factor of seven higher, TNT is a factor of 2.5 higher,

indicates that the second hypothesis is the correct RDX is a factor of 3.5 lower, TNB is about the same,

one. For the Windsor and Charlton soils (Fig. 3), no and concentrations of 3,5-DNA, 4-Am-DNT and 2-

chromatographic peaks other than those for the Am-DNT range from0.64 to 1.85 ýtg/g. The concenfive added analytes were observed in the day zero tration of 2,4DNT in the Crane soil was too low to

extracts except for a small peak corresponding to accurately quantify. Relative standard deviations

the retention time of 3,5--DNA and a background for the analytes in the Crane soil range from 8.3%

peak eluting just before TNB. This latter peak has for 4-Am-DNT to 37.4% for HMX, indicating that

often been observed in acetonitrile extracts of blank attempts to homogenize the soil prior to subsoils (Walsh et al. 1993). However, peaks corre- sampling were not completely successful. This

7
X

Frozen Zz Room Temperature

•- Refrigerated

I zz ZZ

Reenio Tim (rain)edRomTmpr~u~

zI C:

z . 2

CF 4e

.0 4 E

C S2

Z cV

0- S

0 4 2 8 00 2 1 4 6 8 I

condition is not unusual for field-contaminated Behavior of analytes in fortified soil

soils. Further, the limited amount of soil available as a function of holding time

made it necessary to use 2.0-g test samples and this The myean concentrations of the five fortified

small size undoubtedly contributed to the poor analytes and three transformation products are

precision by increasing the sampling error. presented in Tables 7-9 as a function of holding

Chromatograms for the extracts from the Crane time and storage condition for the Windsor,

soil also reveal small peaks corresponding to the Charlton and Ft. Edwards soils, respectively. Of

presen~ce of 1,3-dintitrobenzene (1,3-DNB) and 2- the five fortified analytes, TNB shows the most

amino-4-nitrotoluene (Fig. 4). The identification of rapid rate of degradation. For all three soils TNB

this variety of nitroaromatics; and nitramines in the concentration rapidly decreases at room temperafield-contaminated soil from Crane is consistent ture with only an average of 6.5% remaining in

with what has been reported elsewhere (Walsh and these soils after three days. This result reinforces

Jenkins 1992, Walsh et al. 1993) although the iden- our conclusion that the low initial value found for

tification of 2-amino-4-nitrotoluene has not been TNB in the Ft. Edwards clay was due to degradapreviously reported. The reason for the presence of tion in the first two hours of exposure. For refrigthese compounds in soils initially contaminated erator storage, the rate of disappearance of TNB is

with production grade TNT and RDX wastes is slower than at room temperature, but even so, only

discussed elsewhere (Walsh 1990, Walsh and an average of 15.3% remains after 7 days. Further

Jenkins 1992, Walsh et al. 1993). reduction of TNB occurs by 14 days, and by 28 days

Since the stability of these chemicals in the for- the concentration of TNB is below its detection

tified soils and the field-contaminated soils was limit. This disappearance is accompanied by the

found to be quite different, the two cases are dis- appearance of an increased level of 3,5-DNA, the

cussed separately. expected initial Microbiological transformation (rex I I8
Frozen Refrngerateod Room Temperature

X

a0

0

I

I I I I I i I I I I I i l I I I , I i 1 l I I

0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10

Retention Time (min)

Figure 5. Chromatograms for extracts of Windsor soil after three days of soil storage at different temperatures.

duction) product (McCormick et al. 1976). These the concentration decreased to the lower 99% conchanges can be seen in four chromatograms for the fidence interval representing a 15% decrease, but it

Windsor soil (Fig. 5-8) and they are summarized was no lower after 56 days. When estimates from

for all three soils at refrigerator storage in Figure 9. the five storage times were averaged and comOn a molar basis, a maximum of 36%,47% and 15% pared to day zero estimates, the mean percent

of the TNB lost could be accounted for as 3,5-DNA recovery of TNB for freezer storage of the three

for the Windsor, Charlton and Ft. Edwards soils, fortified soils was 99.3% and the average for the 56-

respectively. It is also interesting to note the slow day test was 98.6%. Given the considerable varidecrease in 3,5-DNA concentration in all three soils ability in texture among these soils, and the unonce the TNB precursor is gone. Clearly this is a avoidable daily calibration error, the overall mean

very dynamic system even under refrigeration, recovery is surprisingly close to 100%.

In contrast to the rapid degradation found at The behavior of TNT in these fortified soils

room temperature and under refrigeration, TNB is parallels that of TNB except that the rate of disapquite stable in tite frozen soils (Tables 7-9). Accord- pearance is reduced. The expected transformation

ingtoourmodifiedASTMtest, TNB is stable for the products, 2- and 4-Am-DNT (McCormick et al.

entire 56-day testperiod in Windsorsoil. In Charlton 1976), are observed to increase as TNT concentrasoil, TNB concentration does rise slightly above the tions decline. The rate of loss of TNT varies from

upper 99% confidence interval after 14 days of soil to soil in the following order: Ft. Edwards >

storage. This finding is due to a very small pooled Charlton > Windsor, the same order that was found

standard deviation for this data set, and it is of no for TNB. However, thedifference between Charlton

practical importance because the concentration and Windsor was very small. The rapid loss of TNT

change is still only 7.6% after 56 days. The greatest for the room temperature storage condition paralchange occurred in Ft. Edwards soil. After 28 days lels that observed by Maskarinec et al. (1991), in

9
Frozen Refrigerated Room Temperature

I .

1 I JAU LL

I I I a a I I a I I a a I a I I = I a I

0 2 4 6 8 10 a 2 4 6 8 10 0 2 4 6 8 10

Retention Time (rmin)

Figure 6. Chromatograms for extracts of Windsor soil after seven days of soil storage at different temperatures.

0 Ca

Frozen Refigerated Room Temperature

0r 0

XQ

0 CD 

0 2 4 6 8 10 0 4 6 8 10 0 2 4 6 8 10

Retention Time (rain)

Figure 7. Chromatograms for extracts of Windsor soil after 14 days of soil storage at different temperatures.

10
Table 7. Concentrations of analytes and transformation products as a function of holding time and storage condition,

Windsor sandy loam.

MeWuz concentration (pgig) sndard deviation (g/g)

Holding time

00 Days 03 Days 14_Dags 28 DQa 56 Daw

Compound Storage X S X S X S X S X S X S

HMX Room. temp. 0.373 0.003 0.353 0.007 0.385 0,012 0.377 0.001 0.392 0.009 0.349 0.008

Refrigerator 0.373 0.003 0.360 0.004 0.375 0.004 0.381 0.009 0.379 0-004 0.350 0.009

Freezer 0.373 0.003 0.354 0.003 0.377 0.011 0.377 0.005 0.399 0.018 0.354 0-008

RDX Room. temp. 1.500 0.007 1.355 0.019 1.608 0.005 1.568 0.003 1.622 0o015 1.572 0.016

Refrigerator 1.500 0.007 1.374 0.006 1.612 0.009 1.590 0.015 1.597 0.006 1.570 0.046

Freezer 1.500 0.007 1.368 0.008 1.600 0.023 1.575 0.004 1.633 0.025 1.586 0010

TNB Room. temp. 0.914 0.004 0.013 0.023

Refrigerator 0.914 0.004 0.598 0.020 0.300 0.030 0,090 0.027 0.013 0.001

Freezer 0.914 0.004 0.885 0.008 0.946 0,037 0.952 0,001 0.937 0.054 0.949 0.010

3,5-DNA Room. temp. 0.277 0.007 0.274 0.016 0.238 0.007 0.191 0.008 0.127 0,009

Refrigerator 0.116 0.005 0.220 0.013 0.283 0.003 0.277 0.007 0.255 0.022

Freezer 0.014 0.024 0.028 0.001

TNT Room. temp. 0.969 0.005 0.465 0,030 0.067 0.010

Refrigerator 0.969 0,005 0.861 0.005 0.777 0.013 0.637 0.043 0.309 0.026 0.086 0.017

Freezer 0.%9 0.005 0.926 0.006 0.975 0.026 0.978 0.003 0.980 0.024 0.954 0.010

4-Am-DNT' Room. temp. 0.109 0.005 0.202 0.006 0.215 0.005 0.211 0.010 0.169 0.004

Refrigerator 0.025 0.002 0.041 0.006 0.067 0.010 0.124 0.002 0.169 0.003

Freezer 0.004 0.003

2-Am-DNT Room. temp. 0.037 0.002 0.074 0.004 0.088 0.002 0.092 0.000 0.079 0.003

Refrigerator 0.010 0,004 0.016 0.004 0.031 0.002 0.051 0.002 0.065 0.018

Freezer 0.004 0.007

2,4-DNT Room. temp. 0.850 0.002 0.741 0.016 0.716 0.021 0.626 0.006 0.573 0.004 0.419 0.020

Refrigerator 0.850 0.002 0.802 0.004 0.837 0.007 0.828 0.013 0.772 0.005 0.675 0.015

Freezer 0.850 0.002 0.799 0.006 0.863 0.020 0.856 0.005 0.857 0.017 0.808 0.007

their holding time study, and also by Pennington period. With Ft. Edwards soil the TNT concentraand Patrick (1990) and Cragin et al. (1985) (Fig.10) tion reached the lower 99% confidence interval

for their low concentration spiked soils. Our results (15% change) after about 20 days. However, the

are quite different from those found by Maskarinec total decrease was still only 16.1% after 56 days.

for refrigerated storage, however. We found that When averaged across the five storage times and

for seven days of storage, the concentrations of three soils, the mean percent recovery of TNT for

TNT remaining were only 8 0%, 72% and 0%, re- freezer storage was 95.6% of the day zero concenspectively, for Windsor, Charlton and Ft. Edwards tration and the average for the 56 day test was

while Maskarinec et al. (1991) found no significant 94.4%.

TNT loss until after day 7 for the three soils studied. The stability of 2,4-DNT in these fortified soils is

The accumulation of TNT biodegradation prod- much greater than that of either TNB or TNT. At

uctsis shown in Figure 11, where the sumsof 2-and room temperature an average of 68.4% remained

4-Am-DNT are plotted along with TNT concentra- after three days. This increased stability of 2,4-DNT

tions. In contrast to 3,5-DNA, the Am-DNT concen- relative to TNT at room temperature agrees with

trations continue to increase throughout the stor- the resultsof Maskarinecet al. (1991). Under refrigage period, albeit at a slow rate after 28 days. eration an average of 86% remained after seven

When soils were frozen our modified ASTM days of storage. A slow rate of loss continued

criterion showed no significant change for TNT in throughout the study and, by 14 days, peaks correWindsor or Charlton soils during the 56-day test sponding to the expected reduction products,

11
a x× XX

Frozen a Refrigerated Cr Room Temperature

Icc

I- Z

0 CD

= 00I"• .. a IE (D

I -6

0 10 00E 

S0FE E w 9 t

0 WindsorE

Figure 8. Chroratogramsfor extracts ofCWindsorsoilafter8days i L peraures."

1.000 II

Z0 , 0 Windsor

U1 M Charlton 3 5-DNA

•l 0.600 A Ft. Edwards

"C

cr1

Z

!- 0.400

0

U0

S0.200

0 1 

0 10 20 30 40 50 60

Refrigerator Holding Time (days)

Figure 9. Refrigerator storage effects on TNB for three fortified soils.

12
Table 8. Concentrations of analytes and transformation products as a function of holding time and storage condition,

Chariton silty loam.

Mean concentration (pg/g) ± standard deviation (pg/g)

Holding time00 Days 03 Days 07 Days 14 Days__ 28 Days 56 Days

Compound Storage X S X S X S X S X S X S

HMX Room. temp. 0.387 0.010 0.414 0.032 0.389 0.026 0,399 0.027 0.378 0.009 0.358 0.008

Refrigerator 0.387 0.010 0.400 0.006 0.369 0.013 0.403 0.005 0.387 0.017 0.363 0.009

Freezer 0.387 0.010 0.389 0.010 0.391 0,006 0.409 0.016 0.390 0.003 0.371 0.013

RDX Room. temp. 1.618 0.038 1.448 0.025 1.604 0.043 1.570 0.004 1.500 0.058 1.349 0.051

Refrigerator 1.618 0.038 1.475 0.013 1.588 0.069 1.672 0.026 1.654 0.017 1.542 0.128

Freezer 1.618 0.038 1.439 0.064 1.666 0.010 1.682 0.021 1.633 0.041 1 668 0.028

TNB Room. temp. 0.817 0.014 0.119 0.036 0.059 0.004

Refrigerator 0.817 0.014 0.320 0.030 0.108 0.001 0.054 0.003 0.013 0.012

Freezer 0.817 0.014 0.820 0.034 0.854 0.012 0.884 0.012 0.833 0.020 0.879 0.029

3,5-DNA Room. temp. 0,016 0.028 0.282 0.005 0.278 0.008 0.258 0.016 0.218 0.005 0.166 0.006

Refrigerator 0.016 0.028 0.178 0.006 0.224 0.011 0.270 0.008 0.274 0.002 0.252 0.017

Freezer 0.016 0.028 0.014 0.024 0.044 0.004

TNT Room. temp. 0.977 0.021 0.437 0.028 0.190 0.006 0.072 0.008 0.008 0.007

Refrigerator 0.977 0.021 0.876 0.018 0.702 0.029 0.601 0.014 0.372 0.021 0.225 0.005

Freezer 0.977 0.021 0.940 0.041 0,963 0.014 0.993 0.018 0.944 0.037 0.984 0.026

4-Am-DNT Room. temp. 0.130 0.007 0.175 0.010 0.190 0.009 0.179 0.009 0.132 0.010

Refrigerator 0.021 0.005 0.041 0.004 0.077 0.009 0.111 0.004 0-135 0.010

Freezer

2-Am-DNT Room. temp. 0.061 0.003 0.081 0.004 0.096 0.007 0.097 0.004 0.087 0.007

Refrigerator 0.011 0.004 0.018 0.003 0.037 0,009 0.051 0.002 0.071 0.003

Freezer

2,4-DNT Room. temp. 0.860 0.021 0,793 0.009 0.751 0.024 0.667 0.040 0.574 0.011 0,426 0.027

Refrigerator 0.860 0.021 0.828 0.013 0.783 0.037 0.843 0.021 0.803 0.005 0.726 0.057

Freezer 0.860 0.021 0,813 0.037 0.850 0.014 0.869 0.016 0.825 0.032 0.839 0.018

100

80 TNT(ppm)-

. too

60 o 50-

A 10

40 5-

-a 20

>0

:100

0

roo

a:iO0l I I I I /

I--

ij- 80 -

40-

20 SEOIMENT

Figure 10. Time variation of recoveries of TNT

0 2 4 6 8 tO from spiked Charlton soil and Kewanee sediment

Storage Time (days) samples (from Cragin et al. 1985).

13
Table 9. Concentrations of analytes and transformation products as a function of holding time and storage condition,

Ft. Edwards clay.

Mean concentration (lpg/g) ± standard deviation (pug/g)

Holding time

00 Days 03 Days " 7 DaysDays 1 Days 28__a_ 56 Days

Compound Storage X S X S X S X S X S X S

HMX Room. temp.

Refrigerator

Freezer

RDX Room. temp. 1.335 0.085 1.241 0,005 1.365 0.125 1.240 0.123 1.214 0.071 1.343 0.011

Refrigerator 1.335 0.085 1.206 0.054 1.324 0.037 1.220 0.089 1.372 0.081 1.375 0.017

Freezer 1.335 0.085 1.180 0.004 1.259 0.097 1.220 0.086 iL279 0.046 1.296 0.039

TNB Room. temp. 0.566 0.148 0.020 0.034

Refrigerator 0.566 0.148 0.102 0.018

Freezer 0.566 0.148 0.609 0.310 0.477 0.221 0.538 0.108 0.480 0.041 0.477 0.081

3,5-DNA Room. temp. 0.027 0.046 0.082 0.010 0.042 0.037 0.028 0.001

Refrigerator 0.027 0.046 0.166 0.008 0.144 0.013 0.112 0.009 0.099 0.009 0.080 0.005

Freezer 0.027 0.046 0.131 0.055 0.062 0.009 0.065 0.018 0.054 0.047 0.070 0.017

TNT Room. temp. 0.5% 0.089

Refrigerator 0.596 0.089 0.130 0.025

Freezer 0.596 0.089 0.530 0.015 0.479 0.215 0.553 0.077 0.504 0.007 0.500 0.060

4-Am-DNT Room. temp. 0.110 0.035 0.205 0.034 0.143 0.018 0.109 0.006 0.086 0.024 0.056 0.021

Refrigerator 0.110 0.035 0.226 0.031 0.208 0.008 0.174 0.019 0.223 0.008 0.187 0.023

Freezer 0.110 0.035 0.210 0.054 0.087 0.013 0.106 0.027 0.106 0.030 0.135 0.036

2-Am-DNT Room. temp. 0.049 0.017 0.027 0.010 0.029 0.006 0.032 0.004

Refrigerator 0.044 0.005 0.028 0.012 0.052 0.006 0.117 0.005

Freezer 0.011 0.010 0.004 0.006

2,4-DNT Room. temp. 0.875 0.013 0.226 0.063 0.194 0.024 0.113 0.025 0.060 0.052 0.047 0.005

Refrigerator 0.875 0.013 0.768 0.024 0.586 0.057 0.426 0.050 0.391 0.036 0.315 0.017

Freezer 0.875 0.013 0.840 0.006 0.719 0.152 0.783 0.090 0.749 0.062 0.697 0.010

"a 1.000

0 Windsor

Z . Charlton TNT 0.800 . Ft Edwards

E

0i * Windsor

Charion Am NT "V Ft. Edwards A , 0.600

z

--0.400

o 0.200 A

0 10 20 30 40 50 60

Refrigerator Holding Time (days)

Figure 11. Refrigerator storage effects on TNT for three fortified soils.

14
2-amino-4-nitrotoluene and 4-amino-2-nitrotoluene 56-day study. The overall mean recoveries for HMX

(McCormick et al. 1976), were observed (Fig. 7). and RDX were, respectively, 99.8% and 97.1% for

With freezer storage, 2,4-DNT was quite stable. room temperature storage, 99.1% and 99.5% for

For the Windsor soil, our modified ASTM criterion refrigerator storage, and 100.3% and 99.0% for

was exceeded on the low side after 42 days. Once freezer storage. When HMX and RDX are the only

again, this occurred because of a very small pooled analytes of interest, these data indicate that all

standard deviation and, at 56 days, the concentra- three storage conditions are acceptable for at least

tion decrease was only 4.9%. Charlton soil showed 56 days.

no significant change during the 56-day test period. However, the Ft. Edwards soil produced a Holding time behavior of analytes in

significant 2,4-DNT decrease after 30 days and the a field-contaminated soil

loss after 56 days was 20.3%. Still, the overall mean The mean concentrations of four explosives (2,4-

recovery relative to day zero for the three soils was DNT concentration was too low to quantitate) and

94.2% and the mean for the 56-day time was 90.8%. three transformation products in Crane-Rockeye

The stability of HMX and RDX in these three soil are presented in Table 10 as a function of hold

fortified soils is much greater than that of TNB, time and storage condition. Several differences

TNT or 2,4-DNT. This agrees with the results ob- from the fortified soils are evident from these data.

tained by Maskarinec et al. (1991) and Harvey et al. First, it is apparent that triplicate analysis of 2.0-g

(1991) and is consistent with results from Hoffsom- samples of this field-contaminated soil failed to

mer et al. (1978) and Spanggord et al. (1980) who yield satisfactory precision despite efforts at hoshowed that RDX does not biodegrade under aero- mogenization. RSDs often exceeded ±25%, with

bic conditions. Regardless of storage conditions, no the poorest results found for HMX and TNT. Secloss of HMX or RDX was observed over the entire ondly, and most important, the rapid loss of

Table 10. Concentrations of analytes and transformation products as a function of holding time and storage condition,

Crane-Rockeye soil.

Mean concentration (pug/g) ± standard deviation (/ygS.-

Holdingt.tirme

00 Days 03 Days 07 Day.i 14 Days 28 Days . ._ 56Day.

Compound Storage X S X S X S X S X S X S

HMX Room. temp. 2.478 0.927 1.882 0.302 1.936 0.030 2.534 1.351 2.475 0.853 2.199 0.780

Refrigerator 2-478 0.927 2.188 0.711 1.850 0.067 1.986 0.475 1.848 0.410 2.101 0.894

Freezer 2.478 0.927 2.668 0.850 1.938 0.516 3.068 1.888 1.946 0.432 1.915 0.784

RDX Room. temp. 0.421 0.052 0.398 0.033 0.432 0.070 0.335 0.027 0.380 0.015 0.399 0.034

Refrigerator 0.421 0.052 0.421 0.035 0.447 0.100 0.355 0.037 0.467 0.042 0.351 0.058

Freezer 0.421 0.052 0.390 0.039 0.383 0.044 0.403 0.090 0.404 0.029 0.375 0.071

TNB Room. temp. 0.794 0.213 0.912 0.133 0.671 0.320 0.406 0.103 0.701 0.084 0.563 0.084

Refrigerator 0.794 0.213 0.842 0.122 1.010 0.012 0.559 0.009 0.927 0.310 0.636 0.122

Freezer 0.794 0.213 1.035 0.238 0.672 0.170 0.689 0,027 0.825 0.133 0.644 0.071

3,5-DNA Room. temp. 0.643 0.079 0.770 0,038 0.692 0.162 0.493 0.051 0.512 0.074 0.416 0.041

Refrigerator 0.643 0.079 0.705 0.110 0.786 0.050 0.625 0.010 0.775 0.133 0.633 0.041

Freezer 0.643 0.079 0.762 0.085 0.650 0.100 0.639 0.017 0.707 0.052 0.604 0.047

TNT Room. temp. 2.209 0.334 2.346 0.313 2.085 0.510 1.520 0.135 2.400 0,469 1.692 0.371

Refrigerator 2.209 0.334 2.085 0.032 2.348 0.383 2.369 0.584 2.137 0.171 1.718 0.321

Freezer 2.209 0.334 2.631 0.271 2.642 0.449 2.117 0.035 2.044 0.183 2.421 0.241

4-Am-DNT Room. temp. 1.765 0.147 2.034 0.134 1.909 0.308 1.587 0.061 1.667 0.087 1.501 0.097

Refrigerator 1.765 0.147 1.848 0.099 2,068 0.091 1.699 0.068 1.939 0.155 1.818 0.142

Freezer 1.765 0.147 1.955 0.158 1.754 0.130 1.700 0.053 1.802 0.129 1.695 0.090

2-Am-DNT Room. temp. 1.130 0.099 1.285 0.129 1.205 0.187 1.077 0.055 1.149 0.056 1.087 0.105

Refrigerator 1.130 0.099 1.167 0,090 1,262 0.086 1.059 0.044 1.204 0.086 1.137 0.081

Freezer 1.130 0.099 1.236 0.074 1,083 0.062 1.081 0.032 1.155 0.082 1.085 0.061

15
1.000 , I I I I f I

Crane

40.800 TNB

Zi

0

Crane

3, 5-DNA

00.600

Z

Z 0.400

I 0.200

0

0 10 20 30 40 50 60

Refrigerator Holding Time (days)

Figure 12. Refrigerator storage effects on TNB and 3,5-DNA for fieldcontaminated soil.

nitroaromatics observed with the fortified soils is soil or storage condition. We doubt these empirical

not evident in the field contaminated soil. Also, fits imply any fundamental relationships. The probthere is no significant increase with time for the lem is further exacerbated by 1) nonrandom caliconcentrations of degradation products. However, bration errors (day-to-day) that cannot be sepathese compounds are initially present at much rated from real changes in analyte concentrations,

higher concentrations than ever found in the forti- and 2) by distortion of experimental errors caused

fied soils. These points are illustrated in Figure 12 by transformations of data (Motulsky and Ransnas

for TNB and 3,5-DNA. The striking similarity in the 1987). We believe that our modified ASTM procepatterns of vaiiationwith time forthese two analytes dure using a pooled standard deviation is an acoffers convincing evidence that random sampling ceptable way to estimate MHTs. However, future

errors are a dominant factor controlling results. work should employ preliminary studies to estiIn view of the poor precision, our modified mate the required number of replicates for acceptASTM procedure to estimate MHTs was not ap- able precision of means, and more replicates should

plied. Only the isomers of Am-DNT yielded 99% be used for day zero data. For organic analytes in

confidence intervals that were less ±15%. How- field-contaminated soils, consideration should be

ever, overall mean recoveries relative to day zero given to relaxing the limits for 99% confidence

were calculated. For freezer storage, mean recover- from ±15% to ±20 or 25% to accommodate insuries were 93.1%, 92.9%, 97.4% and 107.3% for HMX, mountable sample heterogeneity problems.

RDX, TNB and TNT, respectively. Comparable Results from fortified soils appear most applivalues for refrigerator storage given in the same cable to freshly contaminated soils such as one

order were 80.5%, 97.0%, 100.8% and 96.5%. If we might find near the front of a moving groundwater

consider the large uncertainties in the day zero plume. If we assume that future studies will conmeans, these recovery estimates do not suggest firm the difference observed here between fortified

rapid degradation of these analytes in the Crane and field-contaminated soils, MHTs for sites with

soil. long-standing contamination could be based on

soils from similar sites because this offers the potential to extend MHTs. Where this is impractical,

ADDITIONAL COMMENTS MHTs should be based on worst case results, which

appear to result from fortified soils. Based on reLeast squares models were not fitted to our data. suits for fortified soils, when HMX and RDX are the

Maskarinec et al. (1991) required five different only analytes of interest, either refrigeration or

models to fit all their data and there was no consis- freezing are acceptable storage conditions for at

tent pattern of the "best fit" models as a function of least eight weeks. When nitroaromatics are to be

16
determined, soil differences become important, as will lead to unnecessarily restrictive sample hannoted from the muchmore rapid degradation found dling procedures and storage times. At some time

in Ft. Edwards clay compared to Windsor and these comparisons should be extended to other

Charlton loams. Samples should be immediately biodegradable organic compounds with different

frozen. At -15°C, TNB and TNT remain acceptably binding strengths to soils.

stable for eight weeks and 2,4-DNT is stable for

four weeks. From practicalconsiderations, 2,4-DNT

will still give acceptable results after being frozen LITERATURE CITED

for eight weeks.

There remains a further unresolved issue rela- Association of Official Analytical Chemists (1990)

tive to the effects of air drying. In an earlier study, Munition residues in soil, liquid chromat, graphic

Bauer et al. (1989) spiked air-dried soils with a method. Official First Action, September, 1990.

series of nitroaromatics and nitramines in ACN, Method 991.09, Second Supplement to the 15th

evaporated the ACN and studied the stability of Edition of Official Methods of Analysis, pp. 78-80.

these analytes over a 62-day period under refrig- Association of Official Analytical Chemists.

eration. Their results indicate that these analytes American Society forTesting and Materials (1986)

are stable once the soil is dry. In our study, how- Standard practice for estimation of holding time

ever, we find substantial degradation in only two for water samples containing organic and inorhours for TNB and TNT added to wet soils main- ganic constituents. American Society for Testing

tained at room temperature. This raises the issue and Materials, Method D4841-88.

of when to dry soils for homogenization and American SocietyforTesting and Materials (1991)

subsampling. Regardless of whether soils are air Method foranalysisof nitroaromatic and nitramine

dried after sampling and before freezing, or not explosives in soil by high performance liquid chrountil they are thawed, significant loss of TNB and matography. American Society for Testing and

TNT is possible. Furthermore, there is some evi- Materials, Method D5143-90.

dence to suggest more microbiological activity on Bauer, C.F., S.M. Koza and T.F. Jenkins (1990)

thawing than before freezing (Skagland et al. 1988). Collaborative test results for a liquid chromatoIs it possible that freezing would be unnecessary if graphic method for the determination of explosive

soils are first dried? Is there any way to speed the residues in soil. Journal of the Association of Official

drying process to minimize microbiological degra- Analytical Chemists, 73: 541-552.

dation? Although freeze drying could possibly Cragin, J.H., D.C. Leggett, B.T. Foley and P.W.

minimize the problem (Cragin et al. 1985), a ques- Schumacher (1985) TNT, RDX, and HMX explotion remains as to the practicality of this alternative sives in soils and sediments: Analysis techniques

for general usage. Will drying have a different and drying losses. USA Cold Regions Research and

effect on fortified samples compared to field- Engineering Laboratory, CRREL Report 85-15.

contaminated samples? Must we avoid drying and, Environmental Protection Agency (1992)

therefore, homogenization, in order to prevent large Nitroaromatics and nitramines by HPLC. Second

losses of analytes such as TNB and TNT? Separate Update SW846 Method 8330.

samples could beair dried for moisture correction.AUl Harvey, S.D., R.J. Fellows, D.A. Cataldo and R.M.

of these issues should be addressed in future work. Bean (1991) Fate of the explosive hexahydro-1,3,5-

The large difference in the behavior of nitro- trinitro-1,3,5-triazine (RDX) in soil and bioaccumaromatics behavior between fortified and field- ulation in bush bean hydroponic plants. Environcontaminated soils is extremely important. Even mental Toxicology and Chemistry, 10: 845-855.

with avoidance of the addition of unnatural sol- Hoffsommer, J.C., L.A. Kaplan, D.J. Glover, D.A.

vents during fortification, the behavior of nitroaro- Kubose, C. Dickinson, H. Goya and M.E. Sitzmann

matics appears not to accurately mimic soils con- (1978) Biodegradablity of TNT: A three-year pilot

taminated in the field over extended periods of study. Silver Spring, Maryland: Naval Surface

time. To better define these differences, there is Weapons Center, NSWC/WOL-77-136.

need for more extensive studies of field-contami- Jenkins, T.F., M.E. Walsh, P.W. Schumacher, P.H.

nated soils under conditions where sample size Miyares, C.F. Bauer and C.L. Grant (1989) Liquid

and replicate numbers are not arbitrarily limited. If chomatographic method for the determination of

analytes in field-contaminated soils areconsistently extractable nitroaromatic and nitramine residues

found to be much more stable than in fortified soils, in soil. Journal of the Association of Official Analytical

continued use of fortified soils to estimate MHTs Chemists, 72: 890-899.

17
Maskarinec, M.P., C.K. Bayne L.H. Johnson, S.K. Skagland, T.S., S. Lomeland and J. Goksoyr (1988)

Holladay, R.A. Jenkins and B.A. Tomkins (1991) Respiratory burst after freezing and thawing of

Stability of explosives in environmental wa.'r and soil: Experiments with soil bacteria. Soil Biology and

soil samples. Oak Ridge National Laboratory Re- Biochemistry, 20: 851-856.

port ORNL/TM-11770, Oak Ridge, Tennessee, Spanggord, R.J., T. Mill, T. Chou, W. Mabey, J.

37831. Smith and S. Lee (1980) Environmental fate studMcCormick, N.G.,F.E.FeeherryandH.S.Levinson ies on certain munition wastewater constituents-

(1976) Microbial transformation of 2,4,6-trinitrotolu- lab studies. Menlo Park, California: SRI Intemaene and other nitroaromatic compounds. Applied tional. ADA099256.

and Environmental Microbiology, 31(6): 949-958. Walsh, M.E. (1990) Transformation products of

Moltulsky, H.J. and LA. Ransnas (1987) Fitting nitroaromatics and nitramines: Literature review

curves to data using nonlinear regression: a practical and recommendations for analytical method deand nonmathematical review. FASEB J., 1: 365-374. velopment. USA Cold Regions Research and EngiPennington, J.C. and W.H. Patrick, Jr. (1990) Ad- neering Laboratory, Special Report 90-2.

sorption and desorption of 2,6,4-trinitrotoluene by Walsh, M.E. and T.F. Jenkins (1992) Identification

soils. Journal of Environmental Quality, 19: 559-567. of TNT transformation products in soil. USA Cold

Prentice, H.S., J.E. Singley, L.J. Bilello, J.T. Regions Research and Engineering Laboratory,

McClare, K.C. Tuttle, E.M. Kellar and M.G. Special Report 92-16.

Schultz (1986) Development of preservation tech- Walsh, M.E., T.F. Jenkins, P.S. Schnitker, J.W.

niques and establishment of maximum holding Elwell and M.H. Stutz (1993). Evaluation of SW856

times. Inorganic Constituents of the National Pol- Method 8330 for characterization of sites contamilutant Discharge Elimination System and Safe nated with residues of high explosives. USA Cold

Drinking Water Act. EPA/600/4-86/043, Decem- Regions Research and Engineering Laboratory,

ber 1986, EMSL, Cincinnati, Ohio, 45268. CRREL Report 93-5.

18
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4, TITLE AND SUBTITLE 5. FUNDING NUMBERS

Experimental Assessment of Analytical Holding Times for

Nitroaromatic and Nitramine Explosives in Soil

6. AUTHORS

Clarence L. Grant, Thomas F. Jenkins, Susan M. Golden

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION

REPORT NUMBER

U.S. Army Cold Regions Research and Engineering Laboratory

72 Lyme Road Special Report 93-11

Hanover, New Hampshire 03755-1290

9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING/MONITORING

AGENCY REPORT NUMBER

U.S. Army Environmental Center

Aberdeen Proving Ground, Maryland 21010 CETHA-'IS-CR-93045

11. SUPPLEMENTARY NOTES

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Approved for public release; distribution is unlimited.

Available from NTIS, Springfield, Virginia 22161

13. ABSTRACT (Maximum 200 words)

A study was conducted to experimentally evaluate the maximum acceptable preextraction analytical holding times (MHTs)

for three nitroaromatic compounds and two nitramines in soil. Three spiked soils and a field-contaminated soil were utilized

in the study. Analytes investigated were HMX, RDX, TNB, TNT and 2,4-DNT, all at the low ig/g level. Subsamples of each

soil were extracted with acetonitrile in an ultrasonic bath after being held for periods of 0, 3, 7, 14, 28 and 56 days at either

room temperature (22°C), under refrigeration (2QC) or frozen (-15'C). Extracts were analyzed by RP-HPLC. The two

nitramines, HMX and RDX, were stable over the entire period for all soils under all storage temperatures. For the three

nitroaromatics (TNB, TNT and 2,4-DNT) the results were very different, in that all three analytes rapidly degraded in spiked

soils at room temperature, more slowly degraded under refrigerator temperature and remained quite stable when frozen. Of

the three, TNB degraded most rapidly, followed by TNT and 2,4-DNT. The degradation at room temperature and in the

refrigerator was much faster for one soil than for the others. Even when frozen there was a small loss of 2,4--DNT in the soil

showing the most rapid degradation. For the field-contaminated soil, the nitroaromatics were much more stable, even at room

temperature, although some degradation occurred. Because of the large stability difference between fortified and fieldcontaminated soils, the efficacy of using fortified soils to estimate MHTs is discussed. The recommended MHT for soils

containing only nitramines is eight weeks under refrigeration. When nitroaromatics are present, refrigeration is inadequate

and soils should be frozen to preserve analyte integrity. When frozen, an MHT of eight weeks is recommended.

14. SUBJECT TERMS 15. NUMBER 0 PAGES

Analyte holding time Nitramine explosives 16. PRICE CODE

Nitroaromatic explosives Soil contamination

17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT

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